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Ann Microbiol (2017) 67:157–163 DOI 10.1007/s13213-016-1245-z ORIGINAL ARTICLE Co-fermentation of cassava waste pulp hydrolysate with molasses to ethanol for economic optimization 1 2 3 Thippawan Wattanagonniyom & Wen-Chien Lee & Vasana Tolieng & 4 1 Somboon Tanasupawat & Ancharida Akaracharanya Received: 21 July 2016 /Accepted: 30 November 2016 /Published online: 24 December 2016 Springer-Verlag Berlin Heidelberg and the University of Milan 2016 Abstract Cassava waste pulp (CWP)–enzymatic hydrolysate Introduction was co-fermented with molasses (CWP-EH/molasses mixture) with the aim to optimize ethanol production by Saccharomyces Cassava waste pulp (CWP), a waste product from the cassava cerevisiae TISTR 5606 (SC 90). The optimal fermentation con- starch manufacturing industry, is produced at approximately 7 ditions for ethanol production using this mixture were 245 g/L million tons annually in Thailand (Office of the National initial total sugar supplemented with KH PO (8 g/L), at 30 °C Economic and Social Development Board 2006). Although 2 4 for 48 h of fermentation under an oxygen-limited condition with the predominant component of CWP is starch (67.8% w/w, agitation at 100 rpm, producing an ethanol concentration of dry weight; Akaracharanya et al. 2011), this waste product is 70.60 g/L (0.31 g ethanol/g total sugar). The addition of cassava not suitable as an animal feed due to its low nitrogen content tuber fiber (solid residue of CWP after enzymatic hydrolysis) at and is not an economically viable substrate for ethanol pro- 30 g/L dry weight to the CWP-EH/molasses mixture increased duction on its own because its saccharified sugar content is too ethanol production to 74.36 g/L (0.32 g ethanol/g total sugar). dilute. Despite efforts to improve the technology to extract as Co-fermentation of CWP-EH with molasses had the advantage much starch from cassava tubers as possible, at the present of not requiring any supplementation of the fermentation mixture time a considerable amount of starch remains in CWP. The with reduced nitrogen. dumping of CWP leads to environmental problems, including air pollution due to offensive odor and smells due to microbial deterioration of the residual starch in CWP. . . Keywords Cassava waste pulp Co-fermentation Molasses, a waste product from the sugar industry, is a . . Molasses Ethanol Saccharomyces cerevisiae major substrate for ethanol production in Thailand. Because it contains a high concentration of sucrose (30–40%, w/w) and other nutrients necessary for microbial growth and ethanol production (Chotineeranat et al. 2010), pretreatment and sac- charification steps are not required for fermentation with mo- lasses. However, it has been reported that supplementation of * Ancharida Akaracharanya molasses with urea, MgSO ,MnCl or soybean powder im- 4 2 email@example.com proves the production of ethanol from molasses. Specifically, urea and soybean powder supplement a reduced nitrogen for Department of Microbiology, Faculty of Science, Chulalongkorn 2+ improvement of yeast growth, Mg is a cofactor of some University, Phayathai Rd., Pathumwan, Bangkok 10330, Thailand 2+ enzymes in the ethanol fermentation pathway and Mn in- Department of Chemical Engineering, National Chung Cheng creases the activity of invertase, an enzyme that hydrolyzes University, 168 University Road, Minhsiung, Chiayi 621, Taiwan sucrose to glucose and fructose, which is necessary for sucrose Institute of Biotechnology and Genetic Engineering, Chulalongkorn fermentation by Saccharomyces cerevisiae (Takeshige and University, Phayathai Rd., Pathumwan, Bangkok 10330, Thailand Ouchi 