For practical applications of bioethanol, the uses of both highly concentrated biomass materials and their effective fermentation by yeasts are indispensable in order to produce ethanol at low costs. However, as the saccharified products of those biomass generally contain abundant sugars, the yeasts are affected by the compounds and are inclined to decrease their physiological activities. In the process of fermentation, ethanol is gradually produced by the yeasts in the culture; the concentrated metabolic product also damages itself, and inhibition of the fermentation frequently occurs. The application of yeasts with high fermentative activities under stress pressures such as sugars and ethanol is thus desired for bioethanol production. In this study, various types of high-fermentative yeasts under stress pressures were isolated mainly from coastal waters in Japan and characterized. All yeast strains with high fermentative activities under 20% v/v ethanol were found to be Saccharomyces cerevisiae. The HK21 strain isolated from Tokyo Bay and identified as S. cerevisiae had the highest fermentation activity under 30% w/v sorbitol and under 20% v/v ethanol, and it produced approx. 70 g/l (9% v/v) ethanol from the 15% w/v glucose solution at 25 oC within 5 days.
Today, the United States is the largest bioethanol producer in the world, with corn and wheat as the main materials used to make bioethanol. The starch from corn and wheat is saccharified with both α-amylase and glucoamylase, followed by yeast fermentation; the final ethanol concentration reaches 8% - 10% v/v [
Globally, various types of bioethanol production systems have been developed that use unutilized biomass such as seaweeds, i.e., Undaria pinnafida [
However, as the first-step saccharified products from a biomass generally contain abundant sugars and yeasts are added for fermentation in the second step, the cells are affected by the concentrated compounds and their physiological activities are inclined to decrease. With the progress of fermentation, ethanol is gradually produced by the yeasts in the culture, and the cells are also seriously affected themselves by the concentrated ethanol, and inhibition of the fermentation frequently occurs. The application of yeasts with high fermentative activity under stress pressure such as that provided by sugars and ethanol is thus desired for bioethanol production. In our present study, we isolated various types of high-fermentative yeasts against both sugar and ethanol stresses, gathered mainly from Japan's coasts, and we characterized their fermentation behaviors in detail.
Saccharomyces cerevisiae C-19 from Tokyo Bay with high fermentative activity [
We first assayed the yeast fermentation activity under a no-stress condition. Each yeast strain was precultured at 25˚C for 24 hr in 10 ml of YPD2 liquid medium (D-glucose 20 g/l, peptone 20 g/l, and yeast extract 10 g/l). The growing cells were then centrifuged at 800 g for 5 min, and the cell pellet was gathered. A portion of each pellet was inoculated into a test tube containing 10 ml of YPD2 with a Durham pipe and then statically incubated at 25˚C for 7 days. The yeast fermentative activity in the culture was examined by the naked eye based on the storage of gas evolving from the cells in the Durham pipe.
Next, the yeast fermentation activity under stress pressure was assayed. Yeast cells were precultured as described above, and a portion of each pellet was inoculated into a test tube containing 10 ml of YPD2 plus 20% v/v ethanol or 30% w/v sorbitol with a Durham pipe and then statically incubated at 25˚C for 7 days. The yeast fermentative activity in the culture was also examined by naked eye based on the storage of gas evolving from the cells in the Durham pipe.
The 26S rRNA genes of the fermentative yeasts assayed under stress pressure as described above in Section 2.2. were amplified by polymerase chain reactions (PCRs) using the forward primer NL-1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') and the reverse primer NL-4 (5'-GGTCCGTGTTTCAAGACGG-3'). The D1/D2 domain sequences of the 26S rRNA genes in the yeasts were deposited in DDBJ, EMBI, and GenBank.
Each yeast was precultured at 25˚C for 24 hr in 10 ml of YPD10 liquid medium (D-glucose 100 g/l, peptone 20 g/l, and yeast extract 10 g/l). The growing cells were centrifuged at 800 g for 5 min, and the cell pellet was gathered. A portion of the pellet was cultured at 25˚C for 48 hr in 100 ml of YPD15 liquid medium (D- glucose 150 g/l, peptone 20 g/l, and yeast extract 10 g/l).
The yeast culture was centrifuged at 800 g for 5 min, and the cell pellet was washed with physiological saline and gathered by centrifugation three times. The pellet (0.1 g wet cells) was inoculated into 10 ml of 15% w/v glucose solution in the test tube. Fermentation by the yeast in the tube was anaerobically carried out at 25˚C for 5 days using the anaero Pack System (Mitsubishi Gas Chemical Co., Tokyo). The amounts of ethanol production and glucose consumption in the fermentative suspension were measured by a high-performance liquid chromatography (HPLC) system (Prominence, Shimadzu, Kyoto, Japan). The HPLC conditions were as follows: a refractive index detector (RID-10A, Shimadzu), column (Shim-pack SPR-Pb, particle 8 μm, size 250 × 7.8 mm, Shimadzu); mobile phase: distilled water (Wako Pure Chemical Industries, Osaka, Japan), flow rate: 0.6 ml/min, column temperature: 80˚C.
From the total of 89 strains tested as described above in Section 2.1, the screening results regarding the fermentative yeasts under stress pressures (sugar and ethanol) are summarized in
Second, we selected 24 strains with fermentative activity under 30% w/v sorbitol (
Sampling area | Strain | Fermentation |
---|---|---|
Hakkeijima (Kanagawa) | HK6 | +++ |
HK21 | +++ | |
HK27 | +++ | |
Tokyo University of Marine Science and Technology (Tokyo) | K6-38 | +++ |
Tokyo Bay (Tokyo) | RB59 | + |
RB60 | + | |
Miura Peninsula (Kanagawa) | M39 | ++ |
M46 | + | |
Tokyo Bay (Tokyo) | C-19 | +++ |
Yeast Fermentation in 1 day. +++: strong activity, CO2 was completely pooled in the Durham pipe; ++: moderate activity, CO2 was half pooled in the Durham pipe; +: weak activity, CO2 was pooled a little in the Durham pipe.
