Bioethanol potential of raw and hydrothermally pretreated banana bulbs biomass in simultaneous saccharification and fermentation process with Saccharomyces cerevisiae
Residual banana bulbs (RBB) were characterized and assessed as a potential starch and cellulose-based feedstock for bioethanol production. To facilitate the enzymatic digestibility, hydrothermal pretreatment was performed on RBB prior to simultaneous saccharification and fermentation (SSF) with Saccharomyces cerevisiae. Composition of RBB was similar to traditional starch and cellulose-based feedstocks with high glucan (60 g/100 gDM) and relatively low lignin content (7 g/100 gDM). Both amylase and cellulase were needed to efficiently hydrolyze RBB. The highest ethanol yield (310 kg EtOH/ton_DM_RBB, 93% of theoretical production based on total available glucose) was obtained with non-pretreated RBB. SSF can be carried out at lower RBB concentrations. Hydrothermal pretreatment affected negatively the bioethanol potential due to the loss of fermentable carbohydrates. In a case study of an African leading producer of bananas and plantains (Cameroon), the energy derived from bioethanol was 80 GWh ethanol/year and corresponded to 1.6% of the annual transportation requirement. This study shows that RBB is a promising alternative feedstock for bioethanol production.
KeywordsBanana bulbs Hydrothermal pretreatment Bioethanol Fermentation Biorefinery
Banana (Musa acuminata and Musa balbisiana) is an important crop widely cultivated in Asia (continent of origin), South America, Caribbean countries, and Africa, where its fruit contributes to food security and socio-economical stability. Currently, banana is the second most produced fruit in the world with a worldwide annual production of approximately 125 million tons [1, 2], and which generates about 250 million tons of fresh lignocellulosic biomass residues . In Cameroon, the production of bananas and plantains represents the second agricultural economic resource of the country after wood . For the year 2012, banana production reached 1.4 million tons in Cameroon, resulting in about 90,000 tons as dry matter of post-harvest agro-industrial residues such as pseudo-stems, peduncles, bulbs, leaf sheath, and rachis . Among these post-harvest residues, banana bulbs represent a potential renewable feedstock for a variety of biorefinery applications, thanks to its specific composition approximately 50% of starch and 20% of lignocellulose, on a dry matter basis (Awedem et al. (unpublished results)).
Bioethanol is nowadays increasingly used as an alternative liquid biofuel for transportation in high- and medium-income countries like the USA and Brazil, respectively [5, 6]. Both countries produce a total of about 16 billion liters of ethanol per year mainly from sugar cane (Saccharum L.) and corn (Zea mays L.) as feedstocks . However, the use of these important sources of food and feed for biofuel production is currently criticized due to the use of arable lands for fuel production to the detriment of food production. The current increased worldwide ethanol demand in transportation sector will lead to the expansion of fuel ethanol production using these crops and could consequently lead to shortages and price increase in food and feed . Using post-harvest wastes like residual banana bulbs as a feedstock for ethanol production could be an effective alternative. Banana bulbs are non-food biomass resources and therefore do not compete with human food supply. When compared to other post-harvest banana biomass wastes (e.g., banana rachis, pseudo-stems, peduncles, leaves, etc.) that are visually green and fibrous, banana bulbs are rather white and less fibrous. Banana bulbs are one of the main residues (11% of the total dry residues) of the large quantities of post-harvest banana wastes . They have a good potential as a raw material for ethanol production because of their apparent high starch content and low lignin content that can be hydrolyzed and fermented to produce ethanol. Despite the availability and the attractive chemical composition of banana bulbs biomass for a variety of biorefinery applications, their bioethanol potential has not been published in the scientific literature.
