Supplementary Material
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Title:
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Butyric acid production from red algae by a newly isolated Clostridium sp. S1
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Kyung Min Lee1, 2, Okkyoung Choi1, Ki-Yeon Kim1, Han Min Woo1,4, Yunje Kim1, Sung Ok
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Han2, Byoung-In Sang3 and Youngsoon Um1, 4*
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Republic of Korea
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School of Life Science and Biotechnology, Korea University, Seoul, Republic of Korea
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Department of Chemical Engineering, Hanyang University, Seoul, Republic of Korea
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Daejeon, Republic of Korea
Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Seoul,
Clean energy and Chemical engineering, Korea University of Science and Technology,
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Corresponding Author:
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*Dr. Youngsoon Um, Tel.: +82. 2. 958. 5819, Fax: +82. 2. 958. 6858, Email: yum@kist.re.kr
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Supplementary Table 1. Comparison of cell growth and butyric acid production of C.
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tyrobutyricum strains in MP2Gal after 48 h. One experiment was done.
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Cell growth
(OD at 600 nm)
Galactose
consumption (g/l)
C. tyrobutyricum ATCC 25755
1.34
N.D.
a
0.27
C. tyrobutyricum NCIMB 9582
0.39
N.D.
0.30
C. tyrobutyricum NCIMB 9583
1.21
N.D.
0.13
C. tyrobutyricum NCIMB 9584
1.05
N.D.
0.42
C. tyrobutyricum NCIMB 12573
1.11
N.D.
0.26
C. tyrobutyricum NCIMB 701715
0.89
N.D.
0.28
C. tyrobutyricum NCIMB 701753
0.67
N.D.
0.41
C. tyrobutyricum NCIMB 701755
1.12
N.D.
0.55
C. tyrobutyricum NCIMB 701756
0.87
N.D.
0.14
C. tyrobutyricum NCIMB 701757
1.17
N.D.
0.46
C. tyrobutyricum NCIMB 701790
1.51
N.D.
0.59
C. tyrobutyricum DSM 664
0.69
N.D.
0.56
C. tyrobutyricum DSM 1460
0.89
N.D.
0.46
Clostridium sp. S1
2.93
13.1
7.01
Strain
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a
N.D.: not determined due to little or no galactose consumption.
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Butyric acid
production (g/l)
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Supplementary Table 2. Composition of G. amansii hydrolysate. One experiment was done
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under each condition.
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H2SO4 (% v/v)
Galactose (g/l)
Glucose (g/l)
HMF (g/l)
Levulinic acid (g/l)
0.5
7.95
0.50
2.15
0.07
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11.32
1.22
2.69
0.41
1.5
10.98
1.15
2.49
0.73
2
11.21
1.29
2.30
1.11
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(a)
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(b)
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Supplementary Figure 1. GC-MS spectrums of butyric acid after 48 hours cultivation of
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Clostridium sp. S1 on MP2Gal supplemented with (a) 12C2-acetate (12CH3-12COOH) and (b)
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Aldrich. GC-TOF MS system (GC: Model 6890, Agilent Technologies, USA; TOF-MS: Model
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Pegasus III, USA) was used to confirm
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comparison to the mass spectral library in the National Institute of Standards and Technology
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(NIST). Acetic and butyric acids in fermented broth were extracted with ethyl acetate by the
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liquid/liquid extraction method, and the prepared sample was injected into the GC using a J
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and W Scientific DB-5 column (J and W Scientific, USA). Helium carrier gas was used at a
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constant flow rate of 1.2 mL/min. In the MS analysis, impact with ionization mass spectra was
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recorded at 70 eV electron energy in scan mode. The ionization source temperature was set at
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230 °C.
C2-acetate (13CH3-13COOH).
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C2-acetate (12CH3-12COOH) was purchased from Sigma-
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C-labeled butyric acid from
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C-acetate by
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Clostridium aciditolerans JW/YJL-B3T(DQ114945)
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Clostridium nitrophenolicum 1DT(AM261414)
Clostridium magnum DSM 2767T(X77835)
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Clostridium drakei SL1T(Y18813)
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Clostridium acidisoli CK74T(AJ237756)
Clostridium akagii CK58T(AJ237755)
Clostridium pasteurianum ATCC 6013T(M23930)
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Clostridium arbusti SL206T(EU816420)
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Clostridium sp. S1 (JN241679)
Clostridium tyrobutyricum ATCC 25755T(M59113)
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Clostridium ljungdahlii DSM 13528T(CP001666)
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Clostridium kluyveri DSM 555T(CP000673)
Bacillus subtilis subsp. subtilis NCIB 3610T(ABQL01000001)
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0.02
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Supplementary Figure 2. Neighbour-joining tree showing the phylogenetic positions of Clostridium S1 and other related species. Bootstrap
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values (%) are indicated at branch points based on neighbour-joining analyses of 1000 resampled datasets.
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DCW
Butyric acid
DCW and Butyric acid (g/l)
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1
0
0
0.5
1
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Levulinic acid (g/l)
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Supplementary Figure 3. Effect of levulinic acid on cell growth and butyric acid production
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by Clostridium sp. S1 in MP2Gal medium. Error bars represent 1 standard deviation of
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triplicate experiments.
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