Feasibility of Biofuel Production from Banana Waste in Peru and Colombia

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Assessing the Feasibility of Biofuel Production from Lignocellulosic Banana Waste in Rural Agricultural Communities in Peru and Colombia

Article in BioEnergy Research · September 2013

DOI: 10.1007/s12155-013-9333-4

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Assessing the Feasibility of Biofuel Production

from Lignocellulosic Banana Waste in Rural Agricultural Communities in Peru and Colombia

Monica Santa-Maria_&A. A. Ruiz-Colorado_&

Gaston Cruz_&Tina Jeoh Published online: 16 April 2013

#Springer Science+Business Media New York 2013

Abstract Banana cultivation is widespread in tropical and subtropical regions where many rural agricultural communities exist. The banana plant bears fruit once in its life cycle, leaving behind a significant amount of usable agricultural residue. Current practice leaves this residue to decompose in the field spreading diseases and polluting water supplies. We evaluated the technological feasibility of converting lignocellulosic banana residue to ethanol as a localized biofuel production strategy to improve the liveli- hoods of rural agricultural communities in Peru and Colombia. Liquid hot water (LHW) and steam explosion pretreatment followed by saccharification and fermentation using commercial cellulolytic enzymes and yeast strain were evaluated for three different lignocellulosic residues independently (pseudostems, leaves, and rachis). Stems and rachis, with higher glucan conversion, appeared more prom- ising for biofuel production than leaves (up to 93 and 77 % glucose yields for rachis and pseudostems, respectively).

Steam explosion pretreatment allowed higher glucan conversion for stems and leaves, while LHW was better suited for rachis. Pseudostem is the most abundant residue generated with 306,000 tons/year in Uraba Province (Colombia) and 15,000 tons/year in the Chira Valley (Peru) on a dry

weight basis. Potential ethanol production in the Chira Valley was estimated in 4.8 and 76.8 ML year⁻¹ in Colombia, processing stems and rachis combined. This study indicated that there is potential for biofuel production using the lignocellulosic banana residue, which could be expanded to other banana growing communities around the world. Process improvements such as increasing solids loading, water recycling, and optimizing fermentation are still required.

Keywords Banana biomass . Steam explosion pretreatment . Liquid hot water pretreatment . SSF . Sustainable development

Introduction

Concerns over climate change due to the massive burning of fossil fuels have triggered continued research on renewable forms of energy for their replacement [1]. Biofuels (alcohols and biodiesel) can be used as cleaner alternative to fossil fuels for transportation because their combustion is carbon neutral and they are compatible with existing engine technology [2]. First-generation biofuels produced from sugar or starch crops currently dominate the market, but second- generation biofuels obtained from lignocellulosic biomass, such as agricultural residues or dedicated energy crops, appear as a more viable and sustainable alternative in the long term [3,4]. In this respect, ongoing research to under- stand the structure and transformation of lignocellulosic biomass is being held to attain economic viability [5].

Bioenergy systems including local biofuel production from agricultural residues are gaining relevance worldwide as a sustainable development strategy [3,6]. For instance, studies in sub-Saharan Africa and Southeast Asia indicate that bioconversion of biomass to fuels could replace a significant portion of the energy imports while diversifying Electronic supplementary material The online version of this article

(doi:10.1007/s12155-013-9333-4) contains supplementary material, which is available to authorized users.

A. A. Ruiz-Colorado

Bioprocesos y Flujos reactivos, Facultad de Minas, Universidad Nacional de Colombia–Sede Medellin, Medellin, Colombia G. Cruz

Chemistry Laboratory, Universidad de Piura, Piura, Peru M. Santa-Maria

:

T. Jeoh (*)

Biological and Agricultural Engineering, University of California, Davis, Davis, CA, USA

e-mail: [email protected] DOI 10.1007/s12155-013-9333-4

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rural economies and creating employment; reducing poverty, and providing net energy gains and positive environmental impacts [6, 7]. These schemes are particularly suitable for banana cultivation given its widespread occurrence in tropical and subtropical regions where many poor agricultural communities exist [7,8]. Furthermore, the banana plant bears fruit once in its life cycle, leaving behind a significant amount of usable agricultural residue, which is generally left in the field to decompose spreading diseases and polluting water supplies [7, 8]. Alternative uses for the banana residue that could improve the livelihood of local populations while preserving the environmental health are thus necessary.

The banana residue includes unmarketable banana fruit (pulp and peels) and lignocellulosic biomass (pseudostem and flower stalk). Banana fruit waste has been traditionally used for animal feed and/or composted, while lignocellulosic residue has been used to produce various handcrafts, eating utensils, food wrapping, in fiber and textile applications, pulped for the paper industry, and used as adsorbent material for waste water treatment schemes [7, 9]. Only recently has the banana residue been considered for energy production [7,10, 11]. Due to its high starch content, the banana fruit has been used almost directly for ethanol production after enzymatic hydrolysis with amylolytic and cellulolytic enzymes for pulp and peel, respectively [12,13].

The banana peels have also been converted to biogas by anaerobic fermentations with relative success [11,14,15].

However, using the lignocellulosic residue has remained a bottleneck for whole banana waste utilization with yet low glucan to ethanol conversion and high energy demand for transformation [7,10]. Given the abundance and widespread distribution of the lignocellulosic banana residue, efforts to optimize bioconversion routes should be explored.

In this work, we evaluate bioconversion of different lignocellulosic residues generated in the banana cultivation aiming at small-scale operations in rural agricultural communities.

