Volume 3 - Year 2016 - Pages 1-15
DOI: 10.11159/jbb.2016.001

Phenolic Inhibitors of Saccharification and Fermentation in Lignocellulo-Starch Prehydrolysates and Comparative Efficacy of Detoxification Treatments

Madhanamohanan G. Mithra and Gourikutty Padmaja

Division of Crop Utilization, ICAR-Central Tuber Crops Research Institute
Thiruvananthapuram-695 017, Kerala, India

Abstract - The generation of phenolic inhibitors in the prehydrolysates from steam, dilute sulphuric acid (DSA) and lime pretreated root wastes (peels from sweet potato, elephant foot yam, tannia, beet root and greater yam) and vegetable processing residues (peels from ash gourd, pumpkin and vegetable banana and mixed vegetable waste) was monitored. Further, the effect of detoxification agents such as Tween 20, PEG 4000, active charcoal and sodium borohydride in reducing their levels was compared. The aqueous extracts from native biomass contained soluble phenolics at levels of 1.03% to 3.02%. Highest release (12.5-16.2% increase from the values in aqueous extracts of native biomass) of total soluble phenolics (TSPs) into the prehydrolysates was found in steam (60 min.) and DSA (60 min; 121 ⁰C) pretreatments, irrespective of the type of biomass, while lime pretreatment resulted in only 1.7-3.1% increase in the phenolics. Maximum quantity of TSPs was removed in 2 h at room temperature (30±1 ⁰C) by Tween 20 (70-81%) or its combination with PEG (73-82%). Active charcoal (2%; 45 ⁰C; 1 h) or sodium borohydride (40 mM; 20 min; 30±1 ⁰C) removed only 40-59% and 37-53% phenolics respectively. Loss of reducing sugars was the least (2-3%) in Tween 20 (0.50%) treatment, indicating its superiority over the other treatments.

Keywords: Lignocellulo-starch prehydrolysates,Phenolic inhibitors, Detoxification, Surfactants, Charcoal, Borohydride, Reducing sugar loss.

© Copyright 2016 Authors - This is an Open Access article published under the Creative Commons Attribution License terms. Unrestricted use, distribution, and reproduction in any medium are permitted, provided the original work is properly cited.

Date Received: 2016-01-23
Date Accepted: 2016-06-18
Date Published: 2016-07-06

1. Introduction

Lignocellulosic biomass has been globally recognized as the most attractive source for bioethanol production, owing to the cheap and abundant availability. Nevertheless, the sustainable production depends on the successful overcoming of biomass recalcitrance and other technological barriers such as high enzyme costs, low conversion rate, generation of saccharification/fermentation inhibitors etc. [1-3]. Pretreatment of biomass is the key step to breaking biomass recalcitrance and enhancing the accessibility of cellulose to enzymatic hydrolysis [3]. Besides disrupting the crystalline structure of cellulose, pretreatment also helps the lignin shield to be broken down and part of the hemicelluloses to be solubilised [4, 5]. Pretreatment operations generally adopted include steam, acid or alkaline exposures at high temperatures [4] and result in a solid fraction enriched with amorphous cellulose that is easily susceptible to enzymatic hydrolysis along with partially degraded lignin and a soluble prehydrolysate rich in monomeric sugars released from hemicellulose along with lignin degradation products [6]. Most of the lignocellulose derived by-products are inhibitory to fermentation organisms and based on the origin, they have been grouped into weak acids, furan derivatives and phenolic compounds [7, 8]. The extent of formation of these inhibitors depends on the composition of biomass, nature and severity of pretreatment conditions etc. [9].

Phenolic inhibitors are mainly formed during pretreatment from the degradation of lignin and to a small extent from the extractives as well as the phenolic ester groups of hemicellulose and include hydroxycinnamic acid, vanillin, syringaldehyde, tannic acid, gallic acid etc. [10-12]. Tejirian and Fu [13] reported the inhibition of cellulolytic enzymes by simple and oligomeric phenols generated during pretreatment from lignin modification/degradation. Phenolic compounds in the soluble fraction have been reported to inhibit microbial growth by possibly interfering with the cell membrane [14, 15]. Clarke and Mackie [16] found that lower molecular weight phenols had the strongest toxicity to yeast growth. Detoxification of prehydrolysates has been attempted by several researchers using chemical, physical or biological methods [17-19]. Surfactants such as Tween and polyethylene glycol have been reported to reduce the levels of lignin degradation products and enhance the saccharification rate [13, 20, 21]. Adsorption on active charcoal has been attempted by others to reduce the toxicity of hydrolysates [22, 23]. In situ detoxification with sodium borohydride has been reported to tremendously improve the fermentation of sugarcane bagasse hydrolysate [24].

Although lignocellulosic biomasses generally comprise of agricultural residues, woody forest biomass and dedicated grasses such as switchgrass or Miscanthus sp., a major contribution is currently from processing residues also due to increased industrial and domestic activities. We had earlier reported the importance of root and vegetable processing residues as promising feedstocks for bioethanol production, capable of reducing environmental pollution from their inadequate disposal strategies [25-27]. As different from the typical lignocellulosic biomasses (LCBs), these residues also contain appreciable quantities of starch, enabling them to be grouped as lignocellulo-starch wastes. Pretreatment techniques such as steam, dilute sulphuric acid (DSA) and lime (calcium hydroxide) treatments were found to deconstruct the cellulose by partial solubilisation of lignin and moderate to high hydrolysis of hemicellulose and starch.  It was reported earlier that steam pretreatment brought about Ca. 53% and 25% hydrolysis of hemicelluloses and starch, while DSA hydrolyzed 83-94% starch and 42-48% hemicelluloses, with 27-29% delignification in the former [25]. On the contrary, lime pretreatment solubilised hemicellulose to a limited extent (Ca. 12%), with minimal depolymerisation of starch [26]. Being rich in fermentable sugars, the liquid phase or prehydrolysates from the processing residues of roots (sweet potato, elephant foot yam, tannia, beet root and greater yam) and vegetables (ash gourd, pumpkin, vegetable banana and mixed vegetable wastes) is also a valuable feedstock and combined or whole slurry fermentation has to be considered in these cases. Hence, the monitoring of build-up of fermentation inhibitors and their effective removal from the prehydrolysates become obligatory to enhance the fermentable sugar yield at the saccharification stage. In the present study, the generation of phenolic inhibitors in steam, DSA and lime pretreated root and vegetable processing residues was studied. Besides, the comparative efficacy of various detoxification approaches including surfactant and sodium borohydride application, active charcoal adsorption etc. in reducing their levels in the prehydrolysates was also investigated.

