Saccharification Stage Research Articles

Applying huaimi (flos sophorae immaturus) as a brewing adjunct and its impact on sensory properties of beer

Huaimi is a precious Chinese herbal medicine mainly containing rutin. In this study, Cyan Huaimi (CH) and Golden Huaimi (GH) were introduced at various stages of beer production. A total of 8 beer groups and 1 control group were set up to compare their physicochemical, bioactivity, volatile compounds and sensory characteristics. The results indicated that compared with the control group, adding Huaimi significantly reduced the pH and real extract of beer, and improved the other Physicochemical parameters. Furthermore, the addition of Huaimi brought higher levels of total polyphenols, flavonoids, and phenols compounds in 8 beer groups, particularly rutin content, reaching as high as 212.69 mg/L. Moreover, through the evaluation of DPPH, ABTS, and reducing power methods revealed that adding Huaimi at the boiling stage benefited most in improving antioxidant capacity. However, the results of sensory evaluation demonstrated that panelists preferred the beer with Huaimi added at the saccharification stage because of its smoother foam. Volatiles analysis revealed that adding Huaimi introduced twelve new volatiles to the beer, enhancing its flavor and taste complexity.

Effect of Hydrolysis Reactor Design on Maltodextrin Production

Maltodextrin is a short-chain polysaccharide compound resulting from the hydrolysis of starch, which is easily soluble in water, so it has very wide applications for food and health. The production of maltodextrin uses the method of reaction of starch hydrolysis with enzymes carried out using fermenters. The temperature used in the fermenter is 95 °C with a residence time of about 12 hours to reduce the risk of failure in the enzymatic reaction. The stirrer used is a turbine with 6 flat blades. The selection of this type because it has an excellent mixing rate, especially for microscale products. The enzymes that react are alpha-amylase at the liquification stage, and the glucoamylase at the saccharification stage. The yield of maltodextrin is 95% and 5% is the remaining water produced based on the starch hydrolysis process that occurs in the fermenter.

Aflatoxin Decontamination in Maize Steep Liquor Obtained from Bioethanol Production Using Laccases from Species within the Basidiomycota Phylum.

Maize (Zea mays L.) is an important crop in Argentina. Aspergillus section Flavi can infect this crop at the pre-harvest stage, and the harvested grains can be contaminated with aflatoxins (AFs). During the production of bioethanol from maize, AF levels can increase up to three times in the final co-products, known as, dry and wet distiller's grain with solubles (DDGS and WDGS), intended for animal feed. Fungal enzymes like laccases can be a useful tool for reducing AF contamination in the co-products obtained from this process. The aim of the present study was to evaluate the ability of laccase enzymes included in enzymatic extracts (EE) produced by different species in the Basidiomycota phylum to reduce AF (AFB1 and AFB2) accumulation under the conditions of in vitro assays. Four laccase activities (5, 10, 15, and 20 U/mL) exerted by nine isolates were evaluated in the absence and presence of vanillic acid (VA), serving as a laccase redox mediator for the degradation of total AFs. The enzymatic stability in maize steep liquor (MSL) was confirmed after a 60 h incubation period. The most effective EE in terms of reducing AF content in the buffer was selected for an additional assay carried out under the same conditions using maize steep liquor obtained after the saccharification stage during the bioethanol production process. The highest degradation percentages were observed at 20 U/mL of laccase enzymatic activity and 1 mM of VA, corresponding to 26% for AFB1 and 26.6% for AFB2. The present study provides valuable data for the development of an efficient tool based on fungal laccases for preventing AF accumulation in the co-products of bioethanol produced from maize used for animal feed.

Characterization of a New Glucose-Tolerant GH1 β-Glycosidase from Aspergillus fumigatus with Transglycosylation Activity.

