Immobilised Sarawak Malaysia Yeast Cells for Production of Bioethanol

Zain, Masniroszaime Mohd; Kofli, Noorhisham Tan; Abdullah, Siti Rozaimah Sheikh

INTRODUCTION

Interest in bioethanol has been growing since it has considered as an alternative fuel for future due to depleting fossil fuel energy resources (Lin and Tanaka, 2006; Yu et al., 2009; Giordano et al., 2000). Ethanol can be produced by microbial fermentation using renewable substrates such as corn and sorghum, thus economic and profitable production emphasizing on substrates, processes and the microbes used deserved a proper attention from researchers (Ibeto et al., 2011; Millati et al., 2011; Sherief et al., 2010; Nadir et al., 2009).

Indigenous fermented foods are essential components of the diet and represent identity culture of the people or countries. Fermented food include alcoholic and non alcoholic beverages developed well traditionally with village-art methodologies that normally produced at household or cottage industry scales (Dung et al., 2007; Sujaya et al., 2002; N’Guessan et al., 2008; Valyasevi and Rolle, 2002). In Malaysia, various fermented foods being produced and amongst the popular are tapai and tuak. Tapai is a food made from tapioca (cassava), glutinous rice, rice or banana can be eaten raw after few days of fermentation with ragi. Similar to Indonesian tape ketan (black rice fermentation), Malaysian tapai is partially liquefied, sweet sour and mildly alcoholic rice paste served as dessert or snack. Whereas, tuak is rice wine for native peoples (especially in the state of Sarawak) usually serve during special and important occasions. Tuak is prepared similarly like tapai but in longer fermentation period, resulting in greater liquefaction of the rice and consumed as an alcoholic beverage. Both tapai and tuak produced by fermentation of rice from starter ragi, dry flattened circular cakes, about 3-5 cm in diameter, consisted of microflora of microorganism. These microbial exist in the traditional starter associated with the raw materials which is rice flour mixed with grounded spices (Gandjar, 2003; N’Guessan et al., 2008).

Traditionally, ethanol fermentation includes the usage of free cells of any suitable species and strains mainly involving Saccharomyces cerevisiae (Somda et al., 2011). The recuperation and reutilization of these cells require onerous steps (Rivaldi et al., 2008) resulted in separation of cells normally achieved by a unit procedure and discarded after the process ended. This also will escalate burden to a fermentation plant in order to discard such volume of biomass. The growing demands for bioethanol needing alternative optimization such as immobilization of cells in an inert support (Goksungur and Zorlu, 2001). Since the cells are entrapped inside the inert support, separation of bioethanol from the medium should be easier and cost saving since it is omitting a unit procedure than those of free cells. Apart from that, cells entrapment also allowing reutilization of entrapped cell, protection against adverse environmental conditions, utilizations of high cell densities that usually make higher processing rate and high dilution in continuous operation (Da Cunha et al., 2006).

There are many support materials and carrier that is used in cell immobilization and give the different entrapment including alginate beads entrapment, biocapsules and PVA particles (Rakin et al., 2009; Peinado et al., 2006). Alginate in food, pharmaceutical, textile and paper production industry used for thickening, stabilizing, gel and film forming (Najafpour et al., 2004). Entrapment in calcium alginate beads has been one of the most used matrices for whole cell entrapment due to its simplicity and non-toxic character. This simple and mild immobilization technique involves the drop-wise addition of cells suspended in sodium alginate onto a solution of calcium chloride whereby the cells are immobilized in precipitated calcium alginate gel in the form of beads (Goksungur and Zorlu, 2001).

Local Brown Sugar (LBS), also known as ‘gula merah’ in Malay is the names of jaggery, a traditional unrefined sugar consumed in Asia, Africa, Latin America and Caribbean (Rathnasabapathy et al., 2009; Rajvanshi and Nimbkar, 1996). It is a natural sweetening substance made by concentrating sugarcane juice without any preservatives and colourings. LBS normally used in preparing cakes, syrups and desserts and sold cheaply at local market. There are many published studies on bioethanol production using sweet juice as a substrate from sorghum and sugar cane using Saccharomyces cerevisiae have been reported in producing bioethanol manipulating unsterilized juice substrate (Rajvanshi and Nimbkar, 1996; Yu et al., 2009). Thus, the usage of local yeast in production of ethanol has the potential to be explored. In this study, the ability of yeast isolated from cottage industry in Sarawak, Malaysia to produce ethanol using LBS and calcium alginate entrapment method were investigated.

