An effective acid pretreatment of agricultural biomass residues for the production of second-generation bioethanol
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Currently, the potential for energy recovery of plant biomass by biotechnological processes is a preferred solution for the use of agricultural products of low commercial value in order to produce bioenergy that is alternative to fossil fuels. The objective of this study was to obtain second-generation bioethanol by valorization of sugar beet and common dates of low quality. This involved separate hydrolysis and anaerobic fermentation process using the yeast strain Saccharomyces cerevisiae. Physicochemical and biochemical analyzes were carried out on beet and date substrates before and after alcoholic fermentation to determine their effect on yeast activity. The results showed that palm date was a good substrate for the microorganisms in contrast to sugar beet which required a high pretreatment in order to hydrolyze sucrose into fermentable sugars. After distillation, it was possible to recover bioethanol with a quality and a concentration depending on the substrate nature. For an initial sugar concentration of 12.0 °Brix, 74.7 g/kg DM of bioethanol (92.4 g/L) was produced by Saccharomyces from date syrup. In the presence of concentrated date syrup (22.2 °Brix), a lower efficiency of ethanol production was observed (78.2 g/L). It can be conclude that the diluted date substrate led to a good quality bioethanol with a high production yield.
KeywordsAlcoholic fermentation Beet juice Bioethanol Date syrup Energy recovery Saccharomyces cerevisiae
Fossil fuels (oil and natural gas) provide fast and efficient tools of transport as well as a good source of several industrial activities. However, their reserves are limited and security of supply is problematic for many countries importing this kind of fuels. On the other hand, their contribution to environmental pollution is a major problem . Contemporary societies are always interested in finding substitutes for these fuels which must be renewable and environmentally sustainable. Thus, the waste-based energy rich in renewable organic matter (biomass) constitutes good raw materials for many industries and the recovery of biomass by biotechnological processes would be an economical and sustainable solution of energy supply. Special attention should be paid to agricultural by-products (sugar beet, palm date, sugar cane, wheat, fruits, vegetables, etc.) since some fruits and vegetables of low quality cannot be integrated into human nutrition as they may have colors, tastes and aromas that are not appreciated [2, 3, 4]. Thus, a considerable part of this biomass is recognized as waste and not conflicting with food availability.
Agricultural residues are rich in organic matter but are unfortunately underutilized and often end up being polluters of the environment. These by-products can be valorized in order to produce bioethanol which can be used as alone or can be blended with gasoline as transportation fuels [5, 6, 7]. Bioethanol can be produced through fermentation of any raw materials as long as it contains sugar [8, 9]. Vegetables and fruits are very rich in carbohydrates. A successful conversion of these carbohydrates is considered as the most crucial step for bioethanol production. Thus, an efficient pretreatment should increase the content of fermentable sugars (glucose, fructose, galactose, etc.) and will results in a good yield of fermentative bioethanol [6, 10, 11]. Palm date is very rich in sucrose, fructose and glucose. Other simple sugars (oses) were identified in some date varieties; it is arabinose, xylose, galactose and mannose present at very low concentrations . Palm date can therefore be good candidates for the production of bioenergy. In Algeria, one of the leading countries in the production and export of dates in the world, palm date cultivars are numerous and poorly exploited, with the exception of Deglat-Nour, Ghars, Deglat- Beida and Mech-Deglat, which are of major economic importance . Thousands of tons of dates remain unused and can exceed 50% of year-round production, which could be valorized [13, 14]. Sugar beet is also a typical feedstock for bioethanol production which is rich in hemicelluloses (24–32%), and cellulose (22–30%) (It has very low lignin content) . The juice is fermented by yeast or bacteria. The chemical composition of sugar beet reveals a high content of fermentable sugars (arabinose, mannose and xylose) .The pulp, once drained, is used as animal feed or sold to the chemical, pharmaceutical or food industry.
The yeast Saccharomyces cerevisiae has long been an efficient agent for ethanol production at laboratory and industrial level with high efficiency [17, 18, 19]. It is considered as the world’s premier industrial microorganisms being the best exploited microorganism in terms of both old and new biotechnologies . The yeast tolerates a wide range of pH with an optimum under acidic conditions, which makes its fermentation less susceptible to infection than bacteria . It also tolerates ethanol and inhibitory compounds better than other ethanol producing microorganisms. S. cerevisiae is considered as of major economic and social significance in human culture since it has long been used to produce alcoholic beverages (beer and wine) and ferment bread.
The objective of the present project was to propose a method of valorization of agricultural products in order to produce second-generation bioethanol. We opted for Algerian palm dates and sugar beets of low quality. Saccharomyces cerevisiae was the microorganism used to perform the alcoholic fermentation. The main goal was to develop an effective pretreatment of the selected raw material and alternative and efficient process for bioethanol production from a large variety of agro-industrial wastes.
2 Materials and methods
All the chemicals used in the present work (acids, nutrient salts, etc.) were purchased from Merck.
2.2 Selection and pretreatment of raw material (palm date and sugar beet)
The sugar beet, Beta vulgaris from Algeria was used in the present work. It is a vegetable with a tuberous root, in which high amounts of sugar accumulate. The palm date used was Deglet-Nour which is the edible fruit of the date palm Phoenix dactylifera. It is highly coveted inside and outside the country because of its flavor and nutritional quality; it is a fleshy fruit containing an elongated core marketed mostly in the form of dry dates. The two products used in this work are of lower quality to be taken as residues of agriculture.