2. Materials and Methods

2.1. Raw Materials

Peels from five root crops such as sweet potato (Ipomoea batatas), elephant foot yam (Amorphophallus paeoniifolius), tannia (Xanthosoma sagittifolium), beet root (Beta vulgaris) and greater yam (Dioscorea alata) and three vegetables such as ash gourd (Benincasa hispida), pumpkin (Cucurbita moschata) and vegetable (cooking) banana (Musa spp. ABB) were selected for the study. Besides, mixed vegetable waste (comprising peels, seeds, non-edible pulp, rotten vegetables etc.) collected from households and local restaurants was also subjected to the study. Samples were dried and powdered to particle size of Ca. 2-3mm, as described earlier [25, 27]. The unscreened powders were subjected to steam, dilute sulphuric acid (DSA) and lime pretreatments.

2.2. Pretreatments

The most effective pretreatments with regard to hemicellulose and starch hydrolysis coupled with lignin degradation from earlier studies [25-27] were selected for the present study. In simple steam treatment (ST), the dry biomass powders (5.0 g) were moistened to 40% moisture content with water and then exposed to steam in a Vegetable Steamer (M/s TTK Prestige India Ltd., India) for 45 min. and 60 min. After steam exposure, volume was made up to 50 ml with distilled water, mixed well and centrifuged at 3000 rpm for 20 min. The clear supernatant was stored at 4 ⁰C till use. In the DSA treatment, 5.0 g biomass powders were suspended in 50 ml DSA (0.5% v/v) and treated at 121 ⁰C and 0.102 MPa pressure in a Pressure cooker (M/s TTK Prestige India Ltd., India) for 60 min. (time after pressure build-up). Samples after cooling were treated as described before. Dry biomass residues (10% w/v) were also subjected to lime pretreatment at room temperature (30 ± 1 ⁰C) for 24 h and 48 h and at high temperature (121 ⁰C; 0.102 MPa) for 60 min., using calcium hydroxide (0.1g/g biomass) as described elsewhere [26]. The clear supernatants from the various treatments (hereinafter referred to as prehydrolysates) were used for the detoxification experiments. In the case of all the prehydrolysates and the aqueous extracts from native biomass, the pH was adjusted to 6.0 and volume increased to the nearest before subjecting to the detoxification treatments.

2.3. Detoxification Treatments

Two non-ionic surfactants, i.e., Tween 20 [Poly (oxyethylene)20 sorbitan-monolaurate] and PEG 4000 [poly (ethylene glycol) 4000] as well as active charcoal and sodium borohydride or their combinations were supplemented to the prehydrolysates and their comparative efficacy in removing the soluble phenolics was investigated. Tween 20 (0.50% or 0.75% v/v) and PEG (0.25% or 0.50% w/v) were added to separate lot of prehydrolysates and incubated at room temperature for 2 h with occasional shaking. Immediately after 2 h, the total soluble phenolics (TSP) content in each of the surfactant added sample was assayed. In the combination treatment, the level of Tween 20 was reduced to 0.25% (v/v), while retaining the concentration of PEG as 0.25% (w/v).

Four types of detoxification treatments were attempted using active charcoal such as 1% and 2% (w/v) levels at room temperature and at 45 ⁰C for 1 h each, after which the clear supernatants obtained through centrifugation were used for quantification of phenols in the prehydrolysates and native extracts. Sodium borohydride was also added at two levels such as 20 mM and 40 mM with occasional shaking at room temperature for 20 min. and the TSPs were quantified.  In the combination treatments, active charcoal (1% w/v) and sodium borohydride (20 mM) were added and kept for 1 h at room temperature and the TSPs were assayed. A parallel set of native extracts was also kept in all cases. The levels of the detoxification chemicals were finalised based on the earlier studies using Tween 20 from our laboratory as well as from other research reports [20, 22, 24, 28].

2.4. Total Soluble Phenolics in the Prehydrolysates

Total soluble phenolics (TSPs) were determined in the non-detoxified and detoxified prehydrolysates using Folin-Ciocalteu reagent [29] and expressed as gallic acid equivalents (g/L) computed using pure gallic acid standard (SIGMA). Original (native) biomass samples were also extracted with distilled water in the same solid: liquid ratio (1:10 w/v) at room temperature (30 ± 1 ⁰C), with occasional stirring and the TSPs were determined by the same method. Any interference from the surfactants or the other detoxification agents in the assay was nullified by keeping a blank containing the same concentration of detoxification chemicals as in the test samples.

2.5. Reducing Sugar Loss

The loss of reducing sugars (RS) brought about by each of the detoxification agents was studied by assaying the RS in the sample extracts prior to and after the detoxification treatment by the Nelson Somogyi method [30]. Interference from the detoxification agents was nullified using blank systems containing the same concentration of the detoxification chemicals for each sample.

2.6. Statistical Analysis

Three replicates were kept for each experiment and duplicate analyses were performed on each replicate. The data were subjected to Analysis of Variance (ANOVA) for statistical testing of the mean values and least significant difference (LSD) for pair-wise comparison of mean values was found out using the statistical package, SAS 9.3 [31] Each value is considered significant at p <0.05.

3. Results and Discussion

3.1. Generation of Soluble Phenolics During Pretreatment

The concentration of soluble phenolic compounds in the prehydrolysates from root and vegetable processing residues subjected to steam, DSA and lime treatments was monitored and compared with that released by aqueous extraction from native (untreated) biomass. The original biomass extracts were found to contain high levels of soluble phenolics and the total concentration ranged from 1.03-3.02 g/L (Table 1). Least concentration was found in the aqueous extracts from beet root peel, while very high concentration of 2.9-3.02 g/L was found in ash gourd, pumpkin and vegetable banana peels. Phenolics in higher plants range from low molecular weight compounds to polymeric lignin type compounds and these have definite defensive role in the plants, accumulating in higher levels in peels than in edible parts [32]. Many of the simple phenolics have high solubility in water and could therefore exert appreciable inhibition on cellulases unless otherwise they are effectively removed [33].