MATERIALS AND METHODS

Microorganism: The yeast strain used in fermentation (2009) was isolated from two different of starter ragi which is used in making tuak and tapai are bought at the local market in Kuching, Sarawak (Malaysia). The starter ragi is dry flattened circular cakes, about 3-5 cm in diameter, prepared from rice flour and packed in small plastic bag. It was subcultured and screened from tapai fermentation followed the method of Sujaya et al. (2002) and isolated on Sabouraud agar (Koehler et al., 1999) to produce a single colony. The strain selected was named as ST1 (tuak) and ST3 (tapai) and was kept on YPD medium/agar at 4°C.

Substrates: The medium of Yeast-extract-peptone-dextrose (YPD) consisted of yeast extract 20 g L^-1, Peptone 10/L and dextrose 20 g L^-1. The local brown sugar used as a substrate in immobilisation fermentation was bought at the local supermarket in a packed plastic. The composition of the medium is LBS (20-50 g L^-1) which was diluted and filtered with addition of 5.19 g L^-1 (NH₄)₂SO_4, 1.53 g L^-1 KH₂PO₄ and 0.55 g L^-1 MgSO₄(Bravo and Gonzalez, 1991).

Cell immobilization: ST1 and ST3 cells were grown at 30°C for 10 h. Two hundred and fifty milliliter culture broths were harvested by centrifuge at 13000 rpm for 5 min. Fifty milliliter of this growth medium was mixed with an equal volume (1:1,v/v) of 4% (w/v) Na-alginate (Sigma, A-2033) solution (Goksungur and Zorlu, 2001). One hundred milliliter aliquot of alginate-cell suspension containing 2% Na-alginate (unless otherwise stated) was added drop wise to 1000 mL of 2% CaCl₂ with a syringe (Tataridis et al., 2005). Alginate drops solidified upon contact with CaCl₂, forming beads and thus entrapping yeast cells. The beads were allowed to harden for 30 min and then were washed with sterile saline solution (0.85% NaCl) to remove excess calcium ions and cells (Rakin et al., 2009). The fermentation using immobilized beads were executed in a 250 mL flask with agitation at 75 rpm to avoid bead breakage.

Analytical method: Ethanol and glucose concentrations were determined using biochemical analyzer, YSI Select (Yellow Spring Ltd.). The data were analyzed using Microsoft excel.

RESULTS AND DISCUSSION

Free cell: The experiment started with fermentation of free yeast cell ST1 and ST3 in 500 mL shake flask using Yeast extract-peptone-dextrose (YPD) as medium incubated at 30°C and Fig. 1a, b showed the early results with 12 h fermentation time. The growth rate, μ of ST1 and ST3 were 0.396 and 0.38 h^-1, respectively and the yield of ethanol production ST1 and ST2 in free cell fermentation were Y_p/s0.29±0.03 g ethanol/g substrate and 0.287±0.033 g ethanol/g substrate, respectively indicate that the choice of ST1 and ST3 used in this study were achieved.


Fig. 1a:	Growth of ST1 and ST3 in YPD medium using free-suspension system


Fig. 1b:	Glucose consumption and ethanol production profiles by ST1 and ST3 in YPD medium using free-suspension system

Table 1:	Bioconversion of glucose to ethanol by ST1 and ST3 strain in free cell fermentation

The Table 1 summarized the yield of ST1 and ST3 yeast cell, Y_p/s for free cells fermentation. It is found that ST1 and ST3 produce similar results in term of μ and Y_p/s.These results suggest both strain has the capability to produce ethanol. It is expected since both starter ragi are used in food and alcoholic beverages (tuak and tapai).

The optical density data patterns were identical to both strains and the yeast cells adapted to the substrate in the first two hours. The maximum values reached at 8 h fermentation and correspond well with the maximum ethanol concentration (also at 8th h fermentation) values of 7.36 g L^-1 while the biomass, X reached maximum at 10th h, at 15.1 g L^-1 both in ST1 and ST3 strains showed in Fig. 2.

Figure 1b showed the sugar was quickly consumed and almost depleted after 10 h of fermentation.


Fig. 2:	Alginate beads

Sugar were utilized as energy source by yeast cell as during the fermentation to produce bioethanol. Therefore, the consumption rate of sugar can be related to the concentration of yeast cells (N’Guessan et al., 2008) and ethanol production. After 8 h fermentation, the production of ethanol declined since the sugar had almost 98% consumed. Moreover, there were factors which affect the production rate such as nitrogen limitation and ethanol inhibitory effect (Nikolic et al., 2009). The maximum ethanol production by ST1 and ST3 strain is 7.36 and 7.06 g L^-1, respectively. These results seemed to be similar as reported by Lin and Tanaka (2006) saying that the range of ethanol concentration produced by yeast cells is 2.4-91.8 g L^-1.