Concern over environmental impacts has spurred many efforts to replace fossil fuels with biofuels such as ethanol. However, for this to be possible, it is necessary to invest in other production technologies, such as second generation (2G) ethanol, in order to raise the levels of this product and meet the growing demand. Currently, this type of production is not yet economically feasible, due to the high costs of the enzyme cocktails used in saccharification stage of lignocellulosic biomass. In order to optimize these cocktails, the search for enzymes with superior activities has been the goal of several research groups. For this end, we have characterized the new β-glycosidase AfBgl1.3 from A. fumigatus after expression and purification in Pichia pastoris X-33. Structural analysis by circular dichroism revealed that increasing temperature destructured the enzyme; the apparent Tm value was 48.5 °C. The percentages of α-helix (36.3%) and β-sheet (12.4%) secondary structures at 25 °C were predicted. Biochemical characterization suggested that the optimal conditions for AfBgl1.3 were pH 6.0 and temperature of 40 °C. At 30 and 40 °C, the enzyme was stable and retained about 90% and 50% of its activity, respectively, after pre-incubation for 24 h. In addition, the enzyme was highly stable at pH between 5 and 8, retaining over 65% of its activity after pre-incubation for 48 h. AfBgl1.3 co-stimulation with 50-250 mM glucose enhanced its specific activity by 1.4-fold and revealed its high tolerance to glucose (IC50 = 2042 mM). The enzyme was active toward the substrates salicin (495.0 ± 49.0 U mg-1), pNPG (340.5 ± 18.6 U mg-1), cellobiose (89.3 ± 5.1 U mg-1), and lactose (45.1 ± 0.5 U mg-1), so it had broad specificity. The Vmax values were 656.0 ± 17.5, 706.5 ± 23.8, and 132.6 ± 7.1 U mg-1 toward p-nitrophenyl-β-D-glucopyranoside (pNPG), D-(-)-salicin, and cellobiose, respectively. AfBgl1.3 displayed transglycosylation activity, forming cellotriose from cellobiose. The addition of AfBgl1.3 as a supplement at 0.9 FPU/g of cocktail Celluclast® 1.5L increased carboxymethyl cellulose (CMC) conversion to reducing sugars (g L-1) by about 26% after 12 h. Moreover, AfBgl1.3 acted synergistically with other Aspergillus fumigatus cellulases already characterized by our research group-CMC and sugarcane delignified bagasse were degraded, releasing more reducing sugars compared to the control. These results are important in the search for new cellulases and in the optimization of enzyme cocktails for saccharification.

Non-ionic surfactant formulation sequentially enhances the enzymatic hydrolysis of cellulignin from sugarcane bagasse and the production of Monascus ruber biopigments

The effect of a non-ionic surfactant optimized formulation (SOF) obtained from an experimental design was evaluated for different influencing variables in the processing of sugarcane bagasse cellulignin to produce biopigments. The major findings in the saccharification stage using the SOF point that: at same enzyme loading, the highest glucan hydrolysis yield was 63 % (2-fold higher compared to control); the enzyme loading of 2.5 FPU/g resulted in similar yield compared to 10 FPU/g (control); 15 % (m/v) of total solids loading maintained the yield in fed-batch configuration; the hydrolysis yield is maintained at high shear force stress (800 rpm of stirring rate) and temperatures (50–70 °C). Besides, under separate and semi-simultaneous hydrolysis and fermentation, the maximum biopigments production were of 10 AU510nm/mL and 17.84 AU510nm/mL, respectively. The SOF used in this study was found to be a promising additive either in a single or sequential steps to produce biopigments in biorefineries.

Optimization of a Simultaneous Enzymatic Hydrolysis to Obtain a High-Glucose Slurry from Bread Waste.

Bread and bakery products are among the most discarded food products in the world. This work aims to investigate the potential use of wasted bread to obtain a high-glucose slurry. Simultaneous hydrolysis of wasted bread using α-amylase and glucoamylase was carried out performing liquefaction and saccharification at the same time. This process was compared with a traditional sequential hydrolysis. Temperature and pH conditions were optimized using a response surface design determining viscosity, reducing sugars and glucose concentration during the enzymatic processes. The optimal conditions of pH and temperature in the saccharification stage and the simultaneous hydrolysis were pretty similar. Results show that the slurry produced with simultaneous process had a similar glucose yield at 2 h, and at 4 h a yield higher than that obtained by the sequential method of 4 h and could reduce time and energy.

Fermentasi Singkong Karet (Monihot carthagenensis MÜII) menjadi Etanol dengan Pemanfaatan Albumin Dari Ikan Gabus (Channa striata) Sebagai Sumber Nitrogen Bagi Mikroba

Has been cassava rubber fermentation (M. carthagenensisMÜll) as a raw material for making ethanol with the utilization of fishcork albumin (Channastriata) as nitrogen Saccharomyces cerrevisiae with enzymatic hydrolysis result through 2 stages ieliquification using α-amylase and saccharification stage using gluco -amilase. The best reducing sugar content was obtained at 0.1307%. Based on this research, maximum ethanol content was obtained with 0.5% concentration of nutrient at fermentation time for 232 hours, obtained by result of 12,688% at 5.6 mL distillate volume using gas chromatography method.