1995; Pradeep and Reddy 2010). Department of Biochemistry and Microbiology, Faculty of In the study reported here we studied the value addition of Pharmaceutical Science, Chulalongkorn University, Phayathai Rd., Pathumwan, Bangkok 10330, Thailand CWP by co-fermentation of its saccharified starch with 158 Ann Microbiol (2017) 67:157–163 molasses to ethanol using S. cerevisiae. The composition and Enzymatic hydrolysis The CWP at a substrate loading level optimal concentrations of supplemented nutrients required for of 250 g/L was hydrolyzed at the optimized condition maximizing ethanol production were determined. (Thongchul et al. 2010) by suspension in deionized water (1 g CWP/4 mL deionized water) and autoclaving at 121 °C, 15 lb/in for 15 min. The suspension was then sequentially hydrolyzed by cellulase (1.41 CMC U/g) at 50 °C for 24 h, α- Materials and methods amylase (48 U/g) at 85 °C for 1 h and GC (4.8 TGA U/g) at 60 °C for 3 h. The resultant CWP slurry was filtered, and the Materials filtrate is hereafter defined as the CWP-enzymatic hydrolysate (CWP-EH), and its reducing sugar content was analyzed. The CWP was collected from the Sa-nguan Wong Industry Co., Ltd., Nakhon Ratchasima province, Thailand and kept at All experiments described above were performed based on wet −20 °C until use. The chemical composition of the CWP is weight. In some experiments the solid residue of the CWP sep- shown in Table 1. Just before use the CWP was thawed to arated from the filtrate. This solid residue, designated as cassava room temperature. The molasses sample was collected from tuber fibers (CTF), was dried at 65 °C and remixed into CWP- the Khonburi Sugar Co., Ltd., Nakhon Ratchasima province, EH at concentrations of 0, 25, 30, and 35 g/L. Thailand and kept at 4 °C until use. The enzymes cellulase [Accellerase™ 1500; 2500 car- Ethanol production from CWP-AH and CWP-EH boxymethylcellulose (CMC) units (U)/g], 650 p-nitrophenyl- mixtures glucoside, α-amylase (Spezyme alpha; 13,775 U/g) and the glucoamylase (GC 147; 580 U/g) were obtained from Inoculum preparation A single colony of S. cerevisiae TISTR Genencor, Danisco US Inc., Rochester, NY). 5606 grown on YPD agar at 30 °C for 48 h was inoculated into Saccharomyces cerevisiae TISTR 5606 (SC 90) was ob- 50 mL YPD broth in a 250-mL Erlenmeyer flask and incubated tained from the Thailand Institute of Science and Technology at 30 °C on a gyrotary shaker (100 rpm) for 24 h. The culture was and was maintained on YPD agar slant (in g/L: yeast extract, then transferred into 100 mL of fresh YPD broth in a 500-mL 10; peptone, 20; glucose, 20; agar, 20; pH 5.5) at 4 °C. Erlenmeyer flask at 1% (v/v) and incubated at the same condition until the late log phase (18 h). The cell number per milliliter of culture was determined using a haemacytometer under a light Hydrolysis of CWP microscope prior to harvesting by centrifugation (4 °C, 8000 rpm, 15 min), and the culture was used as the inoculum Acid hydrolysis The CWP at the designated substrate loading by suspension in fermentation medium. level of 100 g/L was hydrolyzed at the optimized condition as described by Thongchul et al. (2010). Specifically, 1 g CWP Fermentation medium preparation The CWP fermentation was added to 9 mL of 1 N HCl (or 1 g CWP/0.33 g HCl) and medium was prepared by adding 2 g/L (NH ) SO to 35 mL 4 2 4 the mixture autoclaved at 121 °C, 15 lb/in (0.1034 MPa) for of the CWP-AH or the CWP-EH mixture in a 50-mL 15 min and then filtered. The filtrate was harvested and is Erlenmeyer flask, adjusting the medium to pH 5.5. The medi- hereafter defined as the CWP-acid hydrolysate (CWP-AH); um was sterilized by autoclaving at 110 °C (10 min). its