Bioresource Technology 187 (2015) 91–96
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Production of cellulosic ethanol from cotton processing residues after pretreatment with dilute sodium hydroxide and enzymatic hydrolysis Douglas Henrique Fockink a, Marcelo Adriano Corrêa Maceno a, Luiz Pereira Ramos a,b,⇑ a b
Research Center in Applied Chemistry (CEPESQ), Department of Chemistry, Federal University of Paraná, P.O. Box 19032, Curitiba, Paraná 81531-980, Brazil INCT Energy & Environment (INCT E&A), Department of Chemistry, Federal University of Paraná, Brazil
h i g h l i g h t s A mild alkaline pretreatment enhanced the ethanol production from cotton gin residues. Total ethanol productions as high as 232 L ton
1
were achieved from cotton gin dust. 1 of ethanol under similar conditions. Substrate hydrolysates had no inhibitory effects on ethanol fermentation.
More lignified cotton gin wastes produced 129 L ton
a r t i c l e
i n f o
Article history: Received 2 March 2015 Received in revised form 20 March 2015 Accepted 21 March 2015 Available online 27 March 2015 Keywords: Cotton gin waste Cotton gin dust Alkaline pretreatment Enzymatic hydrolysis Fermentation
a b s t r a c t In this study, production of cellulosic ethanol from two cotton processing residues was investigated after pretreatment with dilute sodium hydroxide. Pretreatment performance was investigated using a 22 factorial design and the highest glucan conversion was achieved at the most severe alkaline conditions (0.4 g NaOH g1 of dry biomass and 120 °C), reaching 51.6% and 38.8% for cotton gin waste (CGW) and cotton gin dust (CGD), respectively. The susceptibility of pretreated substrates to enzymatic hydrolysis was also investigated and the best condition was achieved at the lowest total solids (5 wt%) and the highest enzyme loading (85 mg of Cellic CTec2 g1 of dry substrate). However, the highest concentration of fermentable sugars – 47.8 and 42.5 g L1 for CGD and CGW, respectively – was obtained at 15 wt% total solids using this same enzyme loading. Substrate hydrolysates had no inhibitory effects on the fermenting microorganism. Ó 2015 Published by Elsevier Ltd.
1. Introduction Cotton is the main supply of natural fibers for textile industries and one of the largest sources of agro-industrial residues that can be utilized to produce fuels and chemicals (Howard et al., 2004; Ojumu et al., 2003). In 2014, the total world production of refined cotton fibers was 25.9 million ton, with Brazil and India, the largest world producers, contributing with 1.5 and 6.7 million ton, respectively. The world total area destined to cotton production was 34.4 million hectares across 80 countries, being China, India, United States, Pakistan and Brazil the most important contributors to these very impressive numbers (USDA, 2014). The high total world production capacity of cotton fibers also equates to the production of high amounts of cotton gin residues ⇑ Corresponding author at: Research Center in Applied Chemistry (CEPESQ), Department of Chemistry, Federal University of Paraná, P.O. Box 19032, Curitiba, PR 81531-980, Brazil. Tel.: +55 41 33613175. E-mail address: [email protected] (L.P. Ramos). http://dx.doi.org/10.1016/j.biortech.2015.03.096 0960-8524/Ó 2015 Published by Elsevier Ltd.
(McIntosh et al., 2014). In Brazil, 900 thousand hectares are destined to cotton production (USDA, 2014). The residues from cotton processing are of two types: the cotton gin dust (CGD) and the cotton gin waste (CGW). CGW arises from ginning process and is composed of cottonseed residues, hulls, sticks, leaves, and dirt, whereas CGD contains short fiber residues that are recovered from filter screens during the spinning/weaving processes. Slight differences in their composition are usually found among various mechanical harvesting methods. The major advantage of these cotton residues compared to other lignocellulosic materials is their high cellulose content. Hence, these renewable feedstocks have been tentatively used in a number of bioenergy applications to avoid its disposal by incineration or landfilling, such as in the case of cellulosic ethanol (Agblevor et al., 2003; McIntosh et al., 2014), pyrolysis (Zabaniotou et al., 2000), gasification (Sadaka, 2013), anaerobic fermentation (Isci and Demirer, 2007) and catalytic conversion to value-added chemicals (Grilc et al., 2015). However, the supporting evidence for the application of cotton gin residues for ethanol production is relatively scarce in comparison to other notable
92
D.H. Fockink et al. / Bioresource Technology 187 (2015) 91–96
agricultural residues such as corn stover, sugarcane bagasse and wheat straw (McIntosh et al., 2014). For conversion of cotton gin residues to cellulosic ethanol, a pretreatment method is needed to increase the accessibility of cellulose to enzymatic hydrolysis (Mosier et al., 2005). Among the various chemical and physical pretreatments used for cotton gin residues, acid pretreatments including steam explosion have been more often used (Agblevor et al., 2003; Ibrahim et al., 2010; Jeoh and Agblevor, 2001; Shen and Agblevor, 2008, 2011) while alkali pretreatments seems to be more effective for other cellulosic materials (Balat et al., 2008; Hendriks and Zeeman, 2009) and has not been studied inasmuch depth. The effectiveness of alkaline pretreatment depends on the biomass chemical composition and the conditions used for pretreatment (Singh et al., 2015). Deejing and Ketkorn (2009) have shown that this pretreatment technology is usually more effective for agricultural residues and herbaceous crops rather than for wood materials. This study was developed to evaluate the cellulosic ethanol production from CGD and CGW after pretreatment with dilute sodium hydroxide and enzymatic hydrolysis. The alkaline pretreatment performance was investigated using a 22 factorial design with variations in the catalyst amount (NaOH) and the pretreatment temperature. The enzymatic hydrolysis was also tentatively improved by using the same type of factorial design in which the process variables were the substrate total solids (TS) and the enzyme loading. Finally, the fermentability of sugar hydrolysates was evaluated using an industrial strain of Saccharomyces cerevisiae. 2. Methods 2.1. Material Cotton gin dust (CGD) and cotton gin waste (CGW) were provided by Hantex Textile Residues Ltd. (Gaspar, Brazil) with a 7 wt% moisture content (wet basis). The commercial enzyme preparation Cellic CTec2Ò was obtained from Novozymes Latin America (Araucária, Brazil) and the microorganism used in the fermentation experiments was the ThermosaccÒ Dry S. cerevisiae strain from Lallemand (Milwaukee, USA). 2.2. Pretreatment factorial design Pretreatment of the cotton gin residues CGD and CGW was investigated through a 22 factorial design in which the following conditions were used: catalyst concentration (NaOH) of 2 and 4 wt% (0.2 and 0.4 g g1 of dry biomass, respectively) and temperature (100 and 120 °C) (Table 1). Also, three replicates were performed at 110 °C and 3 wt% NaOH, which corresponds to the center point of the factorial design. The pretreatment TS (or the solid-to-liquid ratio in relation to the dry biomass) and the residence time at the desired temperature were established at 10 wt% and 60 min as recommended by Silverstein et al. (2007). After pretreatment the materials were separated by filtration in a Büchner funnel: the alkali-soluble (mostly lignin and alkali-soluble polysaccharides) and the alkali-insoluble (mostly cellulosic fibers) fractions. The fibers were washed with water until neutral pH and drained in a Büchner funnel to the lowest water retention level. These materials were stored in vacuum-sealed plastic bags at 4 °C with a moisture content around 70 wt%. Both glucans and lignin recoveries in the alkali-treated materials were obtained according to the following equation:
Table 1 Factorial designs (22) used for the alkaline pretreatment of cotton gin residues and for the enzymatic hydrolysis of the best alkali-treated substrates derived from pretreatment (4% NaOH at 120 °C). Experimenta
Alkaline pretreatment
Enzymatic hydrolysis
NaOH (%)b
Temperature (°C)
Total solids (%)
Enzyme (mg g1)c
CGD-1 CGD-2 CGD-3 CGD-4 CGD-5
4 2 4 2 3
(+1) (1) (+1) (1) (0)
120 120 100 100 110
(+1) (+1) (1) (1) (0)
15 (+1) 5 (1) 15 (+1) 5 (1) 10 (0)
85 85 55 55 70
(+1) (+1) (1) (1) (0)
CGW-6 CGW-7 CGW-8 CGW-9 CGW-10
4 2 4 2 3
(+1) (1) (+1) (1) (0)
120 120 100 100 110
(+1) (+1) (1) (1) (0)
15 (+1) 5 (1) 15 (+1) 5 (1) 10 (0)
85 85 55 55 70
(+1) (+1) (1) (1) (0)
a
CGD – cotton gin dust; CGW – cotton gin waste. Concentrations of 2%, 3%, and 4% correspond of a NaOH loading of 0.2, 0.3, and 0.4 g g1 dry substrate. c Enzyme loadings of 55, 70, and 85 mg g1 correspond to 7.5, 9.5, and 11.5 FPU g1, both in relation to the substrate dry mass. b
