Page 1
JMB Papers in Press. First Published online Mar 12, 2019
DOI: 10.4014/jmb.1801.01005
Manuscript Number: JMB19-01005
Title: Cellulosic nanomaterial-production via fermentation by Komagataeibacter
sp. SFCB22-18 isolated from ripened persimmons
Article Type: Research article
Keywords: Cellulosic nanomaterial, bacterial nanocellulose, Komagataeibacter,
ripened persimmon, fermentation
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Submitted to Journal of Microbiology and Biotechnology 1
2
Cellulosic nanomaterial-production via fermentation by Komagataeibacter sp. SFCB22-3
18 isolated from ripened persimmons 4
5
Myung Soo Park1,a, Young Hoon Jung2,a, Seung-Yoon Oh1, Min Ji Kim1, Won Yeong Bang2, 6
Young Woon Lim1* 7
8
9
1School of Biological Sciences and Institution of Microbiology, Seoul National University, 10
Seoul 08826, South Korea. 11
2School of Food Science and Biotechnology, Food and Bio-industry Institute, Kyungpook 12
National University, Daegu 41566, South Korea 13
14
15
* Corresponding author: Young Woon Lim 16
Tel: +82-2-880-6708; Fax: +82-2-871-5191 17
E-mail address: [email protected] 18
19
a M.S. Park and Y. H. Jung contributed equally to this work. 20
21
Running title 22
Isolation of bacterial cellulose producing strain from persimmon 23
24
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Abstract 1
Bacterial nanocellulose (BC) has a wide variety of industrial uses, particularly in food 2
and material industry. BC has synthesized by several species of bacteria during 3
vinegar or Nata de coco fermentation. However, the low levels of BC production 4
during the fermentation process should be overcome to reduce its production cost. 5
Therefore, in this study, we screened and identified a new cellulose-producing 6
bacterium, optimized production of the cellulose, and investigated morphological 7
properties of the cellulosic materials. Out of 147 bacterial isolates from ripened fruits 8
and traditional vinegars, strain SFCB22-18 showed the highest capacity for BC 9
production, which was identified as Komagataeibacter sp. based on 16S rRNA 10
sequence analysis. During 6-week fermentation of the strain at optimized medium 11
containing 3.0% glucose, 2.5% yeast extract, 0.24% acetic acid, 0.27% Na2HPO4, and 12
0.5% ethanol at 30 °C for 6 weeks, about 5 g/L of cellulosic material was produced. 13
Both imaging and IR analysis proved that the produced cellulose would be nanoscale 14
bacterial cellulose. 15
16
Keywords: Cellulosic nanomaterial; Bacterial nanocellulose; Komagataeibacter; 17
Ripened persimmon; Fermentation18
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Introduction 1
Cellulose is one of the most abundant polymers in the world [1] and is composed 2
of β-1,4-linked glucose units. It is generally synthesized by plants; however, 3
bacteria are also capable of producing cellulose that is structurally similar to plant 4
cellulose through fermentation [2]. This cellulose type is named bacterial 5
nanocellulose (BC), one of cellulosic nanomaterials. It is produced by a number 6
of bacterial species, including Acetobacter, Azotobacter, Rhizobium, 7
Pseudomonas, Salmonella, Alcaligenes, and Sarcina [3], which can be often 8
observed during vinegar fermentation or fruit spoilage. However, the cultivation 9
of cellulose-producing bacteria is inefficient since these bacteria require high 10
oxygen levels and a long adaptation period, thereby giving high production cost 11
in commercial scale production [4]. Hence, significant cost reduction by 12
improvement of fermentation efficiency is required to make the cellulose 13
production a commercially viable option. 14
Until now, cellulose producing bacteria have been isolated from various 15
sources, such as fruits, flowers, fermented foods, and vegetable wastes [5-7]. 16
However, the strategies like isolation and identification of new strains producing 17