Pretreatments such as steam (45 and 60 min.), DSA (60 min.) and lime [24 and 48 h at room temperature (30 ± 1 ⁰C) and 60 min. at 121 ⁰C] resulted in the release of phenols into the liquid phase in varying amounts. Highest release was observed in the case of steam (60 min.) and DSA (60 min.) and the percentage increase ranged from 12.5% to 16.2% in the different biomasses (Table 1). Lime treatment led to an increase of only 1.7% to 3.1% which was negligible. During pretreatment, native lignin is demethylated, solubilised (resulting in the release of monomeric and oligomeric phenols) or undergoes degradation changes [34, 35]. Although insoluble lignin is reported to remain in the solid fraction, many simple and oligomeric phenols move into the liquid fraction [15, 36]. The low molecular weight phenols have a stronger inhibitory effect on fermentative fungi or bacteria and also have deactivating effect on cellulases and β-glucosidases [11-13].  Soluble phenolic levels of 1.3 g/L and 2.0 g/L have been reported in pretreated maple pinchip liquid [37] and Eucalyptus globulus prehydrolysate [38] respectively, which was lower than that obtained in the present study. Jönsson et al [39] found that soluble phenolics such as 4-hydroxyl benzoic acid, vanillin and catechol were present in the untreated willow hemicellulose extracts, which corroborated with our findings that the native aqueous extracts could also exert potential toxicity to cellulases, by virtue of its high content of soluble phenolics. Besides lignin degradation, phenolic ester groups of hemicellulose are also reported to be sources of soluble phenolics in prehydrolysates [10]. Although dilute acid pretreatment led to solubilisation of hemicelluloses and fracture of lignin, much of the lignin was reported to remain in the residue itself [40] and we also found that only 12.5-14.4% increase occurred in soluble phenols in the prehydrolysates from the DSA pretreated biomasses under study. Similarly, formation of lignin-Ca-lignin linkages in lime pretreated residues has been reported preventing the release of lignin fragments into the soluble fraction [41]. The small increase in phenols in the lime pretreated root and vegetable residues might be due to such cross linking with lignin.

3.2. Detoxification of Prehydrolysates

In the case of most of the lignocellulosic feedstocks, subjected to pretreatments other than acid, the solid fraction is separated from the liquid and subjected to enzymatic saccharification. Removal of the bulk of the liquid phase could also eliminate the inhibition from lignin and carbohydrate degradation products. However, in the case of the root and vegetable processing residues, most of the starch also gets hydrolyzed/depolymerised to lower molecular weight fractions along with hemicelluloses solubilisation/hydrolysis and hence removal of the prehydrolysate could amount to considerable loss of fermentable sugars. Detoxification of prehydrolysate is thus obligatory in this case to improve saccharification and fermentation performance. Some of the detoxification methods such as use of non-ionic surfactants (Tween 20 and PEG 4000), active charcoal and sodium borohydride either alone or in combination were tried in the present study to understand their comparative efficacy in channelling out the soluble phenolics from prehydrolysates.

3.2.1. Non-ionic surfactants

Irrespective of the type of biomass or pretreatment techniques, 70-80% phenols were removed by Tween 20 (0.50%). Although slightly higher percentage removal of phenols was observed with 0.75% Tween 20, the effect could not justify the use of higher levels compared to 0.50% (Tables 2 a and b). Poly (ethylene glycol) (PEG 4000) exerted much less effect and the percentage removal ranged from 48-65% in the various pretreated biomass hydrolysates. Total soluble phenolics (TSPs) were removed to the highest extent by both Tween 20 and PEG from the prehydrolysates of steam treated (60 min.) residues (Table 2 a and b), which also had the highest level of TSPs in the prehydrolysates. Kim et al [37] found that the most inhibitory compound formed in lignocellulosic hydrolysates from hot water, steam explosion and dilute acid treatments were phenolics which reduced the cellulose hydrolysis by 50% and they could recover the full cellulase activity by removing the phenols. Water soluble phenolic acids such as tannic acid and gallic acid were reported to inhibit and deactivate β- glucosidase enzymes from Trichoderma reesei and Aspergillus niger [11, 12]. These studies stressed the need to remove soluble phenolics from the prehydrolysates which could also help reduce the enzyme loading. Phenolic compounds were also reported to exert toxicity to the fermenting organisms by partitioning into biological membranes and causing loss of integrity [42]. Hardwood hydrolysates rich in 4-hydroxybenzoic acid were inhibitory to the growth of Saccharomyces cerevisiae at 1g/L [43]. Enhanced enzymatic conversion of lignocellulose has been reported by several researchers, by supplementing with non-ionic surfactants such as Tween or PEG [20, 21] and the major effect reported was the prevention of adsorption of cellulases to exposed lignin surfaces.

Table 1. Total soluble phenolic (TSP) inhibitors in the prehydrolysates (g/L) from root and vegetable processing residues subjected to various pretreatments.

Biomass residues Native*   ST
45 min.
60 min.
60 min.
24 h
48 h
60 min.
Root crop processing residues
Sweet potato peel 2.746c 2.864b
Elephant foot yam peel 2.548c 2.661b
Tannia peel 2.780c 2.879bc
Greater yam peel 2.057c 2.135b
Beet root peel 1.026ab 1.067ab
Vegetable processing residues
Ash gourd peel 3.016c 3.132b
Pumpkin peel 2.961d 3.053c
Vegetable banana peel 2.917cd 3.041b
Mixed vegetable waste 2.653c 2.769b

* Aqueous extract from the original biomass; ** figures in parentheses indicate percentage increase from the original;  ST: steam; DSA: dilute sulphuric acid; LRT: lime room temperature (30±1 ⁰C); LHT: lime high temperature (121 ⁰C); Means with different superscripts in each row are significant at p<0.05.

Table 2a. Percentage removal of total soluble phenolic (TSP) inhibitors from prehydrolysates of root crop processing residues by non-ionic surfactants (Tween 20 and PEG).

