Supplementary Information
Title
Evaluation of microbial diversity in the pilot-scale beer brewing process by culture-dependent
and culture-independent method
Authors and Affiliation
Masayuki Takahashi,* Yasuko Kita, Kazutaka Kusaka, Akihiro Mizuno, Nami Goto-Yamamoto
National Research Institute of Brewing (NRIB), Higashi-Hiroshima 739-0046, Japan
*
Corresponding author. Tel.: +81-82-420-8227; Fax: +81-82-420-8228; E-mail:
m.takahashi@nrib.go.jp
Journal
Journal of Applied Microbiology
Figure S1 – Beer fermentation profile. Alcohol content and apparent extracts were analyzed
using a ServoChem Automatic Beer Analyser (SCABA, Tecator AB, Sweden). Measurements of
pH and titratable acidity were conducted in accordance with the BCOJ Beer Analysis Methods.
Optical density at 660 nm was measured using a UV spectrometer, UVmini-1240 (Shimadzu,
Kyoto, Japan). Black open circles, black triangles and black crosses indicated the profiles of allmalt, half-malt and low-malt beer, respectively.
Figure S2 – Fluorescent in situ hybridization using a PF2 probe for eukaryote and a EUB338
probe for bacteria. (A) Escherichia coli was used as a positive control of EUB338. (B)
Saccharomyces cerevisiae was used as a positive control of PF2.
Figure S3 – Dotplot obtained by flow cytometry for the quantification of eukaryotic
microorganisms.
Figure S4 – Heatmap analysis of eukaryotic microbial community during beer brewing process.
Color key is indicated on the top of the heatmap diagram.
Table S1 – Primer sets and oligonucleotides used in this study.
Table S2 – Multiplex identifier (MID) tags used in this study.
Table S3 – Quantitation of total eukaryotic microorganisms in beer using qPCR.
Table S4 – Bacterial diversity observed in this study.
Table S5 – Eukaryotic microbial diversity observed in this study. Genera marked with an
asterisk were identified to the genus level using a BLAST search in the GenBank database
(http://blast.ncbi.nlm.nih.gov/).
Figure S1.
All-malt
(A) Apparent extract (sucrose w/w%)
(B) Alcohol (v/v%)
12
6
10
5
8
4
6
3
4
2
2
1
0
0
(C) Titratable acidity
2.5
2
(D) pH
6
5
4
1.5
3
1
2
0.5
1
0
0
(E) Optical density at 660 nm
8
7
6
5
4
3
2
1
0
Half-malt
Low-malt
Figure S2.
(A) Escherichia coli
Non-EUB338
EUB338
Pf2
EUB338
Pf2
Fluorescence
DIC
(B) Saccharomyces cerevisiae
Non-EUB338
Fluorescence
DIC
Figure S3.
Pf2
104
Standard particle
104
104
Non-EUB338
10 3
103
103
R1
101
102
PMT 1 LOG
103
104
10 0
100
101
102
PMT 1 LOG
103
100
104
102
103
104
104
104
101
PMT1 LOG
104
100
10 2
10 1
PI LOG
PMT2 LOG
102
101
100
100
101
Day 1
R1
PMT2 LOG
102
R1
10 3
103
103
R1
R1
100
101
102
PMT 1 LOG
103
100
104
101
102
PMT 1 LOG
103
100
104
102
103
104
104
104
101
PMT1 LOG
104
100
10 2
PI LOG
10 1
100
10 0
101
PMT2 LOG
102
101
Day 4
PMT2 LOG
102
R1
10 3
103
103
R1
PI LOG
PMT2 LOG
102
100
101
102
PMT 1 LOG
103
104
10 0
100
10 1
101
PMT2 LOG
