Waste cassava tuber fibers (wCTF), derived from the ethanolic fermentation of cassava tubers, have potential use as anatural adsorption immobilization carrier. Ethanol fermentation was conducted using 15% (w/v) glucose-containing mediumat 40 °C for 48 h by Saccharomyces cerevisiae G6-2-2 (1.3 x 1010cells). Ethanol concentration produced by free, wCTF (1.2 g dry weight) adsorbed, wCTFadsorbed-calcium alginate entrapped,and calcium alginate entrapped cellswere 42.10 ± 0.61, 67.35 ± 0.53, 52.10 ± 0.40, and 46.45 ± 0.18 g/L (0.34, 0.45, 0.35, and 0.31 g ethanol/g reducing sugar), respectively. The wCTF adsorbed cells produced a maximum ethanol yield of 82.15 ± 0.48 g/L (0.43 g ethanol/g total sugar) from molasses (20% w/v initial total sugar) after 48 h, compared to 74 g/L to 76 g/L and 48 h to 100 h for the free suspension cells. The increase in ethanol produced by the wCTF adsorbed cells compared to free cells reflected that the cells were protected from environmental stresses and received amino nitrogen from the wCTF that supported growth and ethanol tolerance.
Waste Cassava Tuber Fibers as an Immobilization Carrier of Saccharomyces cerevisiae for Ethanol Production
Sineenath Kunthiphun,a Pongphannee Phumikhet,a Vasana Tolieng,b Somboon Tanasupawat,c and Ancharida Akaracharanya a,*
Waste cassava tuber fibers (wCTF), derived from the ethanolic fermentation of cassava tubers, have potential use as anatural adsorption immobilization carrier. Ethanol fermentation was conducted using 15% (w/v) glucose-containing mediumat 40 °C for 48 h by Saccharomyces cerevisiae G6-2-2 (1.3 x 1010cells). Ethanol concentration produced by free, wCTF (1.2 g dry weight) adsorbed, wCTFadsorbed-calcium alginate entrapped, and calcium alginate entrapped cells were 42.10 ± 0.61, 67.35 ± 0.53, 52.10 ± 0.40, and 46.45 ± 0.18 g/L (0.34, 0.45, 0.35, and 0.31 g ethanol/g reducing sugar), respectively. The wCTF adsorbed cells produced a maximum ethanol yield of 82.15 ± 0.48 g/L (0.43 g ethanol/g total sugar) from molasses (20% w/v initial total sugar) after 48 h, compared to 74 g/L to 76 g/L and 48 h to 100 h for the free suspension cells. The increase in ethanol produced by the wCTF adsorbed cells compared to free cells reflected that the cells were protected from environmental stresses and received amino nitrogen from the wCTF that supported growth and ethanol tolerance.
Keywords: Waste cassava tuber fiber; Natural immobilization carrier;Immobilization Ethanol
Contact information: a: Department of Microbiology, Faculty of Science, Chulalongkorn University, 254 Phayathai Rd., Pathumwan, Bangkok 10330, Thailand; b:Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, 254 Phayathai Rd., Pathumwan, Bangkok 10330, Thailand; c:Department of Microbiology and Biochemistry, Faculty of Pharmaceutical Science, Chulalongkorn University, 254 Phayathai Rd., Pathumwan, Bangkok 10330, Thailand;*Corresponding author: email@example.com
The key fundamental raw materials used for ethanol production in Thailand are molasses and cassava tuber (tapioca root) (Department of Alternative Energy Development and Efficiency Thailand 2015). The advantage of molasses over cassava tuber is that the former can be fermented into ethanol without prior saccharification. Although molasses is a low-priced fermentable sugar resource, it has high demand inseveral industries that may result in a shortage of supply (Balat and Balat 2009). If there is a molasses shortage, the ethanol industry would be required to shift to using cassava tubers as an alternative substrate (Sriroth et al. 2010). After cassava tubers are fermented, the liquid contains insoluble cassava tuber fibers (CTF) as the waste product. Waste CTF (wCTF) is removed from the fermentation liquid before ethanol distillation and is then disposed by landfilling.
