1
Supplementary data
2
3
Bacteria isolation and identification
4
5
Soil samples were collected from the rice fields in Jeollanam-Do, Korea. The dried samples
6
were ground to powder, and passed through 150 μm sieves. One gram of prepared soil was
7
diluted in 50 ml 0.9% NaCl solution, shaken at 150 rpm and 37oC for 1 h, and 1 ml of the
8
suspension was spread on LB agar. The plates were incubated at 37oC for 24 h. Slimy
9
bacterial colonies recovered from LB agar were inoculated onto a glutamic acid-containing
10
medium comprised of (g/l): glutamic acid 10, citric acid 12, glycerol 80, NH4Cl 7, K2HPO4
11
0.5, MgSO4·7H2O 0.5, MnSO4 0.1, CaCl2 0.15, and FeCl3 0.04, which was slightly modified
12
from that of Yoon et al. (2000). The isolates were subcultured on the glutamic acid medium at
13
37oC for 96 h. For initial identification of bacteria, morphological characteristics of colony
14
morphology, spore, and culture medium color were observed. These characteristics were
15
compared with identification keys from Bergey’s Manual. The pure isolates were stored at
16
4oC and were subcultured every 2 months or stored at -80oC for long term preservation.
17
Biochemical profiles of the bacteria were identified using an API 50CHB test kit
18
(BioMérieux, Marcy l'Etoile, France), following the manufacturer’s instruction. Results were
19
interpreted using the Analytical Profile Index (API) database of the API web software version
20
4.0. The isolated bacteria were also identified by phylogenetic analysis (Mori et al. 1997).
21
The 16S rDNA sequence was amplified using PCR with the universal primers, forward 8F:
22
5'-AGA GTT TGA TCC TGG CTC AG-3' and reverse primer 1492R: 5'-ACG GCT ACC
23
TTG TTA CGA GTT-3'. The PCR reaction was performed with premix (Bioneer, Daejeon,
24
Korea) in a gradient thermo block (Bioneer) according to the manufacturer’s instruction. PCR
25
product was purified using a PCR product purification kit (Bioneer) and was cloned into
26
pGEM-T vector (Promega, Madison, WI, USA). The nucleotide sequence of 16S rDNA was
27
determined using a DNA automated sequencer (PE Applied Biosystems, Foster City, CA,
28
USA) and was aligned with reference sequences obtained from the GenBank databases
29
(NCBI, Bethesda, MO, USA). Phylogenetic analysis was carried out using the BioEdit
30
sequence alignment editor program and GBlocks program, as well as the MEGA 4.0 program
31
using the neighbor-joining method.
32
33
References
34
Mori K, Yamazaki K, Ishiyama T, Katsumata M, Kobayashi K, Kawai Y, Inoue N, Shinano
35
H (1997) Comparative sequence analyses of the genes coding for 16S rRNA of
36
Lactobacillus casei-related taxa. Int J Syst Evol Microbiol 47: 54-57
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Yoon SH, Do JH, Lee SY, Chang HN (2000) Production of poly-γ-glutamic acid by fedbatch culture of Bacillus licheniformis. Biotechnol Lett 22: 585-588
51
Supplementary Table 1. Biochemical characterizations of MJ80 by API 50CHB test