where DMf is the dry mass after pretreatment, DMi is the dry mass before pretreatment, Cf is the glucan or lignin content after pretreatment and Ci is the glucan or lignin content before pretreatment. Delignification was estimated by subtracting the lignin recovery from the maximum attainable yield which is set at 100%. The cellulosic materials were characterized before and after pretreatment with regard to their total moisture, ash and total extractives content as recommended by the NREL/TP-510-42621 (Sluiter et al., 2008a), NREL/TP-510-42622 (Sluiter et al., 2008b) and NREL/TP-510-42619 (Sluiter et al., 2008c) methods, respectively. Acid-insoluble lignin and carbohydrates were determined after a two-stage sulfuric acid hydrolysis to its component sugars as recommended by NREL/TP-510-42618 method (Sluiter et al., 2012), while acid-soluble lignin was quantified by UV spectrophotometry according to NREL/TP-510-42617 method (Hyman et al., 2008). Total carbohydrate content was analyzed in acid hydrolysates by high performance liquid chromatography (HPLC) using an Aminex HPX-87H column (Bio-Rad) that was preceded by a cation-H pre-column. Analyses were performed at 65 °C with 5 mmol L1 H2SO4 as the mobile phase at a flow rate of 0.6 mL min1. Quantitative analyses were performed by external calibration using primary standard solutions of cellobiose, glucose, xylose and arabinose, as well as acetic acid and furfural and hydroxymethylfurfural as carbohydrate dehydration by-products. The standard procedure for enzymatic hydrolysis was carried out in two replicates for 96 h at 150 rpm using the substrate at 5 wt% TS and the enzyme loading at 55 mg of Cellic CTec2 g1 of dry substrate, which corresponded to a total of 7.5 FPU g1 dry substrate as determined by the I.U.P.A.C. method (Ghose, 1987) with adaptations (Schwald et al., 1988). Erlenmeyer flasks containing the reaction mixture in 50 mmol L1 acetate buffer pH 4.8 were incubated at 50 °C in a rotary shaker incubator. Aliquots of approximately 1 mL were collected in several reaction times, heated for 5 min in a boiling water bath, centrifuged at 10,000g and subjected to analysis in the same HPLC system mentioned above for determining the substrate chemical composition. In this case, the glucose was the only component monitored by external calibration, which was then converted to anhydroglucose. Hydrolysis yields were always calculated in relation to the amount of glucan (cellulose) present in the original pretreated material. 2.3. Enzymatic hydrolysis factorial design
DMf C f Recovery ð%Þ ¼ 100 DMi C i
ð1Þ
After selecting the best pretreatment condition, the enzymatic hydrolysis of the corresponding cellulosic substrates was
93
D.H. Fockink et al. / Bioresource Technology 187 (2015) 91–96
investigated through a 22 factorial design with six replicates at the center point and two replicates at the other remaining experimental conditions. The factorial design conditions are listed in the Table 1 for each pretreated substrate. Two variables were defined in two levels: enzyme loading (85 and 55 mg of enzyme g1 of dry substrate) and substrate TS (5 and 15 wt%). The experiments 5 and 10 represent the center point of this factorial design (70 mg of enzyme g1 of dry substrate and 10 wt% substrate TS). 2.4. Fermentation of CGD and CGW hydrolysates The S. cerevisiae strain Thermosacc Dry was maintained in a YM medium containing 10 g L1 glucose, 3 g L1 yeast extract, 3 g L1 malt extract, 5 g L1 peptone and 20 g L1 agar at pH 7.0. The inoculum was prepared by transferring S. cerevisiae cells into a flask containing 100 mL of culture medium (with the same components of the maintenance medium but without agar) and incubating it at 30 °C for 18–24 h. For fermentation, the substrate hydrolysates derived from enzymatic hydrolysis were centrifuged to eliminate any suspended solids and the glucose concentration of the hydrolysates was adjusted to approximately 42.7 g L1. The hydrolysates were supplemented with 1 g L1 yeast extract, 0.5 g L1 (NH4)2PO4 and 0.025 g L1 MgSO4.7H2O (Mesa et al., 2011). The inoculum rate was 4.106 cells mL1 and the fermentation was carried out at 37 °C and 150 rpm. Fermentation aliquots were filtered through a 0.45 lm filter and glucose, glycerol, acetic acid and ethanol were quantified by HPLC using the same procedure mentioned above. By expressing the ethanol concentration (g L1) in relation to the time (h) used for fermentation, the ethanol volumetric productivity (g L1 h1) was calculated, while the ethanol yield (YE) was determined according to the following equation:
Y E ð%Þ ¼
½EtOH 100 0:51 ½Glc
ð2Þ
where [EtOH] is the final ethanol concentration, [Glc] is the initial glucose concentration and 0.51 is the maximum theoretical yield of ethanol from glucose (g g1). 2.5. Statistical analysis The enzymatic hydrolysis data and ethanol yields were submitted to analysis of variance (ANOVA) and the average data were compared by Tukey’s test at a 95% confidence level (a = 0.05) using the Statistica 8.0 software. 3. Results and discussion 3.1. CGD and CGW chemical composition The native CGD and CGW were characterized for their chemical composition (Table 2), revealing high contents of anhydroglucose (glucans) of 76.0 ± 0.1 and 57.4 ± 0.2 wt%, respectively. However, CGD contained less total lignin (9.7 ± 0.2 wt%) and extractives (6.5 ± 0.5 wt%) when compared to CGW, which presented 17.0 ± 0.2 wt% of total lignin and 11.2 ± 0.6 wt% of total extractives content. Agblevor et al. (2003) reported that the cellulose composition varied from 20% to 38% for five cotton gin residues. These variations in glucan could be attributed to the ginning methods, since some gins had higher cotton lint content than others and may contain variable amounts of hulls (16–48%), seeds (6–24%), motes (16–24%), and leaves (14–30%). McIntosh et al. (2014) also found low cellulose quantities (24–36%) and high levels of extractives (26–28%) in three types of cotton gin residues.
Table 2 Chemical characterization of cotton gin residues before and after the alkaline pretreatment. Experiment
Glucan (%)
Pentosan (%)
Total lignin (%)
Ash (%)
Total (%)
Native CGD-1 CGD-2 CGD-3 CGD-4 CGD-5
76.0 ± 0.1 89.7 ± 0.1 88.9 ± 0.1 90.0 ± 0.6 89.9 ± 0.8 88.1 ± 1.0
3.0 ± 0.1 5.0 ± 0.2 5.1 ± 0.4 3.0 ± 0.1 4.0 ± 0.5 4.8 ± 0.2
9.7 ± 0.2 3.4 ± 0.1 4.1 ± 0.4 5.1 ± 0.1 5.1 ± 0.2 4.4 ± 0.6
4.8 ± 0.1 0.8 ± 0.2 1.1 ± 0.1 1.0 ± 0.1 1.2 ± 0.1 0.9 ± 0.1
93.5 98.9 99.2 99.2 99.0 98.2
Native CGW-6 CGW-7 CGW-8 CGW-9 CGW-10
57.4 ± 0.2 82.7 ± 0.2 81.6 ± 0.3 78.4 ± 0.2 78.6 ± 0.5 82.6 ± 0.5
7.9 ± 0.1 8.1 ± 0.1 8.9 ± 0.4 8.4 ± 0.7 8.1 ± 0.6 8.2 ± 0.4
17.0 ± 0.2 5.0 ± 0.5 6.2 ± 0.5 9.7 ± 0.7 9.8 ± 0.3 6.1 ± 1.0
6.5 ± 0.1 0.9 ± 0.1 1.4 ± 0.4 1.8 ± 0.1 2.0 ± 0.1 1.5 ± 0.2
88.8 96.7 97.1 98.3 98.5 98.4
CGD – cotton gin dust; CGW – cotton gin waste.
The ash content was similar for both residues, being 4.8 ± 0.1 wt% for CGD and 6.5 ± 0.1 wt% for CGW. These contents were lower than those observed by Agblevor et al. (2006), who reported a wide variation in the ash content (10.8–21.9%) for samples derived from four ginning processes. The ash content in CGD and CGW was also lower than what is found in other biomass types such as rice straw (17.5%) and wheat straw (11.0%) (Barcelos et al., 2013), while industrial samples of sugarcane bagasse (6.5%) and straw (6.2%) (Szczerbowski et al., 2014) contained similar amounts of inorganic compounds. For both alkali-pretreated substrates the cellulose content was higher compared to the native material, regardless of the conditions used for pretreatment (Table 2). For CGD, the glucan content increased from 76.0 ± 0.1% to 89% in average, whereas for CGW, this same increase was from 57.4 ± 0.2% to 80% in average. These results are mainly due to lignin removal since the main effect of dilute sodium hydroxide pretreatment is biomass delignification, which occurs primarily by breaking down the ester bonds in lignin carbohydrate complexes and promoting the diffusion and dissolution of lignin fragments to the aqueous phase, thus increasing surface area of the resulting fibers to enzymatic hydrolysis (Tarkow and Feist, 1969). The mass recovery that was obtained under each pretreatment condition of the factorial design is shown in Table 3. For pretreated CGD, the mass yields and glucan recovery decreased slightly with an increase in the NaOH concentration used for pretreatment. Also, delignification was more efficient at the most severe pretreatment conditions, with 75.0% of lignin removed at 120 °C using 0.4 g NaOH g1 of dry substrate (CGD-1). Considering the average mass yield and the chemical composition of CGD before and after pretreatment, nearly 14% of its glucan component was solubilized in alkali at the conditions used in this study. Table 3 Mass yield, cellulose recovery and delignification after alkaline pretreatment of cotton gin residues. Experiment
Mass yield (%)
Glucan recovery (%)
Delignification (%)
CGD-1 CGD-2 CGD-3 CGD-4 CGD-5
71.2 74.5 71.9 73.2 72.1 ± 1.7
84.0 87.1 85.2 86.6 86.1 ± 2.2
75.0 68.4 62.4 61.8 67.0 ± 3.9
CGW-6 CGW-7 CGW-8 CGW-9 CGW-10
53.1 61.3 49.4 67.0 57.5 ± 2.5
76.5 87.1 67.5 91.6 82.7 ± 3.4
84.3 81.3 71.8 61.2 79.2 ± 0.6
CGD – cotton gin dust; CGW – cotton gin waste.