BC as well as optimization of fermentation process are still promising to obtain 18
high yield of BC. Therefore, researchers have continued to investigate the 19
isolation of microorganisms capable of greater BC production. This goal is 20
particularly vital since BC nanomaterial has many possible applications arising 21
from its diverse traits. For example, its gelation ability has made BC a source of 22
traditional foods like “Nata de coco” [8]. BC is also expected to be an additive for 23
use as a thickener, texturizer, and/or dietary fiber in food such as “Kombucha 24
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tea” [9,10]. Beside food industry, this cellulose nanomaterial can be used as a 1
valuable material in the medical field in reconstructive surgery, in cell and tissue 2
engineering as a drug carrier, and as electronic paper due to its high purity, high 3
water content, high bio-compatibility, high mechanical strength, and gas 4
permeability [6, 11-14]. In the present study, the isolation and identification of 5
cellulose-producing bacteria were performed from various sources including 6
ripened fruits and traditional vinegars. Then, the fermentation conditions were 7
optimized and the properties of the cellulose pellicles were identified. 8
9
Materials and Methods 10
Isolation and screening of cellulose-producing bacteria 11
To isolate bacteria that produce cellulose pellicles, we collected ripened fruits 12
(citrus, persimmon, and tomato) and traditional vinegars made from apricot, 13
banana, grape, lespedeza, peach, persimmon, Sanghwang mushroom, and rice. A 14
piece of tissue from each fruit was dissected and placed in a 50-mL conical tube 15
with 5 mL of distilled water. The tubes were vortexed and 100 μL of the resultant 16
liquid was spread on Hestrin-Schramm medium (HS medium; pH 6.0) containing 17
2.0% glucose, 0.5% yeast extract, 0.5% peptone, 0.27% Na2HPO4, 0.115% citric 18
acid, and 2.0% agar [15]. Traditional vinegars (100 μL) were diluted 1/5 and 1/10 19
and spread on HS plates. After incubation at 30 °C for 5 days, individual isolates 20
were transferred to new HS agar plates and further incubated at 30 °C. The strains 21
were also stored in 20 % (w/w) glycerol at -80 °C in the Seoul National 22
University Fungal Collection (SFC, Seoul, South Korea) for further use. 23
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1
Selection and identification of BC producing strains 2
To investigate BC producing ability, all strains isolated from ripened fruits and 3
traditional vinegars were individually inoculated in 20 mL HS media. The 4
formation of pellicles on the surface of the culture media was visually verified 5
and the BC producing ability was compared after cultivation at 30 °C for 7 days 6
[16]. Then, the strain that produced the highest amount of cellulose was selected 7
for further experiments. 8
The selected bacterial strain was identified based on 16S rRNA sequence 9
analysis. Bacterial 16S rDNA was amplified using a Maxime PCR PreMix kit 10
(iNtRON Biotechnology, Seongnam, South Korea) with 27F forward and 1492R 11
reverse primers [17]. Colony PCR amplification was performed with a PCR 12
mixture containing 1.0 μL of each primer, 23.0 μL sterilized distilled water, and 13
one colony of a bacterial strain. PCR conditions were as follows: denaturation at 14
95 °C for 10 min, 35 cycles of elongation at 95 °C for 40 sec, at 55 °C for 40 sec, 15
and at 72 °C for 60 sec, and final extension at 72 °C for 5 min. PCR products 16
were separated and visualized via 1% agarose gel electrophoresis, and purified 17
using an ExpinTM PCR purification kit (GeneAll, Seoul, South Korea). 18
Sequencing was performed using an automated DNA analyzer system (PRISM 19
3730XL DNA analyzer, Applied Biosystems, USA) from Macrogen (Seoul, 20
South Korea). The sequences were checked and edited using MEGA v.5 [18]. 21