Modeling Based Identifiability and Parametric Estimation of an Enzymatic Hydrolysis Process of Amylaceous Materials.

This work presents the modeling of an enzymatic hydrolysis process of amylaceous materials considering the parameter identification problem as a basis for the construction of the model. For this, a modeling methodology is modified in order to apply the identifiability property and improve the proposed model structure. A brief theoretical explanation of the identifiability is described. This concept is based on the observability property of a nonlinear dynamic system. The used methodology is based on the phenomenological based semiphysical model (PBSM). This methodology visualizes that the structure of a dynamic model can only improve with new mass or energy balances suggested by model suppositions. Additionally, a computer algorithm is included in the methodology to validate if the model is structurally locally identifiable or know if the parameters are unidentifiable. Also, an optimization algorithm is used to obtain the numeric values of the identifiable parameters and, hence, guarantee the validity of the result. The methodology focuses on the liquefaction and saccharification stages of an enzymatic hydrolysis process. The results of the model are compared with experimental data. The comparison shows low errors of 7.96% for liquefaction and 7.35% for saccharification. These errors show a significant improvement in comparison with previous models and validate the proposed modeling methodology.

RETRACTED: Unraveling the composition and succession of microbial community and its relationship to flavor substances during Xin-flavor baijiu brewing

A novel baijiu type, Xin-flavor, has been developed via combining Qu and craftsmanship from three famous baijiu types including light-flavor, sauce-flavor, and strong-flavor. However, the microbial community and its relationship to physicochemical factors and flavor substances were little known during the three-stage brewing processes including saccharification, stacking, and fermentation. In this study, 21 phyla, 407 genera, and 615 species distributed in bacteria and fungi were identified via high-throughput sequencing (HTS). For bacterial community, the dominant bacteria at saccharification stage were Weissella and Leuconostoc, which were superseded by Acetobacter and Gluconobacter at stacking stage, then quickly replaced by Lactobacillus at fermentation stage. For fungal community, Rhizopus, dominant at saccharification stage, was partially replaced by Thermomyces at stacking stage, then was completely superseded by Thermoascus and three yeasts (Saccharomyces, Kazachstania and Apiotrichum) at fermentation stage. The moisture, ethanol, and acidity, as key factors affecting the microbial community, explained this microbial succession. Interestingly, Apiotrichum was firstly detected in fermented grains. Furthermore, the rapid accumulation of ethanol mainly occurred in the first 10days of fermentation stage, and highly correlated with Saccharomyces, while Kazachstania and Apiotrichum were related to the formation of flavor substances (acetic acid, lactic acid, caproic acid, ethyl lactate and ethyl caproate). Therefore, this study provides fundamental basis for the rational control of baijiu production and improving the craft and quality of Xin-flavor baijiu.

Bioethanol Production from Wastewater of Brown Sugar Home Industry in Kediri via Enzymatic Hydrolysis and Fermentation

Bioethanol is ethanol whose main ingredients are from plants and generally use a pharmaceutical process. Therefore, Indonesia still needs a more effective source of bioethanol as fuel. Bioethanol production from vegetable waste is a realistic solution, one example is waste from the brown sugar home industry. The purpose of this research is to innovate the production of bioethanol by utilizing sap sugarcane waste in a brown sugar industry home using the Enzyme Hydrolysis and Fermentation Method. The process of making bioethanol is the raw material preparation stage, the hydrolysis stage, and the fermentation stage. The first stage, the preparation of raw materials is done by filtering the molasses waste and then dissolving it with distilled water in 2000 mL Erlenmeyer. the second stage, the hydrolysis stage, is to hydrolyze the molasses solution according to the predetermined ratio variables, by going through two stages in the hydrolysis stage, namely liquefaction and saccharification. In the liquefaction process, -amylase is added at a temperature of 90oC and heated on a hot plate stirrer for 2 hours. Then the saccharification stage was carried out by adding the enzyme gluco-amylase at a temperature of 65oC for 4 hours. The third stage, the fermentation stage, was carried out with variations of bread and yeast tape with the additional variables of 5%, 10%, and 15% nutrient (2% urea and 3%). Fermentation will be carried out for 3 days. The results showed that the maximum sugar content was achieved by using a concentration ratio of sugarcane juice: water = 1:0 with an enzyme hydrolysis process of 196.08 g/L. The maximum bioethanol content after fermentation was 18.6% and reducing sugar of 10.98 g/L was achieved by using 10% baker's yeast at a concentration of sugar cane juice: water ratio = 1:1. The maximum bioethanol content after the fermentation process was 12.96% and the reducing sugar was 27.78 g/L was achieved using 10% tape yeast at a concentration ratio of sugarcane juice: water = 1:1.

