Ethanol production by fermentation process has been studied extensively for
several decades. Over the last few years, new approach with great potential
for ethanol production from starchy materials and lignocellulosic biomass have
been used, that is Simultaneous Saccharification Fermentation (SSF) process
(Wingren et al., 2003) and Separate Hydrolysis
and Fermentation (SHF) process (Montesinos and Navarro,
2000). Furthermore, it was found that SSF has been shown to be more efficient
than SHF in terms of overall ethanol yield (Soderstrom et
SSF process is the double step fermentation by the saccharification of starchy
or cellulosic materials and conversion to fermentation product such as ethanol
and lactic acid (John et al., 2009). Generally
the hydrolysis step, the substrates were treated with enzymes (Tomás-Pejó
et al., 2009; Nikolic et al., 2009;
Srichuwong et al., 2009) acids (Li
et al., 2009) or include stream explosion (Chen
et al., 2008). Hydroxymethylfurfural (HMF), is a harmful product
from D-glucose during stream explosion or acid hydrolysis of starch. Furthermore,
various inhibitors such as furfural, levulinic acid, formic acid and acetic
acid are also formed during steam pretreatment of wood chips (Sassner
et al., 2006). These inhibitory substances including 0.1% HMF have
an effect on growth and alcohol production by yeast.
Due to lack of starch-decomposinsdg enzyme, many microorganisms including Saccharomyces
cerevisiae is not able to produce ethanol from starch. It is necessary to
add specific enzyme such as amylase, isomerase, amyloglucoamylase, and pulluanase
for hydrolyzing starch slurry (Montesinos and Navarro, 2000;
Bandaru et al., 2006; Jamai
et al., 2007; Ebrahimi et al., 2008).
For enzymatic hydrolysis the starch kernels, the slurry should be brought to
high temperature at 90-100°C.
In order to reduce the cost and avoid high energy consuming, the possible of hydrolyzing starch at low temperatures is being investigated. Therefore, the basic knowledge in traditional style for making beverage from tan-koji was applied to use in this study. Tan-koji (loog-pang), known as dried solid starter, is widely used to produce alcoholic beverage in Asia for long time ago. It is a mixed culture of alcohol fermented yeast and starch hydrolysis fungi. Starch materials especially rice grains are to be used for Thai traditional beverage. The effective fungi can convert starch to sugar by enzymatic hydrolysis. Then the fermentable sugars are used and converted to ethanol by yeast.
The purpose of this study was to isolate, select and couple use starch hydrolysis fungi from tan-koji and S. cereviseae 5088 for alcohol production from cooked cassava starch slurry in SSF process.
MATERIALS AND METHODS
Organisms: Ten strains of Rhizopus sp. that were isolated from loog-pang at various sources (Table 1) were screened for starch saccharification. They were maintained on Potato Dextrose Agar (PDA) slants which were incubated at 30°C for 7 days and then stored at 4°C. Saccharomycetes cereviceae 5088 (TISTR, Thailand) was used as the ethanol fermenting organism. It is stored on malt extract-glucose-yeast extract-peptone (MGYP) growth medium (malt extract, 3 g L-1: glucose; 10 g L-1: yeast extract; 3 g L-1: peptone; 5 g L-1). Agar (1.5%) was used for preparing agar slants.
Enzyme preparation: Enzymes from 10 strains of Rhizopus sp. that were prepared in wheat soy bean broth (WSB: 3 g; wheat bran, 2 g; soybean, 2 g; distilled water, 100 mL) were collected from 5 days culture medium. Filtrated supernatant solution was stored at -20°C before used. Twenty microlitres of enzyme were dropped in small hold on starch agar plate. Clear zone were detected by adding iodine solution (iodine crystal, 10 g; KI, 20 g; distilled water, 3 L) and compared with 1.15 U μL-1 of amyloglucosidase.
Enzymatic assayed: Enzymatic activities of 10 Rhizopus sp. strains were tested on 0.1% starch agar plate at pH ranging from 3 to 10. Starch substrate plate were prepared as following: solution A consisted of 0.1 g starch, 1.5 g agar, 50 mL distilled water. Solution B, Britten-Robinson universal buffer (2x concentration contained 85% H3PO4 9.16 mL, glacial acetic acid 9.16 mL, H3PO3 9.9 g) adjusted the pH to 3, 4, 6, 8 and 10 with 4 N NaOH and adjusted volume to 1 L) Equal volume of solution A and B were mixed and autoclaved at 121°C for 15 min.
fungi from various sources of tan-koji
Starch hydrolysis: Reducing sugar liberated by Rhizopus sp. was
determined in cassava starch (1-6 g cassava starch, 0.5 g yeast extract, 100
mL distilled water pH 4.5) on 200 rpm rotary shaking at 30°C.
SSF process: Starch hydrolysis fungi was used in the saccharification process of 6% cassava starch medium. After 1, 2 and 3 days of saccharification step followed by fermentation using 10% by volume of S. cerevisiae of YM medium (3 g L-1 yeast extract, 3 g L-1 malt extract, 5 g L-1 peptone, 10 g L-1 glucose). The amount of alcohol formed and reducing sugar remaining in the medium were determined as in analytical methods.