94
D.H. Fockink et al. / Bioresource Technology 187 (2015) 91–96
Table 4 Enzymatic conversion of alkali-pretreated cotton gin residues in experiments carried out with 7.5 FPU g1 of dry substrate. Experiment
Glucan conversion (%) in a time-course hydrolysis 3h
6h
24 h
48 h
72 h
96 h
CGD-1 CGD-2 CGD-3 CGD-4 CGD-5
12.1 ± 0.1ª 12.3 ± 0.1ª 11.9 ± 0.2ª 12.6 ± 0.4ª 11.9 ± 0.5ª
17.7 ± 0.1ª 17.9 ± 0.1ª 17.2 ± 0.2ª 17.2 ± 0.2ª 17.2 ± 0.6ª
34.7 ± 0.1ª 33.3 ± 0.1ab 32.7 ± 0.4b 32.6 ± 0.1b 33.2 ± 0.8ab
43.8 ± 1.1ª 39.9 ± 1.8ab 39.0 ± 1.0b 37.4 ± 0.2b 39.6 ± 1.5b
50.0 ± 1.5ª 44.6 ± 1.7ab 43.2 ± 1.6b 39.6 ± 0.5b 43.8 ± 1.7b
54.4 ± 2.0ª 49.2 ± 1.8ab 46.8 ± 2.1b 39.9 ± 0.9c 47.2 ± 2.1b
CGW-6 CGW-7 CGW-8 CGW-9 CGW-10
13.1 ± 0.1ª 12.7 ± 0.4ab 13.0 ± 0.1ab 10.7 ± 0.1c 12.2 ± 0.3b
17.9 ± 0.1ª 17.1 ± 0.5ab 17.2 ± 0.1ab 14.6 ± 0.1c 16.4 ± 0.3b
33.0 ± 0.4ª 30.4 ± 0.2ab 30.6 ± 1.1ab 24.7 ± 1.0c 29.1 ± 1.2b
40.9 ± 1.6ª 35.0 ± 0.5b 36.3 ± 1.9ab 27.2 ± 1.7c 33.3 ± 1.6b
45.0 ± 1.9ª 35.8 ± 0.8b 39.0 ± 2.6ab 27.6 ± 1.6c 34.9 ± 2.0b
46.1 ± 0.8ª 36.7 ± 0.4bc 41.1 ± 1.6b 28.0 ± 2.1d 36.3 ± 1.2c
CGD – cotton gin dust; CGW – cotton gin waste. Mean values with the same letter in the same column do not differ significantly according to the Tukey’s test (p > 0.05).
Pretreatment of CGW led to the most evident changes in the mass yield and glucan recovery in the pretreated substrate. For instance, assays that employed lower alkali concentrations, such as in the case of CGW-7 and CGW-9, reached 87.1% and 91.6% of glucan recovery, respectively, but this value dropped considerably when a higher alkali concentration of 4 wt% was used. However, delignification was more efficient at higher temperatures, giving 84.3% for CGW-6 and 81.3% for CGW-7. Compared to CGD, lower mass yields and glucan recoveries for CGW were attributed to its higher total extractives, pentosan and lignin contents (Table 2). Varga et al. (2002) reported a 95% reduction in lignin content as a result of pretreatment for corn stover with 10% NaOH for 60 min in autoclave. These high reduction levels may be attributed to the use of a higher NaOH concentration. On the other hand, Silverstein et al. (2007) obtained a 65.6% lignin reduction after pretreatment of cotton stalks with 2% of NaOH for 90 min at 121 °C. The susceptibility of the pretreated substrates to enzymatic hydrolysis was evaluated at 5 wt% TS using 55 mg of Cellic CTec2 g1 of dry substrate (Table 4). For alkali-treated CGD, differences in glucan conversion were only statistically different after 24 h of hydrolysis (p < 0.05 in the ANOVA). On the other hand, regardless of the time used for hydrolysis, all glucan conversion values described in Table 4 for alkali-treated CGW were statistically different (p < 0.05). The Tukey’s test was used to indicate which substrate hydrolysis yields could be considered statistically different at a 95% confidence level. These results are also shown in Table 4, in which the use of the same letter in the same column reveals mean values that are not different (p > 0.05) from one another. In the first 6 h of hydrolysis, all of the pretreatment conditions for CGD did not differ in glucan conversion (p > 0.05) but, after 24 h, the experiment CGD-1 reached the highest conversion of 54.4 ± 2.0% in 96 h. However, this value was not different (p > 0.05) than that obtained
in experiment CGD-2 (49.2 ± 1.8%). A net increase of 384% in glucan conversion was observed for CGD-1 in relation to the native material (used as control), which had only 11.2 ± 0.4% of its glucan content converted to glucose. For CGW alkaline pretreatment, the differences in conversion appeared in the first 3 h of enzymatic hydrolysis. Also, pretreatment performance at the most severe alkaline condition (CGW-6) was significantly different (p < 0.05) than all other pretreatment conditions, reaching 46.1 ± 0.8% of glucan conversion in 96 h. Hence, CGW-6 had a 620% increase in glucan conversion compared to the native material, whose hydrolysis reached only 6.4 ± 0.1% in 96 h. Thus, on the basis of the enzymatic hydrolysis performance, the best cellulosic substrate were produced when both residues were pretreated with 0.4 g NaOH g1 of dry substrate at 120 °C (experiments CGD-1 and CGW-6 in Table 4). 3.2. Improvements in enzymatic hydrolysis In an attempt to improve the enzymatic hydrolysis of the best CGD and CGW pretreated substrates, a new set of experiments was organized in a typical 22 factorial design as shown in Table 1. Fig. 1 shows the gradual release of glucose from both alkali-treated residues as a function of time. The results obtained after 24 and 92 h of hydrolysis under different experimental conditions are shown in Table 5 and these are presented as conversion (%) in relation to the substrate glucan content and as the corresponding glucose concentration (g L1) in the substrate hydrolysate. Since these values were statistically different for different pretreatment conditions (p < 0.05 in the ANOVA), the Tukey’s test was used to identify which of the individual responses were different from one another. The largest glucan conversion after 24 h was obtained with the lowest substrate total solids (5 wt%) and the highest enzyme
Glucan Conversion (%)
70
70
A
60
B
60
50
50
40
40
30
30
20
20
10
10
0
0 0
24
48
Time (h)
72
96
0
24
48
72
96
Time (h)
( )15%, 85 mg g-1 ss (x) 5%, 85 mg g-1 ss ( ) 15%, 55 mg g-1 ss ( ) 5%, 55 mg g-1 ss ( ) 10%, 70 mg g-1 ss
Fig. 1. Enzymatic hydrolysis of the best alkali-treated cotton gin substrates at different total solids (%) and enzyme loadings (mg g1 ss): cotton gin dust (A) and cotton gin waste (B).
D.H. Fockink et al. / Bioresource Technology 187 (2015) 91–96
Table 5 Glucan conversion and glucose concentration after 24 and 92 h enzymatic hydrolysis of the best alkali-pretreated substrates (0.4 g of NaOH g1 of dry substrate and 120 °C) derived from cotton gin residues. Experiment
24 h
92 h
Glucan conversion (%)
Glucose (g L1)
Glucan conversion (%)
Glucose (g L1)
CGD-1 CGD-2 CGD-3 CGD-4 CGD-5
18.2 ± 0.3d 40.8 ± 1.0ª 16.2 ± 0.7d 35.9 ± 0.4b 24.4 ± 0.6c
24.1 ± 0.4a 18.1 ± 0.4bc 21.2 ± 0.9ab 15.8 ± 0.2c 21.4 ± 1.3ª
37.1 ± 0.8d 62.1 ± 1.6ª 30.2 ± 0.1d 52.4 ± 0.2b 46.0 ± 2.2c
47.8 ± 1.1a 27.1 ± 0.7c 38.8 ± 0.1b 22.7 ± 0.1c 39.5 ± 2.4b
CGW-6 CGW-7 CGW-8 CGW-9 CGW-10
17.3 ± 0.6d 36.5 ± 0.9ª 14.8 ± 0.6d 26.3 ± 1.1b 22.7 ± 0.4c
22.9 ± 0.8ª 16.4 ± 0.4c 19.4 ± 0.7b 11.7 ± 0.5d 20.0 ± 0.4b
32.8 ± 0.6d 50.3 ± 0.8ª 26.4 ± 0.1e 42.8 ± 0.1b 40.2 ± 0.9c
42.5 ± 0.8ª 22.2 ± 0.3c 34.1 ± 0.1b 18.7 ± 0.1d 34.7 ± 0.8b
CGD – cotton gin dust; CGW – cotton gin waste. Mean values with the same letter in the same column do not differ significantly according to the Tukey’s test (p > 0.05).
Table 6 Theoretical yield, concentration and volumetric productivity of ethanol after 12 h of fermentation of glucose (control) and enzyme hydrolysates derived from alkalitreated cotton gin residues. Substrate
Theorical yield (%)
Concentration (g L1)
Volumetric productivity (g L1 h1)
Control (glucose) CGD hydrolysate CGW hydrolysate
85.5 ± 1.7ª 86.9 ± 1.6ª 87.5 ± 0.7ª
18.4 ± 0.1ª 19.5 ± 0.4ª 20.0 ± 0.2ª
1.5 ± 0.1ª 1.6 ± 0.1ª 1.7 ± 0.1ª
Mean values in the same column with the same letter do not differ significantly according to the Tukey test (p > 0.05).
loading (11.5 FPU g1 of dry substrate), as demonstrated by CGD-2 and CGW-7 in Table 5. These experiments resulted in 40.8 ± 1.0 and 36.5 ± 0.9% of glucan conversion, respectively, and these values were statistically different (p < 0.05) when compared to all other hydrolysis conditions. However, the highest concentration of fermentable sugars was obtained at 15 wt% total solids with 11.5 FPU g1 of dry substrate (or 85 mg of enzyme g1) for both pretreated materials, being 24.1 ± 0.4 g L1 of glucose for CGD-1 and 22.9 ± 0.8 g L1 of glucose for CGW-6. Similar results were also observed after 92 h of hydrolysis. Experiments CGD-2 and CGW-7 displayed the largest glucan conversion upon enzymatic hydrolysis (62.1 ± 1.6 and 50.3 ± 0.8%, respectively), being significantly different (p < 0.05) than the conversions achieved at all other hydrolysis conditions. However, the highest glucose concentrations of 47.8 ± 1.1 and 42.5 ± 0.8 g L1 were obtained in experiments CGD-1 and CGW-6, respectively. These results were much better than those reported by Jeoh and Agblevor (2001), who obtained glucan conversions of 28–40% from different steam-exploded CGW materials in 24 h at 1 wt% total solids using 70 FPU g1. Despite the use of very high enzyme loadings, these authors attributed the slow hydrolysis rate after 5 h to the sample heterogeneity, since the cotton gin waste used in that study contained undetermined amounts of cotton lint, immature bolls and cotton seeds. Lower glucan conversions were also obtained by Plácido et al. (2013), who achieved values of only 23.4% in 96 h for CGW pretreated sequentially by ultrasound, hot water extraction and ligninolytic enzymes. For microbial (Shi et al., 2009) and sulfuric acid (Silverstein et al., 2007) pretreatments of cotton stalks, glucan conversions were 18.0% and 23.8% in 72 h, respectively. Silverstein et al. (2007) also investigated the NaOH pretreatment (2% NaOH, 90 min and 121 °C) of cotton stalks and had a glucan conversion of 60.8% in 72 h using 40 FPU g1 cellulose.