reducing sugar content was analyzed by the Somogyi– The CWP-EH/molasses fermentation medium was prepared Nelson method (Somogyi 1952). by mixing molasses (26.1 g) into CWP-EH (100 mL). The initial total sugar content of the resultant supernatant, determined using the phenol sulfuric acid method (Dubois et al. 1956), was 265 g/ Table 1 Chemical L. The CWP-EH/molasses mixture was supplemented with 2 g/L Composition % (w/w DW) composition of cassava (NH ) SO ,2g/LKH PO ,0.75g/L MgSO 7H O and 10 g/l waste pulp 4 2 4 2 4 4 2 Starch 67.8 yeast extract and then sterilized by autoclaving (110 °C, 10 min); Protein 2.1 this mixture was used to determine nutrient requirements for Fat 1.5 maximization of ethanol production. Moisture 80.0 Ash 3.7 Ethanol fermentation The different fermentation media [CWP-AH, CWP-EH, CWP-EH/molasses (standard or modi- DW, Dry weight fied)] were inoculated with the inoculum cells to a final con- The cassava waste pulp (CWP) was ana- 8 centration of 10 cells/mL and incubated at 30 °C with agita- lyzed by the Food research and testing lab- tion at 100 rpm for 48 h under an oxygen-limited condition. oratory (FRTL), Faculty of Science, Chulalongkorn University, Thailand The oxygen-limited condition was obtained by sealing the Ann Microbiol (2017) 67:157–163 159 50-mL Erlenmeyer flask with a rubber stopper connected to an Results and discussion airlock containing saturated copper sulfate solution. At the end of the 48-h fermentation period the culture medium was Acid and enzymatic hydrolysis of CWP clarified by centrifugation and the ethanol concentration in the supernatant analyzed by gas chromatography as described by Hydrolysis of CWP at a 100 g/L substrate loading by HCl Jutakanoke et al. (2012). yielded 31.6 g/L reducing sugar or 0.42 g reducing sugar/g [dry weight (DW)] CWP. When the CWP was hydrolyzed by Nutrient requirements for maximum ethanol production cellulase, α-amylase or GC at a substrate loading level of 250 g/L, 34.9 g/L reducing sugar (0.28 g reducing sugar/g CWP Fermentation of the CWP-EH/molasses mixture to produce DW) was obtained. CWP comprises 11.2% (w/w) lignocellu- ethanol was studied by varying the composition of the lose (Akaracharanya et al. 2011), and these fibers efficiently CWP-EH/molasses fermentation medium by adding or not both protect the starch from enzymes and bind the enzymes, adding the nutrient supplements shown in Table 2 and analyz- leading to a non-productive situation of reduced hydrolytic ing the supernatant for ethanol content following the ethanol efficiency. fermentation process. The CWP-EH/molasses fermentation media was then further optimized for highest ethanol produc- tion by univariate sequential analysis of the optimal concen- Ethanol production from the CWP-AH and CWP-EH tration of KH PO , initial total sugar and CTF in the fermen- 2 4 tation medium. Specifically, the CWP-EH/molasses medium Ethanol fermentation using the CWP-AH or the CWP-EH fer- mentation medium was assessed by inoculating the respective was supplemented with various concentrations of KH PO (0, 2 4 2, 4, 6, 8 and 10 g/L final) and then fermented for 48 h prior to medium with S. cerevisiae TISTR 5606 (final concentration the analysis of the ethanol level. Then the initial concentration 10 cells/mL) and incubating the inoculated media under an of total sugar in the CWP-EH/molasses mixture supplemented oxygen-limited condition at 30 °C for 48 h with agitation at with the determined optimal level of KH PO was varied at 100 rpm. The ethanol yield was lower with CWP-AH medium 2 4 205, 225, 245 and 265 g/L and fermented for 48 h prior to the (0.149 g ethanol/g reducing sugar) than with CWP-EH medium analysis of the ethanol level. Finally, the CWP-EH/molasses (0.242 g ethanol/g reducing sugar). Based on this result, we medium containing the determined optimal level of KH PO selected the CWP-EH medium for further study. 