Identification was conducted using both of BLAST against the EzBioCloud 22
database [19] and phylogenetic analysis based on neighbor joining method with 23
Kimura-2 parameter and 1000 bootstrap replicates. The sequence was deposited 24
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into GenBank under accession number MH045739. 1
2
Optimization of culture conditions 3
Growth of the selected strain was examined by inoculating the strain in HS 4
medium in 250-mL flasks and incubating at 30 °C for 7 days with shaking at 200 5
rpm. Culture viability was examined by plating daily on HS agar after vigorous 6
shaking and filtering through sterile four-layered gauze to detach cells from the 7
cellulose, and counting the number of colony forming units (CFU) of viable 8
bacterial cells. For BC production, a colony of the selected strain was inoculated 9
in 100 mL HS medium in a 250-mL flask and incubated at 30 °C for 3 days with 10
shaking at 200 rpm. Five percent (v/v) of the culture broth supernatant was then 11
inoculated in 100 mL of medium in 250-mL flasks for the optimization of culture 12
conditions. 13
To optimize culture conditions, the effect of various temperatures (20 °C, 14
25 °C, 30 °C, and 37 °C) and agitation on BC production for 7 days was 15
compared. BC amounts were also compared to that of Komagataeibacter xylinus 16
(ATCC10245), which is a well-known cellulose-producing bacteria [20]. In 17
addition, the composition of the growth medium was optimized by adding various 18
carbon sources (glucose, mannitol, fructose, sucrose, maltose, and lactose; 19
concentrations 1%–5%), nitrogen sources (yeast extract, corn steep liquor, beef 20
extract, malt extract, and peptone; 0.5%–3%), acids (acetic acid, lactic acid, and 21
succinic acid; 0%–0.3%), phosphate sources (KH2PO4, K2HPO4, NaH2PO4, and 22
(NH4)2HPO4; 0.27%) and ethanol 0%–2.5%. 23
To determine statistical significance, a one-way analysis of variance with 24
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post hoc testing and a least significant difference test were performed using 1
Statistica software (Version 7.1, StatSoft, Tulsa, OK). 2
3
Bacterial cellulose purification 4
BC pellicles produced at the air-liquid interface of the medium were immersed in 5
0.3 N NaOH at 90 °C for 20 min, placed on filter paper (No. 2; Advantec, Tokyo, 6
Japan), and rinsed with distilled water until the pH of filtrate became neutral to 7
ensure the complete removal of sodium hydroxide. The total solids content of the 8
filtered pellicles was obtained by drying at 80 °C for 48 h in triplicate. 9
10
Analysis of BC properties by Scanning electron microscopy (SEM) and Fourier 11
transform infrared spectroscopy (FTIR) 12
To evaluate conformational characteristics of BC fibrils obtained from the 13
medium, BC pellicles were analyzed by FTIR (Nicolet iS5 FTIR Spectrometer, 14
Thermo Scientific, Waltham, MA) and SEM (SU8220; Hitachi, Tokyo, Japan). 15
For FTIR analysis, the lyophilized sample was cut into pieces of a predetermined 16
size and spectra in the range of 500 to 4000 cm-1 were obtained at a resolution of 17
4 cm-1, with 16 scans for each sample in the transmission mode. For SEM 18
imaging, the lyophilized sample was coated with a thin platinum film and all 19
images were taken at an accelerating voltage of 15 kV. 20
21
Results and Discussion 22
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Screening of cellulose producing bacteria and identification of the isolate 1
A total of 147 bacterial strains were isolated from various fruits (citrus fruits-11 2
strains, persimmons-92, and tomatoes-9) and traditional vinegars (apricot-4, 3
banana-1, Chinese matrimony vine-2, lespedeza-10, peach-3, persimmon-6, 4
Sanghwang mushroom-2, and rice-7) using HS medium. Among them, 24 strains 5
isolated from ripened persimmons only produced extracellular compounds, which 6