95
3.3. Fermentation of CGD and CGW hydrolysates The effect of alkaline pretreatment on the fermentability of both CGD and CGW hydrolysates was evaluated in relation to their ethanol yield (YE), final ethanol concentration (g L1) and the resulting volumetric productivity (g L1 h1). The fermentation results showed that there is no significant difference (p > 0.05) between the ethanol production derived from substrate hydrolysates and the glucose control (Table 6). The ethanol yield in 12 h was 85.5 ± 1.7% for the glucose control, 86.9 ± 1.6% for CGD hydrolysate and 87.5 ± 0.7% for CGW hydrolysate with ethanol volumetric productivities of 1.5 ± 0.1, 1.6 ± 0.1 and 1.7 ± 0.1 g L1 h1, respectively. These data are consistent with those of McIntosh et al. (2014), who also tested the ethanol fermentation of CGW hydrolysates using the same S. cerevisiae Thermosacc Dry industrial strain. The fermentations were completed within 9 h, yielding 85% of the ethanol theoretical yield (16 g L1) with an overall maximum ethanol volumetric productivity of 2.3 g L1 h1. Plácido et al. (2013) studied the fermentation of CGW hydrolysates that were obtained by a combination of three pretreatment technologies and obtained an ethanol yield of 31.6% in 72 h using the commercial S. cerevisiae strain Ethanol RedÒ (Fermentis, USA). Kumari and Pramanik (2012) produced a series of hybrid yeast strains by fusing protoplasts from S. cerevisiae and xylose-fermenting yeasts and the fermentation of CGW hydrolysates with the best fusant resulted in an ethanol concentration of 7.1 ± 0.1 g L1 and productivity of 0.4 g L1 h1. These fermentation performances were much lower than those observed in our study. By contrast, Jeoh and Agblevor (2001) obtained 83% of the ethanol theoretical yield in 72 h using the Escherichia coli strain KO11 for fermentation, which corresponded to an estimated production of 270 L ton1 from both hexoses and pentoses found in CGW hydrolysates. 4. Conclusion Both residues derived from cotton ginning had high cellulose contents and a good potential for ethanol production after a mild alkaline pretreatment. The glucan content of these materials was easily converted to fermentable sugars at high yields and the fermentability of their hydrolysates was equivalent to that of the glucose control. Total ethanol productions of 232 and 129 L ton1 were achieved from cotton gin dust and cotton gin waste under the conditions used in this study, respectively. Also, both substrate hydrolysates had no inhibitory effects on the performance of the fermenting microorganism. These results are promising due to the simplicity of the pretreatment method used in this study. Acknowledgements The authors are grateful to the Araucária Foundation (Grant 490/2011-19197) and to the INCT of Energy and Environment for providing financial support to carry out this study, as well as to Hantex Textile Residues Ltd. (Blumenau, SC, Brazil) and to Novozymes Latin America (Araucária, PR, Brazil) for providing the raw materials and the enzyme preparation used for hydrolysis, respectively. References Agblevor, F.A., Batz, S., Trumbo, J., 2003. Composition and ethanol production potential of cotton gin residues. Appl. Biochem. Biotechnol. 105–108, 219–230. Agblevor, F.A., Cundiff, J.S., Mingle, C., Li, W., 2006. Storage and characterization of cotton gin waste for ethanol production. Resour. Conserv. Recycl. 46, 198–216. Balat, M., Balat, H., Öz, C., 2008. Progress in bioethanol processing. Prog. Energy Combust. Sci. 34, 551–573.
96
D.H. Fockink et al. / Bioresource Technology 187 (2015) 91–96
Barcelos, C.A., Maeda, R.N., Betancur, G.J.V., Pereira Jr, N., 2013. The essentialness of delignification on enzymatic hydrolysis of sugar cane bagasse cellulignin for second generation ethanol production. Waste Biomass Valor. 4, 341–346. Deejing, S., Ketkorn, W., 2009. Comparison of hydrolysis conditions to recover reducing sugar from various lignocellulosic materials. Chiang Mai J. Sci. 36, 384–394. Ghose, T., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257–268. Grilc, M., Veryasov, G., Likozar, B., Jesih, A., Levec, J., 2015. Hydrodeoxygenation of solvolysed lignocellulosic biomass by unsupported MoS2, MoO2, Mo2C and WS2 catalysts. Appl. Catal. B 163, 467–477. Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 100, 10–18. Howard, R., Abotsi, E., Van Rensburg, E.J., Howard, S., 2004. Lignocellulose biotechnology: issues of bioconversion and enzyme production. Afr. J. Biotechnol. 2, 602–619. Hyman, D., Sluiter, A., Crocker, D., Johnson, D., Sluiter, J., Black, S., Scarlata, C., 2008. Determination of Acid Soluble Lignin Concentration Curve by UV–Vis Spectroscopy. Technical report NREL/TP-510-42617. National Renewable Energy Laboratory, Golden. Ibrahim, M.M., Agblevor, F.A., El-Zawawy, W.K., 2010. Isolation and characterization of cellulose and lignin from steam-exploded lignocellulosic biomass. BioResources 5, 397–418. Isci, A., Demirer, G.N., 2007. Biogas production potential from cotton wastes. Renew. Energy 32, 750–757. Jeoh, T., Agblevor, F., 2001. Characterization and fermentation of steam exploded cotton gin waste. Biomass Bioenergy 21, 109–120. Kumari, R., Pramanik, K., 2012. Improved bioethanol production using fusants of Saccharomyces cerevisiae and xylose-fermenting yeasts. Appl. Biochem. Biotechnol. 167, 873–884. McIntosh, S., Vancov, T., Palmer, J., Morris, S., 2014. Ethanol production from cotton gin trash using optimised dilute acid pretreatment and whole slurry fermentation processes. Bioresour. Technol. 173, 42–51. Mesa, L., González, E., Romero, I., Ruiz, E., Cara, C., Castro, E., 2011. Comparison of process configurations for ethanol production from two-step pretreated sugarcane bagasse. Chem. Eng. J. 175, 185–191. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y., Holtzapple, M., Ladisch, M., 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96, 673–686. Ojumu, T.V., Solomon, B.O., Betiku, E., Layokun, S.K., Amigun, B., 2003. Cellulase Production by Aspergillus flavus Linn Isolate NSPR 101 fermented in sawdust, bagasse and corncob. Afr. J. Biotechnol. 2, 150–152. Plácido, J., Imam, T., Capareda, S., 2013. Evaluation of ligninolytic enzymes, ultrasonication and liquid hot water as pretreatments for bioethanol production from cotton gin trash. Bioresour. Technol. 139, 203–208. Sadaka, S., 2013. Gasification of raw and torrefied cotton gin wastes in an auger system. Trans. ASABE 29, 405–414.
Schwald, W., Chan, M., Breuil, C., Saddler, J., 1988. Comparison of HPLC and colorimetric methods for measuring cellulolytic activity. Appl. Microbiol. Biotechnol. 28, 398–403. Shen, J., Agblevor, F.A., 2011. Ethanol production of semi-simultaneous saccharification and fermentation from mixture of cotton gin waste and recycled paper sludge. Bioprocess. Biosyst. Eng. 34, 33–43. Shen, J., Agblevor, F.A., 2008. Kinetics of enzymatic hydrolysis of steam-exploded cotton gin waste. Chem. Eng. Commun. 195, 1107–1121. Shi, J., Sharma-Shivappa, R.R., Chinn, M., Howell, N., 2009. Effect of microbial pretreatment on enzymatic hydrolysis and fermentation of cotton stalks for ethanol production. Biomass Bioenergy 33, 88–96. Silverstein, R.A., Chen, Y., Sharma-Shivappa, R.R., Boyette, M.D., Osborne, J., 2007. A comparison of chemical pretreatment methods for improving saccharification of cotton stalks. Bioresour. Technol. 98, 3000–3011. Singh, J., Suhag, M., Dhaka, A., 2015. Augmented digestion of lignocellulose by steam explosion, acid and alkaline pretreatment methods: a review. Carbohydr. Polym. 117, 624–631. Sluiter, A., Hames, B., Hyman, D., Payne, C., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Wolfe, J., 2008a. Determination of total solids in biomass and total dissolved solids in liquid process samples. Technical report NREL/TP-51042621. National Renewable Energy Laboratory, Golden. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008b. Determination of ash in biomass. Technical report NREL/TP-510-42622. National Renewable Energy Laboratory, Golden. Sluiter, A., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008c. Determination of extractives in biomass. Technical report NREL/TP-510-42619. National Renewable Energy Laboratory, Golden. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2012. Determination of structural carbohydrates and lignin in biomass. Technical report NREL/TP-510-42618. National Renewable Energy Laboratory, Golden. Szczerbowski, D., Pitarelo, A.P., Zandoná Filho, A., Ramos, L.P., 2014. Sugarcane biomass for biorefineries: comparative composition of carbohydrate and noncarbohydrate components of bagasse and straw. Carbohydr. Polym. 114, 95– 101. Tarkow, H., Feist, W.C., 1969. A mechanism for improving the digestibility of lignocellulosic materials with dilute alkali and liquid ammonia. In: George, J.H., Elwyn, T.R. (Eds.), Cellulases and their Applications. American Chemical Society, Washington, pp. 197–218. USDA – United States Department of Agriculture, 2014. World Cotton Production. Foreign Agricultural Service. Available online: (accessed January 20th 2015). Varga, E., Szengyel, Z., Réczey, K., 2002. Chemical pretreatments of corn stover for enhancing enzymatic digestibility. Appl. Biochem. Biotechnol. 98, 73–87. Zabaniotou, A.A., Roussos, A.I., Koroneos, C.J., 2000. A laboratory study of cotton gin waste pyrolysis. J. Anal. Appl. Pyrolysis 56, 47–59.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Production of cellulosic ethanol from cotton processing residues after pretreatment with dilute sodium hydroxide and enzymatic hydrolysis Douglas Henrique Fockink a, Marcelo Adriano Corrêa Maceno a, Luiz Pereira Ramos a,b,⇑ a b
Research Center in Applied Chemistry (CEPESQ), Department of Chemistry, Federal University of Paraná, P.O. Box 19032, Curitiba, Paraná 81531-980, Brazil INCT Energy & Environment (INCT E&A), Department of Chemistry, Federal University of Paraná, Brazil
h i g h l i g h t s A mild alkaline pretreatment enhanced the ethanol production from cotton gin residues. Total ethanol productions as high as 232 L ton
1
were achieved from cotton gin dust. 1 of ethanol under similar conditions. Substrate hydrolysates had no inhibitory effects on ethanol fermentation.