Native   ST
45 min.
60 min.
60 min.
24 h
48 h
60 min.
Sweet potato peel
T1 73.54b 74.38b 78.05ab 76.25b 73.58b 70.04b 71.34b
T2 76.65a 76.69a 78.75a 77.45a 76.29a 72.78a 72.45a
T3 55.35d 56.56cd 57.29d 56.66c 55.93d 55.11c 55.15c
T4 56.45c 57.26c 60.47c 57.30c 56.92c 55.90c 56.11c
Elephant foot yam peel
T1 74.06b 74.90b 78.37a 76.77a 74.10b 70.56b 71.86a
T2 76.24a 76.28a 78.57a 77.04a 75.88a 72.37a 72.04a
T3 52.59d 52.99d 54.31c 53.41c 52.95d 51.85c 52.20c
T4 54.00c 54.11c 64.49b 54.45b 54.06c 52.35c 53.93b
Tannia peel
T1 74.57b 75.41b 79.08a 77.28b 74.61b 71.07b 72.37a
T2 77.13a 77.17a 79.23a 77.93a 76.77a 73.26a 72.93a
T3 53.96d 54.18d 55.84c 54.49d 53.98d 52.70d 53.84bc
T4 55.03c 55.22c 62.04b 55.75c 55.06c 54.45c 54.86b
Beet root peel
T1 74.60b 75.44b 79.11a 77.31a 74.64b 71.10b 72.40a
T2 77.20a 77.24a 79.30a 78.00a 76.84a 73.33a 73.00a
T3 49.70d 50.15c 50.48c 50.41b 49.99c 49.56c 49.64b
T4 50.29c 50.90c 58.90b 51.26b 50.37c 49.96c 50.21b
Greater yam peel
T1 74.04b 74.88b 78.32a 76.75a 74.08b 70.54b 71.84a
T2 76.22a 76.26a 78.55a 77.02a 75.86a 72.35a 72.02a
T3 52.57d 52.97d 54.29c 53.39b 52.93d 51.83c 52.18c
T4 53.98c 54.09c 64.47b 54.43b 54.04c 52.33c 53.91b

T1: Tween 20 (0.50%); T2: (0.75%); T3: Polyethylene glycol (PEG 4000) (0.25%); T4: PEG (0.5%); ST: steam; DSA: dilute sulphuric acid; LRT: lime room temperature (30±1 ⁰C); LHT: lime high temperature (121 ⁰C); Means with different superscripts in each column for each biomass are significant at p< 0.05.

Table 2b. Percentage removal of total soluble phenolic (TSP) inhibitors from prehydrolysates of vegetable processing residues by non-ionic surfactants (Tween 20 and PEG)*.

Native   ST
45 min.
60 min.
60 min.
24 h
48 h
60 min.
Ash gourd peel
T1 75.60b 76.44b 80.11b 78.31b 75.64b 72.10b 73.40b
T2 79.20a 79.24a 81.30a 80.00a 78.84a 75.33a 75.00a
T3 54.04d 54.57d 56.96d 55.46d 54.71d 53.51d 53.90d
T4 57.35c 58.12c 59.81c 58.38c 57.89c 57.06c 57.15c
Pumpkin peel
T1 73.65b 74.49b 78.16a 76.36b 73.69b 70.15b 71.45b
T2 76.72a 76.76a 78.82a 77.52a 76.36a 72.85a 72.52a
T3 57.07c 57.32c 58.41c 57.46c 57.10c 55.56c 56.86c
T4 58.02c 58.31c 64.25b 58.35c 58.02c 56.22c 57.72c
Vegetable banana peel
T1 74.54b 75.38b 79.05a 77.25a 74.58b 71.04b 72.34a
T2 77.22a 77.26a 79.32a 78.02a 76.86a 73.35a 73.02a
T3 57.95c 58.53c 59.05c 58.76c 58.03c 56.50d 57.93b
T4 58.91c 59.19c 64.95b 59.96b 58.95c 58.17c 58.46b
Mixed vegetable waste
T1 74.04b 74.88b 78.55a 76.75b 74.08b 70.54b 71.84b
T2 77.07a 77.11a 79.17a 77.87a 76.71a 73.2a 72.87a
T3 50.13c 50.20d 51.89c 50.44d 50.16d 48.11d 49.85c
T4 50.99c 51.65c 61.62b 51.76c 51.04c 49.22c 50.26c

* Footnotes as in Table 2a.

Surfactants with high hydrophilic-lipophilic balance (HLB) such as Tween 20 (HLB 16.7) and PEG 4000 (HLB 18.5) were reported to be more effective in extracting hydrophobic lignin degradation products into the soluble phase [44]. We have found that out of the two surfactants, Tween had a better action in removing phenolics from the prehydrolysates, by possible complex formation with them. This could be advantageous in the case of root and vegetable biomass prehydrolysates rich in fermentable sugars as it could exert a dual effect on the soluble and solid fractions simultaneously.

3.2.2. Active charcoal

The effect of active charcoal at two levels such as 1% and 2% at room temperature (RT; 30± 1 ⁰C) and at 45 ⁰C in removing phenols from the prehydrolysates was investigated. When compared to the non-ionic surfactants, the extent of phenol removal was significantly low (Tables 3 a and b) and ranged from 28-51% in the prehydrolysates treated with active charcoal at RT. Enhancing the temperature to 45 ⁰C resulted in significantly higher removal of phenolics (38-59%), which still was much less than  that removed by Tween 20. The extent of removal of inhibitors especially phenolics by active charcoal was highly dependent on pH and pH 2.0 favoured maximum removal [22].

Table 3a. Percentage removal of total soluble phenolic (TSP) inhibitors from prehydrolysates of root crop processing residues by active charcoal.