could be cellulose polymer. Particularly, SFCB22-18 produced the greatest 7
amount of polymer (1.70 g/L) (Fig. 1). 8
Strain SFCB22-18 was identified based on its 16S rRNA sequence. 9
Primary identification using BLAST against the EzBioCloud database showed 10
similarities of 99.7%, 99.6%, and 99.6% to K. intermedius TF2T, K. oboediens 11
DSM 11826T, and K. medellinensis NBRC 3288T, respectively. Phylogenetic 12
analysis based on the neighbor joining method showed that strain SFCB22-18 13
was close to K. medellinensis NBRC 3288T with a high support value (80%), 14
while it was clustered with several Komagataeibacter spp. (Fig. 2). Thus, the 15
isolated strain remained as Komagataeibacter sp. SFCB22-18 because of its 16
unclear phylogenetic relationship with closely related species. 17
So far, either acetic acid-producing species or polysaccharide producing 18
species have been isolated from persimmons and persimmon vinegars. For 19
example, Acetobacter syzygii, K. intermedius (= Gluconacetobacter intermedius) 20
and K. europaeus were observed during the acetification of persimmon [21]. In 21
addition, Gluconacetobacter sp. RKY5, K. xylinus TJU-S8, K. xylinus KJ-1 (= A. 22
xylinum), and K. intermedius TF2 were observed in persimmon vinegars [22,23]. 23
Mostly, they are producing insoluble cellulose polymers and soluble extracellular 24
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polysaccharides. In this study, we have found that some of bacterial stains 1
isolated from ripened persimmons were able to produce insoluble extracellular 2
polysaccharide. The extracellular polysaccharide which would be assumed as 3
cellulose was examined with FTIR (Fig. 3). A typical absorption spectrum of BC 4
was observed, such as OH stretching at 3400 cm-1, CH stretching at 2897 cm-1, 5
COC stretching at 1030 cm-1, etc. [24,25]. Thus, we assumed that the produced 6
extracellular pellicles from Komagataeibacter sp. SFCB22-18 are mainly 7
composed of cellulose. 8
9
Optimization of culture parameters 10
BC is a major form of exopolysaccharide that is produced on the surface of 11
bacterial cultures in a slow fermentation process along with acid production. In 12
addition, BC production is dependent on the strain type and on favorable 13
cultivation conditions [26]. Thus, in this study, the growth and cellulose 14
production in Komagataeibacter sp. SFCB22-18 under various fermentation 15
conditions were investigated. The growth curve of Komagataeibacter sp. 16
SFCB22-18 cultivated on HS media at 30 °C showed a steep increase to 3.8×106 17
CFU in 3 days. Since there was no further significant increase in cell growth 18
during the 7 days of cultivation, the inoculums cultured for 3 days were used to 19
optimize the batch fermentation conditions, including medium components, on 20
BC production. 21
Initially, the effects of fermentation temperature and agitation on BC 22
production were investigated (Fig. 4). As the culture temperature increased from 23
20 to 30 °C, BC concentration after 7 days fermentation increased from 1.2 g/L to 24
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1.6 g/L (Fig. 4A). However, a further increase in temperature to 37 °C reduced 1
BC production, which is similar to previous results for BC production in K. 2
xylinus 0416 and Komagataeibacter sp. PAP1 [27,28]. The fermentation 3
temperature of 30 °C was found to be optimal for efficient cellulose production in 4
Komagataeibacter sp. SFCB22-18. Meanwhile, agitation during cultivation also 5
affected the quantity and quality of the synthesized cellulose. When 6
Komagataeibacter sp. SFCB22-18 was cultured with vigorous shaking at 200 7
rpm, BC production significantly decreased by approximately 40% (Fig. 4B). 8
These results suggested that agitation is not necessary to obtain a high amount of 9