Leading biomass pretreatments with low environmental impact such as uncatalyzed steam explosion and liquid hot water were explored, along with simultaneous saccharification and fermentation using commercial enzymes and yeast strain. The results on biomass composition, effect of pretreatments and bioconversion potential, along with potential ethanol production for fuel replacement in agricultural communities from Peru and Colombia are presented.

Materials and Methods

Banana Lignocellulosic Biomass Banana leaves, rachis, and pseudostems were collected from banana-growing communities near the villages of Samán (4°52′22″S, 80°46′35″W) and Salitral (4°51′08″ S, 80°40′47″ W), Chira Valley, Sullana Province (Piura, Peru), and banana plantations sites

located between 07°40′37″N and 08°05′00″N, and 76°38′

05″ W and 76°44′00″ W, Uraba Province (Colombia).

Banana cultivars in both locations consist ofMusa acuminate.

In the Chira Valley, about 10 kg of each residue type was collected from four different plantation sites. In Colombia, residues were collected from different areas within the plantation sites. All the residues collected were pooled, washed in situ with tap water, chopped, and dried in a convection oven at 45 °C for 3–5 days. Moisture content was recorded. Residues were Wiley-milled with a 0.5-mm diameter mesh and stored at room temperature until use.

Chemical Composition Analysis Chemical composition of the different pretreated and nonpretreated banana residues was obtained following National Renewable Energy (NREL) Laboratory Analytical Procedures (LAPs) [16].

Non-pretreated biomass was Wiley-milled to 20 mesh, moisture and ash content were measured, and a two-step (water-ethanol) extraction was done to remove soluble non- structural components. Extracted biomass was vacuum- filtered and dried in a convection oven at 40 °C until moisture content was below 10 %.Pretreated biomasswas lyophilized, samples were Wiley-milled to 20 mesh and moisture and ash content was measured. Structural carbohydrates and lignin of extracted nonpretreated and pretreated biomass was obtained following NREL-LAP guidelines using a two-step sulfuric acid hydrolysis [16].

Hydrolyzed sugars were quantified by high-performance liquid chromatography (HPLC) using an Aminex HPX- 87P column (BioRad). Acid insoluble lignin was corrected for ash content (no protein was measured). Acid-soluble lignin was determined spectrophotometrically usingλ=240 nm and extinction coefficient (25) for bagasse NIST (see NREL LAP

“Determination of Structural Carbohydrates and Lignin”) [16].

Biomass Pretreatment Banana leaves, rachis, and pseudostems collected in Peru and Colombia were pretreated independently by either LHW or steam explosion (SE) without added catalyst. LHW pretreatment was done in a 1-L stirred reactor (Parr Instruments Company, Illinois, USA) for 10 min at 180 °C and 10 % solids (w/v). Pretreated slurry was vacuum-filtered using glass fiber filters (Fisher Scientific, USA) and the flow-through collected; both fractions were stored at−20 °C until use. Moisture content of LHW pretreated residues was determined by oven drying at 105 °C.

SE pretreatments were conducted at the University of Colombia in Medellin (Colombia) in a 100-L reactor with 150 g solids at (1) lower severity (10 min at 190 °C) and (2) higher severity (76.4 min at 200 °C). Pretreated residues were lyophilized and sent to UC Davis for further processing. Detailed pretreatment parameters are available online as Supplementary Information1.

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Saccharification of Lignocellulosic BiomassSaccharification (SAC) reactions were performed according to NREL LAP

“Lignocellulosic Biomass Hydrolysis and Fermentation” with some modifications [17]. A volume of 250 mL SAC flasks were loaded with 1 % (w/w) glucan usingunwashed pretreated biomass samples, 1 % (w/v) yeast extract, 2 % (w/v) peptone, 0.05 M Na-citrate (pH 5), and cellulase enzyme in 50 mL total volume. All reactions were autoclaved prior to enzyme addition. Cellulolytic enzymes (Novozymes Biomass kit) were diluted 1:5 with Na-citrate buffer (0.05 M, pH 5) and filtered right before use. Enzyme loadings were as follows: NS 50013 (cellulase) 19.05 mg protein or 27.36 FPU/ g glucan; NS 50010 (β-glucosidase) 1.76 mg protein or 178.86 U/g glucan (defined as mmoles of p-nitrophenyl-glucoside hydrolyzed per minute, measured as previously described [18]); and NS 20022 (xylanase and β-glucanase) 4.035 mg protein or 140.28 U/g glucan (defined as micromoles of glucose equivalents released per minute, measured as previously described [19, 20]). Filter paper units (FPU) were measured according to NREL LAP- 006 [16] with the DNS reagent prepared as previously described [21].

All SAC reactions were performed in duplicate with one reaction control (without enzyme). A cellulose control, using α-celullose (Sigma-Aldrich catalogue number C8002) as substrate, and an enzyme control (without substrate) were run in parallel. Flasks were incubated at 50 °C and 130 rpm. Three milliliters slurry samples was taken at different time points and stored at −20 °C until analysis.

YPD plates (yeast extract, 1 %; peptone, 2 %; dextrose, 2 %;

and agar, 2 %) were inoculated at the last time-point to verify absence of microbial contamination in the SAC reactions. Released sugars were quantified by HPLC using an Aminex HPX-87P column (BioRad).

Simultaneous Saccharification and FermentationSimultaneous saccharification and fermentation (SSF) reactions were performed according to NREL LAP “Lignocellulosic Biomass Hydrolysis and Fermentation”with some modifications [17]. A volume of 250 mL SSF flasks was loaded with 1 % glucan usingunwashedpretreated biomass samples, 1 % (w/v) yeast extract, 2 % (w/v) peptone, 0.05 M Na- citrate (pH 5), and cellulase enzyme in 100 mL total volume.