The extraction was operated in a 500-ml flask equipped with a reflux condenser at a temperature of 65 °C during 30 min. This allowed a better extraction of sugars and a good consumption of the vegetable pulp.
2.3 Characteristics of Saccharomyces cerevisiae
Saccharomyces cerevisiae selected in the present work was that marketed in Algeria. It was inoculated in nutrient broth (supplemented with glucose and salts) at 30 °C under shaking conditions to ensure the growth and activity conditions of the yeast. Cell density refered to the number of living cells per unit volume was measured at 600 nm (see Analytical techniques).
2.4 Alcoholic fermentation experiments
The fermentation assays were carried out in a 1 L-batch bioreactor under magnetic stirring. The temperature was maintained at 30 ± 2 °C. A volume of 500 ml of slightly heated juice or syrup (pH = 4.5) was placed in the fermenter supplemented with nutrients necessary to promote rapid cell growth. Two grammes of yeast were then added to the reactor which must be quickly closed with a hermetic plug to ensure anaerobic conditions. Fermentation began once the release of CO2 was observed. The fermentation time was fixed at 72 h after which the bioethanol was recovered by distillation of the fermented mixture. The distillation temperature was of 78 °C. It was carried out in conventional equipment containing a heating bottle, refrigeration columns and a distillate recovery bulb.
2.5 Analytical techniques
2.5.1 Characterization of beet juice and date syrup
The Brix, measured by a refractometer, is a measure of total soluble solids (including sugars). It is based on the ability of a juice’s sugar to deflect light. In the present work, the Brix of beet juice and date syrup was made by measuring the refractive index at 20 °C using an ATAGO RX-5000 type refractometer.
The pH which is essential for the control of the substrate before and during fermentation is an indicator of the metabolic activity of the yeast during the transformation of sugars. Its measure was carried out by a pH-meter Inolab pH 7110.
The dry matter (DM) was determined by drying in an oven at a temperature of 105 °C until a constant weight a sample of 10 ml of juice or syrup. It is expressed as:
Total sugars were determined by the phenol–sulfuric acid method based on the absorption of light at 448 nm . A Secomam Prime-type spectrophotometer was used for this purpose.
2.5.2 Cell density
Cell density was measured at 600 nm (A600) using a Secomam Prime-type spectrophotometer. A600 values were converted into cell density (103 kcell/mL) by using a standard curve .
2.5.3 Characterization of bioethanol
The fermentative bioethanol was characterized by its physical appearance, smell and flammability. Its density, weight and concentration were also measured. Common techniques were used for this purpose.
2.5.4 Pressure of CO2
The biological conversion was followed by measuring the CO2 release resulting from the fermentation following the degradation of sugars; CO2 production was measured by displacement of water (Fig. 2). The reactor was connected via a tube to an Erlenmeyer flask filled with water at pH 2. The latter was connected by a second tube to an empty container. The CO2 produced in the reactor enters the flask exerting a pressure that will expel a quantity of water proportional to this pressure. This quantity of water recovered in the second container has a volume equivalent to the CO2 pressure.
The results were analyzed using a one-way analysis of variance (ANOVA). Comparison between the different treatments was statistically analyzed and the validity of investigation was expressed as probability value of p < 0.05.
3 Results and discussion
3.1 Physicochemical proprieties of beet juice and date syrup
Physicochemical proprieties of beet juice and date syrup before fermentation
pH (20 °C)
Density (20 °C)
Total sugars (mg/L)
Very light red
3.2 Fermentation monitoring
3.3 Physicochemical and biochemical characterization of fermentative products
Physicochemical and biochemical characteristics of fermentative products
pH (20 °C)
Density (20 °C)
Total soluble solids (°Brix)
Total sugars (mg/L)
Cell density (103 kcell/mL)
Dry matter (g/L)
3.4 Bioethanol yield and concentration
Characterization of bioethanol produced by distillation
Yieldb (g/kg DM)
Beet juice (4.8 °Brix)
Beet juicea (3.1 °Brix)
Beet juice (2.2 °Brix)
Date syrup (22.1 °Brix)
Date syrup (12 °Brix)
Regarding the efficiency of the fermentation, in respect to S. cerevisiae growth and ethanol yield, the results seemed to be promising. The ethanol yield was 74.67 g/kg dry matter (DM) of original date material with a concentration of 92.4 g/L. Syrup extracted from Algerian dates of lower quality has the potential to be an industrially useful substrate for producing second-generation bioethanol. However, sugar beet requires high pretreatments to improve biomass digestibility by hydrolyzing complex sugars into fermentable sugars. Finally, to increase even more the productivity of this alcoholic fermentation process, factors inhibiting the bioethanol production should be identified and overcome. Thus, the extrapolation of the present process to the semi-pilot and pilot scale will be possible. Also, the search for other microorganisms capable of accelerating the process of alcoholic fermentation, increasing the conversion rate and producing a good ethanol is strongly encouraged.
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Ibtissem FENNOUCHE and Nabila KHELLAF. The first draft of the manuscript was written by Nabila KHELLAF and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that there is no conflict of interest.
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