More lignified cotton gin wastes produced 129 L ton
a r t i c l e
i n f o
Article history: Received 2 March 2015 Received in revised form 20 March 2015 Accepted 21 March 2015 Available online 27 March 2015 Keywords: Cotton gin waste Cotton gin dust Alkaline pretreatment Enzymatic hydrolysis Fermentation
a b s t r a c t In this study, production of cellulosic ethanol from two cotton processing residues was investigated after pretreatment with dilute sodium hydroxide. Pretreatment performance was investigated using a 22 factorial design and the highest glucan conversion was achieved at the most severe alkaline conditions (0.4 g NaOH g1 of dry biomass and 120 °C), reaching 51.6% and 38.8% for cotton gin waste (CGW) and cotton gin dust (CGD), respectively. The susceptibility of pretreated substrates to enzymatic hydrolysis was also investigated and the best condition was achieved at the lowest total solids (5 wt%) and the highest enzyme loading (85 mg of Cellic CTec2 g1 of dry substrate). However, the highest concentration of fermentable sugars – 47.8 and 42.5 g L1 for CGD and CGW, respectively – was obtained at 15 wt% total solids using this same enzyme loading. Substrate hydrolysates had no inhibitory effects on the fermenting microorganism. Ó 2015 Published by Elsevier Ltd.
1. Introduction Cotton is the main supply of natural fibers for textile industries and one of the largest sources of agro-industrial residues that can be utilized to produce fuels and chemicals (Howard et al., 2004; Ojumu et al., 2003). In 2014, the total world production of refined cotton fibers was 25.9 million ton, with Brazil and India, the largest world producers, contributing with 1.5 and 6.7 million ton, respectively. The world total area destined to cotton production was 34.4 million hectares across 80 countries, being China, India, United States, Pakistan and Brazil the most important contributors to these very impressive numbers (USDA, 2014). The high total world production capacity of cotton fibers also equates to the production of high amounts of cotton gin residues ⇑ Corresponding author at: Research Center in Applied Chemistry (CEPESQ), Department of Chemistry, Federal University of Paraná, P.O. Box 19032, Curitiba, PR 81531-980, Brazil. Tel.: +55 41 33613175. E-mail address: [email protected] (L.P. Ramos). http://dx.doi.org/10.1016/j.biortech.2015.03.096 0960-8524/Ó 2015 Published by Elsevier Ltd.
(McIntosh et al., 2014). In Brazil, 900 thousand hectares are destined to cotton production (USDA, 2014). The residues from cotton processing are of two types: the cotton gin dust (CGD) and the cotton gin waste (CGW). CGW arises from ginning process and is composed of cottonseed residues, hulls, sticks, leaves, and dirt, whereas CGD contains short fiber residues that are recovered from filter screens during the spinning/weaving processes. Slight differences in their composition are usually found among various mechanical harvesting methods. The major advantage of these cotton residues compared to other lignocellulosic materials is their high cellulose content. Hence, these renewable feedstocks have been tentatively used in a number of bioenergy applications to avoid its disposal by incineration or landfilling, such as in the case of cellulosic ethanol (Agblevor et al., 2003; McIntosh et al., 2014), pyrolysis (Zabaniotou et al., 2000), gasification (Sadaka, 2013), anaerobic fermentation (Isci and Demirer, 2007) and catalytic conversion to value-added chemicals (Grilc et al., 2015). However, the supporting evidence for the application of cotton gin residues for ethanol production is relatively scarce in comparison to other notable
92
D.H. Fockink et al. / Bioresource Technology 187 (2015) 91–96
agricultural residues such as corn stover, sugarcane bagasse and wheat straw (McIntosh et al., 2014). For conversion of cotton gin residues to cellulosic ethanol, a pretreatment method is needed to increase the accessibility of cellulose to enzymatic hydrolysis (Mosier et al., 2005). Among the various chemical and physical pretreatments used for cotton gin residues, acid pretreatments including steam explosion have been more often used (Agblevor et al., 2003; Ibrahim et al., 2010; Jeoh and Agblevor, 2001; Shen and Agblevor, 2008, 2011) while alkali pretreatments seems to be more effective for other cellulosic materials (Balat et al., 2008; Hendriks and Zeeman, 2009) and has not been studied inasmuch depth. The effectiveness of alkaline pretreatment depends on the biomass chemical composition and the conditions used for pretreatment (Singh et al., 2015). Deejing and Ketkorn (2009) have shown that this pretreatment technology is usually more effective for agricultural residues and herbaceous crops rather than for wood materials. This study was developed to evaluate the cellulosic ethanol production from CGD and CGW after pretreatment with dilute sodium hydroxide and enzymatic hydrolysis. The alkaline pretreatment performance was investigated using a 22 factorial design with variations in the catalyst amount (NaOH) and the pretreatment temperature. The enzymatic hydrolysis was also tentatively improved by using the same type of factorial design in which the process variables were the substrate total solids (TS) and the enzyme loading. Finally, the fermentability of sugar hydrolysates was evaluated using an industrial strain of Saccharomyces cerevisiae. 2. Methods 2.1. Material Cotton gin dust (CGD) and cotton gin waste (CGW) were provided by Hantex Textile Residues Ltd. (Gaspar, Brazil) with a 7 wt% moisture content (wet basis). The commercial enzyme preparation Cellic CTec2Ò was obtained from Novozymes Latin America (Araucária, Brazil) and the microorganism used in the fermentation experiments was the ThermosaccÒ Dry S. cerevisiae strain from Lallemand (Milwaukee, USA). 2.2. Pretreatment factorial design Pretreatment of the cotton gin residues CGD and CGW was investigated through a 22 factorial design in which the following conditions were used: catalyst concentration (NaOH) of 2 and 4 wt% (0.2 and 0.4 g g1 of dry biomass, respectively) and temperature (100 and 120 °C) (Table 1). Also, three replicates were performed at 110 °C and 3 wt% NaOH, which corresponds to the center point of the factorial design. The pretreatment TS (or the solid-to-liquid ratio in relation to the dry biomass) and the residence time at the desired temperature were established at 10 wt% and 60 min as recommended by Silverstein et al. (2007). After pretreatment the materials were separated by filtration in a Büchner funnel: the alkali-soluble (mostly lignin and alkali-soluble polysaccharides) and the alkali-insoluble (mostly cellulosic fibers) fractions. The fibers were washed with water until neutral pH and drained in a Büchner funnel to the lowest water retention level. These materials were stored in vacuum-sealed plastic bags at 4 °C with a moisture content around 70 wt%. Both glucans and lignin recoveries in the alkali-treated materials were obtained according to the following equation:
Table 1 Factorial designs (22) used for the alkaline pretreatment of cotton gin residues and for the enzymatic hydrolysis of the best alkali-treated substrates derived from pretreatment (4% NaOH at 120 °C). Experimenta
Alkaline pretreatment
Enzymatic hydrolysis
NaOH (%)b
Temperature (°C)
Total solids (%)
Enzyme (mg g1)c
CGD-1 CGD-2 CGD-3 CGD-4 CGD-5
4 2 4 2 3
(+1) (1) (+1) (1) (0)
120 120 100 100 110
(+1) (+1) (1) (1) (0)
15 (+1) 5 (1) 15 (+1) 5 (1) 10 (0)
85 85 55 55 70
(+1) (+1) (1) (1) (0)
CGW-6 CGW-7 CGW-8 CGW-9 CGW-10
4 2 4 2 3
(+1) (1) (+1) (1) (0)
120 120 100 100 110
(+1) (+1) (1) (1) (0)
15 (+1) 5 (1) 15 (+1) 5 (1) 10 (0)
85 85 55 55 70
(+1) (+1) (1) (1) (0)
a
CGD – cotton gin dust; CGW – cotton gin waste. Concentrations of 2%, 3%, and 4% correspond of a NaOH loading of 0.2, 0.3, and 0.4 g g1 dry substrate. c Enzyme loadings of 55, 70, and 85 mg g1 correspond to 7.5, 9.5, and 11.5 FPU g1, both in relation to the substrate dry mass. b