All reactions were autoclaved prior to enzyme addition.

Cellulolytic enzymes (Novozymes Biomass kit) were diluted 1:5 with Na-citrate buffer (0.05 M, pH 5) and filtered right before use. Enzyme loading was the same as used in the saccharification experiments: NS 50013 (cellulase), 38.11 mg protein or 27.36 FPU/g glucan, measured as previously described [16, 21]; NS 50010 (β-glucosidase), 3.52 mg protein or 178.86 U/g glucan; and NS 20022 (xylanase and β-glucanase), 8.07 mg protein or 140.28 U/g glucan.

All SSF reactions were run in duplicates. Cellulose control containing α-celullose (Sigma-Aldrich catalogue number C8002) as substrate and enzyme control (without substrate) were run in parallel. Two SSF experiments were done (SSF1 and SSF2). In SSF1, flasks were incubated 17 h at 37 °C followed by 9 h at 50 °C (SAC stage), yeast was added, and reactions were further incubated for 135 h at 37 °C (SSF stage). In SSF 2, flasks were incubated 24 h at 50 °C (SAC stage), yeast was added, and reactions were further incubated for 120 h at 37 °C (SSF stage). Anaerobic condition was maintained using one-way valves (Fisher Scientific); flasks were agitated at 130 rpm throughout.

Slurry samples (4 mL) were taken at different time points and stored at−20 °C until analysis. YPD plates were inoculated at the last time point to verify absence of microbial contamination in the SSF reactions. Released sugars and ethanol produced in fermentation were quantified by HPLC and gas chromatography (GC) analysis, respectively.

Yeast Preparation Bio-Ferm® XR (North American Bioproducts Corporation, Georgia, USA) was used in the SSF experiments. A thawed 1 mL yeast glycerol stock was grown in 100 mL YPD medium (yeast extract, 1 %; peptone, 2 %; dextrose, 2 %;w/v), incubated overnight at 37 °C and 130 rpm. Yeast was subcultured into fresh YPD medium using 1 % (v/v) inoculum and grown until OD600≈1.0 (late log phase). Cells were centrifuged (10 min at 4,500 rpm), rinsed with sterile H2O, centrifuged again, and resuspended in 1/10th of its initial volume using sterile ultrapure water.

Cells were added to saccharification flasks to obtain a starting yeast concentration of OD600=0.5.

HPLC Analysis Sugars produced from banana biomass by acid hydrolysis (compositional analysis) or enzymatic hydrolysis (saccharification and SSF) were quantified in a Shimatzu HPLC system with RID detection, separated in an Aminex HPX-87P column (BioRad). Mobile phase was HPLC-grade water with 0.6 mL/min isocratic flow and 85 °C column temperature. Sugar standards were cellobiose, glucose, xylose, arabinose, galactose, and mannose.

GC Analysis Ethanol produced in SSF experiments was quantified in an Agilent 6890 GC with flame ionization detection and ChemStation data analysis, following NREL LAP-011 with modifications [16]. The column was an Agilent DB-wax 30 m×0.530 mm diameter× 1.0 μm film.

Methanol was used as internal standard.

Yeast Growth Curves The effect of potentially inhibitory compounds generated during LHW pretreatment on yeast growth was investigated. Pretreatment flow-through solutions, obtained from vacuum-filtering LHW pretreatment slurries, were filter-sterilized and stored at 4 °C.Saccharomyces

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cerevisiaestrains“Bio-Ferm® XR”and“NREL D5A”were used. Cells were grown from glycerol stocks in YPD medium.

Grown broth was subcultured into fresh YPD medium with 1 or 5 % (v/v) pretreatment flow-through solutions and other- wise according to BioTek Application Note guidelines [22].

Yeast strains were grown in flat-bottomed optically clear 96- well plates (Corning) sealed with optically clear tape for real- time PCR applications (Applied Biosystems). Samples were run in triplicates with the corresponding controls: a control without yeast inoculum and a control without pretreatment solution. Plates were incubated at 37 °C and continuous orbital shaking (slow setting) in a Synergy^TM 4 Hybrid Microplate Reader (BioTek). Cell growth was assessed by light scatter measurements made at 600 nm every 10 min.

Results and Discussion

Chemical Composition of Lignocellulosic Residues from Banana

The chemical composition of the different lignocellulosic residues from banana plants collected in Peru and Colombia is summarized in Table1. Residues from both locations had similar chemical composition, with pseudo-stems having the highest glucan content (54–60 %) followed by rachis (42–

52 %) and leaves having the highest ash content (10–11 %).

Hemicellulose content was slightly lower in all residues from Peru compared to those from Colombia. This was more pronounced for pseudostems, which had lignin and hemicellulose content almost half of their Colombian counterpart. While this seems to be the trend, a more precise comparison of biomass composition from either location is necessary to confirm this observations since differences in mass balance in the compositional analysis (i.e., 96 and 85 % for pseudostems from Colombia and Peru, respectively) could have influenced these results. In any case, banana species cultivated in both locations consist ofM. acuminate.

One major difference among the two locations is water availability; while banana plantations in Colombia have plenty of water supply, water is limiting for the Chira Valley (Peru) [23].