cellulose, probably due to a limitation in binding between newly synthesized BC 10
and existing BC since cells might not have had enough time to anchor BC at such 11
high agitations [29]. Furthermore, higher amount of cellulose was produced by 12
Komagataeibacter sp. SFCB22-18 than the reference strain K. xylinus 13
(ATCC10245). As a result, static culture condition was chosen for further 14
experiments. 15
Next, the effects of different sources of carbon and nitrogen and their 16
concentrations on BC production were investigated (Fig. 5A-D). Among the 17
various types of carbon sources used (glucose, fructose, lactose, maltose, 18
mannitol, and sucrose), glucose addition (2% w/v, standard glucose concentration 19
in HS media) led to the highest production of BC, followed by the addition of 20
same concentrations of fructose and mannitol (Fig. 5A). According to previous 21
studies, the optimal carbon source depends on the bacterial strain since bacteria 22
have diverse metabolic activities. For example, K. medellinensis preferred 23
glucose over sucrose and fructose, however, the amount of cellulose produced by 24
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G. xylinus were higher in the presence of fructose or glycerol than in the presence 1
of glucose [30,31]. When the glucose concentration increased to 3% (w/v), BC 2
production improved by 160% (Fig. 5B). When glucose is readily available, 3
bacteria do not need to convert accessible carbohydrate sources into glucose 4
molecules before polymerization into BC, a process that uses energy. Thus, it is 5
likely that glucose addition to the culture medium may lead to a greater 6
production of cellulose. However, a further increase in glucose concentration to 7
5% did not show any significant increase in BC production. Thus, a glucose 8
concentration of 3% (w/v) was selected as the optimal carbon source for efficient 9
BC production by Komagataeibacter sp. SFCB22-18. 10
Among the various nitrogen sources used (yeast extract, corn steep liquor, 11
beef extract, malt extract, and peptone), yeast extract (1% addition) led to the 12
highest BC production (approximate increase of 120% over BC production in HS 13
media) (Fig. 5C), which is in good agreement with results from previous studies 14
for BC production in Acetobacter sp. V6 and A. lovaniensis HBB5 [32,33]. As 15
the yeast extract concentration in the culture media increased to 2.5%, the amount 16
of BC produced increased to around 220% of that of the 0.5% yeast extract found 17
in typical HS medium (Fig. 5D). Since further additions of yeast extract (3.0%) 18
did not show beneficial effects in BC production, 2.5% was chosen as the optimal 19
yeast extract concentration for efficient cellulose production by 20
Komagataeibacter sp. SFCB22-18. 21
Third, the effects of different sources of acid and the concentrations on 22
BC production were investigated (Fig. 5E-F). Various acid sources such as citrate, 23
acetate, lactate, and succinate at concentrations of 0.115% were used individually 24
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to maintain the pH of the modified HS medium. Among them, the acetate-1
containing medium showed the highest BC production, which was approximately 2
140% of that in standard HS medium (Fig. 5E). It is probably attributed by higher 3
pKa value of acetate than the other acid sources; thereby indicating that acetate is 4
the least toxic to cells during fermentation at pH 6.0. As the acetate concentration 5
increased from 0–0.24%, the concentration of BC pellicles continued to increase 6
up to 149% of that produced in standard HS medium containing 0.115% citrate 7
(Fig. 5F). Thus, 0.24% of acetate concentration was selected as optimal for 8
efficient BC production by Komagataeibacter sp. SFCB22-18. 9
Fourth, the effects of the different sources of phosphate and the 10
concentrations on BC production were investigated (Fig. 5G). HS medium 11