102
101
100
Day 12
10 2
R1
R1
100
101
102
PMT 1 LOG
103
104
100
101
102
PMT1 LOG
103
104
Figure S4
1
2
3
4
5
B01
Start time of mashing
B02
Before boiling
B03
Day 1 (after adding yeast)
DayB042
DayB054
DayB067
12
Day B07
B08
Before filtration
B09
After filtration
B10
Stored for 6 months
Start time of mashing
B11
B12
Before boiling
B13
Day 1 (after adding yeast)
DayB142
DayB154
DayB167
12
Day B17
B18
Before filtration
B19
After filtration
B20
Stored for 6 months
B21
Start time of mashing
B22
Before boiling
B23
Day 1 (after adding yeast)
DayB242
DayB254
DayB267
DdayB27
12
B28
Before filtration
B29
After filtration
B30
Stored for 6 months
0
All-malt beer
Half-malt beer
Low-malt beer
Other (order Cantharellales)
genus Cochliobolus
Other (order Capnodiales)
Other (family Sclerotiniaceae)
Other (order Malasseziales)
Other (family Amphisphaeriaceae)
Other (class Agaricomycetes)
genus Gibberella
Other (family Pichiaceae)
Other (order Sporidiobolales)
genus Trichosporon
genus Sporidiobolus
Other (family Dipodascaceae)
Other (class Dothideomycetes)
Other (order Xylariales)
genus Cystofilobasidium
Other (family Tremellaceae)
genus Dioszegia
genus Sporobolomyces
Other (order Sporidiobolales, family Incertae sedis)
Other (phylum Basidiomycota)
genus Aureobasidium
genus Cryptococcus
Other (family Cystofilobasidiaceae)
Other (family Phyllachoraceae)
genus Holtermanniella
genus Saturnispora
Other (class Saccharomycetes)
genus Fusarium
genus Kazachstania
Other (kingdom Fungi)
genus Acremonium
genus Wickerhamomyces*
genus Kazachstania*
genus Arthroascus
genus Dipodascus
genus Galactomyces*
genus Pichia*
genus Candida*
genus Geotrichum*
Other (order Entylomatales)
Other (order Pleosporales)
genus Udeniomyces
Other (order Tremellales)
genus Metschnikowia
genus Dissoconium
Other (family Exidiaceae)
genus Eurotium
genus Alternaria
Other (family Trichocomaceae)
genus Bullera
Other (class Eurotiomycetes)
genus Exophiala
Other (family Saccharomycetaceae)
genus Exidia*
genus Penicillium*
genus Wallemia*
Other (order Agaricales)
genus Malassezia*
Other (class Sordariomycetes)
Other (order Saccharomycetales)
genus Saccharomyces*
genus Torulaspora
genus Schizosaccharomyces
genus Haplotrichum
Other (phylum Ascomycota)
genus Hanseniaspora
Other (order Saccharomycetales, family Incertae sedis)
genus Kluyveromyces
genus Aspergillus*
genus Didymosphaeria*
genus Cladosporium*
genus Eurotiomycetes*
Other (class Tremellomycetes)
genus Rhodotorula
genus Hannaella
Table S1. Primer sets and oligonucleotides used in this study.
No.
Sequence 5’ – 3’ *
Comment
Target
Application §
Reference
5’-TCCTACGGGAGGCAGCAGT-3’
Forward primer
Bacteria
qPCR
Nadkarni et al. 2002
5’-GGACTACCAGGGTATCTAATCCTGTT-3’
Reverse primer
(SSU-rRNA gene)
(6-FAM)-5’-CGTATTACCGCGGCTGCTGGCAC-3’-(TAMRA)
Dual-labeled probe
5’-GGRAAACTCACCAGGTCCAG-3’
Forward primer
Eukaryote
qPCR
Liu et al. 2012