Consequently, a common practice among farmers in the Peru region is to leave banana trees from previous seasons planted in the field to serve as water reservoirs for nascent banana plants. The pseudostems collected for this study thus correspond to 2–3-year-old banana plants that were left planted in the field, not the case for leaves and rachis, which were freshly harvested. Furthermore, the chemical composition of banana residues obtained in this work is comparable to Cordeiro et al.

[24] for pseudostems, who reported cellulose content of 34–

40 %, 12 % lignin, and 25 % hemicellulose (percentage accounting for the glucan fraction in hemicellulose). Our results indicate higher cellulose content and less ash content than Oliveira et al. [25], who reported cellulose content of 31, 37.3, and 31 % for leaves (foliage), pseudo-stems (leave sheath), and rachis, respectively. Variations in the chemical composition of a biomass feedstock due to location, harvesting season, weather conditions, and/or precipitation has been previously noted [26].

Effect of Pretreatment on Biomass Composition

Biomass pretreatment is primarily conducted to overcome the lignin–hemicellulose barrier that limits enzyme access to cellulose for its depolymerization [5]. Pretreatment can thus enhance the rate of enzymatic hydrolysis and increase the overall conversion yield [4]. The cost of pretreatment can have a significant impact on biorefinery costs and the choice of pretreatment is extremely important; however, deciding upon a particular treatment for a particular feedstock is not straightforward. Many pretreatment strategies, including physical, chemical, biological, and solvent fractionation, have been investigated, but so far, thermochemical treatments are the most commonly used [5]. Furthermore, the effect of different pretreatment conditions on the digestibility of various biomass feedstocks has been extensively

Table 1 Chemical composition of banana biomass collected in Peru and Colombia

Data (corresponding to water- and ethanol-extracted biomass) is percentage of total on a dry weight basis (dwb), average±

standard deviation (n=4)

aAcid-soluble and insoluble lignin combined

Pseudostems Leaves Rachis

Colombia Peru Colombia Peru Colombia Peru

Ash 5±0.0 5±0.8 10±1.0 11±0.9 3±0.6 7±1.2

Lignin^a 21±2.5 12±0.1 25±4.4 26±6.0 16±3.0 21±4.5

Glucan 54±2.3 60±0.6 34±1.0 27±1.2 53±0.9 42±1.5

Xylan 11±0.2 5±0.5 11±0.5 7±0.4 11±0.8 8±0.4

Galactan 1±1.2 1±0.4 3±0.2 1±0.2 1±1.0 2±0.1

Arabinan 3±0.2 2±0.1 4±0.6 3±0.3 3±0.1 4±0.1

Mannan 1±0.8 0.2±0.3 1±0.9 0 2±0.1 2±0.1

Total 96 85 88 75 89 86

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studied [27]. Due to the structural complexity and compositional variability of lignocellulosic biomass, the enzymatic digestibility of biomass is highly substrate and pretreatment specific; therefore, different pretreatment chemistries and conditions have to be evaluated for any new biomass feedstock that is aimed for bioprocessing [4].

In this work, we evaluated two leading pretreatment technologies: uncatalyzed SE and LHW. Both treatments are relatively mild and avoid the use of hazardous catalysts, catalyst recycling, and effluent treatment. For instance, uncatalyzed steam explosion is a popular pretreatment that has been proven cost effective and advanced to pilot-scale demonstration [4]. In SE, biomass is rapidly heated by high- pressure saturated steam for a period of time followed by a swift release of pressure that causes an explosive decompres- sion in the chamber. Under these conditions, acetic acid and other organic acids are formed from acetyl or other functional groups released from biomass that hydrolyze hemicellulose [4]. In addition, the rapid thermal expansion in SE opens up the biomass particle structure leading to particle size reduction and increased pore volume [28]. Thus, the result of SE pretreatment is mostly hemicellulose and some lignin removal depending on pretreatment severity. Similarly, LHW pretreatment involves high temperature cooking of biomass by liquid water under high pressure. LHW is another preferred treatment because it allows for increased cellulose digestibility while producing fewer fermentation inhibitors, has no need for neutralizing liquid streams, and there is no need for biomass size reduction since particles are broken during pretreatment [4,29]. Similar to uncatalyzed SE, acetic acid and other organic acids are produced from functional groups in biomass, which catalyze hemicellulose removal;

thus, LHW can also result in hemicellulose and some lignin removal depending on pretreatment severity [4, 30]. In both cases, acidic conditions could be generated that could result in some sugar degradation products, especially furfural from xylose degradation [30].

In our study, selective removal of hemicellulose was more evident under the high severity steam explosion pretreatment with around 80 % xylan removal, followed by lower severity SE pretreatment with about 20 % xylan removal (Table2). In contrast, LHW pretreatment had no apparent effect on the chemical composition of all the banana residues evaluated (Table3). The LHW-pretreated slurry was not washed prior to compositional analysis; thus, some solu- bilized components might have remained, affecting the structural carbohydrates estimation in the pretreated solids [26]. In any case, the higher hemicellulose removal observed for the SE pretreatment in this work is possibly due to the higher severity in these treatments (Supplementary information1). In general, increasing pretreatment severity (e.g., increasing temperature, time, or decreasing pH) results in higher xylan removal from biomass [30]. Additional studies will be

required to further compare the effect of pretreatment modal- ity on hemicellulose removal and downstream processing.