containing Na2HPO4 led to the greatest production of BC. HS media 12
supplemented with K2HPO4 and (NH4)2HPO4 also showed comparable BC 13
concentration. However, KH2PO4 and NaH2PO4 inhibited BC production. This 14
may be due to the fact that Na2HPO4 is more basic than NaH2PO4, which can 15
result in greater buffering activity during fermentation. 16
Finally, the effect of ethanol concentration was also investigated (Fig. 17
5H) because some acetic acid bacteria are capable of oxidizing ethanol [34], 18
which might be used as an energy source. When 0.5% ethanol was added, BC 19
concentration in the medium after 7 days of cultivation increased 1.15-fold over 20
production in HS medium without ethanol. However, further ethanol additions 21
did not enhance BC production probably because of the toxicity of ethanol and 22
thus, 0.5% ethanol concentration was considered as optimal for efficient BC 23
production by Komagataeibacter sp. SFCB22-18. 24
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1
Time profile of bacterial nanocellulose production under optimized conditions 2
BC production by the newly isolated Komagataeibacter sp. SFCB22-18 was 3
explored under the optimized conditions of 3.0% glucose, 2.5% yeast extract, 4
0.24% acetic acid, and 0.5% ethanol with 0.27% Na2HPO4 as a phosphate source 5
at 30 °C without shaking. In comparison with BC production in HS medium, 6
Komagataeibacter sp. SFCB22-18 grown on the optimized medium produced 7
about 2-fold higher BC amounts (Fig. 6). In addition, BC produced by 8
Komagataeibacter sp. SFCB22-18 in the optimized medium increased 9
continuously to up to 5 g/L during the six-week cultivation period, which is 10
comparable to other studies showing 2-5 g/L by Gluconacetobacter sp. RKY5 11
and K. sucrofermentans DSM 15973 [22,35,36]. SEM images also showed that 12
BC was composed of thread-like cellulosic nanofibrils of which diameter were 13
about 20-70 nm in the regardless of cultured medium according to random 14
measurement of the fibrils (Fig. 7). Therefore, the strain, Komagataeibacter sp. 15
SFCB22-18 has a high potential for the production of bacterial nanocellulose. 16
In conclusion, we isolated an extracellular compound-producing strain, 17
Komagataeibacter sp. SFCB22-18 from ripened persimmons. The material was 18
identified as nanocellulosic material with SEM and FTIR analysis and optimized 19
the fermentation conditions to facilitate the highest production. Approximately a 20
2-fold increase in BC production was investigated using the optimized medium of 21
3.0% glucose, 2.5% yeast extract, 0.24% acetic acid, and 0.5% ethanol with 22
0.27% Na2HPO4 at 30 °C without shaking and 7 days of cultivation. The highest 23
BC concentration (4.9 g/L) was obtained for 6 weeks of cultivation. Therefore, 24
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the newly isolated strain, Komagataeibacter sp. SFCB22-18, was identified as a 1
good producer of BC, which can be used in food and material industry. 2
3
Acknowledgements 4
This work (Grant No. C0004405) was supported by Business for Cooperative R&D 5
among Industry, Academy, and Research Institute, the Korean Small and Medium 6
Business Administration in 2012. This research was also supported by the Basic Science 7
Research Program through the National Research Foundation of Korea (NRF), the 8
Ministry of Education (Grant No. 03030504). 9
10
Conflict of Interest 11
The authors declare that they have no conflict of interest. 12
13
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20
Figure Captions 1
2
Fig. 1. Comparison of bacterial nanocellulose production by strains isolated from 3
ripened persimmons. Box with asterisk indicates significantly different at p<0.05. 4
Fig. 2. Neighbor-joining tree inferred from 16S rRNA showing the relationship 5