On the one hand, ethanol production from starchy feedstocks by conventional fermentation requires saccharification with hydrolytic enzymes after low temperature (< 90 °C) pretreatments  and subsequent fermentation using the yeast Saccharomyces cerevisiae (S. cerevisiae). On the other hand, ethanol production from lignocellulosic feedstocks requires physico-chemical pretreatment prior to fermentation to decrease the recalcitrance of the biomass by breaking up the lignocellulosic structure of the pretreated materials and thus enabling enzymatic hydrolysis. These pretreatments usually require high severity and temperature to initiate the deconstruction of biomass [5, 10, 11, 12, 13]. Ethanol production from a starch and cellulose-based feedstock like banana bulbs biomass would require a compromise between low- and high-temperature pretreatment. When compared to other pretreatment processes like chemical (acid and alkaline) pretreatments that are expensive, hydrothermal pretreatment has been proven to be an effective and cost-efficient method for a wide range of starch and cellulose-based materials including cassava pulp and corn, producing highly digestible fiber fractions [5, 6, 7, 9, 11, 14, 15]. Hydrothermal pretreatment fulfills the following potential advantages as compared to chemical (acid and alkaline) pretreatments: (1) no chemicals (or only small amounts of mineral acids/alkali as catalysts) are required, (2) hemicelluloses are converted into soluble compounds (usually, a mixture of mono- and oligosaccharides), and (3) the pretreated solid (enriched in cellulose and lignin) usually present high susceptibility for subsequent enzymatic hydrolysis [5, 10, 14, 15, 16]. Moreover, hydrothermal pretreatment as compared to chemical (acid and alkaline) pretreatments shows favorable features in terms of environmental impact, selectivity of component separation (hemicelluloses can be dissolved without causing significant effects on cellulose and acid-insoluble lignin), limitation of the residence times, limitation of chemicals and sludges, limitation of equipment corrosion, and reduced capital and operational costs [16, 17, 18, 19, 20, 21]. Hydrothermal treatment is defined commonly as “reactions occurring under the conditions of high temperature and high pressure in aqueous solutions in a closed system” . The two forms of hydrothermal pretreatment are liquid hot-water pretreatment and steam explosion. However, steam explosion is known to lead to the loss of some part of biomass during the explosive decomposition [8, 22]. Liquid hot-water (LHW) treatment is a traditional hydrothermal pretreatment practice. As other pretreatment processes, LHW is known to generate fermentation inhibitors during the process. However, S. cerevisiae, the most commonly used microorganism for ethanol production [9, 23], has been found to resist such fermentation inhibitors [5, 6, 24, 25, 26, 27, 28]. After hydrothermal pretreatment, the fiber fractions left are mainly composed of cellulose, hemicellulose, and starch that can be enzymatically hydrolyzed to release monomeric sugars, which subsequently become available for microbial conversion into ethanol. Different hydrolytic enzyme mixtures are generally used based on the main components of the feedstocks.
The present research investigates banana bulbs biomass as a potential starch and cellulose-based bioethanol feedstock. A hydrothermal process was chosen as a pretreatment method applied prior to enzymatic hydrolysis in order to increase the efficiency of both hydrolysis and alcoholic fermentation. The effect of hydrothermal pretreatment and the use of enzyme mixture (amylase and cellulase) on bioethanol potential were investigated. We assessed the influence of both enzyme loading and substrate concentration on ethanol yield. The obtained results were used to determine the renewable energy potential as bioethanol from residual banana bulbs biomass in Cameroon.
2 Materials and methods
2.1 Raw material
Residual banana bulb biomass (RBB) was collected from an industrial banana plantation (Plantations Haut Penja, PHP) in Cameroon after the mature fruits had been harvested. The variety selected for this study was “Grande Naine” (Musa AAA group) as extensively described previously [29, 30, 31]. The harvested biomass was chopped using a garden mill into particles with maximum diameter of 25 mm. The samples were then oven-dried at 50 °C until constant weight, resulting in a 105 °C dry matter (DM) content of approximately 95% w/w. Dried samples were ground to particle size of less than 2 mm using FOSS CYCLOTEC_1093 SAMPLE MILL. Ground samples were stored in air-tight plastic bags until use.
2.2 Simultaneous saccharification and fermentation
2.2.1 Enzymes used for SSF
Cellulase (commercial Cellic CTec2; activity: 74 FPU/ml_cellulase) and amylase (endo-alpha-amylase–Termamyl; activity: 14 μmol_alpha_1_4_glucose_bond_hydrolyzed/min/ml_amylase) were kindly supplied by Novozymes A/S, Denmark.
2.2.2 SSF reference protocol
About 5 gDM of substrate was mixed with 50 ml of distilled water (pH adjusted at 4.8 with 0.01 M H2SO4) in 250-ml Schott Duran GL 45 bottle flasks. The mixture was then supplied with 1 ml of cellulase + 1 ml of amylase and incubated for 24 h at 50 °C under constant orbital shaking (300 rpm) as a pre-hydrolysis step. This enzyme mixture was based on the manufacturers (Novozymes A/S, Denmark) recommended loading range and will be further referenced as loading “C”.