where DMf is the dry mass after pretreatment, DMi is the dry mass before pretreatment, Cf is the glucan or lignin content after pretreatment and Ci is the glucan or lignin content before pretreatment. Delignification was estimated by subtracting the lignin recovery from the maximum attainable yield which is set at 100%. The cellulosic materials were characterized before and after pretreatment with regard to their total moisture, ash and total extractives content as recommended by the NREL/TP-510-42621 (Sluiter et al., 2008a), NREL/TP-510-42622 (Sluiter et al., 2008b) and NREL/TP-510-42619 (Sluiter et al., 2008c) methods, respectively. Acid-insoluble lignin and carbohydrates were determined after a two-stage sulfuric acid hydrolysis to its component sugars as recommended by NREL/TP-510-42618 method (Sluiter et al., 2012), while acid-soluble lignin was quantified by UV spectrophotometry according to NREL/TP-510-42617 method (Hyman et al., 2008). Total carbohydrate content was analyzed in acid hydrolysates by high performance liquid chromatography (HPLC) using an Aminex HPX-87H column (Bio-Rad) that was preceded by a cation-H pre-column. Analyses were performed at 65 °C with 5 mmol L1 H2SO4 as the mobile phase at a flow rate of 0.6 mL min1. Quantitative analyses were performed by external calibration using primary standard solutions of cellobiose, glucose, xylose and arabinose, as well as acetic acid and furfural and hydroxymethylfurfural as carbohydrate dehydration by-products. The standard procedure for enzymatic hydrolysis was carried out in two replicates for 96 h at 150 rpm using the substrate at 5 wt% TS and the enzyme loading at 55 mg of Cellic CTec2 g1 of dry substrate, which corresponded to a total of 7.5 FPU g1 dry substrate as determined by the I.U.P.A.C. method (Ghose, 1987) with adaptations (Schwald et al., 1988). Erlenmeyer flasks containing the reaction mixture in 50 mmol L1 acetate buffer pH 4.8 were incubated at 50 °C in a rotary shaker incubator. Aliquots of approximately 1 mL were collected in several reaction times, heated for 5 min in a boiling water bath, centrifuged at 10,000g and subjected to analysis in the same HPLC system mentioned above for determining the substrate chemical composition. In this case, the glucose was the only component monitored by external calibration, which was then converted to anhydroglucose. Hydrolysis yields were always calculated in relation to the amount of glucan (cellulose) present in the original pretreated material. 2.3. Enzymatic hydrolysis factorial design
DMf C f Recovery ð%Þ ¼ 100 DMi C i
ð1Þ
After selecting the best pretreatment condition, the enzymatic hydrolysis of the corresponding cellulosic substrates was
93
D.H. Fockink et al. / Bioresource Technology 187 (2015) 91–96
investigated through a 22 factorial design with six replicates at the center point and two replicates at the other remaining experimental conditions. The factorial design conditions are listed in the Table 1 for each pretreated substrate. Two variables were defined in two levels: enzyme loading (85 and 55 mg of enzyme g1 of dry substrate) and substrate TS (5 and 15 wt%). The experiments 5 and 10 represent the center point of this factorial design (70 mg of enzyme g1 of dry substrate and 10 wt% substrate TS). 2.4. Fermentation of CGD and CGW hydrolysates The S. cerevisiae strain Thermosacc Dry was maintained in a YM medium containing 10 g L1 glucose, 3 g L1 yeast extract, 3 g L1 malt extract, 5 g L1 peptone and 20 g L1 agar at pH 7.0. The inoculum was prepared by transferring S. cerevisiae cells into a flask containing 100 mL of culture medium (with the same components of the maintenance medium but without agar) and incubating it at 30 °C for 18–24 h. For fermentation, the substrate hydrolysates derived from enzymatic hydrolysis were centrifuged to eliminate any suspended solids and the glucose concentration of the hydrolysates was adjusted to approximately 42.7 g L1. The hydrolysates were supplemented with 1 g L1 yeast extract, 0.5 g L1 (NH4)2PO4 and 0.025 g L1 MgSO4.7H2O (Mesa et al., 2011). The inoculum rate was 4.106 cells mL1 and the fermentation was carried out at 37 °C and 150 rpm. Fermentation aliquots were filtered through a 0.45 lm filter and glucose, glycerol, acetic acid and ethanol were quantified by HPLC using the same procedure mentioned above. By expressing the ethanol concentration (g L1) in relation to the time (h) used for fermentation, the ethanol volumetric productivity (g L1 h1) was calculated, while the ethanol yield (YE) was determined according to the following equation:
Y E ð%Þ ¼
½EtOH 100 0:51 ½Glc
ð2Þ
where [EtOH] is the final ethanol concentration, [Glc] is the initial glucose concentration and 0.51 is the maximum theoretical yield of ethanol from glucose (g g1). 2.5. Statistical analysis The enzymatic hydrolysis data and ethanol yields were submitted to analysis of variance (ANOVA) and the average data were compared by Tukey’s test at a 95% confidence level (a = 0.05) using the Statistica 8.0 software. 3. Results and discussion 3.1. CGD and CGW chemical composition The native CGD and CGW were characterized for their chemical composition (Table 2), revealing high contents of anhydroglucose (glucans) of 76.0 ± 0.1 and 57.4 ± 0.2 wt%, respectively. However, CGD contained less total lignin (9.7 ± 0.2 wt%) and extractives (6.5 ± 0.5 wt%) when compared to CGW, which presented 17.0 ± 0.2 wt% of total lignin and 11.2 ± 0.6 wt% of total extractives content. Agblevor et al. (2003) reported that the cellulose composition varied from 20% to 38% for five cotton gin residues. These variations in glucan could be attributed to the ginning methods, since some gins had higher cotton lint content than others and may contain variable amounts of hulls (16–48%), seeds (6–24%), motes (16–24%), and leaves (14–30%). McIntosh et al. (2014) also found low cellulose quantities (24–36%) and high levels of extractives (26–28%) in three types of cotton gin residues.
Table 2 Chemical characterization of cotton gin residues before and after the alkaline pretreatment. Experiment
Glucan (%)
Pentosan (%)
Total lignin (%)
Ash (%)
Total (%)
Native CGD-1 CGD-2 CGD-3 CGD-4 CGD-5
76.0 ± 0.1 89.7 ± 0.1 88.9 ± 0.1 90.0 ± 0.6 89.9 ± 0.8 88.1 ± 1.0
3.0 ± 0.1 5.0 ± 0.2 5.1 ± 0.4 3.0 ± 0.1 4.0 ± 0.5 4.8 ± 0.2
9.7 ± 0.2 3.4 ± 0.1 4.1 ± 0.4 5.1 ± 0.1 5.1 ± 0.2 4.4 ± 0.6
4.8 ± 0.1 0.8 ± 0.2 1.1 ± 0.1 1.0 ± 0.1 1.2 ± 0.1 0.9 ± 0.1
93.5 98.9 99.2 99.2 99.0 98.2
Native CGW-6 CGW-7 CGW-8 CGW-9 CGW-10
57.4 ± 0.2 82.7 ± 0.2 81.6 ± 0.3 78.4 ± 0.2 78.6 ± 0.5 82.6 ± 0.5
7.9 ± 0.1 8.1 ± 0.1 8.9 ± 0.4 8.4 ± 0.7 8.1 ± 0.6 8.2 ± 0.4
17.0 ± 0.2 5.0 ± 0.5 6.2 ± 0.5 9.7 ± 0.7 9.8 ± 0.3 6.1 ± 1.0
6.5 ± 0.1 0.9 ± 0.1 1.4 ± 0.4 1.8 ± 0.1 2.0 ± 0.1 1.5 ± 0.2
88.8 96.7 97.1 98.3 98.5 98.4
CGD – cotton gin dust; CGW – cotton gin waste.
The ash content was similar for both residues, being 4.8 ± 0.1 wt% for CGD and 6.5 ± 0.1 wt% for CGW. These contents were lower than those observed by Agblevor et al. (2006), who reported a wide variation in the ash content (10.8–21.9%) for samples derived from four ginning processes. The ash content in CGD and CGW was also lower than what is found in other biomass types such as rice straw (17.5%) and wheat straw (11.0%) (Barcelos et al., 2013), while industrial samples of sugarcane bagasse (6.5%) and straw (6.2%) (Szczerbowski et al., 2014) contained similar amounts of inorganic compounds. For both alkali-pretreated substrates the cellulose content was higher compared to the native material, regardless of the conditions used for pretreatment (Table 2). For CGD, the glucan content increased from 76.0 ± 0.1% to 89% in average, whereas for CGW, this same increase was from 57.4 ± 0.2% to 80% in average. These results are mainly due to lignin removal since the main effect of dilute sodium hydroxide pretreatment is biomass delignification, which occurs primarily by breaking down the ester bonds in lignin carbohydrate complexes and promoting the diffusion and dissolution of lignin fragments to the aqueous phase, thus increasing surface area of the resulting fibers to enzymatic hydrolysis (Tarkow and Feist, 1969). The mass recovery that was obtained under each pretreatment condition of the factorial design is shown in Table 3. For pretreated CGD, the mass yields and glucan recovery decreased slightly with an increase in the NaOH concentration used for pretreatment. Also, delignification was more efficient at the most severe pretreatment conditions, with 75.0% of lignin removed at 120 °C using 0.4 g NaOH g1 of dry substrate (CGD-1). Considering the average mass yield and the chemical composition of CGD before and after pretreatment, nearly 14% of its glucan component was solubilized in alkali at the conditions used in this study. Table 3 Mass yield, cellulose recovery and delignification after alkaline pretreatment of cotton gin residues. Experiment
Mass yield (%)
Glucan recovery (%)
Delignification (%)
CGD-1 CGD-2 CGD-3 CGD-4 CGD-5
71.2 74.5 71.9 73.2 72.1 ± 1.7
84.0 87.1 85.2 86.6 86.1 ± 2.2
75.0 68.4 62.4 61.8 67.0 ± 3.9
CGW-6 CGW-7 CGW-8 CGW-9 CGW-10
53.1 61.3 49.4 67.0 57.5 ± 2.5
76.5 87.1 67.5 91.6 82.7 ± 3.4
84.3 81.3 71.8 61.2 79.2 ± 0.6
CGD – cotton gin dust; CGW – cotton gin waste.