52
Characteristics
Morphology shape
Cell dimension (μm)
Spore formation
Gram stain
Catalase
Oxidase
Glycerol
Erythritol
D-Arabinose
L-Arabinose
D-Ribose
D-Xylose
L-Xylose
D-Adonitol
β-Methyl-D-xylopyranoside
D-Galactose
D-Glucose
D-Fructose
D-Mannose
L-Sorbose
L-Rhamnose
Dulcitol
Inositol
D-Mannitol
D-Sorbitol
Methyl-α-D-mannopyranoside
Methyl-α-D-glucopyranoside
N-Acetyl-glucosamine
53
54
55
56
57
58
59
60
61
62
63
64
65
Result
Rod
0.5~0.8 x 2~5
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Characteristics
Result
Amygdalin
Arbutin
-
Esculin ferric citrate
Salicin
D-Cellobiose
D-Maltose
D-Lactose
D-Melibiose
Sucrose
D-Trehalose
Inulin
D-Melezitose
D-Raffinose
Starch
Glycogen
Xylitol
Gentiobiose
D-Turanose
D-Lyxose
D-Tagatose
D-Fucose
L-Fucose
D-Arabitol
L-Arabitol
Potassium Gluconate
Potassium 2-ketogluconate
Potassium 5-ketogluconate
+
+
+
+
+
+
+
+
+
+
+
-
1
2
3
Supplementary Table 2. γ-PGA producers and their optimal culture conditions
Strain
Important components in media
Culture
conditions
Productivity
References
(g/ l)
Glutamic acid-dependent γ-PGA producers
B. licheniformis ATCC9945A
Glutamic acid (20 g/l), glycerol (80 g/l),
citric acid (12 g/l), NH4Cl (7 g/l)
37℃, 2 days
23
Cromwick et al. 1996
B. licheniformis ATCC9945A
Glutamic acid (20 g/l), NH4Cl (7 g/l),
citric acid (12 g/l), CaCl2 (0.2 g/l),
MnSO4 ·7H2O (0.3 g/l)
37℃, 2-3days
35
Yoon et al. 2000
B. lichenifomis CCRC12826
Glutamic acid (20 g/l), glycerol (80 g/l),
citric acid (12 g/l), NH4Cl (7 g/l)
37℃, 3 days
19.8
Shih et al. 2002
B. licheniformis WBL-3
Glutamic acid (10 g/l), glycerol,
citric acid (10 g/l), NH4Cl (1 g/l)
37℃, 4 days
19.3
Du et al. 2005
B. licheniformis NCIM2324
Glycerol, citric acid, (NH4)2SO4,
glutamic acid, glutamine, α-ketoglutaric
acid
Glutamic acid (70 g/l), glucose (1 g/l),
30℃, 2-3 days
veal infusion broth (20 g/l)
35.75
Bajaj and Singhal 2009b
50
Kubota et al. 1993
B. subtilis IFO3335
Glutamic acid (30 g/l), (NH4)2SO4 (30 g/l), 37℃, 2 days
citric acid (20 g l-1)
10-20
Kunioka and Goto 1994
B. subtilis(natto) MR-141
Glutamic acid (30 g/l), maltose (60 g/l),
soy sauce (70 g/l)
35
Ogawa et al. 1997
B. subtilis (chungkookjang)
Glutamic acid (20 g l-1), sucrose (50 g l- 30℃, 5 days
13.5 – 16.5
Ashiuchi et al. 2001
B. subtilis F-2-01
40℃, 3-4 days
1),
NaCl (0.5-5.0 g l-1)
Glutamic acid (20 g/l), glucose (20 g/l),
yeast extract (5 g/l),
37℃, 1 day
30.2
Xu et al. 2005
B. subtilis ZJU-7
Sucrose (60 g/l), tryptone (60 g/l),
glutamic acid (80 g/l), NaCl (10 g/l)
37℃, 1 day
54.4
Shi et al. 2006
B. subtilis CGMCC0833
Glutamic acid (40 g/l), (NH4)2SO4 (8 g/l), 32.5℃, 2 days
DMSO, Tween-80, glycerol
34.4
Wu et al. 2008
B. subtilis R23
Glutamic acid, α-ketoglutaricacid,
glucose, citricacid, (NH4)Cl, NaCl
25.38
Bajaj and Singhal 2009a
B. subtilis RKY3
Glycerol (17.6 g/l), glutamic acid (59.6
g/l),
yeast extract (2.7 g/l), K2HPO4 (2.3 g/l)
Glutamate (20 g/l), Glucose (30 g/l),
yeast extract (25 g/l), NH4Cl (10 g/l),
K2HPO4 (0.5 g/l), KH2PO4 (0.5 g/l),
