Microsoft Word - 476hernandez.docx CHEMICAL ENGINEERINGTRANSACTIONS VOL. 43, 2015 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Chief Editors:SauroPierucci, JiříJ. Klemeš Copyright © 2015, AIDIC ServiziS.r.l., ISBN 978-88-95608-34-1; ISSN 2283-9216 Increase in Second Generation Ethanol Production by Different Nutritional Conditions from Sugarcane Bagasse Hydrolysate using a Saccharomyces cerevisiae Native Strain Sebastián Lancheros-Castañeda, Diana Morales-Fonseca, Mario Velasquez- Lozano* Universidad Nacional de Colombia- Sede Bogotá, Department of Chemical and Environmental Engineering, Avenida Carrera 30 No. 45 - 03, 111321, Bogotá, Colombia. mevelasquezl@unal.edu.co The lignocellulosic materials are an alternative feedstock for ethanol production, because their low cost and high availability. Sugarcane bagasse is an agroindustrial residue that has been identified as a feasible option due to its high sugar content. A second generation process of ethanol production consists of three steps: pre- treatment, enzymatic hydrolysis and fermentation. Two of the most critical aspects during hydrolysate fermentation to obtain high ethanol productivity are: yeast selection and nutrient supplementation. Native yeast strains isolated from extreme environments as sugarcane distilleries could have an easy adaptation to new medium as lignocellulosic biomass hydrolysate that could contains high inhibitors concentrations. The objective of this study was to evaluate different nutrients supplementation (ZnSO4, MgSO4, MnSO4 and KH2PO4) in a sugarcane bagasse hydrolysate to increase ethanol productivity during fermentation with a native strain of Saccharomyces cerevisiae (202-3). One central composite design with a response surface methodology (RSM) was performed in order to identify the optimal experimental condition. The best results were 0.480 g ethanol g glucose-1 ethanol yield and 1.72 g-1 L-1 h-1 ethanol volumetric productivity on a hydrolysate supplemented with 1.0 g L-1 KH2PO4, 0.05 g L-1 MgSO4, 0.01 g L-1 ZnSO4 and 0.001 g L-1 MnSO4. 1. Introduction In the last decades, bioethanol has been recognized as an efficient alternative to petrochemical fuels. Renewable resources like biomass are a sustainable feedstock to produce biofuels with a subsequent reduction on environmental impact.Lignocellulosic materials are a renewable source for low-cost ethanol generation, because they are agricultural by-products with a high concentration of cellulose that could be converted in glucose. The use of the whole portion of sugarcane in a sugar refinery included agro-industrial waste named sugarcane bagasse (Andrade, et al., 2014). It is obtained during cane milling, its composition ranges between 19-24% of lignin, 27-32% of hemicellulose, 32 - 44% of cellulose and 4.5 - 9.0% of ashes (Soccol, et al., 2011).To obtain fermentable sugars from lignocellulosic biomass are required two important steps: a pre-treatment and an enzymatic hydrolysis. Pre-treatment stage is applied to facilitate enzyme accessibility to cellulose chains and the subsequent stage to release glucose. In order to obtain ethanol is required a fermentation stage. Saccharomyces cerevisiae is the common microorganism in fermentation processes, thus its high tolerance to inhibitors and ethanol concentration, not requirement of oxygen, low optimum pH. Fermentation industry relies on a small fraction of the yeasts diversity (Steensels, et al., 2014), ignoring potential native strains, that due to their evolution and adaptation could reach higher ethanol productions and productivities than current industrial strains. For this reason it is important to evaluate its potential to be used in industrial process. Though yeasts are versatile microorganisms on the development of fermentation process, they require different elements and micronutrients necessary for an optimum ethanol production. Nitrogen is involved in DOI: 10.3303/CET1543038 Please cite this article as: Lancheros-Castaneda S., Morales Fonseca D., Velásquez Lozano M., 2015, Increase in second generation ethanol production by different nutritional conditions from sugarcane bagasse hydrolysate using a saccharomyces cerevisiae native strain, Chemical Engineering Transactions, 43, 223-228 DOI: 10.3303/CET1543038 223 nitrogenized cell compounds