94
D.H. Fockink et al. / Bioresource Technology 187 (2015) 91–96
Table 4 Enzymatic conversion of alkali-pretreated cotton gin residues in experiments carried out with 7.5 FPU g1 of dry substrate. Experiment
Glucan conversion (%) in a time-course hydrolysis 3h
6h
24 h
48 h
72 h
96 h
CGD-1 CGD-2 CGD-3 CGD-4 CGD-5
12.1 ± 0.1ª 12.3 ± 0.1ª 11.9 ± 0.2ª 12.6 ± 0.4ª 11.9 ± 0.5ª
17.7 ± 0.1ª 17.9 ± 0.1ª 17.2 ± 0.2ª 17.2 ± 0.2ª 17.2 ± 0.6ª
34.7 ± 0.1ª 33.3 ± 0.1ab 32.7 ± 0.4b 32.6 ± 0.1b 33.2 ± 0.8ab
43.8 ± 1.1ª 39.9 ± 1.8ab 39.0 ± 1.0b 37.4 ± 0.2b 39.6 ± 1.5b
50.0 ± 1.5ª 44.6 ± 1.7ab 43.2 ± 1.6b 39.6 ± 0.5b 43.8 ± 1.7b
54.4 ± 2.0ª 49.2 ± 1.8ab 46.8 ± 2.1b 39.9 ± 0.9c 47.2 ± 2.1b
CGW-6 CGW-7 CGW-8 CGW-9 CGW-10
13.1 ± 0.1ª 12.7 ± 0.4ab 13.0 ± 0.1ab 10.7 ± 0.1c 12.2 ± 0.3b
17.9 ± 0.1ª 17.1 ± 0.5ab 17.2 ± 0.1ab 14.6 ± 0.1c 16.4 ± 0.3b
33.0 ± 0.4ª 30.4 ± 0.2ab 30.6 ± 1.1ab 24.7 ± 1.0c 29.1 ± 1.2b
40.9 ± 1.6ª 35.0 ± 0.5b 36.3 ± 1.9ab 27.2 ± 1.7c 33.3 ± 1.6b
45.0 ± 1.9ª 35.8 ± 0.8b 39.0 ± 2.6ab 27.6 ± 1.6c 34.9 ± 2.0b
46.1 ± 0.8ª 36.7 ± 0.4bc 41.1 ± 1.6b 28.0 ± 2.1d 36.3 ± 1.2c
CGD – cotton gin dust; CGW – cotton gin waste. Mean values with the same letter in the same column do not differ significantly according to the Tukey’s test (p > 0.05).
Pretreatment of CGW led to the most evident changes in the mass yield and glucan recovery in the pretreated substrate. For instance, assays that employed lower alkali concentrations, such as in the case of CGW-7 and CGW-9, reached 87.1% and 91.6% of glucan recovery, respectively, but this value dropped considerably when a higher alkali concentration of 4 wt% was used. However, delignification was more efficient at higher temperatures, giving 84.3% for CGW-6 and 81.3% for CGW-7. Compared to CGD, lower mass yields and glucan recoveries for CGW were attributed to its higher total extractives, pentosan and lignin contents (Table 2). Varga et al. (2002) reported a 95% reduction in lignin content as a result of pretreatment for corn stover with 10% NaOH for 60 min in autoclave. These high reduction levels may be attributed to the use of a higher NaOH concentration. On the other hand, Silverstein et al. (2007) obtained a 65.6% lignin reduction after pretreatment of cotton stalks with 2% of NaOH for 90 min at 121 °C. The susceptibility of the pretreated substrates to enzymatic hydrolysis was evaluated at 5 wt% TS using 55 mg of Cellic CTec2 g1 of dry substrate (Table 4). For alkali-treated CGD, differences in glucan conversion were only statistically different after 24 h of hydrolysis (p < 0.05 in the ANOVA). On the other hand, regardless of the time used for hydrolysis, all glucan conversion values described in Table 4 for alkali-treated CGW were statistically different (p < 0.05). The Tukey’s test was used to indicate which substrate hydrolysis yields could be considered statistically different at a 95% confidence level. These results are also shown in Table 4, in which the use of the same letter in the same column reveals mean values that are not different (p > 0.05) from one another. In the first 6 h of hydrolysis, all of the pretreatment conditions for CGD did not differ in glucan conversion (p > 0.05) but, after 24 h, the experiment CGD-1 reached the highest conversion of 54.4 ± 2.0% in 96 h. However, this value was not different (p > 0.05) than that obtained
in experiment CGD-2 (49.2 ± 1.8%). A net increase of 384% in glucan conversion was observed for CGD-1 in relation to the native material (used as control), which had only 11.2 ± 0.4% of its glucan content converted to glucose. For CGW alkaline pretreatment, the differences in conversion appeared in the first 3 h of enzymatic hydrolysis. Also, pretreatment performance at the most severe alkaline condition (CGW-6) was significantly different (p < 0.05) than all other pretreatment conditions, reaching 46.1 ± 0.8% of glucan conversion in 96 h. Hence, CGW-6 had a 620% increase in glucan conversion compared to the native material, whose hydrolysis reached only 6.4 ± 0.1% in 96 h. Thus, on the basis of the enzymatic hydrolysis performance, the best cellulosic substrate were produced when both residues were pretreated with 0.4 g NaOH g1 of dry substrate at 120 °C (experiments CGD-1 and CGW-6 in Table 4). 3.2. Improvements in enzymatic hydrolysis In an attempt to improve the enzymatic hydrolysis of the best CGD and CGW pretreated substrates, a new set of experiments was organized in a typical 22 factorial design as shown in Table 1. Fig. 1 shows the gradual release of glucose from both alkali-treated residues as a function of time. The results obtained after 24 and 92 h of hydrolysis under different experimental conditions are shown in Table 5 and these are presented as conversion (%) in relation to the substrate glucan content and as the corresponding glucose concentration (g L1) in the substrate hydrolysate. Since these values were statistically different for different pretreatment conditions (p < 0.05 in the ANOVA), the Tukey’s test was used to identify which of the individual responses were different from one another. The largest glucan conversion after 24 h was obtained with the lowest substrate total solids (5 wt%) and the highest enzyme
Glucan Conversion (%)
70
70
A
60
B
60
50
50
40
40
30
30
20
20
10
10
0
0 0
24
48
Time (h)
72
96
0
24
48
72
96
Time (h)
( )15%, 85 mg g-1 ss (x) 5%, 85 mg g-1 ss ( ) 15%, 55 mg g-1 ss ( ) 5%, 55 mg g-1 ss ( ) 10%, 70 mg g-1 ss
Fig. 1. Enzymatic hydrolysis of the best alkali-treated cotton gin substrates at different total solids (%) and enzyme loadings (mg g1 ss): cotton gin dust (A) and cotton gin waste (B).
D.H. Fockink et al. / Bioresource Technology 187 (2015) 91–96
Table 5 Glucan conversion and glucose concentration after 24 and 92 h enzymatic hydrolysis of the best alkali-pretreated substrates (0.4 g of NaOH g1 of dry substrate and 120 °C) derived from cotton gin residues. Experiment
24 h
92 h
Glucan conversion (%)
Glucose (g L1)
Glucan conversion (%)
Glucose (g L1)
CGD-1 CGD-2 CGD-3 CGD-4 CGD-5
18.2 ± 0.3d 40.8 ± 1.0ª 16.2 ± 0.7d 35.9 ± 0.4b 24.4 ± 0.6c
24.1 ± 0.4a 18.1 ± 0.4bc 21.2 ± 0.9ab 15.8 ± 0.2c 21.4 ± 1.3ª
37.1 ± 0.8d 62.1 ± 1.6ª 30.2 ± 0.1d 52.4 ± 0.2b 46.0 ± 2.2c
47.8 ± 1.1a 27.1 ± 0.7c 38.8 ± 0.1b 22.7 ± 0.1c 39.5 ± 2.4b
CGW-6 CGW-7 CGW-8 CGW-9 CGW-10
17.3 ± 0.6d 36.5 ± 0.9ª 14.8 ± 0.6d 26.3 ± 1.1b 22.7 ± 0.4c
22.9 ± 0.8ª 16.4 ± 0.4c 19.4 ± 0.7b 11.7 ± 0.5d 20.0 ± 0.4b
32.8 ± 0.6d 50.3 ± 0.8ª 26.4 ± 0.1e 42.8 ± 0.1b 40.2 ± 0.9c
42.5 ± 0.8ª 22.2 ± 0.3c 34.1 ± 0.1b 18.7 ± 0.1d 34.7 ± 0.8b
CGD – cotton gin dust; CGW – cotton gin waste. Mean values with the same letter in the same column do not differ significantly according to the Tukey’s test (p > 0.05).
Table 6 Theoretical yield, concentration and volumetric productivity of ethanol after 12 h of fermentation of glucose (control) and enzyme hydrolysates derived from alkalitreated cotton gin residues. Substrate
Theorical yield (%)
Concentration (g L1)
Volumetric productivity (g L1 h1)
Control (glucose) CGD hydrolysate CGW hydrolysate
85.5 ± 1.7ª 86.9 ± 1.6ª 87.5 ± 0.7ª
18.4 ± 0.1ª 19.5 ± 0.4ª 20.0 ± 0.2ª
1.5 ± 0.1ª 1.6 ± 0.1ª 1.7 ± 0.1ª
Mean values in the same column with the same letter do not differ significantly according to the Tukey test (p > 0.05).
loading (11.5 FPU g1 of dry substrate), as demonstrated by CGD-2 and CGW-7 in Table 5. These experiments resulted in 40.8 ± 1.0 and 36.5 ± 0.9% of glucan conversion, respectively, and these values were statistically different (p < 0.05) when compared to all other hydrolysis conditions. However, the highest concentration of fermentable sugars was obtained at 15 wt% total solids with 11.5 FPU g1 of dry substrate (or 85 mg of enzyme g1) for both pretreated materials, being 24.1 ± 0.4 g L1 of glucose for CGD-1 and 22.9 ± 0.8 g L1 of glucose for CGW-6. Similar results were also observed after 92 h of hydrolysis. Experiments CGD-2 and CGW-7 displayed the largest glucan conversion upon enzymatic hydrolysis (62.1 ± 1.6 and 50.3 ± 0.8%, respectively), being significantly different (p < 0.05) than the conversions achieved at all other hydrolysis conditions. However, the highest glucose concentrations of 47.8 ± 1.1 and 42.5 ± 0.8 g L1 were obtained in experiments CGD-1 and CGW-6, respectively. These results were much better than those reported by Jeoh and Agblevor (2001), who obtained glucan conversions of 28–40% from different steam-exploded CGW materials in 24 h at 1 wt% total solids using 70 FPU g1. Despite the use of very high enzyme loadings, these authors attributed the slow hydrolysis rate after 5 h to the sample heterogeneity, since the cotton gin waste used in that study contained undetermined amounts of cotton lint, immature bolls and cotton seeds. Lower glucan conversions were also obtained by Plácido et al. (2013), who achieved values of only 23.4% in 96 h for CGW pretreated sequentially by ultrasound, hot water extraction and ligninolytic enzymes. For microbial (Shi et al., 2009) and sulfuric acid (Silverstein et al., 2007) pretreatments of cotton stalks, glucan conversions were 18.0% and 23.8% in 72 h, respectively. Silverstein et al. (2007) also investigated the NaOH pretreatment (2% NaOH, 90 min and 121 °C) of cotton stalks and had a glucan conversion of 60.8% in 72 h using 40 FPU g1 cellulose.