After the pre-hydrolysis step, 0.2 g of active dry commercial yeast (Saccharomyces cerevisiae 95% DM, Malteserkors tørgær, De Danske Spritfabrikker A/S, Denmark) [9, 17, 23, 32, 33] was added along with 0.2 ml of a 24% urea solution as nitrogen source. The flask headspace was flushed with nitrogen gas for 1 min and was secured with glycerol-filled yeast locks, to ensure anaerobic conditions, while enabling carbon dioxide release. The fermentation process was performed at 32 °C, with constant agitation (300 rpm) for 5–7 days. The pH was not further controlled. The flasks were weighed daily to measure the weight loss (caused by CO2 release) and thus monitor fermentation. All SSF experiments were performed in duplicate, with a parallel reference experiment without any addition of enzymes.
At the end of fermentation, the total concentration of ethanol produced was determined simultaneously with carbohydrates by high-performance liquid chromatography (PerkinElmer series 200a). The Aminex HPX-87H column (Bio-Rad) at 65 °C using 0.005 M H2SO4 as the mobile phase (eluent) with a flow rate of 0.6 ml/min and refractive index detector (RID) were used to determine the concentrations of ethanol produced and carbohydrates (glucose, xylose, and arabinose).
2.2.3 Influence of enzyme activity
SSF of RBB was performed using both the enzyme mixture of the reference protocol (loading “C”) and individual enzymes, i.e., (1) RBB + 1 ml of cellulase only and (2) RBB + 1 ml of amylase only.
2.2.4 Influence of enzyme loading
The influence of enzyme loading on SSF was assessed with 5 gDM_RBB as described in the reference protocol, with reduced enzyme loading: (1) 0.5 ml of cellulase + 0.5 ml of amylase, further referenced as “A”, and (2) 0.75 ml of cellulase + 0.75 ml of amylase, further referenced as “B”.
2.2.5 Influence of substrate concentration
The reference protocol was performed with 5, 2.5, and 1.25 gDM RBB, further referenced as “RBB”, “½ RBB”, and “¼ RBB”, respectively, and with amylase only, keeping the total amylase activity and other volumes unchanged. The removed substrate was replaced by distilled water (pH adjusted at 4.8 with 0.01 M H2SO4).
2.2.6 Influence of hydrothermal pretreatment on fermentation
SSF of solid fractions recovered after hydrothermal pretreatment was performed following the reference protocol as described above. The fermentation of solid fractions recovered after hydrothermal pretreatment was also performed using their corresponding hydrothermally liquid fractions (pH adjusted at 4.8 with 0.01 M H2SO4) instead of distilled water as fermentation medium.
2.3 Hydrothermal pretreatment
Hydrothermal pretreatment experiments were performed in a Roth high-pressure laboratory autoclave (Model II 300 ml, Carl Roth GmbH Company, Karlsruhe, Germany) fitted with heating mantle and a magnetic stirrer head MRK 10. About 5 gDM of biomass (RBB) and 95 g of distilled water were loaded to the reactor. Pretreatment was performed at 100, 110, or 120 °C for 10 min once the desired temperature was reached (within 5 to 8 min). These pretreatment conditions were selected as upper range of investigation in order to preserve starch from thermal degradation. It was based on preliminary analysis which had shown 50 g starch/100 gDM RBB (Awedem et al., unpublished results). After the pretreatment, the reactor was cooled in ice cold water for 10 to 15 min. The pretreated materials were separated by filtration on 1-mm mesh size sieve to solid fraction (fibers) and liquid fraction (filtrate). The DM content of the recovered solid fractions was 12.4–12.9 gDM/100 g FM (FM: fresh matter). Both fractions were kept in a freezer at − 20 °C and used for further investigations (sugar analysis, pretreatment by-products, simultaneous saccharification, and fermentation).
2.4 Chemical characterization
2.4.1 Dry matter and ash content
The dry matter (DM) content of the samples (solid and liquid material) was determined after drying at 105 °C until constant weight. The dry residue was subsequently burned in a furnace at 550 °C for 5 h to determine the total ash content.
2.4.2 Determination of extractives
where Wdried extracted biomass = weight of the extractives-free biomass remaining in thimble and dried (g).