formation (protein, amino acids, nucleic acids and metabolites).Nitrogen content is usually quantified by Yeast Assimilable Nitrogen (YAN) which minimal value depends specifically of the strain, but ranges between 120 - 257 mg L-1 in the fermentation medium (Aranda, et al., 2011)(Vilanova, et al., 2015). Phosphorous is required in energy generation and metabolism cell, because it is involved on ATP and phosphoric compounds formation. Also cells require a wide range of minerals for their growth and metabolic operation, like magnesium, calcium, manganese, zinc, etc. Zinc has been recognized as an important factor on fermentation, because it acts as an activator in the ethanol dehydrogenase. This enzyme is involved in the conversion of acetaldehyde into ethanol in the Embden-Meyehof-Parnas pathway (Zhao, et al., 2009). Magnesium and manganese are cofactors involved in different metabolic and bioenergetics pathways (Walker, 2004). Due to the absence of micronutrients and minerals in sugarcane bagasse, this study is focused on investigate the effect of nutrient supplementation on sugarcane hydrolysates, using a native strain of S.cerevisiae with the aim to reach higher volumetric productivities with a possible industrial potential. 2. Methods 2.1 Strain A group of S.cerevisiae strain was isolated from a sugarcane distillery in Puerto Lopez, Meta (Colombia). Was made a fermentation study (data not showed) with the aim to isolate the strain with the best performance on the ethanol production and the higher inhibitor tolerance. The selected strain was S. cerevisiae (202-3). 2.1 Preparation of sugarcane hydrolysate The sugarcane bagasse was treated with acid (H2SO4) and then the lignin chains on the material became broken, letting the enzymatic treatment arise higher concentrations of glucose in the hydrolysate. Soaking was made with H2SO4 2% w/w on the crude bagasse (20% weight of Biomass/Acid Volume) at 60°C for 1 h. The biomass was pressed reducing the acid content until 32% weight of Biomass/Acid Volume. Later was made a pressurized heating at 160°C in a Parr Reactor for 10 min. The obtained solution was neutralized with NH4OH and then was developed the enzymatic hydrolysis stage with an enzymatic extract (Novozymes ®)for 5 d at 50°C and 120 rpm in a 3 L reactor with an effective volume of 700 mL.The main composition of the hydrolysate used on the fermentation is shown in (Table 1). Table 1: Characterisation of sugarcane hydrolysate. Property Value Humidity (% w/w) 88.7 Glucose (g L-1 ) 47 Xylose (g L-1) 27 pH 4.963 FAN (Free amino nitrogen) (mg L-1) 1,146 Acetic Acid (g L-1) 5.0 HMF (g L-1) 0.03 Furfural (g L-1) 0.03 2.2 Inoculum culture preparation The inoculums of S.cerevisiae 202-3 was prepared transferring a couple colonies from a solid culture (with composition 20 g L-1 glucose, 20 g L-1 agar-agar, 10 g L-1 yeast extract and 10 g L-1 tryptose with distilled water) into a 250 mL Erlenmeyer with 50 mL of propagation medium (with composition 50 g L-1 glucose, 5 g L- 1 tryptose, 3 g L-1 malt extract, 3 g L-1 yeast extract, 1 g L-1 NH4Cl and 0.4 g L-1 KH2PO4 with distilled water). The culture was incubated for 16 h at 32°C in an incubator-shaker. 2.3 Fermentation Fermentation was developed on a 50 mL flask with 40 mL (previously sterilized at 120°C) of sugarcane bagasse hydrolysate (Previously pasteurized at 80°C for 30 min) at 32°C. It was transferred 400µL of concentrated cellular suspension (300mg mL-1), obtaining a 1% v/v inoculum and 1.3 x 106 cells by mL of hydrolysate. For the anaerobic condition, was used an airlock with sterilized glycerine. The fermentation was developed for 24-72 h and the sampling was made at 6, 11, 14, 24, 36, 48 and 72 h. 2.4 Experimental design Experiments were carried out on sugarcane hydrolysates using different nutrients concentrations (Table 2) with a native strain of Saccharomyces cerevisiae (202-3). A 24 full factorial design with two coded levels was used to develop a statistical model for the highest ethanol productivity. A repetition of the experiment was carried out to estimate the experimental error of the data. 224 Table 2: Minimum and maximum levels of nutrients used in the fermentations. Variable Symbol Minimum Level Maximum Level KH2PO4 A 0.5 g L-1 1 g L-1 MgSO4 B 0.05 g L-1 0.5 g L-1 MnSO4 C 0.001 g L-1 0.01 g L-1 ZnSO4 D 0.001 g L-1 0.01 g L-1 2.5 Analytical Methods The glucose, xylose, ethanol, hydroxymethylfurfural, acetic acid and furfural concentrations were quantified by HPLC (High Pressure Liquid Chromatography), using a BioRadAminex® HPX-87H column eluted at 65°C with 5mM H2SO4 at a flow rate of 0.6 mL/min and a refractive-index detector at 4 °C. The Free Amino Nitrogen was measured by ninhydrin method reported by (Lie, 1973). 