of strain SFCB22-18 with the most closely related members of the genus 6
Komagataeibacter. The scale bar indicates the number of nucleotide substitutions 7
per site. 8
Fig. 3. FTIR spectra of bacterial nanocellulose 9
Fig. 4. Effects of (A) cultivation temperature and (B) agitation on the 10
concentration of bacterial nanocellulose produced by Komagataeibacter sp. 11
SFCB22-18. Box with asterisk indicates significantly different at p<0.05. 12
Fig. 5. Effects of (A) carbon source, (B) glucose concentration, (C) nitrogen 13
source, (D) yeast extract concentration, (E) acid source, (F) acid concentration, 14
(G) phosphate source, and (H) ethanol concentration on the concentration of 15
bacterial nanocellulose (BC) produced by Komagataeibacter sp. SFCB22-18. Box 16
with patterns refers to Hestrin-Schramm (HS) medium. Bars with different letters 17
are significantly different at p<0.05. 18
Fig. 6. Bacterial nanocellulose concentration grown under optimized conditions 19
during the six weeks of fermentation 20
Fig. 7. SEM images of bacterial nanocellulose synthesized by Komagataeibacter 21
sp. SFCB22-18 for 6 weeks cultivation at (A) typical Hestrin-Schramm (HS) 22
medium and (B) modified medium (HSM). Bars with asterisk indicates 23
significantly different at p<0.05. 24
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Fig
. 1
1
SFCB22-03
SFCB22-16
SFCB22-17
SFCB22-18
SFCB22-19
SFCB22-20
SFCB22-21
SFCB22-22
SFCB22-23
SFCB22-24
SFCB22-25
SFCB22-26
SFCB22-27
SFCB22-28
SFCB22-29
SFCB22-31
SFCB22-32
SFCB22-33
SFCB22-34
SFCB22-35
SFCB22-36
SFCB22-37
SFCB22-38
SFCB22-40
BC concentration (g/L)
0.0
0.5
1.0
1.5
2.0
*
2
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Fig. 2 3
4
5
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Fig. 3 6
7
Wavenumber (cm-1
)
5001000150020002500300035004000
% T
ran
sm
itta
nce
OH
CH strHOH
CH ben
COC
8
9
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Page 25
Fig. 4 10
11
Temperature (oC)
20 25 30 37
BC
co
nce
ntr
atio
n (
g/L
)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
SFCB22-18
ATCC10245
A
*
12
13
Culture type
Static culture Agitated culture
Re
lative
BC
yie
ld (
% o
f con
tro
l)
0
20
40
60
80
100
120
SFCB22-18
ATCC10245
B
*
14
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Page 26
15
Fig. 5 16
17
Carbon source
Glucose Mannitol Fructose Sucrose Maltose Lactose
Rela
tive B
C y
ield
(%
of contr
ol)
0
20
40
60
80
100
120
140
A
a
b
b
bb
a
18
19
Glucose conc. (%, w/v)
1 2 3 4 5
Rela
tive B
C y
ield
(%
of contr
ol)
0
50
100
150
200
250
300
B
a a
a
a
a
20
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Nitrogen sourceYeast extract (0
.5%)
Yeast extract (1%)
Corn steep liquor
Beef extract
Malt extract
Peptone
Rela
tive B
C y
ield
(%
of contr
ol)
0
20
40
60
80
100
120
C
a
b
b
b
b
b
21
22
Yeast extract conc. (%, w/v)
0.5 1 1.5 2 2.5 3
Re
lative
BC
yie
ld (
% o
f co
ntr
ol)
0
50
100
150
200
250
D
ab
b
b
b
b
23
24
25
26
27
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Acid source
Citrate Acetate Lactate Succinate None
Re
lative
BC
yie
ld (
% o
f co
ntr
ol)
0
20
40
60
80
100
120
140
160
180E
b b
b
a
a
28
29
Acetic acid concentration (%, w/v)
Citrate 0 0.06 0.12 0.18 0.24 0.3
Re
lative
BC
yie
ld (
% o
f co
ntr
ol)
0
20
40
60
80
100
120
140
160
180
F
a
aa
a
bb
b
(w/o acetic acid)
30
31 ACCEPTED
Page 29
Phosphate source
Na2HPO4KH2PO4
K2HPO4
NaH2PO4
(NH4)2HPO4None
Re
lative
BC
yie
ld (
% o
f co
ntr
ol)
0
20
40
60
80
100
120
G
a
b
b
b
b
b
Na2HPO
4 KH
2PO
4 K
2HPO
4 NaH
2PO
4 (NH
4)HPO
4 None
32
33
Ethanol conc. (%, v/v)
0 0.5 1 1.5 2 2.5
Re
lative
BC
yie
ld (
% o
f co
ntr
ol)
0
20
40
60
80
100
120
140
H
a
b
b
b bb
34
35
36
37
38
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Fig. 6 39
Culture duration
HS (1 w
eek)
HSM (1 w
eek)
HSM (3 w
eek)
HSM (6 w
eek)
BC
co
nce
ntr
atio
n (
g/L
)
0
1
2
3
4
5
6
*
40
41
42
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Fig. 7 43
44
45
46
47
48
49 ACCEPTED