Ethanol is produced from agricultural resources by saccharification of the polysaccharides, followed by fermentation with microorganisms, especially yeast, in the form of free cells (suspension) or immobilized cells (Behera et al. 2010; Yan et al. 2012; Bouallagui et al. 2013). Ethanol fermentation with immobilized cells is typically better (as in a higher ethanol yield and productivity) than that of free cells because the immobilized cells are protected from environmental stresses, such as acidity, osmosis, CO2, and degenerative substances produced from fermentation, including furfural and acetic acid (Ingledew 1999; Bai et al. 2008; Tesfaw and Assefa 2014). In addition, high cell density are loaded. This results in an increase of ethanol tolerance, ethanol production and decrease in ethanol production time (Tian et al. 2015).
Popular methods of immobilizing cells for ethanol fermentation include calcium alginate entrapment and natural adsorption because the techniques are simple and conduct under mild condition (Razmovoski and Vučurović 2011; Zheng et al. 2012; Tian and Chen 2016). When using entrapment, yeast cells inoculated are trapped within a gel matrix and they are maintained throughout fermentation; conversely, the cells trapped inside the calcium alginate beads may suffer from a mass transfer limitation of nutrients and O2, as well as the removal of the CO2 and ethanol produced (Phisalaphong et al. 2007; Yu et al. 2007; Zhu 2007). In natural adsorption, cells are naturally adsorbed on a carrier by physical or chemical adsorption, vander Waals force, ionic force, or electrostatic attraction between negative charge of cell wall and positive charge of carrier (Vericaet al. 2010; Genisheva et al. 2014). The growth of yeast cells is not notably affected, and the new active yeast cells can be adsorbed by the carrier when older dead cells are washed off (Iqbal and Saeed 2005; Bai et al. 2008). On the contrary, there is an inability to maintain a high number of immobilized cells from the beginning to the end of the fermentation process (Phisalaphong et al. 2007; Yu et al. 2007; Geisheva et al. 2011).A combination of alginate entrapment and natural adsorption caused the structure and mass transfer of alginate gel bead to change, which increased the ethanol yield. As yet, only limited studies have reported on the combination of entrapment and adsorption immobilization (Phisalaphong et al. 2007; Razmovski and Vučurović 2011; Alting and Zhaoping 2015; Tian et al. 2015). The objective of this research was to evaluate the efficiency of wCTF as an immobilization carrier of Saccharomyces cerevisiaethrough wCTF natural adsorption, and combination of the natural adsorption and calcium alginate entrapment for the improvement of ethanol production.
The wCTF used in this study was collected from the Saptip Co. Ltd.,Ethanol Fuel Production Plant, located in Lophburi province, Thailand, and was kept at -20 °C. The wCTF was thawed at room temperature prior to use. Molasses was obtained from the Angvian Industry Co. Ltd. in Nakhon Ratchasima province, Thailand and kept at 4 °C until use. The molasses was diluted to 20% (w/v) total sugar and clarified by centrifugation at 4 °C and 8000 rpm for 5 min with harvesting of the supernatant for the preparation of the molasses medium.
The total sugar content was analyzed by the phenol sulfuric acid method (Dubois et al. 1956). Saccharomyces cerevisiae G6-2-2 was isolated from Khonburi sugar PLC, Nakhon Ratchasima province, Thailand, at 40 °C. The maximum ethanol production level was 59.81 g/L (0.40 g ethanol/g glucose) from a 15% (w/v) glucose containing medium at 40 °C for 48 h.
Preparation of the wCTF for immobilization
The thawed wCTF was washed with running water for 30 min and then dried at 80 °C to a constant weight. The dried wCTF was sieved to screen for 450 µm to 850 µm sized particles and sterilized by autoclaving at 121 °C and100 kPa for 15 min.
Determination of water absorption index (WAI) of the wCTF
The WAI (grams of water absorbed per gram of wCTF dry weight) indicated the quantity of water absorption by wCTF, and was determined according to Anderson et al. (1969). The dried wCTF (1.25 g) was suspended in 15 mL of distilled water, stirred for 10 min, and centrifuged (4 °C, 8,000 rpm, and 10 min). The wCTF pellet was harvested, weighed, and the WAI of the wCTF was calculated as the grams of wet weight/grams of dry weight.
