Immobilised Sarawak Malaysia Yeast Cells for Production of Bioethanol
Masniroszaime Mohd Zain,
Noorhisham Tan Kofli
Siti Rozaimah Sheikh Abdullah
Bioethanol production using yeast has become a popular topic due to worrying depleting worldwide fuel reserve. The aim of the study was to investigate the capability of Malaysia yeast strains isolated from starter culture used in traditional fermented food and alcoholic beverages in producing Bioethanol using alginate beads entrapment method. The starter yeast consists of groups of microbes, thus the yeasts were grown in Sabouraud agar to obtain single colony called ST1 (tuak) and ST3 (tapai). The growth in Yeast Potatoes Dextrose (YPD) resulted in specific growth of ST1 at μ = 0.396 h-1 and ST3 at μ = 0.38 h-1, with maximum ethanol production of 7.36 g L-1 observed using ST1 strain. The two strains were then immobilized using calcium alginate entrapment method producing average alginate beads size of 0.51 cm and were grown in different substrates; YPD medium and Local Brown Sugar (LBS) for 8 h in flask. The maximum ethanol concentration measured after 7 h were at 6.63 and 6.59 g L-1 in YPD media and 1.54 and 1.39 g L-1 in LBS media for ST1 and ST3, respectively. The use of LBS as carbon source showed higher yield of product (Yp/s), 0.59 g g-1 compared to YPD, 0.25 g g-1 in ST1 and (Yp/s), 0.54 g g-1 compared to YPD, 0.24 g g-1 in ST3 . This study indicated the possibility of using local strains (ST1 and ST3) to produce bioethanol via immobilization technique with local materials as substrate.
Received: April 18, 2011;
Accepted: August 19, 2011;
Published: August 30, 2011
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;
NGuessan 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; NGuessan 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,
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)2SO4, 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 Yp/s 0.29±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.
||Growth of ST1 and ST3 in YPD medium using free-suspension
||Glucose consumption and ethanol production profiles by ST1
and ST3 in YPD medium using free-suspension system
||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,
Yp/s for free cells fermentation. It is found that ST1 and ST3 produce
similar results in term of μ and Yp/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.
|| 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 (NGuessan 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.
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.
||Production of ethanol and glucose consumption profile of ST1
and ST3 in YPD by immobilized cell system
||Production of ethanol and glucose consumption profile of ST1
and ST3 in LBS using immobilized cell system
||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.
|| Yield, YP/S for YPD and LBS media
||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 YP/S
were 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.
From the results we found that the local isolate, both ST1 and ST3 immobilised-cells have the ability to produce ethanol using both commercial (YP/S; 0.25 and 0.24 g g-1, respectively) and local substrate (YP/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.
The research is funded by University Research Grant (Code: UKM-GUP-BTT-07-25-165) which is duly acknowledged by authors.
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