MgSO4 ·7H2O (0.1 g/l)
38℃, 2 days
48.5
Jeong et al. 2010
45℃, 24hr
19.92
Zeng et al. 2014
30℃, 4 days
20
Ito et al. 1996
B. subtilis NX-2
B. subtilis GXA-28
Glutamic acid-independent γ-PGA producers
B. subtilis TAM-4
NH4Cl (18 g/l), fructose (75 g/l)
B. subtilis C10
Glucose (80 g/l), NH4Cl (10 g/l),
32℃, 32 h
citric acid (20 g/l), MgSO4 ·7H2O (0.5 g/l),
K2HPO4 (0.5 g/l), FeCl3·6H2O (0.04 g/l),
CaCl2 (0.11 g/l)
27.2
Zhang et al. 2012b
B. licheniformis A35
NH4Cl (18 g/l), glucose (75 g/l),
MnSO4 ·7H2O (0.04 g/l), HNO3 (20 g/l)
30℃, 3-5 days
8-12
Cheng et al. 1989
B. licheniformis S173
NH4Cl (4 g/l), citric acid (20 g/l),
Mn2+, Fe2+, Ca2+, Zn2+ (1 mM each)
37℃, 30 h
1.27
Kambourova et al. 2001
B. licheniformis SAB-26
Casein hydrolysate, KH2PO4 (NH4)2SO4,
59.9
Abdel-Fattah et al. 2007
B. amyloliquefaciens LL3
Glucose (20 g/l), NH4Cl (10 g/l), citric
37℃, 48 h
acid
(20 g/l), MgSO4 (0.2 g/l), KH2PO4 (6 g/l),
K2HPO4 (14 g/l), trace elements
4.36
Cao et al. 2011
1
2
References for Supplementary Table 2
3
4
Abdel-Fattah YR, Soliman NA, Berekaa MM (2007). Application of Box-Behnken design for
5
optimization of poly-γ-glutamatic acid production by Bacillus licheniformis SAB-26.
6
Res J Microbiol 2: 664-670
7
Ashiuchi M, Kamei T, Baek DH, Shin SY, Sung MH, Soda K, Yagi T, Misono H (2001)
8
Isolation of Bacillus subtilis (chungkookjang), a poly-γ-glutamate producer with high
9
genetic competence. Appl Microbiol Biotechnol 57: 764-769
10
Bajaj IB, Singhal RS (2009a) Sequential optimization approach for enhanced production of
11
poly (γ-glutamic acid) from Bacillus subtilis of marine origin. Food Technol Biotechnol
12
47:313-322
13
Bajaj IB, Singhal RS (2009b) Enhanced production of poly (γ-glutamic acid) from Bacillus
14
licheniformis NCIM 2324 by using metabolic precursors. Appl Biochem Biotechnol
15
159:133–141
16
Cao M, Geng W, Song C, Xie H, Guo W, Jin Y, Wang S (2011) Glutamic acid independent
17
production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of
18
pgsBCA genes. Bioresour Technol 102: 4251-4257
1
2
Cheng C, Asada Y, Aaida T (1989) Production of γ-poly glutamic acid by Bacillus subtilis
A35 under denitrifying conditions. Agri Biol Chem 53: 2369-2375
3
Cromwick AM, Birrer GA, Gross RA (1996) Effects of pH and aeration on γ-poly(glutamic
4
acid) formation by Bacillus licheniformis in controlled batch fermentor cultures.
5
Biotechnol Bioeng 50: 222-227
6
7
Du G, Yang G, Qu Y, Chen J, Lun S (2005) Effects of glycerol on the production of poly(γglutamic acid) by Bacillus licheniformis. Process Biochem 40: 2143-2147
8
Ito Y, Tanaka T, Ohmachi T, Asada Y (1996) Glutamic acid independent production of
9
poly(γ-glutamic acid) by Bacillus subtilis TAM-4. Biosci Biotechnol Biochem 60:
10
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1239-1242
Jeong JH, Kim JN, Wee YJ, Ryu HW (2010) The statistically optimized production of poly
12
(γ-glutamic acid) by batch fermentation of a newly isolated Bacillus subtilis RKY3.