Preparation of S. cerevisiae inoculum
A single colony of S. cerevisiae G6-2-2 was inoculated in 50 mL of fermentation medium (15% (w/v) glucose, 0.9% (w/v) peptone, 0.6% (w/v) yeast extract, pH 5.0) in a 250 mL flask and incubated at 40 °C with shaking at 200 rpm for 24 h. The obtained culture was inoculated into a new fermentation medium at an initial optical density (OD) at 660 nm of 0.05 and incubated at the same condition until the late log phase. The culture was centrifuged (4 °C, 8000 rpm, and 5 min) to precipitate the cells, which were then suspended in 5 mL of 0.9% (w/v) NaCl.Cellnumber in the cell suspension was counted under light microscope using a haemocytometer and used as inoculum.The inoculum was 1.37×1010 cells/5mL.
Preparation of wCTF adsorbed S. cerevisiae for cell immobilization
The S. cerevisiae G6-2-2 inoculumwas mixed with 1.2 g wCTF (dry weight), and maintained at room temperature for 30 min. After filtration, non-immobilized cells in filtrate were determined by hemocytometer (Table 1).
No. cells immobilized = No. cells inoculated – No. non-immobilized cells
(or cells in filtrate)
Subsequently, the number of yeast cells inoculated (1 x 109 to 3.93 x 1010cells/5 mL) and the amount of wCTF loaded (0.6 g to 2.4 g) were independently and sequentially varied to investigate the optimal dose for ethanol production.
Preparation of calcium alginate entrapped S. cerevisiae and wCTF adsorbed-calcium alginate entrapped S. cerevisiae
The S. cerevisiae G6-2-2 inoculum was mixed with 5 mL of 0.9% (w/v) NaCl or 5 mL of a wCTF suspension (1.2 g wCTF (dry weight) in 0.9% (w/v) NaCl) and kept at room temperature for 30 min. Then, it was further mixed with 10 mL of 2.0% (w/v) sodium alginate (pH 7.0) and dispensed at 86 ± 3 µL/ drop (cell-sodium alginate mixture) or 116 ± 4 µL/drop (cell-wCTF-sodium alginate mixture) into 250 mL of 100 mM CaCl2 with gentle stirring at room temperature using a 10-mL glass dropper. The resultant calcium alginate beads were left to harden for 15 min, harvested by filtration, and washed three times with 0.9% (w/v) NaCl (200 mL). All steps were performed aseptically.
The yeast cell number in the filtrate and in the washing NaCl solution were determined under light microscopy using a hemocytometer, and the derived sum of the yeast cell number in the filtrate and washing NaCl solution was defined as the non-immobilized cell number. The number of cell immobilized was calculated as above.
The S. cerevisiae G6-2-2 suspension and immobilized cells were inoculated into 50 mL of molasses medium (20% (w/v)total sugar, 1.3% (w/v) (NH4)2SO4, 0.27% (w/v) KH2PO4, 0.05% (w/v) MgSO4.7H2O, pH 5.0) in a 100-mL Duran bottle and incubated at 40 °C in an oxygen limited condition with 100 rpm agitation. The oxygen limited condition was performed by tightly closing the screw cap of the Duran bottle. Supernatant obtained after centrifugation (4 C, 8000 rpm, 10 min) was analyzed forethanol by gas chromatography(Hewlett-Packard, HP5890 series, USA) with a flame ionization detector at 150 °C using a Porapak QS (cabowax 20 M) column (2 m x 0.32 m) at an oven temperature of 175 °C. Helium at a flow rate of 35 mL/min was used as the carrier gas (Jutakanoke et al. 2012).
Comparison of the different immobilization systems on the ethanol production level
The free or immobilized S. cerevisiae G6-2-2 cells (wCTF-adsorbed, wCTF adsorbed-calcium alginate entrapped, and calcium alginate entrapped), at a final dose of 1.3 x 1010 cells, were inoculated into the fermentation medium and fermented to ethanol. Likewise, thefree and wCTF adsorbed cells were inoculated into molasses medium and fermented to ethanol.
