Journal of Biomass to Biofuel (JBB)

ISSN: 2368-5964

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.	ST 60 min.	DSA 60 min.	LRT 24 h	LRT 48 h	LHT 60 min.
Root crop processing residues
Sweet potato peel	2.746c	2.864b (4.30)**	3.142a (14.41)	3.141a (14.40)	2.825b (2.88)	2.802bc (2.03)	2.813b (2.42)
Elephant foot yam peel	2.548c	2.661b (4.44)	2.946a (15.63)	2.902a (13.88)	2.625b (3.02)	2.598c (1.95)	2.601bc (2.09)
Tannia peel	2.780c	2.879bc (3.56)	3.224a (15.94)	3.175b (14.18)	2.855bc (2.67)	2.846bc (2.37)	2.842bc (2.21)
Greater yam peel	2.057c	2.135b (3.78)	2.348a (14.14)	2.314a (12.51)	2.121b (3.11)	2.091bc (1.66)	2.103b (2.23)
Beet root peel	1.026ab	1.067ab (3.97)	1.188a (15.83)	1.170a (14.07)	1.052ab (2.55)	1.049ab (2.25)	1.048ab (2.09)
Vegetable processing residues
Ash gourd peel	3.016c	3.132b (3.82)	3.511a (16.41)	3.426a (13.58)	3.111b (3.14)	3.102b (2.84)	3.080c (2.10)
Pumpkin peel	2.961d	3.053c (3.09)	3.424a (15.63)	3.359ab (13.42)	3.047c (2.90)	3.024c (2.11)	3.023c (2.10)
Vegetable banana peel	2.917cd	3.041b (4.28)	3.387a (16.14)	3.336a (14.38)	3.000bc (2.86)	2.99c (2.56)	2.987c (2.40)
Mixed vegetable waste	2.653c	2.769b (4.37)	3.083a (16.22)	3.014a (13.59)	2.731b (2.95)	2.702bc (1.85)	2.700bc (1.77)

* 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).

Detoxification treatments	Native	ST 45 min.	ST 60 min.	DSA 60 min.	LRT 24 h	LRT 48 h	LHT 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)*.

Detoxification treatments	Native	ST 45 min.	ST 60 min.	DSA 60 min.	LRT 24 h	LRT 48 h	LHT 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.

Detoxification treatments	Native	ST 45 min.	ST 60 min.	DSA 60 min.	LRT 24 h	LRT 48 h	LHT 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.

Detoxification treatments	Native	ST 45 min.	ST 60 min.	DSA 60 min.	LRT 24 h	LRT 48 h	LHT 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.

Detoxification treatments	Native	ST 45 min.	ST 60 min.	DSA 60 min.	LRT 24 h	LRT 48 h	LHT 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 NaBH_4, 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.

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.

Acknowledgements

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.

References