2 4 and molasses for the optimal initial total sugar level was sup- Saccharomyces cerevisiae can not ferment the pentose sugars plemented with CTF at 0, 25, 30 or 35 g/L and fermented for xylose and arabinose which are generally found in lignocellulosic 48 h prior to the analysis of the ethanol level. hydrolysate, and it noteworthy that CWP-EH contains glucose (2.84%), xylose and arabinose (<0.1%). The same ethanol yield was obtained from CWP-AH and CWP-EH when Rhizopus Analytical procedure oryzae was used (Thongchul et al. 2010). However, R. oryzae gave a higher ethanol yield and productivity from CWP-EH than Sugar composition of CWP-EH was analyzed by high- with glucose at the same carbon content, which may reflect the performance liquid chromatography (Agilent 1100 Series; promotional effect of the organic nitrogen in the CWP-EH on Agilent Technology, Santa Clara, CA). Sugars were identified R. oryzae population growth, causing a more rapid onset of ox- and quantified by resolution through a Microsorb column ygen limitation in the medium and thereby an increase in the 100–5NH (250 x 4.6 mm; Agilent Technology). Samples ethanol production (Thongchul et al. 2010).. (20 μL) of each sample were injected and eluted with The potential of CWP, which contains 67.8% w/w (DW) acetonitrile:H O (75:25) at a flow rate of 1.5 mL/min using starch, as a raw material for ethanol production was evaluated. a refractive index detector. Akaracharanya et al. (2011) reported that fermentation of the Table 2 Nutrient Supplemented nutrients Medium no. supplementation in the modified (w/v) cassava waste pulp-enzymatic 1 23 456 78 910 11 12 13 14 15 16 hydrolysate/molasses mixture + − + +++ ++ −− − − + −− − 2g/L (NH ) SO 4 2 4 ++ − +++ −− ++ −− − − + − 2g/L KH PO 2 4 ++ + − + − + − + − + −− + −− 0.75 g/L MgSO H O 4 2 ++ ++ −− − + − ++ + −− −− 10 g/L yeast extract 160 Ann Microbiol (2017) 67:157–163 Table 4 Selected nutrient levels in the molasses starch hydrolysate (22.6 g/L glucose) obtained from 30 g CWP (DW) resulted in the production of 9.9 g ethanol Components Content (g/100 g) Analytical method (0.43 g ethanol/g glucose) at 48 h. Due to the low concentra- tion of reducing sugar of CWP-EH, it is not an economically Inorganic nutrients −1 viable ethanol-producing fermentation substrate on its own. In Nitrogen (N) 3.00 × 10 In-house method based on AOAC (2012) 991.20 our study, the CWP-EH was co-fermented with molasses, an- −1 Phosphorus (P) 1.20 × 10 In-house method based other abundant, sustainable and renewable industrial waste Potassium (K) 1.27 on AOAC (2010) 984.27, product in Thailand. −1 975.03 Magnesium (Mg) 2.30 × 10 Trace elements Ethanol production from CWP- EH/molasses mixture Calcium (Ca) 0.68 In-house method based −4 Copper (Cu) <3.60 × 10 on AOAC (2010) 984.27, −4 975.03 The levels of some nutrients present in the CWP-EH and in Zinc (Zn) 1.70 × 10 −3 molasses that are known to potentially influence ethanol pro- Manganese (Mn) 4.43 × 10 duction by S. cerevisiae (Maiorella et al. 1983; Stehlik-Tomas Sugars et al. 2004; Chotineeranat et al. 2010) are shown in Tables 3 Sucrose 31.69 Puwastien et al. (2011), and 4,respectively. Glucose 8.73 pp 27–32 Fructose 8.87 Volatile acid Nutrient requirement for optimal ethanol production level Acetic acid 1.00 AOAC (2010) 935.57, 942.15 Non-volatile acid Various modified CWP-EH/molasses media were prepared Lactic acid 1.50 AOAC (2010) 935.57, 942.15 (Table 2) and then fermented to ethanol for 48 