2.4.3 Determination of carbohydrates and acid-insoluble lignin (Klason lignin) in solid material
To quantify carbohydrates and lignin in the extractives-free material (from RBB or solid fractions recovered after hydrothermal pretreatment), a two-step strong acid hydrolysis procedure based on the National Renewable Energy Laboratory (NREL) protocol was performed . About 0.16 g of dried sample was treated with 1.5 ml of 72% (w/w) sulfuric acid at 30 °C for 1 h, and then, the solution was diluted with deionized water to achieve a 4% (w/w) sulfuric acid concentration. Diluted samples were autoclaved at 121 °C for 1 h. The hydrolysates were filtered through fritted ceramic crucibles, and the Klason lignin content was determined as the weight of the acid-insoluble residue after drying the fritted ceramic crucibles with retained solids at 105 °C for 12 h. The hydrolyzates were analyzed for sugars (glucose, xylose, and arabinose) using high-performance liquid chromatography (PerkinElmer series 200a) as described above for ethanol analysis. The concentrations of the sugars were expressed as their polysaccharide form (glucose in the form of glucan, etc.). Conversion of sugars to polysaccharides was calculated with their dehydration factor of 0.88 for pentoses and 0.90 for hexoses. The concentrations were also corrected for any degradation that may have occurred during the acid hydrolysis steps using a recovery factor calculated from replicates spiked with known concentrations of the sugars analyzed (g/l).
2.4.4 Determination of carbohydrates and degradation products in hydrothermal liquid fraction
Monosaccharides in hydrothermal liquid fraction were directly analyzed by HPLC as described above. To check for the presence of polysaccharides, an 8% (w/w) H2SO4 solution was added to the liquid fraction to reach a final concentration of 4% (w/w) H2SO4. The acidified liquid fractions were then hydrolyzed at 121 °C for 10 min. The total glucose, xylose, and arabinose concentrations were quantified by HPLC as described above.
Furfural and hydroxy-methyl-furfural (HMF) were measured in the liquid fractions recovered after hydrothermal pretreatment using an Agilent HPLC (Agilent 1260 Infinity Bio-inert Binary LC) equipped with a Hypersol Gold column (Thermo Scientific). The column temperature was 30 °C. The solvent A was 90% water with 1% acetic acid and 9% methanol and solvent B was CH3CN (HPLC grade solvents). The gradient was as follows: 0–5′ 0% B, 5–10′ linear gradient up to 100% B, 10–15′ 100% B, 15–20′ linear gradient down to 0% B, 20–30′ 0% B. A UV detector was used to determine the concentrations of furfural and HMF at wavelength of 280 nm. Solutions of known concentration were prepared from 2-furaldehyde (2F, Sigma–Aldrich, ref.181100250) and 5-(hydroxymethyl)-2-furaldehyde (5-HMF, Sigma–Aldrich, ref.121460010) and used for calibration.
2.4.5 Ethanol yield calculation
where Yet = ethanol yield; mglu = amount of glucose available after acid hydrolysis (g).
2.4.6 Mass balance calculation
2.5 Statistical analysis
The data obtained were statistically analyzed with XLSTAT software (Version 2016.02.2). The Tukey and one-way analysis of variance (ANOVA) tests were used for the comparison of ethanol yields from hydrothermally pretreated RBB. Statistical differences were measured at 95% confidence level (p < 0.05).
3 Results and discussion
3.1 Raw material characterization
Chemical composition of residual banana bulbs (RBB)
Content (g/100 gDM RBB)
59.62 ± 0.18
4.28 ± 0.06
2.43 ± 0.04
6.74 ± 0.33
11.19 ± 0.13
14.06 ± 0.15
1.20 ± 0.03
15.26 ± 0.15
Residue (not identified)
0.48 ± 0.43
3.2 Simultaneous saccharification and fermentation of RBB
3.2.1 Ethanol yield after SSF of RBB
3.2.2 Effect of enzyme loading on ethanol yield
3.2.3 Effect of RBB concentration on hydrolysis and ethanol yield
3.3 Hydrothermal pretreatment of RBB
The results obtained from the SSF of RBB have shown that the enzymatic hydrolysis was not complete even with the combined use of both amylase and cellulase (Fig. 1a). With the hope to increase the efficiency of both enzymatic hydrolysis and ethanolic fermentation, RBB was submitted to hydrothermal pretreatment.
3.3.1 Mass balance of hydrothermal pretreatment
Ashes were more and more solubilized and recovered in the liquid fractions as temperature increased (Fig. 4e). However, glucose and xylose that disappeared from the solid fraction were not significantly recovered in the liquid fraction (Fig. 4b, c), neither as polysaccharide nor as monosaccharide. Lignin followed the same trend (Fig. 4d).