Native   ST
45 min.
60 min.
60 min.
24 h
48 h
60 min.
Sweet potato peel
T5 31.32d 34.22d 41.38d 36.61d 31.86d 31.05d 30.72d
T6 36.38c 41.90c 47.74c 43.61c 37.52c 35.94c 35.20c
T7 41.17b 44.07b 51.23b 46.46b 41.71b 40.91b 40.58b
T8 44.81a 50.33a 56.17a 52.04a 45.95a 44.37a 43.63a
Elephant foot yam peel
T5 33.75d 36.83d 44.12d 39.63d 34.29d 33.49d 33.21d
T6 39.21c 43.97c 50.91c 47.21c 40.38c 38.76c 38.06c
T7 43.61b 46.68b 53.98b 49.49b 44.14b 43.35b 43.07b
T8 47.64a 52.40a 59.34a 55.64a 48.81 47.20a 46.49a
Tannia peel
T5 30.93d 34.03d 40.33d 36.22d 31.53d 30.92d 30.40d
T6 36.29c 40.98c 46.53c 43.15c 37.13c 35.38c 34.84c
T7 40.79b 43.89b 50.18b 46.08b 41.38b 40.77b 40.26b
T8 44.72a 49.41a 54.96a 51.58a 45.56a 43.81a 43.27a
Beet root peel
T5 34.11d 36.56d 42.07d 38.45d 34.21d 33.93d 33.03d
T6 39.96c 42.18c 48.80c 45.28c 40.86c 39.08c 39.04c
T7 43.97b 46.41b 51.93b 48.30b 44.07b 43.79b 42.88b
T8 48.39a 50.61a 57.23a 53.71a 49.29a 47.51a 47.47a
Greater yam peel
T5 33.73d 36.81d 44.10d 39.61d 34.27d 33.47d 33.19d
T6 39.19c 43.95c 50.89c 47.19c 40.36c 38.74c 38.04c
T7 43.59b 46.66b 53.96b 49.47b 44.12b 43.33b 43.05b
T8 47.62a 52.38a 59.32a 55.62a 48.79a 47.18a 46.47a

T5: Active charcoal (1.0%; 30±1 ⁰C); T6: (2.0%; 30±1 ⁰C); T7: (1.0%; 45 ⁰C); T8: (2.0%; 45 ⁰C); other footnotes as in Table 2a.

Table 3b. Percentage removal of total soluble phenolic (TSP) inhibitors from prehydrolysates of vegetable processing residues by active charcoal.

Native   ST
45 min.
60 min.
60 min.
24 h
48 h
60 min.
Ash gourd peel
T5 28.51d 31.29d 37.02d 33.57d 28.93d 28.37d 28.05d
T6 33.12c 36.40c 42.72c 39.99c 34.07c 32.46c 32.15c
T7 38.37b 41.15b 46.88b 43.42b 38.79b 38.22b 37.91b
T8 41.55a 44.84a 51.15a 48.42a 42.50a 40.89a 40.58a
Pumpkin peel
T5 29.04d 32.10d 37.97d 34.24d 29.54d 28.77d 28.58d
T6 33.74c 37.34c 43.81c 40.79c 34.79c 33.30c 32.75c
T7 38.90b 41.96b 47.82b 44.09b 39.39b 38.63b 38.43b
T8 42.17a 45.77a 52.24a 49.22a 43.22a 41.73a 41.18a
Vegetable banana peel
T5 29.49d 32.22d 38.38 34.47d 30.00d 29.42d 28.93d
T6 34.25c 39.13c 44.29c 41.07c 35.33c 33.66c 33.15c
T7 39.34b 42.08b 48.24b 44.33b 39.86b 39.27b 38.79b
T8 42.69a 47.56a 52.72a 49.50a 43.77a 42.10a 41.58a
Mixed vegetable waste
T5 32.42d 35.39d 42.16d 38.16d 32.95d 32.19d 31.98
T6 37.65c 42.62c 48.65c 45.46c 38.81c 37.26c 36.64c
T7 42.27b 45.25b 52.02b 48.02b 42.81b 42.05b 41.83b
T8 46.09a 51.05a 57.08a 53.89a 47.24a 45.70a 45.07a

Footnotes as in Tables 2a and 3a.

In the present study the effect of various detoxifying agents was to be compared and hence the same pH of 6.0 was adopted for all the prehydrolysates uniformly, which might have resulted in lower removal in active charcoal treated prehydrolysates. Besides, a uniform contact time of one hour was adopted in the present study for both the concentrations of active charcoal. Parajó et al [23] reported that maximum removal of lignin degradation products from wood hydrolysates occurred at 20 min. and further increase to 90 min. had no significant effect.

Table 4. Percentage removal of total soluble phenolic (TSP) inhibitors from prehydrolysates by sodium borohydride.

Native   ST
45 min.
60 min.
60 min.
24 h
48 h
60 min.
Sweet potato peel
T9 42.53c 43.29bc 46.47a 44.88b 42.86c 41.97c 40.89d
T10 43.44d 45.39c 49.33a 47.43b 44.81c 42.83d 41.56e
Elephant foot yam peel
T9 45.88bc 46.60b 49.55a 48.59a 46.13b 45.27c 44.55d
T10 46.82e 48.85c 52.61a 51.35b 47.05d 46.19e 45.09f
Tannia peel
T9 42.01d 43.06c 45.29a 44.41b 42.42d 41.31e 40.78f
T10 43.01d 45.15c 48.08a 46.93b 44.35c 42.16e 41.38f
Beet root peel
T9 38.01c 39.37bc 41.23a 40.16b 38.96c 37.17cd 36.94d
T10 38.10d 40.31c 44.59a 41.86b 38.96d 38.03d 37.32e
Greater yam peel
T9 45.86cd 46.58c 49.53a 48.57b 46.11c 45.25d 44.53e
T10 46.80e 48.83c 52.59a 51.33b 47.03d 46.17e 45.07f
Ash gourd peel
T9 38.72d 39.60c 43.57a 41.16b 38.93d 37.91e 36.69f
T10 39.55e 41.51c 45.85a 43.49b 40.69d 38.68f 37.96f
Pumpkin peel
T9 39.44c 40.62b 42.64a 41.98a 39.74c 38.89cd 38.33d
T10 40.39e 42.58c 45.27a 44.45b 41.55d 39.68f 38.73g
Vegetable banana peel
T9 40.05c 40.77c 43.10a 42.27b 39.97cd 39.31d 38.81e
T10 41.01d 42.74c 45.76a 44.67b 41.97cd 40.45e 39.21f
Mixed vegetable waste
T9 44.02c 44.78c 47.35a 46.79b 43.97d 43.52d 42.90e
T10 44.97e 46.95c 50.27a 49.44b 45.98d 44.41e 43.34f

T9: Sodium borohydride (20 mM); T10: (40 mM); means with different superscripts in each row are significant at p<0.05; other footnotes as in Table 2a.

Temperature also greatly influenced the removal of phenolics from rice straw prehydrolysates by active charcoal and six fold increase was reported at 45 ⁰C compared to 25 ⁰C [22]. We have however observed that irrespective of the type of biomass, only 30-35% increase in phenolic removal occurred by increasing the temperature to 45 ⁰C. Varying reports are available on the effective concentration of active charcoal and while 1% was sufficient to eliminate 94% of phenolics from pretreated sugarcane bagasse [44], 1:40 (w/w) was found to be optimal for rice straw hemicellulose hydrolysate [22]. We have found that 2% (w/v) significantly improved the removal of phenolics, although the extent was much less than Tween 20.