Deciphering the Shifts in Microbial Community Diversity From Material Pretreatment to Saccharification Process of Fuyu-Flavor Baijiu.

The microbiota of the pretreatment phase is crucial to the assembly of the microbial community in the saccharification of fuyu-flavor baijiu. This study investigates the shifts in microbial community diversity from the pretreatment of raw materials to the end of saccharification. High-throughput sequencing reveals that Lactobacillus, Weissella, and Bacillus in the bacterial community and Rhizopus, Candida, Pichia, and Aspergillus in the fungal community are predominant during raw material pretreatment and saccharification processes. Also, 11 bacterial genera, including Bacillus, Lactobacillus, Leuconostoc, Weissella, Lactococcus, and Acetobacter, and eight yeast genera, including Candida, Pichia, Saccharomyces, and Wickerhamomyces, were isolated from the initial saccharification stage by culture-dependent approaches. Sourcetracker analysis indicates that the cooling grains and rice husks were the main contributors to the bacterial community composition of the saccharification process, and Qu was the main contributor to the shaping of the fungal community structure during the saccharification process. Abundance variation of the predictive functional profiles of microbial communities encoding for key enzymes involved in pyruvate metabolism, starch and sucrose metabolism, and glycolysis/gluconeogenesis during the pretreatment and saccharification phases were inferred by PICRUSt2 analysis. The results of this study will be utilized to produce consistently high-quality fuyu-flavor baijiu via better controlling the shaping of microbial community structures during the pretreatment and fermentation processes.

Optimization of the process of saccharification in ethanol production

The saccharification stage in ethanol production is one of the main ones. Completeness of dextrins and starch ejection considered in the fermentation compartment and, consequently, quantity of production losses, quality and yield of the final product depends on the correctness of this process. Optimization of the process of saccharification of digested mass was carried out by the method of complete factor experiment with the use of the central composite rotatable uniform planning experiment. The following factors were selected as controllable: dosage of the enzyme preparation Biozim 800L; temperature; pH and duration of hydrolysis. The dosage of the enzyme preparation Biozim 800L and the duration of the process have the greatest influence on the process of saccharification. The significance of the regression coefficients was determined by Student's test, the adequacy of the regression equation - by Fisher's test at a significance level of 5%, the convergence of parallel determinations - by Kochner's test. It was found that the use of the enzyme preparation Biozim 800L increased the accumulation of glucose by 40.5% compared with the control. To determine the optimal mode of saccharification the Lagrange multiplier method was used. It was found that the maximum accumulation of glucose in the wort of 12 g/100 ml was achieved at the following regimes: the dosage of the enzyme preparation Biozim 800L 4.8 units GlS/g starch; temperature 60.2 C; pH 4.55 units; duration of saccharification 2.05 h.

Analysis of Saccharification Products of High-Concentration Glutinous Rice Fermentation by Rhizopus nigricans Q3 and Alcoholic Fermentation of Saccharomyces cerevisiae GY-1.

A two-stage process was used to prepare rice alcohol, i.e., saccharification of glutinous rice by Rhizopus nigricans Q3, followed by Saccharomyces cerevisiae’s fermentation. Rhizopus nigricans Q3 was cultured during the saccharification stage, and Saccharomyces cerevisiae GY-1 was added in the fermentation stage. Total sugar content and reducing sugar content in these two stages were analyzed. The relationship between the production proportion and consumption of the reducing sugar in the saccharification interval was analyzed using reducing sugar indices. It is an important rule that the high-concentration syrup and oligosaccharides prepared by glutinous rice could reach 42°Bx and 250 mg/mL by high-concentration fermentation in the growth stage of R. nigricans Q3.