h as described in the BMethods^ section. The lowest ethanol concentration The CWP was analyzed by the Food research and testing laboratory was obtained in the media containing all four supplements (FRTL), Faculty of Science, Chulalongkorn University, Thailand (media no. 1), at approximately 1.05-fold less than that in a Based on a specific gravity of molasses of 1.38 the unsupplemented CWP-EH/molasses (Fig. 1). A slight re- duction in the ethanol concentration was also noted with me- dia supplemented with (NH ) SO +KH PO ,(NH ) SO + 4 2 4 2 4 4 2 4 yeast extract and KH PO +MgSO , respectively,, suggesting 2 4 4 that there was no clear single inhibitory nutrient. High ethanol concentrations were obtained with media no. 2, 10–12 and 15, with the highest ethanol level (62.67 g/L) obtained in the Table 3 Selected nutrient levels in the cassava waste pulp-enzymatic modified CWP-EH/molasses medium that was supplemented hydrolysate with only 2 g/L KH PO (medium no.15), again showing no 2 4 Components Level (g/100 g) Analytical method clear pattern of a single optimal nutrient supplement. Inorganic nutrient: Optimal concentration of KH PO in CWP-EH/molasses 2 4 Nitrogen (N) Not detectable In-house method based on AOAC (2012)991.20 mixture for ethanol fermentation −3 Phosphorus (P) 6.82 × 10 In-house method based −2 Potassium (K) 1.67 × 10 on AOAC (2010) 984.27, Various concentrations of KH PO were then added into the 2 4 −3 975.03 Magnesium (Mg) 2.66 × 10 CWP-EH/molasses mixture and fermented for 48 h to ethanol Trace element: as described in the BMethods^ section. The highest ethanol −3 Calcium (Ca) 1.79 × 10 In-house method based concentration (70.92 g/L) after 48 h of fermentation was found −4 Copper (Cu) <3.60 × 10 on AOAC (2010) 984.27, when the CWP-EH/molasses mixture was supplemented with −4 975.03 Zinc (Zn) 1.10 × 10 8g/L KH PO (Fig. 2), although this was not significantly 2 4 −5 Manganese (Mn) 1.20 × 10 greater than ethanol production at 6 or 10 g/L KH PO ;it 2 4 Sugars was, however, 1.12-fold higher than that at 2 g/L KH PO . 2 4 Sucrose <0.10 Puwastien et al. (2011), Glucose 2.84 pp 27–32 Optimal initial total sugar concentration Fructose <0.10 in CWP-EH/molasses mixture for ethanol fermentation The CWP was analyzed by the Food research and testing laboratory (FRTL), Faculty of Science, Chulalongkorn University, Thailand The level of molasses in the CWP-EH/molasses mixture Based on a specific gravity of the CWP-enzymatic hydrolysate of 1.01 supplemented with 8 g/L KH PO wasvariedtogive a 2 4 Ann Microbiol (2017) 67:157–163 161 Fig. 1 Effect of nutrient abc abcd supplementation on net ethanol ab abcde abc production level in the 48-h cde bcde de fermentation of the cassava waste fg ef pulp-enzymatic hydrolysate fg fg (CWP-EH)/molasses mixture. Medium number refers to composition shown in Table 2. Data are shown as the mean ± standard deviation (SD), derived from three independent replicates. Means with a different lowercase letter are significantly different at p <0.05 (Duncan’s multiple means test) 2+ final initial total sugar concentration that ranged from 205 level similar to the 5.47 and 6.6 g/L Ca reported in molasses to 265 g/L, following which the mixture was fermented by Takeshige and Ouchi (1995)and Chotineeranatetal. for ethanol production for 48 h. At an initial total sugar (2010), respectively. Thus, the requirement for phosphate sup- 2+ content of 245 and 265 g/L a similar ethanol concentra- plementation would depend on the concentration of Ca ions tion was obtained after 48 h—70.6 and 70.92 g/L, respec- in the molasses. tively—which was higher than that obtained with the two lower (225 and 205 g/L) total sugar levels (Fig. 3). Thus, an initial total sugar concentration of 245 g/L was used in Effect of addition of CTF on ethanol production subsequent trials. from CWP-EH/molasses There was no marked effect on ethanol production level when the 8 g/L KH PO was replaced by 8 g/L NaH PO The inclusion of CTF at 0–35 g/L in the CWP-EH/molasses 2 4 2 4 (data not shown). The depletion of phosphate in the CWP- mixture increased net ethanol production level after 48 h of EH/molasses fermentation system, and therefore the need for fermentation by 1.05-fold (Fig. 4), with the highest ethanol its supplementation, might be caused by the precipitation of concentration (74.36 g/L) being found with the addition of an insoluble calcium phosphate. Molasses contains a high 30 g DW/L CTF. However, further increases in the amount 2+ concentration of Ca because calcium oxide is used to clarify of CTF supplemented to the CWP-EH/molasses mixture from the sugarcane juice in the sugar production process. The mo- 30 to 35 g/L DW caused a decrease (1.04-fold) in the amount 2+ lasses used in this study contained 6.8 g/L of Ca (Table 3), a of ethanol produced. Fig. 2 Effect of KH PO 2 4 aa concentrationonethanol production in the CWP-EH/ molasses mixture. Data are shown as the mean ± SD, derived from three independent replicates. Means with a different lowercase letter are significantly different at p <0.05 (Duncan’smultiple means test) 162 Ann Microbiol (2017) 67:157–163 cells (Bai et al. 2008). In addition, the adsorption tech- nique is simple and easy to run (El-Latif et al. 2010). Conclusion Based on the results of this study, co-fermentation of CWP- EH and molasses for ethanol production had an advantage of not requiring any reduced nitrogen supplementation. The ad- dition of CTF in the CWP-EH/molasses mixture increased net ethanol production. Fig. 3 Effect of the initial total sugar concentration on net ethanol production in the CWP-EH/molasses mixture supplemented with 8g/L KH PO . Data are shown as the mean ± SD, derived from 2 4 Acknowledgments The authors thank Dr. Robert Butcher for critical three independent replicates. Means with a different lowercase reading of this manuscript and the Thai Alcohol Public Company, letter are significantly different at p < 0.05 (Duncan’s multiple Thailand for providing the α-amylase and glucoamylase enzymes. This means test) study was financially supported by the Thai Government budget (fiscal year 2015) and Graduate school, Chulalongkorn University to commem- orate the 72nd Birthday Anniversary of His Majesty the King Bhumibol Aduladej. The reason that CTF enhanced the net amount of eth- anol produced during the fermentation is unclear, but sev- eral low-cost plant materials have been reported to be References biomaterials for cell immobilization by natural adsorption in ethanol fermentation, including sugar beet pulp Akaracharanya A, Kesornsit J, Leepipatpiboon N, Srinorakutara T, (Razmovski and Pejin 1996), sugarcane bagasse (Santos Kitpreechavanich V, Tolieng V (2011) Evaluation of the waste et al. 2008), corn cob and grape pomace (Genisheva et al. from cassava starch production as a substrate for ethanol fer- mentation by Sacharomyces cerevisiae. Ann Microbiol 61: 2011). During the fermentation process yeast cells are 431–436 exposed to several stresses, such as ethanol, CO and Association of Official Analytical Chemists (AOAC) (2010) Official oxidative stresses, among others (Tesfaw and Assefa methods of analysis. 19th ed. AOAC International, Gaithersburg, 2014). 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Annals of Microbiology – Springer Journals
Published: Dec 24, 2016
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