3.2.3. Sodium borohydride

The advantages of sodium borohydride (NaBH4) such as direct addition at high concentrations to fermentation vessel and activity at mild conditions of temperature and pH such as 30 ⁰C and 6.0 were utilized for in situ detoxification of sugarcane bagasse and spruce hydrolysates [24].  Its effect in removing phenolics from the prehydrolysates of root and vegetable processing residues was therefore studied. Approximately 37-50% phenolics were removed by 20mM NaBH4, while Ca. 5-8% additional removal was obtained with 40 mM NaBH4 in 20 min. contact time. Phenolic removal was more from steam (60 min.) and DSA pretreated hydrolysates, though these levels were much less than Tween 20 (Table 4). Cavka and Jönsson [24] found that NaBH4 (39-47 mM) significantly enhanced the ethanol yield and productivity of sugarcane bagasse and spruce hydrolysates, the exact mechanism remaining unknown.

3.2.4. Combination treatments

The combined effect of surfactants, Tween 20 at half the dose (0.25%) and PEG at 0.25% was also studied and it was found that as high as 73-82% removal of phenolics was possible (Fig. 1 a-i). The synergistic action could also help reduce the level of Tween 20 addition, thereby reducing the overall process costs. Synergistic effect of NaBH4 (20 mM) and active charcoal (1%) at room temperature for one hour in removing phenolics was also studied and it was found that detoxification was significantly improved compared to either of the two materials alone (Fig. 1 a-i vs. Tables 3 and 4). However, these values were also much lower than the reduction brought about by Tween 20 alone or its combination with PEG, indicating the superiority of these treatments over others.

3.3. Loss of Reducing Sugars

The presence of high amounts of reducing sugars in the prehydrolysates from root and vegetable processing residues [25-27] necessitate whole slurry saccharification to maximize the fermentable sugar yield. Hence the effect of the detoxification treatments in removing reducing sugars along with the inhibitory phenolics was also quantified. While the detoxification treatment effect was evident, there was not much difference between the various prehydrolysates in the extent of sugar removal by each chemical such as Tween 20, PEG 4000, active charcoal or NaBH4. Hence, the mean value from the native and prehydrolysates were taken for each detoxification chemical in the case of each biomass and represented in Table 5. It was found that irrespective of the type of biomass, the least removal of sugars occurred in Tween 20 treatment (T1) followed by T2. Loss of sugars in PEG treated set was 3.7-4.6% in T3 and 5.1-6.0% in T4. Maximum loss of reducing sugars was observed in active charcoal (2%) at 45 ⁰C followed by 2% at room temperature. Nevertheless, Silva et al [45] found that treatment of sugarcane bagasse hydrolysate with active charcoal (1%) resulted in sugar loss of only 0.47%. Sodium borohydride treatment resulted in 6.0-8.8% loss of sugars. Although Tween 20+PEG treatment brought about higher removal of phenolics compared to Tween 20 alone, the loss of reducing sugars was significantly higher in the combination treatment. As 1-2% additional loss of reducing sugars could reduce the ethanol yield also considerably, treatment with Tween 20 alone is considered as the best in removing the toxic phenolic inhibitors from the prehydrolysates of root and vegetable processing residues.

Figure 1. Percentage removal of TSPs from prehydrolysates by combination treatments Bar 1: T11: Tween 20 (0.25%) + PEG (0.25%); Bar 2: T12: Sodium borohydride (20 mM) + active charcoal (1.0%), a - e (peels from sweet potato, elephant foot yam, tannia, beet root and greater yam); f - h (peels from ash gourd, pumpkin and vegetable banana); i- mixed vegetable waste.

Table 5. Mean percentage reduction in reducing sugars in various detoxification treatments*.

Biomass Treatments
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12
Sweet potato peel 2.43h 3.76g 3.87g 5.34ef 7.27d 9.21b 8.26c 10.47a 6.33e 8.15c 4.62f 8.21c
Elephant foot yam peel 3.08h 4.41g 4.61g 6.07e 7.92d 9.87b 8.91c 11.12a 6.98de 8.80c 5.28f 8.87c
Tannia peel 2.82g 4.14f 4.29f 5.75de 7.66cd 9.60b 8.65c 10.86a 6.72d 8.54c 5.01e 8.60c
Beetroot peel 2.29i 3.61gh 3.90gh 5.37f 7.13d 9.07b 8.12c 10.33a 6.18e 8.01c 4.48g 8.07c
Greater yam peel 3.07h 4.39g 4.59g 6.06ef 7.91d 9.85b 8.90c 11.11a 6.97e 8.79c 5.26f 8.85c
Ash gourd peel 2.75h 4.07g 4.25g 5.72f 7.59d 9.53b 8.58c 10.79a 6.64e 8.47c 4.79g 8.53c
Pumpkin peel 2.39h 3.72g 3.91g 5.38e 7.23cd 9.18b 8.22c 10.43a 6.29d 8.11c 4.59f 8.18c
Vegetable banana  peel 2.08h 3.41g 3.67g 5.14ef 6.92cd 8.86b 7.91c 10.12a 5.98e 7.80c 4.27f 7.86c
Mixed vegetable waste 2.63h 3.95fg 4.19f 5.66de 7.47cd 9.41b 8.45c 10.67a 6.52d 8.34c 4.82f 8.41c

* T1: Tween 20 (0.50%); T2: (0.75%); T3: Polyethylene glycol (PEG 4000) (0.25%); T4: PEG (0.5%);T5: Active charcoal (1.0%; 30±1 ⁰C); T6: (2.0%; 30±1 ⁰C); T7: (1.0%; 45 ⁰C); T8: (2.0%; 45 ⁰C); T9: Sodium borohydride (20 mM); T10: (40 mM); T11: Tween 20 (0.25%) + PEG (0.25%); T12: Sodium borohydride (20 mM) + active charcoal (1.0%); means with different superscripts in each row are significant at p < 0.05.