95
3.3. Fermentation of CGD and CGW hydrolysates The effect of alkaline pretreatment on the fermentability of both CGD and CGW hydrolysates was evaluated in relation to their ethanol yield (YE), final ethanol concentration (g L1) and the resulting volumetric productivity (g L1 h1). The fermentation results showed that there is no significant difference (p > 0.05) between the ethanol production derived from substrate hydrolysates and the glucose control (Table 6). The ethanol yield in 12 h was 85.5 ± 1.7% for the glucose control, 86.9 ± 1.6% for CGD hydrolysate and 87.5 ± 0.7% for CGW hydrolysate with ethanol volumetric productivities of 1.5 ± 0.1, 1.6 ± 0.1 and 1.7 ± 0.1 g L1 h1, respectively. These data are consistent with those of McIntosh et al. (2014), who also tested the ethanol fermentation of CGW hydrolysates using the same S. cerevisiae Thermosacc Dry industrial strain. The fermentations were completed within 9 h, yielding 85% of the ethanol theoretical yield (16 g L1) with an overall maximum ethanol volumetric productivity of 2.3 g L1 h1. Plácido et al. (2013) studied the fermentation of CGW hydrolysates that were obtained by a combination of three pretreatment technologies and obtained an ethanol yield of 31.6% in 72 h using the commercial S. cerevisiae strain Ethanol RedÒ (Fermentis, USA). Kumari and Pramanik (2012) produced a series of hybrid yeast strains by fusing protoplasts from S. cerevisiae and xylose-fermenting yeasts and the fermentation of CGW hydrolysates with the best fusant resulted in an ethanol concentration of 7.1 ± 0.1 g L1 and productivity of 0.4 g L1 h1. These fermentation performances were much lower than those observed in our study. By contrast, Jeoh and Agblevor (2001) obtained 83% of the ethanol theoretical yield in 72 h using the Escherichia coli strain KO11 for fermentation, which corresponded to an estimated production of 270 L ton1 from both hexoses and pentoses found in CGW hydrolysates. 4. Conclusion Both residues derived from cotton ginning had high cellulose contents and a good potential for ethanol production after a mild alkaline pretreatment. The glucan content of these materials was easily converted to fermentable sugars at high yields and the fermentability of their hydrolysates was equivalent to that of the glucose control. Total ethanol productions of 232 and 129 L ton1 were achieved from cotton gin dust and cotton gin waste under the conditions used in this study, respectively. Also, both substrate hydrolysates had no inhibitory effects on the performance of the fermenting microorganism. These results are promising due to the simplicity of the pretreatment method used in this study. Acknowledgements The authors are grateful to the Araucária Foundation (Grant 490/2011-19197) and to the INCT of Energy and Environment for providing financial support to carry out this study, as well as to Hantex Textile Residues Ltd. (Blumenau, SC, Brazil) and to Novozymes Latin America (Araucária, PR, Brazil) for providing the raw materials and the enzyme preparation used for hydrolysis, respectively. References Agblevor, F.A., Batz, S., Trumbo, J., 2003. Composition and ethanol production potential of cotton gin residues. Appl. Biochem. Biotechnol. 105–108, 219–230. Agblevor, F.A., Cundiff, J.S., Mingle, C., Li, W., 2006. Storage and characterization of cotton gin waste for ethanol production. Resour. Conserv. Recycl. 46, 198–216. Balat, M., Balat, H., Öz, C., 2008. Progress in bioethanol processing. Prog. Energy Combust. Sci. 34, 551–573.
96
D.H. Fockink et al. / Bioresource Technology 187 (2015) 91–96
Barcelos, C.A., Maeda, R.N., Betancur, G.J.V., Pereira Jr, N., 2013. The essentialness of delignification on enzymatic hydrolysis of sugar cane bagasse cellulignin for second generation ethanol production. Waste Biomass Valor. 4, 341–346. Deejing, S., Ketkorn, W., 2009. Comparison of hydrolysis conditions to recover reducing sugar from various lignocellulosic materials. Chiang Mai J. Sci. 36, 384–394. Ghose, T., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257–268. Grilc, M., Veryasov, G., Likozar, B., Jesih, A., Levec, J., 2015. Hydrodeoxygenation of solvolysed lignocellulosic biomass by unsupported MoS2, MoO2, Mo2C and WS2 catalysts. Appl. Catal. B 163, 467–477. Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 100, 10–18. Howard, R., Abotsi, E., Van Rensburg, E.J., Howard, S., 2004. Lignocellulose biotechnology: issues of bioconversion and enzyme production. Afr. J. Biotechnol. 2, 602–619. Hyman, D., Sluiter, A., Crocker, D., Johnson, D., Sluiter, J., Black, S., Scarlata, C., 2008. Determination of Acid Soluble Lignin Concentration Curve by UV–Vis Spectroscopy. Technical report NREL/TP-510-42617. National Renewable Energy Laboratory, Golden. Ibrahim, M.M., Agblevor, F.A., El-Zawawy, W.K., 2010. Isolation and characterization of cellulose and lignin from steam-exploded lignocellulosic biomass. BioResources 5, 397–418. Isci, A., Demirer, G.N., 2007. Biogas production potential from cotton wastes. Renew. Energy 32, 750–757. Jeoh, T., Agblevor, F., 2001. Characterization and fermentation of steam exploded cotton gin waste. Biomass Bioenergy 21, 109–120. Kumari, R., Pramanik, K., 2012. Improved bioethanol production using fusants of Saccharomyces cerevisiae and xylose-fermenting yeasts. Appl. Biochem. Biotechnol. 167, 873–884. McIntosh, S., Vancov, T., Palmer, J., Morris, S., 2014. Ethanol production from cotton gin trash using optimised dilute acid pretreatment and whole slurry fermentation processes. Bioresour. Technol. 173, 42–51. Mesa, L., González, E., Romero, I., Ruiz, E., Cara, C., Castro, E., 2011. Comparison of process configurations for ethanol production from two-step pretreated sugarcane bagasse. Chem. Eng. J. 175, 185–191. Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y., Holtzapple, M., Ladisch, M., 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour. Technol. 96, 673–686. Ojumu, T.V., Solomon, B.O., Betiku, E., Layokun, S.K., Amigun, B., 2003. Cellulase Production by Aspergillus flavus Linn Isolate NSPR 101 fermented in sawdust, bagasse and corncob. Afr. J. Biotechnol. 2, 150–152. Plácido, J., Imam, T., Capareda, S., 2013. Evaluation of ligninolytic enzymes, ultrasonication and liquid hot water as pretreatments for bioethanol production from cotton gin trash. Bioresour. Technol. 139, 203–208. Sadaka, S., 2013. Gasification of raw and torrefied cotton gin wastes in an auger system. Trans. ASABE 29, 405–414.
Schwald, W., Chan, M., Breuil, C., Saddler, J., 1988. Comparison of HPLC and colorimetric methods for measuring cellulolytic activity. Appl. Microbiol. Biotechnol. 28, 398–403. Shen, J., Agblevor, F.A., 2011. Ethanol production of semi-simultaneous saccharification and fermentation from mixture of cotton gin waste and recycled paper sludge. Bioprocess. Biosyst. Eng. 34, 33–43. Shen, J., Agblevor, F.A., 2008. Kinetics of enzymatic hydrolysis of steam-exploded cotton gin waste. Chem. Eng. Commun. 195, 1107–1121. Shi, J., Sharma-Shivappa, R.R., Chinn, M., Howell, N., 2009. Effect of microbial pretreatment on enzymatic hydrolysis and fermentation of cotton stalks for ethanol production. Biomass Bioenergy 33, 88–96. Silverstein, R.A., Chen, Y., Sharma-Shivappa, R.R., Boyette, M.D., Osborne, J., 2007. A comparison of chemical pretreatment methods for improving saccharification of cotton stalks. Bioresour. Technol. 98, 3000–3011. Singh, J., Suhag, M., Dhaka, A., 2015. Augmented digestion of lignocellulose by steam explosion, acid and alkaline pretreatment methods: a review. Carbohydr. Polym. 117, 624–631. Sluiter, A., Hames, B., Hyman, D., Payne, C., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Wolfe, J., 2008a. Determination of total solids in biomass and total dissolved solids in liquid process samples. Technical report NREL/TP-51042621. National Renewable Energy Laboratory, Golden. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008b. Determination of ash in biomass. Technical report NREL/TP-510-42622. National Renewable Energy Laboratory, Golden. Sluiter, A., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2008c. Determination of extractives in biomass. Technical report NREL/TP-510-42619. National Renewable Energy Laboratory, Golden. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2012. Determination of structural carbohydrates and lignin in biomass. Technical report NREL/TP-510-42618. National Renewable Energy Laboratory, Golden. Szczerbowski, D., Pitarelo, A.P., Zandoná Filho, A., Ramos, L.P., 2014. Sugarcane biomass for biorefineries: comparative composition of carbohydrate and noncarbohydrate components of bagasse and straw. Carbohydr. Polym. 114, 95– 101. Tarkow, H., Feist, W.C., 1969. A mechanism for improving the digestibility of lignocellulosic materials with dilute alkali and liquid ammonia. In: George, J.H., Elwyn, T.R. (Eds.), Cellulases and their Applications. American Chemical Society, Washington, pp. 197–218. USDA – United States Department of Agriculture, 2014. World Cotton Production. Foreign Agricultural Service. Available online: (accessed January 20th 2015). Varga, E., Szengyel, Z., Réczey, K., 2002. Chemical pretreatments of corn stover for enhancing enzymatic digestibility. Appl. Biochem. Biotechnol. 98, 73–87. Zabaniotou, A.A., Roussos, A.I., Koroneos, C.J., 2000. A laboratory study of cotton gin waste pyrolysis. J. Anal. Appl. Pyrolysis 56, 47–59.