Immobilisation: The dropwise method of forming the alginate bead is shown in Fig. 2. Small beads size is favorites because of high surface area thus facilitated better mass transfer of substrate (Margaritis and Kilonzo, 2005; Goksungur and Zorlu, 2001). Using this application, the average sizes obtain was 0.51 cm. But the smaller the alginate beads, it is also prone to breakage. At this diameter and alginate concentration used (2 wt%), the beads were fully active, flexible and hard enough to stand mild agitation and have a good stability (Najafpour et al., 2004). Also, we observed that the method produced that the method produced bud-like shape due to the dragging of the sodium alginate solution. The effect of bud-like shape of alginate bead on the surface area was not investigated and all beads were assumed to be spheres.

Observations by electronic micrographs were taken from the fresh ST1 strain is shown in Fig. 3. These micrographs were used as to observe the yeast cells entrapment in the alginate beads. The cells were found to be attached close to the surface of the alginate beads.


Fig. 3:	Scanning electron microscopy of immobilized ST1-yeast cell

Although, it has been reported that there were no cell in the centre of particle (Ogbonna et al., 1989; Giordano et al., 2000) but from our SEM photograph indicated that the yeast cells were distributed equally throughout the beads.

The results of ethanol production and glucose consumption profiles for immobilisation system of ST1 and ST3 strains are shown in Fig. 4a, b for YPD and LBS, respectively with additional fermentation in medium LBS with two different concentrations showed in Fig. 4c. The maximum ethanol concentration was achieved after 7 h in the immobilised fermentation for both media and ST1 and ST3 strains achieve complete fermentation in same time with 98% glucose consumed well.


Fig. 4a:	Production of ethanol and glucose consumption profile of ST1 and ST3 in YPD by immobilized cell system


Fig. 4b:	Production of ethanol and glucose consumption profile of ST1 and ST3 in LBS using immobilized cell system


Fig. 4c:	Production of ethanol and glucose consumption profile of ST1 in 20 and 50 g L^-1 LBS using immobilized cell system

Ethanol production in YPD media is higher as 6.59 g L^-1 in this immobilized fermentation. In LBS media, the maximum glucose concentration observed at Fig. 4b only 1.54 g L^-1 but when we measured the concentration of glucose in LBS at the beginning of the fermentation (2.57 g L^-1) and it was actually much lower than that in YPD.

Table 2:	Yield, Y_P/S for YPD and LBS media

Table 3:	Summary of theoretical yields of ST1 and ST3 yeast cells in free cell and immobilization fermentation

In the concentration of LBS media, 50 g L^-1 resulted higher ethanol concentration than 20 g L^-1 LBS media showed in Fig. 4c since the initial concentration of glucose is high. The initial glucose concentration might have affected on the yield of ethanol. The yields Y_P/Swere 0.244 and 0.54 g g^-1 for YPD and LBS, respectively showed in Table 2, suggesting that LBS can be used as suitable cheap substrate for production of ethanol. Selection of cheap products as substrate for ethanol production has been investigated thoroughly such as cellulose, cellobiose and xylose but has to overcome of pre treatment methods to obtain the sugar (Fukuda et al., 2009).

The theoretical yield of ethanol production was calculated as the actual ethanol divided by the theoretical maximum on the basis that 1 mol of glucose able to produce 2 mol of ethanolx100 (Goksungur and Zorlu, 2001). Summary of the theoretical yields for ST1 and ST3 yeast cells using these two sugar medium are showed in Table 3. The ST1 yeast cells yielded the highest theoretical yields, 94.5% of bioethanol in immobilised cells fermentation. This indicates that the imobilised yeast cell able to produce high ethanol, as also reported by Lin and Tanaka (2006), saying that the production of bioethanol using immobilised cells were doubled as compared in free cells system.

CONCLUSION

From the results we found that the local isolate, both ST1 and ST3 immobilised-cells have the ability to produce ethanol using both commercial (Y_P/S; 0.25 and 0.24 g g^-1, respectively) and local substrate (Y_P/S; 0.59 and 0.54 g g^-1, respectively). These yields can be increased by studying the optimization parameters involve in the fermentation of ethanol using immobilized system which is ongoing.

ACKNOWLEDGMENT

The research is funded by University Research Grant (Code: UKM-GUP-BTT-07-25-165) which is duly acknowledged by authors.