LHW pretreatment generally solubilizes hemicellulose into oligomers rather than monomeric sugars, especially when the pH is controlled to remain above 5, reducing the amount of degradation products that could be generated [30]. In our study, the pH of LHW-pretreated slurries was between 4.1 and 5.8 (Table3). The observed variation in pH among pretreated slurries is possibly due to differences in acetyl content and/or buffering capacity of the different banana residues evaluated [31]. In general, the pH of LHW pretreated slurries is 4–5 due to the intrinsic buffering capacity of biomass [32]. Since the pretreatment pH did not correlate with ash content in the different banana residues (especially for banana leaves), we can infer that there was minimal buffering effect of the inorganic fraction under the conditions tested.

SE and LHW pretreatment of different biomass feedstock have been extensively studied with various results [30,31, 33]. For instance, Garlock et al. [30] evaluated LHW pretreatment of switchgrass achieving up to 80 % xylan removal treating at 200 °C and 10 min with 15 % solids.

Mosier et al. (2005) optimized LHW pretreatment of corn stover at 190 °C and 15 min with 16 % solids achieving up to 40 % xylan removal and <1 % dissolved carbohydrates lost to degradation products [29, 33]. In our study, up to 80 % xylan removal was obtained with the high severity SE pretreatment (76.4 min at 200 °C) and 20 % xylan removal for the low severity SE pretreatment (10 min at 190 °C) These results for the banana biomass are comparable to those previously reported using similar pretreatment schemes for different feedstock.

Enzymatic Digestibility of Pretreated Lignocellulosic Banana Residues

Enzymatic digestibility of the pretreated banana residues was evaluated using a mixture of cellulase, β-glucosidase, and xylanase activities. Xylanase was supplemented to remove remaining hemicellulose in the pretreated biomass and increase accessibility of cellulose to enzymes. Xylanase supplementation of cellulolytic enzymes has been shown to be more significant than further delignification in steam explosion pretreated corn stover increasing both initial conversion rates and overall glucose and xylose yields [31].

Furthermore, xylanase supplementation has also been shown to be more critical for mildly pretreated substrates compared to more severe treatments of the same feedstock [31]. This is possibly due to a higher fraction of hemicellulose remaining in mildly pretreated substrates compared to more severe ones; xylanases would remove remaining hemi- celluloses, thus increasing accessibility of cellulose molecules to enzymes for their hydrolysis [31].

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Glucose and xylose yields for the different pretreated banana residues are summarized in Fig.1. Various results in terms of glucose yields were obtained for the different residues and pretreatment regimes evaluated. Highest glucose yields were obtained for rachis from Colombia with up to 93 % glucan conversion after 120 h incubation, followed by pseudostems and rachis from Peru with up to 77 % glucan conversion. In general, higher glucose yields were obtained by high severity SE pretreatment, followed by LHW pretreatment for pseudostems and rachis and lower severity SE pretreatment for banana leaves. Glucose yields were lowest for the LHW pretreated leaves both from Peru and Colombia. LHW-pretreated pseudostems from Peru appeared more amenable to enzymatic hydrolysis than its Colombian counterpart. Given that equivalent xylan conversion (percent of theoretical) was observed for both residues (Fig. 1b, h), this difference might be due to the initially lower hemicellulose and lignin content in the pretreated

(and nonpretreated) pseudostems from Peru (Table 3). In general, higher glucose yields followed with higher xylan solubilization for the low-severity SE and LHW pretreatments; this was not the case for the high-severity steam explosion pretreatment (Fig.1c–f). Given that hemicellulose content in the high severity SE pretreated residues was minimal (Table 2), the lower xylan conversion could be due to the recalcitrant hemicellulose fraction remaining after pretreatment or sugar degradation under the higher severity conditions.

In any case, glucan conversion appears to reach a plateau by 44 h incubation for all residues under the different conditions evaluated. Comparable results were obtained by other researchers using different biomass feedstocks treated at similar conditions. For instance, Mosier et al. [29]

reported 80 % glucose yield in 48 h with 30 FPU/g glucan (supplemented with β-glucosidase) using LHW-pretreated corn stover. Kim et al. [26] reported 90 % glucose yield in Table 2 Chemical composition of steam explosion pretreated banana biomass from Colombia

SE-LS SE-HS SE-LS SE-HS SE-LS SE-HS

Ash 6±0.3 7±1.3 6±0.9 15±1.0 6±0.8 12±0.3

Lignin^a 24±1.8 29±2.2 32±2.0 44±1.7 16±4.9 30±2.0

Glucan 52±1.5 60±2.1 38±1.3 28±0.8 58±1.8 52±1.0

Xylan 9±0.3 3±0.03 9±0.9 2±0.2 9±0.4 3±0.1

Galactan 0 0 1±0.8 0 0 0

Arabinan 1±0.03 0 3±0.3 0 2±0.01 0

Mannan 1±0.02 0.2±0.4 1±0.1 0.2±0.4 1±0.02 1±0.02

Total 93 99 90 89 92 98

aChemical composition of the untreated material is presented in Table1for comparison

Data are percentage from total on a dwb, average±standard deviation (n=3). Chemical composition of the untreated material is presented in Table1 for comparison

SE-LSsteam explosion low severity (10 min at 190 °C),SE-HSsteam explosion high severity (76.4 min at 200 °C)

aAcid-soluble and insoluble lignin combined

Table 3 Chemical composition of liquid hot water (LHW) pretreated banana biomass from Peru and Colombia

