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: ywlim@snu.ac.kr 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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33. Esin PÇ, Halil B. 2011. Effect of various carbon and nitrogen sources on 13

cellulose synthesis by Acetobacter lovaniensis HBB5. African J Biotechnol. 14

10:5346-5354. 15

34. Son H-J, Kim H-G, Kim K-K, Kim H-S, Kim Y-G, Lee S-J. 2013. Increased 16

production of bacterial cellulose by Acetobacter sp. V6 in synthetic media under 17

shaking culture conditions. Bioresour Technol. 86:215-219. 18

35. Park JK, Jung JY, Park YH. 2003. Cellulose production by 19

Gluconacetobacter hansenii in a medium containing ethanol. Biotechnol Lett. 20

25:2055-2059. 21

36. Tsouko E, Kourmentza C, Ladakis D, Kopsahelis N, Mandala I, Papanikolaou 22

S, Paloukis F, Alves V, Koutinas A. 2015. Bacterial cellulose production from 23

industrial waste and by-product streams. Int J Mol Sci. 16:14832-14849. 24

ACCEPTED

19

1

2

ACCEPTED

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

ACCEPTED

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

ACCEPTED

Fig. 2 3

4

5

ACCEPTED

Fig. 3 6

7

Wavenumber (cm-1

)

5001000150020002500300035004000

% T

ran

sm

itta

nce

OH

CH strHOH

CH ben

COC

8

9

ACCEPTED

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

ACCEPTED

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

ACCEPTED

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

ACCEPTED

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

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

ACCEPTED

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

ACCEPTED

Fig. 7 43

44

45

46

47

48

49 ACCEPTED