4. Conclusion

Total soluble phenolics (TSPs) ranging from 1-3% were present in the non-treated aqueous extracts of root and vegetable processing residues. Pretreatments such as steam (60 min; 100 ⁰C) and DSA (60 min; 121 ⁰C; 0.102 MPa) enabled the release of 12.5-16.4% more phenolics into the soluble fraction, while very low release was observed in the case of lime pretreatment. Maximum effect on the removal of TSPs was exerted by Tween 20 (0.25%) + PEG 4000 (0.25%) for 2h at room temperature (30± 1 ⁰C) followed by Tween 20 (0.50%) alone. Loss of reducing sugars was however the least in Tween 20 application. Active charcoal and sodium borohydride removed phenolic inhibitors to a much less extent. Unlike in the case of conventional lignocellulosic hydrolysates, the prehydrolysates from root and vegetable processing residues being rich in fermentable sugars, the whole slurry saccharification and fermentation have to be taken. In this context, Tween 20 with its ability to remove soluble phenolic inhibitors from prehydrolysates and prevent the inhibition of cellulases by the lignin held back in the pretreated residue through effective binding with it, stands out as a dual effect detoxification agent.


The financial support from the Kerala State Council for Science, Technology & Environment (KSCSTE Grant No. 853/2015/KSCSTE) is gratefully acknowledged. Authors are also thankful to the Director, ICAR- CTCRI for the facilities provided for the study and to Dr. J. Sreekumar, Principal Scientist (Agricultural Statistics) for the help extended in the statistical analyses.


[1] P. Alvira, E. Tomas-Pejo, M. Ballesteros and M. J. Negro, “Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review,” Bioresource Technology, vol. 101, no. 13, pp. 4851-4861, 2010. View Article

[2] A. K. Chandel, R. Rudravaram, M. L. Narasu, V. Rao and P. Ravindra, “Economics and environmental impacts of bioethanol production technologies: an appraisal,” Biotechnology and Molecular Biology, vol. 2, no. 1, pp. 14-32, 2007. View Article

[3] B. Yang and C. E. Wyman, “Pretreatment: The key to unlocking low cost cellulosic ethanol,” Biofuels, Bioproducts and Biorefining, vol. 2, no. 1, pp. 26-40, 2008. View Article

[4] N. S. Mosier, C. E. Wyman, B. E. Dale, R. T. Elander, Y. Y. Lee, M. Holtzapple and M. R. Ladisch, “Features of promising technologies for pretreatment of lignocellulosic biomass,” Bioresource Technology, vol. 96, no. 6, pp. 673-686, 2005. View Article

[5] C. E. Wyman, B. E. Dale, R. T. Elander, M. Holtzapple, M. R. Ladisch and Y. Y. Lee, “Coordinated development of leading pretreatment technologies,” Bioresource Technology, vol. 96, pp. 1959-1966, 2005. View Article

[6] X. Jing, X. Zhang and J. Bao, “Inhibition performance of lignocellulose degradation products on industrial cellulase enzymes during cellulose hydrolysis,” Applied Biochemistry and Biotechnology, vol. 159, pp. 696-707, 2009. View Article

[7] H. B. Klinke, A. B. Thomsen and B. K. Ahring, “Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pretreatment of biomass,” Applied Microbiology and Biotechnology, vol. 66, pp. 10-26, 2004. View Article

[8] E. Palmqvist and B. Hahn-Hägerdal, “Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition,” Bioresource Technology, vol. 74, no. 1, pp. 25-33, 2000. View Article

[9] L. J. Jönsson, B. Alriksson and N. O. Nilvebrant, “Bioconversion of lignocellulose: inhibitors and detoxification,” Biotechnology for Biofuels, vol. 6, no. 16, 2013. View Article

[10] S. F. Chen, R. A. Mowery, C. J. Scarlata and C. K. Chambliss, “Compositional analysis of water-soluble materials in corn stover,” Journal of Agricultural and Food Chemistry, vol. 55, no. 15, pp. 5912-5918, 2007. View Article

[11] E. A. Ximenes, Y. Kim, N. S. Mosier, B. S. Dien and M. R. Ladisch, “Deactivation of cellulases by phenols,” Enzyme and Microbial Technology, vol. 48, no. 1, pp.  54-60, 2011. View Article

[12] E. A. Ximenes, Y. Kim, N. S. Mosier, B. S. Dien and M. R. Ladisch, “Inhibition of cellulases by phenols,” Enzyme and Microbial Technology, vol. 46, no. 3-4, pp. 170-176, 2010. View Article

[13] A. Tejirian and F. Xu, “Inhibition of enzymatic cellulolysis by phenolic compounds,” Enzyme and Microbial Technology, vol. 48, no. 3, pp. 239-247, 2011. View Article

[14] H. Keweloh, G. Weyrauch, H. J. Rehm, “Phenol-induced membrane changes in free and immobilized Escherichia coli,” Applied Microbiology and Biotechnology, vol. 33, no. 1, pp. 66-71, 1990. View Article

[15] S. Larsson, A. Quintana-Sáinz, A. Reimann, N. O. Nilvebrant and L. J.  Jönsson, “Influence of lignocellulose-derived aromatic compounds on oxygen limited growth and ethanolic fermentation by Saccharomyces cerevisiae,” Applied Biochemistry and Biotechnology, vol. 84, pp. 617-632, 2000. View Article

[16] T. A. Clark and K. L. Mackie, “Fermentation inhibitors in wood hydrolysates derived from the softwood Pinus radiata,” Journal of Chemical Technology and Biotechnology, vol. 34, no. 2, pp. 101-110. 1984. View Article

[17] L. Olsson and B. Hahn-Hägerdal, “Fermentation of lignocelluloses hydrolysates for ethanol production,” Enzyme and Microbial Technology, vol. 18, no. 5, pp. 312-331, 1996. View Article

[18] E. Palmqvist and B. Hahn-Hägerdal, “Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification,” Bioresource Technology, vol. 74, no.1, pp. 17-24, 2000. View Article

[19] W. Parawira and M. Tekere, “Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review,” Critical Reviews in Biotechnology, vol. 31, no. 1, pp. 20-31, 2011. View Article

[20] J. Börjesson, R. Peterson and F. Tjerneld, “Enhanced enzymatic conversion of softwood lignocelluloses by poly (ethylene glycol) addition,” Enzyme and Microbial Technology, vol. 40, no. 4, pp. 754-762, 2007. View Article