Characterization of the wCFT
The wCTF was prepared by soaking in 3.5% glutaraldehyde for 1 h, and dried by sequentially soaking in 30, 50, 70, 95, and 100% ethanol using a critical point dryer (Polaron Range CPD7501, England). The dried wCTF was treated with gold and characterized by scanning electron microscope (SEM; JEOL JSM-5410-LV, Japan)
The data were analyzed using an analysis of variance (ANOVA) and significance was accepted at P< 0.05. All statistical analyses were performed using the SPSS 17.0 software(SPSS Inc., USA).
Table 1. Number of Immobilized S. cerevisiae Cells
wCTF: waste cassava tuber fibers 1.2 g dry weight of wCTFs were used
Data are shown as the mean ± standard deviation, derived from 3 independent replicates
RESULTS AND DISCUSSION
The WAI of wCTF
The wCTF had a WAI value of 5.93 ± 0.22 g water/g wCTF (dry weight), which indicated that it was likely to be suitable for use as a cell immobilization carrier (Orzua et al. 2009; Razmovski and Vučurović 2011). In comparison, the WAI value of wCTF was higher than that previously reported for pecan nut shell, pistachio shell, wheat bran, apple pomace, bean residue, creosote bush leaves (Orzuaet al. 2009), sotol fiber, corn cobs, and candelilla stalks (Flores-Maltos et al. 2014), which have all been reported as agro-industrial wastes that are suitable for use as cell immobilizationcarriers.
Optimal Yeast Cell Number and wCTF Loading Level for Natural Adsorption
Different quantities of S. cerevisiae cells (1.0 x 109, 6.1 x 109, 1.31 x 1010, 2.65 x 1010, and 3.93 x 1010) were immobilized in 1.2 g (dry weight) of wCTF. The wCTF adsorbed cells were inoculated into 50 mL of fermentation medium and the ethanol production level was assayed. The inoculum at 1.31 x 1010 cells yielded the highest ethanol production (67.35 ± 0.4 g/L) after 48 h (Fig. 1a). However, when the wCTF loading was decreased to 0.6 g (dry weight) or increased to 2.4 g (dry weight) the ethanol production level decreased (Fig. 1b). This result agree well with Tian et al. (2015).Therefore, 1.31 x 1010 ofS. cerevisiae G6-2-2 cells were immobilized in 1.2 g wCTF forfuture experiments.
Fig. 1. Effect of the a) immobilized S. cerevisiae cells (1.2 g of wCTF), and b) wCTF loading level (1.31 x 1010 S. cerevisiae G6-2-2 cells) for ethanol production in a 15% (w/v) glucose containing medium at 40 °C for 48 h. Data represent the mean ± standard deviation (0.9), derived from three independent replicates
Comparison of the Immobilization System on the Ethanol Production Level
The free and immobilized S. cerevisiae G6-2-2 cells (1.31 x 1010) were investigated for efficient ethanol production. The wCTF-adsorbed cells produced the highest ethanol level (67.35 ± 0.4 g/L) after 48 h. This result was in agreement with Singh et al. (2013), who reported that the highest ethanol level was produced by cells immobilized on pretreated sugarcane bagasse compared with calcium alginate entrapped cells or free cells. Yeast cells immobilized by the adsorption technique were free to contact the fermentation medium; therefore, their growth was not effected by mass transfer limitations. Meanwhile, the washed out (dead) cells could be replaced by new cells (Braschler et al. 2005; Bai et al. 2008). The porous structure of wCTF increased the mass transfer of wCTF adsorbed-calcium alginate beads, which improved the growth of wCTF adsorbed-calcium alginate entrapped cells. This is because cell biomass and ethanol are co-produced by the ethanolic fermentation-based metabolism of S. cerevisiae. Therefore, the wCTF adsorbed-calcium alginate entrapped cells yielded a slightly higher quantity of ethanol than the calcium alginate entrapped cells (Fig. 2).
However, other co-products, such as CO2 and other degenerative products,created a higher stress level on wCTF adsorbed-calcium alginate cells than thecells adsorbed onto wCTF. Nevertheless, the mass transfer limitationwould likely be the main drawback of the alginate entrapped cells (Kumakura et al. 1992; Groboillot et al. 1994; Iqbal and Saeed 2005). Several high porosity materials have been applied to overcome this drawback (Yu et al. 2010). In addition, several studies have reported that a combination of entrapment and adsorption/immobilization agents, such as alginate and loofa sponge (Phisalaphong et al. 2007), alginate and maize stem ground tissue (Razmovski and Vučurović 2011), alginate and delignified sawdust (Alting and Zhaoping 2015) and alginate and pretreated corn stalk (Tian et al. 2015) can enhance the gel strength and the mass transfer inside the gel carrier.