Valorization of brewer's spent grain by different strategies of structural destabilization and enzymatic saccharification

The valorization of brewer’s spent grain (BSG), the main solid waste from this industry, is a topic of concern. The structural modifications generated by physicochemical pretreatments (autohydrolytic, H2O2 and H2SO4) to promote enzymatic accessibility and thus exploit this residue were assessed by Fourier transform infrared spectroscopy, thermogravimetric analysis and scanning electron microscopy. After the pretreatments, a saccharification stage was conducted by an enzyme cocktail generated with high productivity (1.52 IU/(gds*d) filter paper activity, FPA, and 1.55 IU/(gds*d) endoglucanases, EGA, and 48.37 IU/(gds*d) xylanases) by Trichoderma reesei with BSG as substrate. Biomass recovery and sugar yields after pretreatments were consistent with hemicellulose and lignin removal and structural alterations generated. When the BSG was treated with H2O2 the enzymatic sugar release is efficient, obtaining 193.35 mg sugars/g dry BSG. Thus, conducting this pretreatment with H2O2 at low pH (5.0) and its combination with enzyme saccharification represents a promissory novel strategy to enhance the potential exploitation of this residue, using an economical and environmentally safe process.

TOFU DREGS AS A NUTRIENT FOR Saccharomyces cerevisiae IN THE PRODUCTION OF BIOETHANOL FROM RED RICE (Oryza nivara) THROUGH FERMENTATION

Research on the production of bioethanol from red rice flour starch (Oryza nivara) through enzymatic hydrolysis process and fermentation by Saccharomyces cerevisiae with the addition of tofu dregs as a nutrient source of microorganisms has been conducted. The process of bioethanol production consisted of hydrolysis, fermentation, distillation and ethanol content analysis using gas chromatography instrument. This research was conducted to determine the concentration of nutrients and the duration of fermentation to produce optimal ethanol levels. The hydrolysis process is carried out enzymatically through the liquification stage using ?-amylase and gluco-amylase at the saccharification stage. Then the fermentation process continued with a variation of 7, 8 and 9 days using Saccharomyces cerevisiae with the addition of tofu dregs concentration 0.5 %, 1 % and 1.5 % (w/v). The highest ethanol levels obtained from gas chromatography analysis was the addition of tofu dregs 1 % (w/v) and fermentation time for 8 days, with ethanol content obtained at 59.325 %.

Enzymatic hydrolysis of wheat starch for glucose syrup production

An analysis of the enzymatic hydrolysis of wheat starch was performed. The gelatinization stage was carried out between 90-95°C for 15min. In the liquefaction stage, a commercial α-amylase was used with an enzyme-substrate ratio (E/S ratio) 0.036%w/w at 60°C and pH 5.8 for 4h. In the saccharification stage, a commercial amyloglucosidase was used with an E/S ratio of 0.11% w/w at 60°C and pH 4.3 for 6h. A second hydrolysis was evaluated using a E/S ratio of 0.18%w/w in the saccharification stage. Two methods of enzymatic deactivation, boiling temperatures and pH were evaluated. Inhibitory effects were studied by adding 180g/L of glucose to the process. It is concluded that increases in the E/S ratio decrease reaction times but reaches similar concentrations than lower ratios, the most efficient enzymatic deactivation method is pH. In the inhibition tests, it was determined that there are no glucose inhibitory effects.

Waktu Optimum Hidrolisis Pati Limbah Hasil Olahan Ubi Kayu (Manihot esculenta Crantz var. Lahumbu) Menjadi Gula Cair Menggunakan Enzim α-Amilase Dan Glukoamilase

This study aims to determine optimum time of action of the enzyme α-amylase and glucoamylase needed in hydrolyze of starch from waste processed cassava (Manihot esculenta Crantz var. Lahumbu). This research was conducted through three main stages, namely the gelatinization, liquefaction and saccharification. The method was used method are liquefaction and saccharification. The variation time of the stage liquefaction: 12; 24; 36; 48; 60; and 72 minutes and the saccharification stage are: 9; 18; 27; 36; 45; 54; and 63 hours. The results showed that the optimum time required for stage liquefaction using α-amylase enzyme is 48 minutes on the condition of a temperature of 80 oC with a value of 0.09% amylose levels were measured using UV-Vis spectrophotometer. The optimum time required for saccharification step using a glucoamylase which is 54 hours on the conditions of a temperature of 50oC with the amount of reducing sugar concentration of 9.186 g/L as measured using a UV-Vis spectrophotometer.