[21] T. Eriksson, J. Börjesson and F. Tjerneld, “Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose,” Enzyme and Microbial Technology, vol. 31, no. 3, pp. 353-364, 2002. View Article

[22] S. I. Mussatto and I. C. Roberto, “Alternatives for detoxification of diluted-acid lignocellulosic hydrolysates for use in fermentative processes: a review,” Bioresource Technology, vol. 93, no. 1, pp. 1-10, 2004. View Article

[23] J. C. Parajó, H. Dominguez and J. M. Dominguez, “Charcoal adsorption of wood hydrolysates for improving their fermentability: influence of the operational conditions,” Bioresource Technology, vol. 157, no. 2, pp. 179-185, 1996. View Article

[24] A. Cavka and L. J. Jönsson, “Detoxification of lignocellulosic hydrolysates using sodium borohydride,” Bioresource Technology, vol. 136, pp. 368-376, 2013. View Article

[25] M. G. Mithra and G. Padmaja, “Compositional profile and ultrastructure of selected root and vegetable processing residues subjected to steam and dilute sulphuric acid pretreatment,” Current Biotechnology (In press), 2016.

[26] M. G. Mithra and G. Padmaja, “Lime pretreatment associated compositional and ultrastructural changes in selected root and vegetable processing residues,” International Journal of Renewable Energy Research (In press), 2016.

[27] M. G. Mithra and G. Padmaja. “Comparative alterations in the compositional profile of selected root and vegetable peels subjected to three pretreatments for enhanced saccharification,” Indian Journal of Biotechnology (In press), 2016.

[28] M. P. Divya Nair, G. Padmaja, M. S. Sajeev and J. T. Sheriff, “Bioconversion of cellulo-starch waste from cassava starch industries for ethanol production: Pretreatment techniques and improved enzyme systems,” Industrial Biotechnology, vol. 8, no. 5, pp. 300-308, 2012. View Article

[29] V. L. Singleton and A. Rossi, “Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents,” American Journal of Enology and Viticulture, vol. 16, pp. 144-158, 1965. View Article

[30] N. Nelson, “A photometric adaptation of the Somogyi method for determination of glucose,” Journal of Biological Chemistry, vol. 153, pp. 375-380, 1944. View Article

[31] SAS, SAS/STAT Software Version 9.3, Cary, NC.: SAS Institute Inc., 2010. View Article

[32] M. R. Juntheikki and R. Julkunen-Tiito, “Inhibition of β-glucosidase and esterase by tannins from Betula, Salix and Pinus species,” Journal of Chemical Ecology, vol. 26, no. 5, pp. 1151-1165, 2000. View Article

[33] E. Palmqvist, B. Hahn-Hägerdal, M. Galbe and G. Zacchi, “The effect of water-soluble inhibitors from steam-pretreated willow on enzymatic hydrolysis and ethanol fermentation,” Enzyme and Microbial Technology, vol. 19, no. 6, pp. 470-476, 1996. View Article

[34] B. Du, L. N. Sharma, C. Becker, S. F. Chen, R. A. Mowery, G. P.  Van Walsum, C.K. Chambliss, “Effect of varying feedstock-pretreatment chemistry combinations on the formation and accumulation of potentially inhibitory degradation products in biomass hydrolysates,” Biotechnology and Bioengineering, vol. 107, no. 3, pp. 430-440, 2010. View Article

[35] H. B. Klinke, B. A. Ahring, A. S. Schmidt, A. B. Thomsen, “Characterization of degradation products from alkaline wet oxidation of wheat straw,” Bioresource Technology, vol. 82, no. 1, pp. 15-26. 2002. View Article

[36] J. J. Fenske, D. A. Griffin and M. H. Penner, “Comparison of aromatic monomers in lignocellulosic biomass prehydrolysates,” Journal of Industrial Microbiology and Biotechnology, vol. 20, no. 6, pp. 364-368, 1998. View Article

[37] Y. Kim, E. A. Ximenes, N. S. Mosier and M. R. Ladisch, “Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass,” Enzyme and Microbial Technology, vol. 48, no. 4-5, pp. 408-415, 2011. View Article

[38] A. Romani, H. A.  Ruiz, F. B. Pereira, L. Domingues and J. A. Teixeira, “Effect of hemicelluloses liquid phase on the enzymatic hydrolysis of autohydrolyzed Eucalyptus globulus wood,” Biomass Conversion and Biorefinery, vol. 4, no. 2, 2013. View Article

[39] L. J. Jönsson, E. Palmqvist, N. O. Nilvebrant and B.  Hahn-Hägerdal, “Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor,” Applied Microbiology and Biotechnology, vol. 49, no. 6, pp. 691-697, 1998. View Article

[40] A. T. W. M. Hendriks and G. Zeeman, “Pretreatments to enhance the digestibility of lignocellulosic biomass,” Bioresource Technology, vol. 100, no. 1, pp. 10-18, 2009. View Article

[41] J. Xu, J. J. Chang, R. R. S. Shivappa and J. C. Burns, “Lime pretreatment of switchgrass at mild temperatures for ethanol production,” Bioresource Technology, vol. 101, no. 8, pp. 2900-2903, 2010. View Article

[42] H. J. Heipieper, F. J. Weber, J. Sikkema, H. Kewelo and J. A. M. de Bont, “Mechanism of resistance of whole cells to toxic organic solvents,” Trends in Biotechnology, vol. 12, no. 10, pp. 409-415, 1994. View Article

[43] S. Ando, I. Arai, K. Kiyoto and S. Hanai, “Identification of aromatic monomers in steam-exploded poplar and their influences on ethanol fermentation by Saccharomyces cerevisiae,” Journal of Fermentation Technology, vol. 64, no. 6, pp. 567-570, 1986. View Article

[44] M. Kurakake, H. Ooshima, J. Kato, Y. Harano, “Pretreatment of bagasse by non-ionic surfactant for the enzymatic hydrolysis,” Bioresource Technology, vol. 49, no. 3, pp. 247-251, 1994. View Article

[45] S. S. Silva, M. G. A. Felipe, M. Vitolo, “Xylitol production by Candida guilliermondii FTI 20037 grown in pretreated sugarcane bagasse hydrolysate,” Sustainable Agriculture and Food Energy Industry, pp. 1116-1119, 1998. View Article