Fig. 2. Effect of the immobilization method on the ethanol production. Free (suspension) S. cerevisiae G6-2-2 cells (1.31 x 1010) or those immobilized onto 1.2 g (dry weight) of wCTF and calcium alginate entrapment were employed for the fermentation of 15% (w/v) glucose-containing medium at 40 °C for 48 h. Data represent the mean ± standard deviation (0.6), derived from 3 independent replicates
Comparison of Ethanol Production from Molasses by Free and wCTF-Adsorbed Cells
As shown in Fig. 3, wCTF-adsorbed yeast cells produced a maximum ethanol concentration after 48 h, which was 10.95% higher than that of free cells. The SEM images of wCTF before fermentation showed a rough surface and porous structure (Fig. 4a), including the presence of attached residual yeast cells from the prior commercial fermentation of cassava tubers to ethanol (Fig.4b). After fermentation, the number of yeast cells attached to the porous structure of wCTF increased (Fig. 4c). Analysis of the molasses medium and that containing 5% (w/v) wCTF showed that the molasses medium containing wCTF had a markedly higher content of several amino acids, including an over two-fold higher level of glutamic acid, histidine, lysine, serine, and tyrosine (Table 2). Natural carriers have been recognized as low cost materials with lower mass transfer limitations than entrapment carriers, while some agricultural-based immobilization carriers also provide extra nutrients. For example, maize-stem ground tissue not only protected cells from different stresses during ethanol fermentation, but also provided extra nutrients, such as protein and soluble salt, which promoted ethanol production from molasses by S. cerevisiae (Razmovski and Vučurović 2011).
The use of brewer’s spent grains as a natural carrier in molasses-based ethanol production provided extra nutrients to the system and resulted in an increased ethanol yield (Kopsahelis et al. 2007). Albers et al. (1996) reported that the growth rate and ethanol yield of S. cerevisiae in fermentation medium,containing an amino acids mixture as the nitrogen source,was higher than that of ammonium salt medium. In addition, the thermo-tolerance of yeast was enhanced by the supplementation of soybean flour (Balakumar and Arasaratnam 2012).
Fig. 3. Ethanol production by S. cerevisiae G6-2-2 from molasses medium (20% (w/v) sugar) as (▲) free cells or (●) wCTF-adsorbed cells. Fermentation wasat 40 °C for 48 h. Data represent the mean ± standard deviation (1), derived from 3 independent replicates
Fig. 4.Scanning electron micrographs of wCTF: a and b) before and c) after fermentation. a) The porous structure of the wCTF with attached residual yeast cells (1000X magnification), b) the budding scar of the attached cell (10,000X magnification), andc) the increased cell number attached to wCTF after fermentation (1000X magnification). Images are representative of at minimum of 3 fields of view per sample and 2 independent samples
Table 2.Comparison of the Amino Acid Composition of the Molasses Medium With or Without 5% (w/v) Waste Cassava Tuber Fibers (wCTF)
ND; not detectable.
*Analysed at the ALS Laboratory group (Thailand) Co. Ltd.
1. The ethanol production level of wCTF adsorbed-calcium alginateentrapped S. cerevisiaecellswas 12.16% higher than those of calcium alginate entrapped S. cerevisiaecells.
2. But the ethanol production level of wCTF adsorbed S. cerevisiae cells was 22.64% higher than those ofthe wCTF adsorbed-calcium alginate entrapped S. cerevisiae cells.
3. The ethanol production level of the wCTF adsorbed S. cerevisiaecells was 59.9% higher than those ofS. cerevisiae free cells.
4. It is likely that the wCTF provided an exogenous amino nitrogen supply that
promoted an increased ethanol yield.
This study was ﬁnancially supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0240/2552) and The 90th Anniversary of Chulalongkorn University Fund (Ratchadapisek somphot Endowment Fund).
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Article submitted: July 12, 2016; Peer review completed: August 14, 2016; Revised version received and accepted: November 2, 2016; Published: November 9, 2016.