Influence of Initial pH on ethanol production by the antarctic basidiomycetous yeast mrakia blollopis

Article abstract of DOI:10.1271/bbb.130497

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 fermented ethanol between pH 5.0 and pH 10.0 with optimum pH at 8.0-10.0. Knowledge of ethanol fermentability as to the genus Mrakia remains incomplete. Further experiments are required to elucidate the ethanol fermentability of genus e.g., as to optimum fermentation pH, optimum fermentation temperature, and cell viability during fermentation.

Full text of DOI:10.1271/bbb.130497

Biosci. Biotechnol. Biochem., 77 (12), 2483–2485, 2013
Note
Influence of Initial pH on Ethanol Production
by the Antarctic Basidiomycetous Yeast Mrakia blollopis
y
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1;5;
Masaharu TSUJI,1; Shiv M SINGH,2 Yuji YOKOTA,3; Sakae KUDOH,4 and Tamotsu HOSHINO
1Biomass Refinery Research Center (BRRC), National Institute of Advanced Industrial Science and Technology (AIST),
3-11-32 Kagamiyama, Higashi-hiroshima, Hirosima 739-0046, Japan
2National Centre for Antarctic and Ocean Research, Ministry of Earth Sciences, Vasco-Da-Gama, Goa 403804, India
3Bio-Production Research Institute, National Institute of Advanced Industrial Science and Technology (AIST),
2-17-2-1 Tsukisamu-higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan
4National Institute of Polar Research (NIPR), 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan
5Graduate School of Life Science, Hokkaido University, N10W8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan
Received June 18, 2013; Accepted August 29, 2013; Online Publication, December 7, 2013
[doi:10.1271/bbb.130497]
The Antarctic basidiomycetous yeast Mrakia blollopis
SK-4 fermented ethanol between pH 5.0 and pH 10.0
with optimum pH at 8.0–10.0. Knowledge of ethanol
fermentability as to the genus Mrakia remains incom-
plete. Further experiments are required to elucidate the
ethanol fermentability of genus e.g., as to optimum
fermentation pH, optimum fermentation temperature,
and cell viability during fermentation.
20 g/L of peptone, and 20 g/L of glucose) at 120 rpm for
120 h at 10 ꢀC. After 120 h, 400 mL of culture was
collected by centrifugation at 3;500 ꢁ g for 10 min at
4 ꢀC. The pellet was dissolved in 50 mL of distilled
water, and the resulting culture (OD600 ¼ 170) was
used as an inoculum. OD600 ¼ 1 of the Mrakia blollopis
SK-4 cells, cultured at 10 ꢀC after 120 h was
1:0 ꢁ 107 CFU/mL.
Experiments were performed in 28-mL glass vials.
The fermentation mixture consisted of 40 g/L of
glucose, 5 g/L of yeast extract, 5 g/L of Bacto peptone,
2 g/L of NH4Cl, 1 g/L of KH2PO4, and 0.3 g/L of
Key words: Mrakia; cryophilic yeast; ethanol fermenta-
tion; East Antarctica
.
Cold environments cover much of Earth including the
deep sea, and most biospheres are permanently exposed
to temperatures below 5 ꢀC.1) Microbes adapted to such
cold environments can grow at temperatures below 0 ꢀC
and hence, they can be utilized in de novo biopro-
cesses.2) Cryophilic yeasts,3) Mrakia spp. and Mrakiella
spp., have been found in the Arctic, Siberia, Alaska,
CentralRussia, the Alps, the Apennines, Patagonia, and
Antarctica.4) Di Menna5) reported that the genus Mrakia
accounts for about 24% of culturable yeast in Antarctic
soil. Moreover, we have reported that about 35% of
culturable fungi isolated from lake sediment and soil of
East Antarctica were Mrakia spp.6) These reports
suggest that Mrakia spp. are the dominant culturable
yeasts in Antarctica and the most adapted to the
Antarctic environment.
Mrakia bollopis SK-4, isolated from Naga-ike Lake
in Skarvsnes, East Antarctica, was found to ferment for
typical sugars such as glucose, sucrose, maltose, lactose,
raffinose, and galactose at low temperatures,7) but, little
is known about ethanol production by basidiomycetous
yeasts. Moreover, optimal pH and cell viability for ethanol
fermentation have not yet been studied for basidiomy-
cetous yeasts. Here we report the effects of pH on the
ethanol fermentation and cell viability of the cryophilic
basidiomycetous yeasts Mrakia blollopis SK-4.
MgSO4 7H2O in 50 mM of various buffers as follows:
sodium citrate (pH 2.0–5.0), sodium phosphate (pH 6.0–
8.0), Tris–HCl (pH 8.5), and sodium carbonate (pH 9.0–
11.0). A final concentration of 8:5 ꢁ 107 CFU/mL SK-4
was added to the sterilized fermentation mixture. Ten
mL of each mixture was fermented at 120 rpm at 10 ꢀC.
Six hundred mL of each sample was collected every 24 h,
and the supernatants were used for measurement of
glucose and ethanol concentrations. The collected
samples were diluted in distilled water up to dilutions
of 104–106 and then inoculated on YPD agar plates
(Difcoꢀ, BD Japan, Tokyo), and incubated at 10 ꢀC
for 5 d. Then the colonies that appeared after 5 d were
counted.
The glucose and ethanol concentrations in the
fermentation solutions were measured by high-perform-
ance liquid chromatography (HPLC). All samples were
analyzed by HPLC using an Aminex HP87 cation
exchange column with both UV and RI detection at
0.6 mL/min at 65 ꢀC or 80 ꢀC.8) All experiments were
carried out independently in three vials, and average
results are given.
The results of ethanol fermentation at pH 2.0–11.0 are
shown in Fig. 1. M. blollopis SK-4 fermented between
pH 2.0 and pH 11.0. For maximum ethanol productivity
using SK-4, the optimum pH was 8.0–10.0. At pH 2.0–
4.0 and 10.5–11.0, SK-4 did not completely convert
glucose to ethanol until 168 h of fermentation. When the
Ten mL of M. blollopis SK-4 was inoculated in
400 mL of YPD liquid medium (10 g/L of yeast extract,
y
To whom correspondence should be addressed. Yuji YOKOTA, Tel: +81-11-857-8930; Fax: +81-11-857-8980; E-mail: yu.yokota@aist.go.jp;
Tamotsu HOSHINO, Biomass Refinery Research Center National Institute of Advanced Industrial Science and Technology (AIST), 3-11-32
Kagamiyama, Higashi-hiroshima, Hiroshima 739-0046, Japan; Tel: +81-11-857-8475; Fax: +81-82-420-8291; E-mail: tamotsu.hoshino@
aist.go.jp; Masaharu TSUJI, Tel: +81-11-857-8475; Fax: +81-82-420-8291; masaharu-tsuji@aist.go.jp