Data are percentage of total on a dwb from two replicates. Chem- ical composition of the untreated material is presented in Table1 for comparison

aFiltrate from LHW-pretreated slurry

Peru Colombia Peru Colombia Peru Colombia

Ash 11 / 12 5/6 7/11 14/15 21/20 12/16

Lignin 13/16 24/24 37/32 31/26 24/19 18/10

Glucan 53/52 57/60 31/32 32/30 40/39 44/47

Xylan 6/5 8/8 7/7 9/8 7/7 8/8

Galactan 0/0 0/0 1/0 1/1 1/0 0/0

Arabinan 1/1 1/1 2/2 3/3 2/2 2/2

Mannan 1/1 1/1 1/1 1/1 2/2 1/1

Total 85/87 96/100 86/85 91/84 97/89 85/84

pH in liquid^a 4.17 5.8 4.67 5.4 5.05 5.1

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168 h with 15 FPU/g glucan (supplemented with β- glucosidase) using LHW pretreated switchgrass. Ohgren et al. [31] obtained 70 % glucose yield using 15 FPU/g glucan (supplemented withβ-glucosidase) and 100 % glucose yield using 15 FPU/g glucan when supplemented with β- glucosidase and xylanase, after 72 h incubation using uncatalyzed steam explosion pretreated corn stover. In this work, we obtained 80 % glucose yield in 44 h with 27 FPU/g glucan (supplemented with β-glucosidase and xylanase) using uncatalyzed steam explosion and LHW- pretreated banana rachis. Lower glucose yields were

obtained for the other banana residues. Optimization of pretreatment parameters and the use of more advanced enzyme preparations could help improve conversion yields.

For instance, current developments in enzyme technology have resulted in technologically advanced enzyme preparations that permit the use of lower severity acidic pretreatments with high bioconversion yields [5,31]. These“next generation” enzyme preparations for cellulosic ethanol (compared to the ones used in this study) are currently available in the market, which require lower dosages to achieve high conversion rates and work with a wide range Fig. 1 Glucan and xylan

conversion of pretreated banana biomass from Colombia and Peru.a,c,e,gGlucan conversion.b,d,f,hXylan conversion.SE-lsSteam explosion low severity pretreatment (10 min at 190 °C),SE-hssteam explosion high severity pretreatment (64 min at 200 °C),LHW liquid hot water pretreatment.

Conversion data are percentage from theoretical on a dry weight basis (dwb), average±standard deviation (n=2). Corresponding fermentable sugars

concentrations (g/L, dwb) are plotted to they-axis. Legends given inaapplies tocande;

legends given inbapplies todandf

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of feedstock and pretreatments [34]. Using this improved enzyme systems for lignocelulosic banana waste may also give better and/or faster yields.

Simultaneous Saccharification and Fermentation of Pretreated Biomass

The next step in biomass bioconversion is the reduction of carbohydrate streams to fuel molecules. One of the best- developed and most commonly used reduction chemistry for this aim is fermentation of sugars by yeast and/or bacteria to produce ethanol [5,29]. Major limitations in the fermentation step are the presence of inhibitory compounds (such as furans or phenolics) released during pretreatment and the lack of suitable nutrients to support microbial growth [5]. In addition, the availability of a suitable microorganism capable of utilizing C5 and C6 sugars is still a major limitation for industrial cellulosic ethanol production. Yeast and bacteria strains capable of cofermenting C5 and C6 sugars have been developed by means of metabolic engineering [35], but their suitability and reliability as well as tolerance to inhibitors and high ethanol productivity are still a limiting factor for their use in industrial settings [36]. Process con- figurations to convert pretreated lignocellulosic biomass to ethanol include sequential hydrolysis and fermentation (SHF), SSF, and direct microbial conversion (DMC) [36].

While higher ethanol yields have been reported for SHF compared to SSF or DMC, SSF offer several advantages such as cost reduction using one reactor, shorter processing time, elimination of end product inhibition of cellulolytic enzymes, and lower contamination risk of the sugar hydrolyzate [36]. The main drawback of SSF would be the difference in optimal temperatures for the enzymatic hydrolysis (generally 50 °C) and fermentation (30–35 °C) steps.

In this work, delayed SSF was tested with an initial hydrolysis step at 50 °C for 24 h followed by fermentation at 37 °C upon yeast addition. Shen et al. [37] also reported a prehydrolysis step at 50 °C for 12 h, followed by SSF at 37 °C using aS. cerevisiae strain to decrease viscosity of substrates and facilitate the start of the fermentation process.

Glucan and xylan conversion along with ethanol production (percent based on glucan and xylan content) for pretreated rachis for either location are presented in Fig. 2. Ethanol yields (percent of theoretical) after 122 h incubation for all pretreated residues evaluated are presented in Fig. 3.

Glucose and xylose yields obtained in the prehydrolysis step for each residue and pretreatment type were comparable to those obtained in the previous saccharification study.

Although high glucose yields (up to 90 % of theoretical) were obtained within the 24 h prehydrolysis step, ethanol production was low. Consistent with saccharification data, higher ethanol yields were obtained for banana rachis with up to 49 % of theoretical (Fig.3). Possible explanations for

the low fermentation yields include the high fermentation temperature used, low nutrient content in the fermentation vessel, and possible presence of inhibitory compounds.

Higher ethanol production could be obtained by optimizing fermentation conditions and using recombinant yeast strains than can convert C5 sugars [29].

Very few reports exist on the production of cellulosic ethanol from banana waste. For instance, Sharma et al.

[13] optimized ethanol production from a mixture of banana peels and kinnow waste in India using a steam pretreatment and cofermentation with S. cerevisiae and Pachysolen tannophilus; they obtained a maximum ethanol yield of 0.426 g g⁻¹(dry biomass) and a fermentation efficiency of 83.52 %. Harish et al. [38] investigated DMC of acid or alkali pretreated banana leaves withClostridium thermocellumCT2 and Thermoanaerobacter ethanolicus ATCC 31937; they obtained maximum ethanol yield of 0.41 g g⁻¹(dry substrate) for the 1 % NaOH—alkali pretreated feedstock after 8 days.