Analytical methods: The reducing sugars were measured by the dinitrosalicylic
acid (DNS) method according to Miller (1959). Ethanol
was analyzed by gas chromatography using 100 cm. stainless steel column packed
with Porapack Q, 80-100 mesh. The injector and detector temperature were 200°C
and the column temperature was 180°C. A Shimadzu 14A gas chromatograph equipped
with flame ionization detector was used with nitrogen as a carrier gas. Isopropanol
was used as an internal standard.
Enzymatic hydrolysis of starch: Enzymatic activities of 10 strains of Rhizopus sp. that were isolated from tan-koji at various sources was tested on 0.1% starch agar plate. Table 2 showed average clear zone from 3 replicated at pH ranging from 3 to 10 comparing with amyloglucosidase (1.15 U μL-1). Clear zones were occurred between pH 3-8 and no activity at pH 10. At pH 4 showed the highest activity in all tested including activity of amyloglucosidase. Rhizopus sp. #2Bu and strain #3Su have closely highest clear zone of 0.48±0.076 and 0.5±0.057 mm, respectively. Beside enzymatic activity can be performed by clear zone, this technique also showed the pH optimum of enzymatic activity. This is the advantage of this technique. However, reducing sugar liberated from starch could not detected from starch agar plate.
Sugar liberated from starch hydrolysis: Saccharification process was carried out at various concentration of cooked cassava starch slurry by 2 fungal strains of Rhizopus sp. #2Bu and Rhizopus sp. #3Su. The sugar that was produced at various time courses during starch hydrolysis by fungal enzyme was used for the growth and the rest was detected as reducing sugar liberated (Fig. 1).
clear zone forming by enzymatic activity from 10 strains Rhizopus
sp. that isolated from various source of tan-koji or loog-pang comparing
with commercial amyloglucosidase
cost of reducing sugar liberated by Rhizopus sp. (a) #3Su (a) and
(b) #2Bu from various concentration of cassava starch
It was found that at the higher starch concentration, the higher reducing sugar liberated were gained. Rhizopus sp. #3Su (Fig. 1a) liberated more sugar than Rhizopus sp. #2Bu (Fig. 1b) at various starch concentration. However, the highest sugar concentration was achieved from 6% starch concentration at day 3 about 15.55 g L-1 (Fig. 1a).
SSF process: In order to bring a sufficient amount of sugar liberated
before to start the fermentation, a saccharification for 1, 2, and 3 days were
carried out on 6% cassava starch medium by Aspergilus sp. #3Su as 1°
production from SSF process between Rhizopus sp. #3Su and S.
After saccharification periods (1, 2, 3 days), the fermentation process simultaneous
begun by adding 2° inoculum of S. cerevisiae 5088 (Fig.
2). This study process was applied from the procedure of traditional alcoholic
beverage that made from loog-pang. The results showed that the highest sugar
residue was obtained from 3 day of saccharification and then it was sharp decreased
until equal to 1 and 2 days of saccharification in fermentation process at day
4. However, ethanol production from this experiment was lowest about 11.8 g
L-1 at day 5 and 6. While ethanol production from 1 and 2 days of
saccharification were nearly high around 14.36 g L-1 at day 3 and
day 5, respectively. This result showed that 1 day of saccharification was the
most efficient of SSF process.
In order to select the most efficient fungal strain to hydrolyze starch and provide adequate sugar release in fermentation broth, 2 steps screening were examined. The first step, the enzymatic activities on starch agar plate at pH ranging from 3 to 10 were tested. It was found that the best reaction was at pH 4 and no response at pH 10. Due to closely high activities, the strain of Rhizopus sp. #2Bu and Rhizopus sp. #3Su were both selected for the next tested. Sugar liberated at various starch concentration were studied by these 2 strains. Although, sugar liberated from the 2 fungal strains were nearly equal. However, more sugar residue at 6% starch slurry were found from Rhizopus sp. #3Su. Consequently, SSF processes was conducted between co-culture of Rhizopus sp. #3Su and S. cereviseae 5088. The results showed the highest ethanol yield from the fermentation process which it was begun after 24 h of saccharification process.
Verma et al. (2000) developed their single-step
process for ethanol production from soluble starch using co-culture of amylolytic
yeast (Saccharomyces diastaticus) and Saccharomyces cerevisiae
21. The maximum ethanol of 24.8 g L-1 was produced from 60 g L-1
of soluble starch. With increasing starch concentration in the fermentation
medium the ethanol yield was reduced. Low rate amylolytic yeast (Candida
tropicalis) was used for ethanol production from corn soluble starch in
the presence of α-amylase (Jamai et al., 2007).
They found that more ethanol concentration was produced in corn soluble starch
medium in the presence of α-amylase than fermentation alone with Candida
tropicalis YMEC14. The maximum ethanol yield of 43.1 g L-1 was
gained from 9% soluble starch concentration. While at 6% starch concentration,
ethanol concentration was to 24 g L-1. Furthermore, the highest ethanol
yield of 56 g L-1 was obtained from fed-batch fermentation mode.
SSF process using co-culture between starch decomposing microbe and ethanol producing microbe is an alternative route for ethanol production from starch. Because this procedure leads to save for energy and low cost in fuel ethanol production from renewable feedstock. Ethanol production from an efficient microbes will be an important role in the new era.
The author thanks Dr. Samappito, S. for the fungi as his gift. This study was supported from Mahasarakham University.