13
Bioresour Technol 101: 4533-4539
14
Kambourova M, Tangney M, Priest FG (2001) Regulation of polyglutamic acid synthesis by
15
glutamate in Bacillus licheniformis and Bacillus subtilis. Appl Environ Micorbiol, 67,
16
1004-1007
17
Kubota H, Matsunobu T, Uotani K, Takebe H, Satoh A, Tanaka T (1993) Production of
18
poly(γ-glutamic acid) by Bacillus subtilis F-2-01. Biosci Biotechnol Biochem 57: 1212-
19
1213
20
Kunioka M, Goto A (1994) Biosynthesis of poly(γ-glutamic acid) from L-glutamic acid, citric
21
acid, and ammonium sulfate in Bacillus subtilis IFO3335. Appl Microbiol Biotechnol
22
40: 867-872
23
Ogawa Y, Yamaguchi F, Yuasa K, Tahara Y (1997) Efficient production γ-polyglutamic acid
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by Bacillus subtilis (natto) in jar fermenters. Biosci Biotechnol Biochem 61: 1684–
25
1687
1
2
3
Shi F, Xu Z, Cen P (2006) Efficient production of poly-γ-glutamic acid by Bacillus subtilis
ZJU-7. Appl Biochem Biotechnol 133: 271-281
Shih IL, Van YT, Chang YN (2002) Application of statistical experimental methods to
4
optimize production of poly(γ-glutamic acid) by Bacillus licheniformis CCRC 12826.
5
Enzym Microb Technol 31: 213-220
6
Wu Q, Xu H, Shi N, Yao J, Li S, Ouyang P (2008) Improvement of poly(γ-glutamic acid)
7
biosynthesis and redistribution of metabolic flux with the presense of different additives
8
in Bacillus subtilis CGMCC 0833. Appl Microbiol Biotechnol 79: 527-535
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10
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Xu H, Jiang M, Li H, Lu D, Ouyang P (2005) Efficient production of poly(γ-glutamic acid)
by newly isolated Bacillus subtilis NX-2. Process Biochem 40: 519-523
Yoon SH, Do JH, Lee SY, Chang HN (2000) Production of poly-γ-glutamic acid by fedbatch culture of Bacillus licheniformis. Biotechnol Lett 22: 585-588
13
Zeng W, Chen G, Wang Q, Zheng S, Shu L, Liang Z (2014) Metabolic studies of temperature
14
control strategy on poly(γ-glutamic acid) production in a thermophilic strain Bacillus
15
subtilis GXA-28. Bioresour Technol 155: 104-110
16
Zhang D, Feng X. Li ZS, Chen F, Xu H (2012b) Effects of oxygen vectors on the synthesis
17
and molecular weight of poly(γ-glutamic acid) and the metabolic characterization of
18
Bacillus subtilis NX-2. Process Biochem 47: 2103-2109
19
20
21
22
23
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1
2
3
4
5
6
7
8
9
10
11
12
13
Supplementary Fig. 1. Light microscopy photograph of colonies (A) and electron
microscopy photograph of bacteria (B), and the phylogenic trees (C) of the 16S rDNA
gene of B. subtilis MJ80.
14
15
16
17
18
19
20
8
1
2
3
A
4
5
6
7
8
9
B
10
11
12
13
14
15
Supplementary Fig. 2. Effect of glycerol (A) and citric acid concentration (B) on γ-PGA
16
production. Glycerol (0-120 g/L) was added to glutamic acid medium containing 70 g/L
17
GA, 30 g starch, 7 g urea, 12 g citric acid and minerals, and cultured with 5% inoculum (1 x
18
107 CFU/mL) for 5 days at 37oC with 150 rpm. For the effect of citric acid concentration, 0-
19
20 g/L citric acid was added to glutamic acid medium containing 70 g/L GA, 30 g starch, 9 g
20
urea, 120 g/L glycerol and 12 g citric acid and minerals, and cultured with 5% inoculum (1 x
21
107 CFU/mL) for 5 days at 37oC with 150 rpm. Values are mean ± SD of triplicates.
22
9