El-Zawawy et al. [39] investigated ethanol production from lignocellulosic banana residues pretreated by alkaline pulping, water hydrolysis pulping, microwave alkaline treatment, and steam explosion; residues were hydrolyzed with cellulase enzyme alone and the highest ethanol yield reported was 2.8 g L⁻¹ for the steam-explosion-treated residues but no glucan conversion data were provided. Alternatively, ethanol production by SSF of similarly pretreated biomass has been reported for other feedstock. For instance, Mosier et al. [29]

obtained 88 % fermentation efficiency for both glucose and xylose using recombinantS. cerevisiae 424A (LNH-ST) on LHW-pretreated corn stover hydrolyzed with 11 FPU cellulose (supplemented withβ-glucosidase). Suryawati et al. [40]

reported 72 % ethanol yield using a thermotolerant yeast strain Kluyveromyces marxianusIMB4 on LHW-pretreated switchgrass hydrolyzed with a cellulose preparation. Shen et al. [37]

reported 35 % ethanol yield (of theoretical) for SO2impreg- nated steam pretreated sweetsorghum bagasse hydrolyzed with 7.5 FPU cellulose/g glucan. To our knowledge, this is the first report that systematically evaluates the bioconversion of the different lignocellulosic banana residues to ethanol using well-documented procedures and leading pretreatment technologies. Further studies to increase glucan conversion and ethanol yield could be pursued, such as optimizing pretreatment parameters and using next generation cellulolytic enzyme preparations. Fermentation of C5 and C6 sugars by recombinant yeast strains could be further tested to increase fermentation efficiency and ethanol yields.

Effect of Pretreatment Solutions on Yeast Growth

The type and severity of the pretreatment process can influ- ence the amount, type, and concentration of potential inhibitory compounds that can be released in soluble form from biomass (such as acetic acid from acetylated hemicellulose

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or phenolics or other organic compounds released from lignin) or produced in sugar degradation reactions (such as aldehydes and organic acids). These inhibitory products can impact the fermentation performance and can require that hydrolyzate detoxification or conditioning is conducted,

especially for high solid pretreatments where the concentration of such compounds can be relatively high [27].

Steam explosion and liquid hot water pretreatments in this study resulted in mild acidic conditions (Table3); thus, sugar degradation reactions such as the conversion of xylose to furfural and glucose to 5-HMF compounds, inhibitors to fermenting microorganisms, were less likely than in other more severe acid pretreatments [4]. Nevertheless, the low ethanol yields obtained in the SSF experiments could indicate presence of fermentation inhibitors in the hydrolyzate.

We evaluated the effect of potentially inhibitory compounds generated during LHW pretreatment on yeast growth as a possible explanation of the low ethanol yields observed.

Two yeast strains were tested; a commercial S. cerevisiae

“BioFerm XR”strain and aS. cerevisiaeD5A strain from the NREL. Their growth was evaluated simultaneously in the absence and the presence of 1 and 5 % (v/v) pretreatment liquid from the different banana biomass types.

Yeast growth in the absence and presence of pretreatment liquid is summarized in Fig.4. The commercial yeast strain BioFerm XR had higher overall growth compared to the NREL D5A strain at the temperature tested but appeared more susceptible to the pretreatment liquid at the higher concentration (5 %v/v). No apparent inhibition of yeast growth was noted for either strain when 1 % (v/v) pretreatment liquid was added, compared to the control samples to which water was added instead of pretreatment liquid.

Fig. 2 Simultaneous saccharification and fermentation (SSF) of pretreated banana biomass.

a–cPretreated rachis from Colombia.dPretreated rachis from Peru. Data are percentage from theoretical on a dwb, average±standard deviation (n=2). SSF data of other banana residues are available online as Supplementary Information2

Fig. 3 Ethanol production (percent of theoretical) in SSF of pretreated banana biomass. Data correspond to ethanol production after 122 h incubation of two separate SSF experiments (average±standard deviation,n=4).SE-LSsteam explosion low severity pretreatment,SE-HS steam explosion high severity pretreatment,LHW liquid hot water pretreatment,CbColombia,PrPeru

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In this work, SSF experiments were conducted at 1 % (w/v) glucan content, which corresponded to 1.7–3.5 % solid loadings. Results from the growth inhibition tests indicate that there might have been some inhibitory effect from compounds generated during pretreatment on yeast growth and ethanol yield. Furthermore, they indicate that yeast inhibition will occur at higher glucan loadings.

Therefore, either washing biomass after pretreatment or preconditioning yeast (i.e., by adding pretreatment liquid to the yeast preincubation media) and/or using a more tolerant strain will be needed to increase ethanol yields.