3.3.2 Effect of hydrothermal pretreatment on ethanol yield
To check the presence of potentially inhibitory substances (furfurals, acids) in the liquid fractions recovered after hydrothermal pretreatment, the SSF of the solid fractions was performed using the liquid fractions as fermentation medium. Figure 5 compares the ethanol yield after SSF of solid fractions using liquid fraction and water as fermentation medium. The ethanol yield obtained from the SSF performed with liquid fractions recovered after hydrothermal pretreatment as fermentation medium were always slightly lower than the ethanol yield obtained from the SSF performed with water as fermentation medium (Fig. 5). However, except the SSF performed with liquid fraction of RBB pretreated at 120 °C, no statistical significant difference (p < 0.05) was observed between the ethanol yields obtained from the SSF performed with water and the ethanol yields obtained from the SSF performed with liquid fractions as fermentation medium (Fig. 5).
Concentration of the by-products and inhibitors detected in the liquid fraction (mg/l LF)
52.38 ± 0.33
24.15 ± 0.44
19.02 ± 1.59
0.10 ± 0.00
67.46 ± 2.38
28.29 ± 0.77
32.05 ± 2.80
0.15 ± 0.00
96.10 ± 3.79
46.55 ± 0.80
40.58 ± 4.81
0.23 ± 0.00
3.4 Renewable energy potential of bioethanol from banana bulbs biomass: case study of Cameroon
Estimation of the renewable energy that could be generated annually as bioethanol in Cameroon from residual banana bulbs of the commercially most produced Grande Naine variety
Annual production of fruits (ton_FM/year)a
1.4 × 106
Annual fresh residual biomass production (ton_FM/year)b
2.8 × 106
Annual dry residual biomass production (ton_DM/year)b
Distribution of banana bulb fraction in banana residual biomass (%_DM)c
Annual biomass production of banana bulb fraction (ton_DM/year)
Bioethanol potential of banana bulbs (kg_EtOH/ton_DM)
Bioethanol potential of banana bulbs (kg_EtOH/year)
11 × 106
Bioethanol potential of banana bulbs (liter_EtOH/year)
14 × 106
Energy in bioethanol derived from banana bulbs residues (GWh_EtOH/year)d
Annual transportation requirement of Cameroon (GWh/year)e
When converted to energy, the bioethanol produced could supply an annually estimated 79.9 GWh of energy (Table 3). This is the crude energy content of the produced ethanol, while not subtracting the energy needed by the production process. The energy needed by the process (electricity and heat) could be supplied in the renewable form from the anaerobic digestion of the other banana residues as described by Awedem et al. [30, 31]. The annual Cameroon motor gasoline consumption in 2012 was about 427,285 ton oil equivalents (toe) and corresponded to 4968 GWh/year . If we take this value into account, the residual banana bulbs biomass could cover about 1.6% of the annual Cameroon motor gasoline consumption. The RBB contribution of 1.6% of the transportation requirement of Cameroon may seem small, but this corresponds to about 6880 toe/year, which is certainly more than the amount of fuel consumed in plantations by tractor with trailer during the transportation of RBB from the fields to the bioethanol production plant. Moreover, if we take into account the other banana varieties cropped in Cameroon for local needs, this renewable energy contribution could be doubled [3, 40].
Nevertheless, this simplified assessment does not take into account all the practical constraints (e.g., bulbs harvest and processing, cost of enzymes, ethanol valorization, etc.) of the implementation of a bioethanol production plant. It allows, however, to set the orders of magnitude of the energy potential. The energy derived from bioethanol could contribute to an environmentally and economically sustainable development of the country.
Residual banana bulbs (RBB) showed a good potential as a bioethanol feedstock. Both cellulase and amylase are needed to efficiently hydrolyze RBB. The highest ethanol yield (93% of theoretical production based on glucose) was obtained with RBB and corresponded to 310 kg EtOH/ton_DM_RBB. Hydrothermal pretreatment of RBB affected negatively their conversion to ethanol. When considering the RBB resulting from the Grande Naine variety cropped in Cameroon, the energy derived from bioethanol is about 80 GWh EtOH/year and corresponds to 1.6% of the annual transportation requirement of Cameroon. This energy could contribute to meet the energy requirements of human activities in the tropical banana-producing countries.
They are also grateful to Novozymes A/S (Denmark) for the gift of enzymes. A great acknowledgment to Kathrine Hansen (Department of Energy Technology, Aalborg University, Esbjerg, Denmark) and Linda Madsen (Department of Chemical Engineering, Aalborg University, Esbjerg, Denmark) for their technical assistance. M.D. is Senior Research Associate of the F.R.S.-F.N.R.S. (National Funds for Scientific Research, Belgium).
This work was financially supported by the Wallonia-Brussels International (WBI) and the Ministry of Higher Education, Research and Media of Wallonia-Brussels Federation (FWB) from Belgium.
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