5’-GSWCTATCCCCAKCACGA-3’
Reverse primer
(SSU-rRNA gene)
(6-FAM)-5’-TGGTGCATGGCCGTT-3’-(TAMRA)
Dual-labeled probe
3
(FITC)-5’-GCTGCCTCCCGTAGGAGT-3’
EUB338
Bacteria
FISH
Amann et al. 1990
4
(FITC)-5’-CTCTGGCTTCACCCTATTC-3’
Pf2
Eukaryote
FISH
Kempf et al. 2000
5
(FITC)-5’-ACATCCTACGGGAGGC-3’
Non-EUB338
Negative control
FISH
Wallner et al. 1993
6
5’-(SeqF)-(MID)-AACGCGAAGAACCTTAC-3’
Forward primer (968F)
Bacteria
NGS
Heuer et al. 1997
5’-(SeqR)-GGHWACCTTGTTACGACTT-3’
Reverse primer (1492R)
(SSU-rRNA gene)
5’-(SeqF)-(MID)-GCATATCAATAAGCGGAGGAAAAG-3’
Forward primer (NL1)
Eukaryote
5’-(SeqR)-GGTCCGTGTTTCAAGACGG-3’
Reverse primer (NL4)
(LSU-rRNA gene)
5’-AGAGTTTGATCCTGGCTCAG-3’
Forward primer (27F)
5’-CCTACGGGAGGCAGCAG-3’
Forward primer (341F)
5’- ACCGCGGCTGCTGGC-3’
Reverse primer (531R)
Endo and Okada 2005
5’-GGHWACCTTGTTACGACTT-3’
Reverse primer (1492R)
Modified from Turner et al. (1999)
5’-GCATATCAATAAGCGGAGGAAAAG-3’
Forward primer (NL1)
Eukaryote
General
5’-GGTCCGTGTTTCAAGACGG-3’
Reverse primer (NL4)
(LSU-rRNA gene)
purpose
1
2
7
8
9
Modified from Turner et al. (1999)
NGS
O'Donnell 1993
Bacteria
General
Lane 1991
(SSU-rRNA gene)
purpose
Muyzer et al. 1993
*
SeqF, CGTATCGCCTCCCTCGCGCCATCAG; SeqR, CTATGCGCCTTGCCAGCCCGCTCAG.
§
qPCR, quantitative PCR; FISH, Fluorescent in situ hybridization; NGS, next-generation sequencing
O'Donnell 1993
Table S2. Multiplex identifier (MID) tags used in this study.
No.
MID Sequence
1
ACGAGTGCGT
2
ACGCTCGACA
AGACGCACTC
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
AGCACTGTAG
ATCAGACACG
ATATCGCGAG
CGTGTCTCTA
CTCGCGTGTC
TCTCTATGCG
TGATACGTCT
CATAGTAGTG
CGAGAGATAC
ATACGACGTA
TCACGTACTA
CGTCTAGTAC
TCTACGTAGC
TGTACTACTC
ACGACTACAG
CGTAGACTAG
TACGAGTATG
TACTCTCGTG
TAGAGACGAG
TCGTCGCTCG
ACATACGCGT
ACGCGAGTAT
ACTACTATGT
ACTGTACAGT
AGACTATACT
29
AGCGTCGTCT
30
AGTACGCTAT
Table S3. Quantitation of total eukaryotic microorganisms in beer using qPCR.*
Sample
1
2
3
4
5
6
7
8
9
10
qPCR (log 10 rDNA copy number ml-1)
All-malt beer
7.97 ± 0.09
4.08 ± 0.12
8.27 ± 0.04
8.62 ± 0.05
9.24 ± 0.03
9.41 ± 0.14
8.42 ± 0.04
7.25 ± 0.02
Not detected §
Not detected
Half-malt beer
6.98 ± 0.02
5.30 ± 0.04
8.04 ± 0.35
8.27 ± 0.05
8.55 ± 0.30
9.48 ± 0.06
9.38 ± 0.04
6.51 ± 0.03
Not detected
Not detected
Low-malt beer
7.07 ± 0.10
5.50 ± 0.12
8.25 ± 0.03
8.46 ± 0.13
9.17 ± 0.04
9.57 ± 0.04
8.81 ± 0.23
6.06 ± 0.01
Not detected
Not detected
* 1, Start time of mashing; 2, Before boiling; 3, Day 1 (after adding yeast); 4, Day 2; 5, Day 4; 6, Day 7; 7, Day 12; 8, Before filtration; 9, After filtration; 10, Stored for 6 months
§
“Not detected” indicates that the amount of rDNA copy numbers per ml was below 2.5 × 103 copies per ml (approximately log 10 copy number 3.4).
Tables S4 and S5 – see the Excel file named “Supplementary information (Tables S4, S5).xlsx”.
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