3. Results and discussion The ethanol production (g L-1) is shown (Table 3) for each one of the experimental units. Also are presented the data about productivity (g L-1 h-1) at 11 hours (Maximum productivity identified). The evaluation of the more important factors influencing the ethanol productivity at 11h was made through a Pareto chart (Figure 1), from the results obtained from the experimental design. Table 3: Minimum and maximum levels of nutrients used in the fermentations. Runs KH2PO4 (g L-1) MgSO4 (g L-1) ZnSO4 (g L-1) MnSO4 (g L-1) Ethanol (g L-1) at 11h Ethanol (g L-1) at 24h Ethanol Productivity at 11h (g L-1h-1) 1 0.5 0.05 0.001 0.001 15.67 23.00 1.425 2 0.5 0.05 0.001 0.01 15.60 23.07 1.420 3 0.5 0.05 0.01 0.001 16.57 22.84 1.506 4 0.5 0.05 0.01 0.01 15.72 22.98 1.430 5 1 0.05 0.001 0.001 17.59 23.46 1.599 6 1 0.05 0.001 0.01 17.40 23.06 1.582 7 1 0.05 0.01 0.001 17.82 23.07 1.612 8 1 0.05 0.01 0.01 18.42 22.82 1.675 9 0.5 0.5 0.001 0.001 13.65 22.74 1.240 10 0.5 0.5 0.001 0.01 16.19 23.31 1.471 11 0.5 0.5 0.01 0.001 15.34 23.03 1.394 12 0.5 0.5 0.01 0.01 15.88 22.91 1.443 13 1 0.5 0.001 0.001 18.54 22.92 1.685 14 1 0.5 0.001 0.01 16.69 22.91 1.517 15 1 0.5 0.01 0.001 18.16 23.15 1.650 16 1 0.5 0.01 0.01 17.80 23.03 1.620 The Pareto chart shows that the most important factor influencing the ethanol productivity at 11 h of fermentation is KH2PO4, next to ZnSO4 and the interactions between the KH2PO4 with the other compounds added. Also it is possible to perceive a positive effect on the ethanol productivity at 11 h with the addition of KH2PO4 and ZnSO4. This is explained through the metabolic pathway of conversion of glucose on ethanol. Phosphates are included in the pathway as ATP and ATP-enzymes for the glucose transformation. Zinc as phosphates, are too directly involved in ethanol generation (Ethanol dehydrogenase cofactor) (Walker, 2004), therefore medium’s supplementation with this compounds may achieve a higher productivity on the process. With the data obtained from the experimental design, was created a model to find the optimum condition of supplementation. The response variable in the model was the volumetric productivity (QEth) at 11h. The model obtained is shown on Eq (1), where A is referring to KH2PO4 (g L-1), B to MgSO4 (g L-1), C to MnSO4 (g L-1) and D to ZnSO4 (g L-1). 225 Figure 1: Pareto Chart improving the principal effects of compound supplemented in the ethanol volumetric productivity (g L-1h-1). The model adjust in 83.2% (R-squared), so is not possible to perfectly predict the supplementation effect on the fermentation, but allows to recognize the positive or negative influence of each one of the compounds added in the medium. 1.165 0.457 0.250 20.060 2.079 0.290 7.7900.807 14.109 136.72 8.070 (1) With the model obtained and the Pareto Chart was developed a response surface (Figure 2) with the principal compounds affecting ethanol productivity (KH2PO4 and ZnSO4). The compounds with a less importance on ethanol productivity are MnSO4 and MgSO4 and for this reason was selected the concentrations, where were identified the maximum productivities (MgSO4 (0.05 g L-1), MnSO4 (0.001 g L-1)). Also, was obtained the response surface for higher ethanol productions (g L-1) in base to the KH2PO4 and ZnSO4 concentration (Figure 2). Ethanol Production (g/L) KH2PO4 Concentration (g/L) 0,5 0,6 0,7 0,8 0,9 1,0 Z nS O 4 C on ce nt ra tio n (g /L ) 0,002 0,004 0,006 0,008 0,010 15,5 16,0 16,5 17,0 17,5 18,0 Ethanol Productivity (g/L/h) KH2PO4 Concentration (g/L) 0,5 0,6 0,7 0,8 0,9 1,0 Z nS O 4 C on ce nt ra tio n (g /L ) 0,002 0,004 0,006 0,008 0,010 1,40 1,45 1,50 1,55 1,60 Figure 2: Down: Response surface for Ethanol Volumetric Productivity (g L-1 h-1) in base of ZnSO4 and KH2PO4 concentration. Up: Response surface for Ethanol Production (g L-1) in base of ZnSO4 and KH2PO4 concentration. It