ВЛИЯНИЕ ПРЕДВАРИТЕЛЬНОЙ ОБРАБОТКИ ЭНЕРГЕТИЧЕСКОЙ КУЛЬТУРЫ МИСКАНТУСА НА ВЫХОД БИОЭТАНОЛА

Сellulose containing raw materials are widely recognized to be a promising and ubiquitous type of biomass that can be used to produce ethanol without using additional arable land and without competing with the food sector of the economy. Key factors in determining the efficiency of bioethanol production include the chemical composition of cellulose containing raw materials and methods of their preliminary treatment. In this work, Miscanthus substrates were obtained by preliminary chemical treatment under the conditions of an experimental industrial production laboratory of the Institute for Problems of Chemical and Energetic Technologies of the Siberian Branch оf the Russian Academy of Sciences. It is shown that, at the saccharification stage, the highest concentration of reducing substances was achieved for substrates obtained by one-stage chemical treatment, with the concentrations being equal 43,6 g/l and 43,7 g/l for the substrates after nitrate and alkaline delignification treatments, respectively. The dependence of bioethanol yield on various parameters has been studied. It is shown that, with respect to the amount of bioethanol yield from 1 t of Miscanthus, the substrates obtained can be presented in the following order: product of nitric acid treatment > pulp (technical cellulose, combined method) > product of alkaline delignification > pulp (technical cellulose, nitric acid method).

Waste Bread as a Biomass Source: Optimization of Enzymatic Hydrolysis and Relation between Rheological Behavior and Glucose Yield

Amongst the many forms of food waste, bread is a major contributor to the problem. Two aims of this research investigate waste breads potential of being a bioresource for the production of fermentable sugars which are precursor of valuable bioproducts by fermentation process: finding optimum substrate, water and enzyme ratio to produce the highest amount of fermentable sugars and investigating the rheological behavior of the system during hydrolysis. Two stage waste bread hydrolysis was performed with enzymes α-amylase and amyloglucosiades and response surface methodology was used to optimize substrate, water and enzyme ratio. Discovery Hybrid Rheometer-2 (TA Instruments) fitted with a parallel-plate geometry was used to investigate steady flow viscosity of the slurry during hydrolysis. 99 % of theoretical maximum glucose yield, a main fermentable sugar, is achieved by optimizing enzymatic hydrolysis conditions of waste bread at liquefaction and saccharification stages. Just after the addition of α-amylase enzyme, substantial decrease is observed in viscosity and Casson apparent yield stress of the slurry. During saccharification stage, glucose yield increases dramatically while viscosity of the slurries is very low and does not change considerably. The results imply that utilizing high concentrations of waste bread as a feedstock to produce fermentation products offers economic benefits without causing high power consumption, excessive wear on equipment, and reduced conversion which are generally expected consequences for high-solid processing.

Bioenergy and bioproducts from municipal organic waste as alternative to landfilling: a comparative life cycle assessment with prospective application to Mexico.

A life cycle assessment (LCA) of a four-stage biorefinery concept, coined H-M-Z-S, that converts 1t of organic fraction of municipal solid waste (OFMSW) into bioenergy and bioproducts was performed in order to determine whether it could be an alternative to common disposal of OFMSW in landfills in the Mexican reality. The OFMSW is first fermented for hydrogen production, then the fermentates are distributed 40% to the methane production, 40% to enzyme production, and 20% to the saccharification stage. From hydrogen and methane, up to 267MJ and 204kWh of gross heat and electricity were produced. The biorefinery proved to be self-sustainable in terms of power (95kWh net power), but it presented a deficit of energy for heating services (-155MJ), which was partially alleviated by digesting the wastes from the bioproducts stages (-84MJ). Compared to landfill, biorefinery showed lower environmental impacts in global warming (down to -128kg CO2-eq), ozone layer depletion (2.96 × 10-6kg CFC11-eq), and photochemical oxidation potentials (0.011kg C2H4-eq). The landfarming of the digestates increased significantly the eutrophication impacts, up to 20% below the eutrophication from landfilling (1.425kg PO4-eq). These results suggest that H-M-Z-S biorefinery could be an attractive alternative compared to conventional landfilling for the management of municipal solid wastes, although new alternatives and uses of co-products and wastes should be explored and tested. Moreover, the biorefinery system would benefit from the integration into the market chain of the bioproducts, i.e., enzymes and hydrolysates among others.