Sampling area | Strain | Fermentation |
---|---|---|
Hakkeijima (Kanagawa) | HK6 | + |
HK21 | + | |
HK27 | + | |
Tokyo University of Marine Science and Technology (Tokyo) | K6-38 | + |
Tokyo Bay (Tokyo) | RB59 | + |
Miura Peninsula (Kanagawa) | M5 | + |
M6 | + | |
M7 | + | |
M10 | + | |
M22 | + | |
M39 | + | |
M43 | + | |
M44 | +++ | |
M55 | + | |
M59 | + | |
M63 | ++ | |
M68 | ++ | |
M69 | + | |
M73 | + | |
Shizugawa Bay (Miyagi) | T2 | + |
T5 | + | |
T9 | +++ | |
T13 | + | |
Tokyo Bay (Tokyo) | C-19 | + |
Yeast Fermentation in 1 day. +++: strong activity, CO2 was completely pooled in the Durham pipe; ++: moderate activity, CO2 was half pooled in the Durham pipe; +: weak activity, CO2 was pooled a little in the Durham pipe.
Sampling area | Strain | 20% (v/v) ethanol | 30% (w/v) sorbitol | Accession number |
---|---|---|---|---|
Hakkeijima (Kanagawa) | Saccharomyces cerevisiae HK6 | R | R | LC215954 |
Saccharomyces cerevisiae HK21 | R | R | LC215955 | |
Saccharomyces cerevisiae HK27 | R | R | LC215956 | |
Tokyo University of Marine Science and Technology (Tokyo) | Saccharomyces cerevisiae K6-38 | R | R | LC215947 |
Tokyo Bay (Tokyo) | Candida tropicalis RB59 | R | R | LC215950 |
Candida tropicalis RB60 | R | S | LC215951 | |
Miura Peninsula (Kanagawa) | Kazachstania unispora M39 | R | R | LC215948 |
Candida sake M44 | S | R | LC215952 | |
Kazachstania unispora M46 | R | S | LC215949 | |
Shizugawa Bay (Miyagi) | Metschnikowia bicuspidata T9 | S | R | LC215953 |
Tokyo Bay (Tokyo) | Saccharomyces cerevisiae C-19 | R | R | AB767255 |
R: Fermentative Yeast under Stress Pressure; S: Non-Fermentative Yeast under Stress Pressure.
Kazachstania unispora. The three strains with weak activities were identified as Candida tropicalis (RB59 and RB60) and Kazachstania unispora (M46).
Therefore, all yeast strains with high fermentative activity under 20% v/v ethanol were found to be S. cerevisiae. Under 30% w/v sorbitol, two strains with strong activity, i.e., M44 and T9, were Candida sake and Metschnikowia bicuspidata, respectively. Therefore, all seven of the following strains had fermentative activity under both 20% v/v ethanol and 30% w/v sorbitol: HK6, HK21, HK27, K6-38, and C-19 identified as S. cerevisiae, M39 identified as M. bicuspidata, and RB59 identified as C. tropicalis. We measured the amounts of ethanol production and glucose consumption of these seven strains in 15% w/v glucose solution, as described next in Section 3.3.
In general, bread has been made with baker’s yeast since approx. 2000 BC, and beer has been made using brewer’s yeast since approx. 1500 BC in Mesopotamia. The Japanese alcoholic beverage sake has also been made using sake yeast in Japan since ancient times. Suitable yeasts for various industries have been repeatedly isolated and bred separately according to the types of fermentation for many decades.
However, all industrial yeasts must have the same characteristics of high fermentative activity and high alcohol-tolerance activity. Identification methods for yeasts were developed after the 18th century, and most of the yeasts used in the fermentation industries were found to be Saccharomyces cerevisiae species. In modern times, bioethanol is usually produced by fermentation with S. cerevisiae. From ancient to modern times, S. cerevisiae has been the most important yeast species in the history of humans. However, in the bioethanol industry, various types of biomass materials are used and the screening of novel yeast strains with high fermentative activity under stress pressures is needed, again.
There also seems to be a variety of strains with different characters (even among the S. cerevisiae strains) that are dependent on their living environments. Although the S. cerevisiae species are generally isolated in fermentation factories or from natural terrestrial origins, we isolated highly fermentative yeasts from various marine origins and identified most of them as S. cerevisiae [
From among the 89 strains tested, we isolated and characterized a variety of yeast strains that were highly fermentative under stress pressure (sugar or ethanol), collected mainly from coastal waters in Japan. The seven strains―HK6, HK21, HK27, K6-38, and C-19 identified as S. cerevisiae, M39 identified as M. bicuspidata, and RB59 identified as C. tropicalis―had fermentative activity under both 20% v/v ethanol and 30% w/v sorbitol. Most notably, strain HK21 (isolated from Tokyo Bay and identified as S. cerevisiae) had high fermentation activity under 30% w/v sorbitol and under 20% v/v ethanol, and it produced approx. 70 g/l (9% v/v) ethanol from the 15% w/v glucose solution at 25˚C within 5 days. The fermentation activity shown by S. cerevisiae strains from marine origins was observed to be very high under stress pressure.
Urano, N., Shirao, A., Okai, M. and Ishida, M. (2017) High Ethanol Production by Marine-Derived Yeasts-Saccharomyces cerevisiae under Stress Pressures. Advances in Microbiology, 7, 349-357. https://doi.org/10.4236/aim.2017.75029