is important to recognize that the selected strain of Saccharomyces cerevisiae is able to consume the 98% of initial glucose at 34 h in hydrolysate without supplementation, however adding the enricher compounds to 226 the broth the strain is are able to consume the 98% of initial glucose at the 24 h. In this way, the strain is able to consume 58 – 87% of initial glucose at 11 h depending on the factors added to the broth. With the experimental optimal supplementation obtained from the response surface - MgSO4 (0.05 g L-1), MnSO4 (0.001 g L-1), KH2PO4 (1.0 g L-1) and ZnSO4 (0.010 g L-1) - was developed a fermentation to analyze the differences between the supplementation and the single hydrolysate. The experimental evaluation of fermentation was made through the volumetric productivity of ethanol at different times (Figure 3), where is possible to observe the increasing in the volumetric productivity at 11 h in the supplemented medium. Figure 3: Experimental evaluation of ethanol Volumetric Productivity (g L-1 h-1) in the time for the optimized medium (circles) and the broth without supplementation (squares). The requirement of supplementation is identified with KH2PO4 and ZnSO4 in hydrolysates for the increasing of the ethanol volumetric productivity, due to the absence of compounds rich in zinc and phosphorous in lignocellulosic materials as the sugarcane bagasse. Nitrogen enrichment (More important than phosphorous and Zinc) is supplied in the pretreatment, because the neutralization with NH4OH enriches the medium in nitrogen (Evident on the FAN value). There are many requirements for Saccharomyces cerevisiae. strains to be improved at industrial scale like ethanol productivity, inhibitor tolerance, ethanol yield and inexpensive medium formulations. Different authors consider as necessary in the ethanol biotechnological production, ethanol productivity higher than 1 g L-1 h-1 (Dien, et al., 2003)(Hanh-Hägerdal, et al., 2007). All industrial strains of S. cerevisiae are able to generate ethanol at higher yields (Superior to 90%) (Hanh-Hägerdal, et al., 2007) then the strain evaluation for batch processes is already done in terms of maximum volumetric productivity. The strain of S.cerevisiae 202-3 analyzed in this study is able to reach elevate yields of ethanol (98 - 99%) at 24 hours and higher productivities (approx. 1.1 g L-1 h-1), but supplementation with KH2PO4 and ZnSO4 in hydrolysates, allows the improvement of the strain at industrial scale due to the higher volumetric productivity achieved (approx. 1.72 g L-1 h-1). The volumetric productivity is superior to many of the maximum volumetric productivities reported on the literature for industrial strains of S. cerevisiae and other recombinant strains (0.97 – 1.16g L-1 h-1) (Bothast, et al., 1999) and even superior to other optimized mediums (Martín, et al., 2002)(Singh & Bishnoi, 2013). The inhibitor tolerance is an important factor on strain selection, due to the elevate generation of by-products as acetic acid, phenolic compounds and furfurals on pretreatment stage that could inhibit fermentation(Taherzadeh, et al., 2011) at concentrations of 1.0 g L-1 of furaldehydes and 5.0 g L-1 of aliphatic acids (Martín, 2007) . However the fermentative capacity (yield of ethanol) of this strain is greater than other reported with S.cerevisiae in rich medium without inhibitors (Paciello, et al., 2014 and Putra, et al., 2014). Selection of inhibitors resistant strains avoid the detoxification stages on hydrolysates (Dussán, et al., 2014).Add to the higher productivities achieved, the strain is able to ferment with elevate concentrations of inhibitors (5.0 g L-1 Acetic Acid, 0.03 g L-1 HMF, 0.03 g L-1 Furfural) generated in the hydrolysis and pre- treatment stages. 4. Conclusion The sugarcane hydrolysates are media rich on glucose that allows elevate productions of ethanol through the use of strains of Saccharomyces cerevisiae, however is necessary the supplementation with different compounds with the aim to achieve higher volumetric productivities and then make rentable a fermentation 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 10 20 30 40 50 V o lu m e tr ic P ro d u c ti v it y ( g /L /h ) Time (h) 227 process with the selected strain. 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