2484
M. TSUJI et al.
A
B
E
F
I
J
C
D
G
H
K
L
Fig. 1. Effect of Initial pH on Ethanol Production
A, pH 2.0; B, pH 3.0; C, pH 4.0; D, pH 5.0; E, pH 6.0; F, pH 7.0; G, pH 8.0; H, pH 8.5; I., pH 9.0; J, pH 10.0; K, pH 10.5; L, pH 11.0. All
rounds of fermentation were performed at 10 ꢀC. Triangles indicate viable cell counts, circles denote ethanol concentrations, and diamonds
indicate glucose concentrations. Vertical axes denote glucose and ethanol concentrations (g/L) and second vertical axes indicate viable cell
counts (CFU/mL). Horizontal axes indicate time (h). All experiments were carried out in triplicate and error bars indicate standard deviation.
Table 1. Summary of Ethanol Fermentation at Various pH Values by
Mrakia blollopis
yeast was fermented at pH 6.0–7.0, the glucose con-
sumption speed was slower than at pH 5.0 and glucose
was completely consumed at 168 h. Moreover, when we
Initial glucose
concentration
(g/L)
EtOHM
(g/L)
YE/G
(g/g)
YE/EY
(%)
QE
(g/Lꢂh)
performed a fermentation test, 3 times at pH 5.0,
glucose was completely consumed by 168 h of fermen-
tation. At pH 8.0–10.0, the ethanol fermentation rate
was fastest. The details of maximum ethanol concen-
tration (EtOHM), ethanol yield based on total glucose
content in the ethanol fermentation mixture (YE/G),
theoretical ethanol yield (YE/EY) and ethanol production
rate at 24 h of ethanol fermentation (QE) are summarized
in Table 1. The theoretical yield of ethanol production
was calculated as follows:
pH 2.0
pH 3.0
pH 4.0
pH 5.0
pH 6.0
pH 7.0
pH 8.0
pH 8.5
pH 9.0
pH 10.0
pH 10.5
pH 11.0
44.9
43.0
45.2
44.0
44.2
43.8
44.5
45.9
46.4
45.3
43.2
43.2
12.8
15.7
17.2
19.1
18.2
17.9
19.3
20.4
19.5
19.2
16.6
15.9
0.29
0.36
0.38
0.43
0.41
0.41
0.43
0.44
0.42
0.42
0.38
0.38
55.9
71.5
74.5
85.3
80.9
80.2
85.1
87.0
82.4
83.0
75.3
72.1
0.15
0.17
0.15
0.19
0.16
0.18
0.25
0.20
0.23
0.23
0.14
0.13
%Theoretical yield [YE/EY (%)]
¼ EtOHM/(Initial glucose concentration ꢁ 0.51).
We also measured the pH of during fermentation. At
pH 2.0–4.0, the pH of the mixtures did not change after
168 h of fermentation. However, in the cases of pH 5.0–
7.0, 8.0–9.0 and 10.0–11.0, the pHs of fermentation
mixtures were 7.0, 7.5 and 8.0–8.5 respectively after
24 h of fermentation, and these pH remained steady after
168 h of fermentation (data not shown). The colony
EtOHM, maximum ethanol concentration after 168 h of ethanol fermenta-
tion. YE/G, ethanol yield based on total glucose content in the ethanol
fermentation mixture. YE/EY, The theoretical yield of ethanol is 0.51
g ethanol/g glucose. QE, volumetric ethanol production rate after 24 h of
ethanol fermentation.