Available Lignocellulosic Banana Waste and Potential Ethanol Production in Agricultural Communities in Peru and Colombia

To assess the impact of cellulosic ethanol production in rural agricultural communities in Peru and Colombia, we estimated

the annual lignocellulosic residue generation and potential ethanol production for either location using the bioconversion routes and ethanol yields obtained in this study. These find- ings are summarized in Table 4. The Province of Antioquia (Uraba) is an intensive banana-growing region in Colombia, whose residue generation accounts for 62 % of country-wide total. Similarly, banana cultivation in Peru is concentrated in the Piura Region, where organized communities are devoted to organic banana production [41]. In both locations, the average income of banana growers is approximately 10 USD/day; local populations live in rural conditions and mostly consume fuels from fossil sources (personal commu- nication, Gaston E. Cruz-Alcedo, Director of the Agro- industrial Innovation Center of Piura, Peru; personal commu- nication; Angela A. Ruiz-Colorado, Bioprocesses and Reactive Flows Group, University of Colombia at Medellin). Therefore, using the residual banana biomass for fuel production to be used locally or sold in local markets could help improve the livelihood of local populations as well as benefit the environment [6]. In any case, the data summarized here will serve as reference for process improvements (i.e., fermentation yields) and for feasibility studies of localized cellulosic ethanol generation. Current efforts to design a

“suitable”scale cellulosic ethanol production facility as well as to estimate the socioeconomic impact of local ethanol production in the targeted communities are currently underway.

Conclusion

Cellulosic ethanol is a carbon neutral alternative to fossil fuels for transportation that is gaining increased relevance.

Significant research has been done on the utilization of agricultural residues from major commodities, such as wheat, rice, corn stover, or wood chips; however, little is known about bioconversion of other major nonstaple crops such as banana. Due to the high amount of usable residue that is generated in banana cultivation, along with its widespread distribution in tropical and subtropical regions, Fig. 4 Effect of LHW pretreatment liquid on S. cerevisiae strains

BioFerm XR and NREL D5A growth.ControlYeast strains grown with ultrapure water added instead of pretreatment liquid, XR S.

cerevisiae BioFerm XR strain,D5A S. cerevisiae D5A strain. Data correspond to OD600nmmeasured after 320 min minus the initial OD measured at 10 min from incubation, average±standard deviation,n=3

Table 4 Lignocellulosic banana residue available at Chira Valley (Peru) and Uraba Province (Colombia) and potential ethanol production using bioconversion data from this study

Location Banana residues available (1,000 tons/year)^a

Potential ethanol production (ML/year)^b

Ethanol/biomass (g g⁻¹,dwb)

Stems Leaves Rachis Stems Leaves Rachis Stems Leaves Rachis

Chira Valley (Peru) 15 13 0.81 4.6 1.2 0.16 0.24 0.07 0.15

Uruba Province (Colombia) 306 2.5 75 52.8 0.14 24.0 0.14 0.04 0.25

aOn a dry weight basis

bUsing glucan content in untreated biomass (Table 1) and glucan conversion of LHW-pretreated biomass (Fig. 1), and assuming 100 % fermentation efficiency

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banana waste bioconversion could allow for local cellulosic ethanol production as a sustainable development strategy for rural agricultural communities. Within that aim, the bioconversion potential of three different lignocellulosic banana residues (pseudostems, leaves, and rachis) obtained from agricultural communities in the Sullana Province (Peru) and Uraba Province (Colombia) was investigated.

The chemical composition varied among the different banana residues evaluated; pseudostems had the highest glucan content (up to 60 %) followed by rachis and leaves.

This trend was consistent among residues from either location.

Mild acidic pretreatments resulted in partial hemicellulose removal, more noticeable at the higher severity regimes, with glucan and lignin content remaining unchanged. Biomass saccharification using a mixture of cellulolytic and xylanolytic activities resulted in up to 90 % glucan conversion for pretreated rachis from Colombia, and 77 % for pseudostems and rachis from Peru. LHW pretreatment appeared more suitable for rachis, while higher severity SE schemes gave better results for pseudostems and leaves. Overall, mild acidic pretreatments appeared suitable for treating of lignocellulosic banana waste. SSF of pretreated substrates using commercial S. cerevisiaestrain BioFerm XR resulted in similar sugar conversions in the prehydrolysis step (with up to 90 % glucan conversion for some substrates), but ethanol yields were yet too low for commercial viability (42 % of theoretical for pretreated rachis). The low ethanol yields obtained were possibly due to inhibitory compounds generated during pretreatment, suboptimal fermentation conditions (i.e., low nutrients and high temperature) and the inability of fermenting microorganisms to utilize C5 sugars. Growth inhibition ofS.

cerevisiae strains “BioFerm XR” and “NREL D5A” by pretreatment liquids was further evaluated and found to be significant at 5 %v/vpretreatment liquid concentration in the growth media.

Finally, the amount of banana residue generated and potential cellulosic ethanol production using the bioconversion routes and results from this work estimated for the two communities targeted in this study indicated that there is potential for cellulosic ethanol production from pseudostems and rachis due to their relative abundance, especially for Colombia and the high glucan conversion rates obtained. Nevertheless, fermentation yields will need to be improved to attain economic viability. For instance, this could be done by (1) increasing solids loadings, (2) using tolerant or preconditioned yeast strains, and (3) optimizing fermentation conditions. In any case, we believe that the results from this work constitute a stepping stone for future improvements and feasibility studies for cellulosic ethanol production from banana waste, a technology that could become a game changer for many agricultural communities in tropical and subtropical regions.

Acknowledgments This research was funded by a Faculty Initiative grant from the Pacific Rim Research Program of the University of California. The authors would like to thank Kameron Chun, Nardrapee Karuna, and Cassy Gardner (Biological and Agricultural Engineering, University of California, Davis) for their help in conducting the experiments and sample analyses, the National Renewable Energy Laboratory and Steven Zicari (Biological and Agricultural Engineering, University of California, Davis) for providingS. cerevisiaeyeast strains and Mr. Juan Carlos Quintana for preparing the Colombian residues.

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