Influence of pH on Ethanol Production by Mrakia blollopis
2485
counts of SK-4 dramatically decreased during first 24 h
under all fermentation conditions, and then they grad-
ually decreased, through the fermentation time. At
pH 5.0–10.0, the cell viability of the yeast was higher
than that at other pH values, but we do not know why
SK-4 cell viability dramatically decreased in 24 h of
fermentation.
SK-4 was isolated from an algal mat in lake sediment
of Naga-ike, a lake in the Skarvsnes ice-free area of East
Antarctica. Strain SK-4 secretes extracellular enzymes
such as cellulase, ꢀ-glucosidase, catalase, and amylase
as well as lipase under low temperature conditions (data
not shown). SK-4 has stable lipase against metal ions
and organic solvents, and the optimum pH of SK-4
lipase is 8.5–9.0.7) Naga-ike is an oligotrophic lake, and
pH is 8.5 (Tanabe, Ph. D. thesis, Graduate University for
Advanced Studies, 2009). Knowledge as to ethanol
fermentability of the genus Mrakia remains incomplete.
Further experiments are required to elucidate the ethanol
fermentability of this genus Mrakia, e.g., optimum
fermentation pH, optimum fermentation temperature,
and cell viability during fermentation. This is the first
report on the influence of initial pH on ethanol
fermentation by cryophilic basidiomycetous yeast at
low temperature.
To the best of our knowledge, little is known about
the fermentation by basidiomycetous yeast. Some
species have been reported to have fermentative ability,
such as Mrakiella spp.,9) Rhodotorula spp.,10) Xantho-
phyllomyces spp.,11) and Bandoniozyma spp.12) Seven
species of Mrakia have been reported: Mrakia frigida,
Mrakia gelida, Mrakia stokesii, Mrakia nivalis, Mrakia
psychrophila, Mrakia robertii, and Mrakia blollopis.4)
Species in this basidiomycetous yeast genus are known
for their ability to ferment sugars. Actually, all species
could ferment glucose and sucrose. M. frigida, M. blol-
lopis, M. gelida, and M. robertii were used for fermen-
tation tests with a home brewing kit. Thomass-Holl et
al.4) reported that all of those strains fermented sucrose,
but did not completely convert sucrose to ethanol,
and that cell growth was stopped in the presence of
over 2% (v/v) ethanol. Strain SK-4 fermented
raffinose, galactose, lactose, and maltose at low temper-
ature, while CBS8921T could not ferment raffinose,
galactose, lactose, or maltose.7) Moreover, it fermented,
at ꢃ1–20 ꢀC and the optimum ethanol fermentation
temperature was 10–15 ꢀC (data not shown). Maximally,
48.7 g/L of ethanol was produced from 120 g/L of
glucose by SK-4 at 10 ꢀC at 19 d fermentation.13)
We had little information about SK-4 fermentability,
and hence we tested ethanol production by SK-4 at
various pH values. When it was used for fermentation at
Acknowledgment
This work is a part of the Science Program of JARE-
48. It was supported by NIPR under MEXT.
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