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Research article

J. Microbiol. Biotechnol. 2019; 29(4): 617-624

Published online April 28, 2019 https://doi.org/10.4014/jmb.1801.01005

Copyright © The Korean Society for Microbiology and Biotechnology.

Cellulosic Nanomaterial Production Via Fermentation by Komagataeibacter sp. SFCB22-18 Isolated from Ripened Persimmons

Myung Soo Park 1, Young Hoon Jung 2, Seung-Yoon Oh 1, Min Ji Kim 1, Won Yeong Bang 2 and Young Woon Lim 1*

1School of Biological Sciences and Institution of Microbiology, Seoul National University, Republic of Korea., 2School of Food Science and Biotechnology, Food and Bio-industry Institute, Kyungpook National University, Republic of Korea.

Correspondence to:Young Woon  Lim
ywlim@snu.ac.kr

Received: January 3, 2019; Accepted: March 11, 2019

Abstract

Bacterial nanocellulose (BNC) which is generally synthesized by several species of bacteria has a wide variety of industrial uses, particularly in the food and material industries. However, the low levels of BNC production during the fermentation process should be overcome to reduce its production cost. Therefore, in this study, we screened and identified a new cellulose-producing bacterium, optimized production of the cellulose, and investigated the morphological properties of the cellulosic materials. Out of 147 bacterial isolates from ripened fruits and traditional vinegars, strain SFCB22-18 showed the highest capacity for BNC production and was identified as Komagataeibacter sp. based on 16S rRNA sequence analysis. During 6-week fermentation of the strain using an optimized medium containing 3.0% glucose, 2.5% yeast extract, 0.24% acetic acid, 0.27% Na2HPO4, and 0.5% ethanol at 30°C, about 5 g/l of cellulosic material was produced. Both imaging and IR analysis proved that the produced cellulose would be nanoscale bacterial cellulose.

Keywords: Cellulosic nanomaterial, bacterial nanocellulose, Komagataeibacter, ripened persimmon, fermentation

Introduction

Cellulose is one of the most abundant polymers in the world [1] and is composed of β-1,4-linked glucose units. It is generally synthesized by plants; however, bacteria are also capable of producing cellulose that is structurally similar to plant cellulose through fermentation [2]. This cellulose type is known as bacterial nanocellulose (BNC), which can be categorized as a cellulosic nanomaterial. It is produced by a number of bacterial species, including Acetobacter, Alcaligenes, Azotobacter, Rhizobium, Pseudomonas, Salmonella, and Sarcina [3], which can be often observed during vinegar fermentation or fruit spoilage. However, the cultivation of cellulose-producing bacteria is inefficient since these bacteria require high oxygen levels and a long adaptation period, thereby resulting in high cost when produced on a commercial scale [4]. Hence, significant cost reduction by improvement of fermentation efficiency is required to make cellulose production a commercially viable option.

Until now, cellulose-producing bacteria have been isolated from various sources, such as fruits, flowers, fermented foods, and vegetable wastes [5-7]. However, the strategies like isolation and identification of new strains producing BNC as well as optimization of the fermentation process are still promising to obtain a high yield of BNC. Therefore, researchers have continued to investigate the isolation of microorganisms capable of greater BNC production. This goal is particularly vital since this nanomaterial has many possible applications arising from its diverse traits. For example, its gelation ability has made BNC a source of traditional foods like “Nata de coco” [8]. BNC is also expected to be an additive for use as a thickener, texturizer, and/or dietary fiber in food such as “Kombucha tea” [9, 10]. Besides the food industry, this cellulose nanomaterial can be used as a valuable material in the medical field in reconstructive surgery, in cell and tissue engineering as a drug carrier, and as electronic paper due to its high purity, high water content, high bio-compatibility, high mechanical strength, and gas permeability [6,11-14]. In the present study, the isolation and identification of cellulose- producing bacteria were performed from various sources including ripened fruits and traditional vinegars. Then, the fermentation conditions were optimized and the properties of the cellulose pellicles were identified.

Materials and Methods

Isolation and Screening of Cellulose-Producing Bacteria

To isolate bacteria that produce cellulose pellicles, we collected ripened fruits (citrus, persimmon, and tomato) and traditional vinegars made from apricot, banana, grape, lespedeza, peach, persimmon, Sanghwang mushroom, and rice. A piece of tissue from each fruit was dissected and placed in a 50-ml conical tube with 5 ml of distilled water. The tubes were vortexed and 100 µl of the resultant liquid was spread on Hestrin-Schramm medium (HS medium; pH 6.0) containing 2.0% glucose, 0.5% yeast extract, 0.5% peptone, 0.27% Na2HPO4, 0.115% citric acid, and 2.0% agar [15]. Traditional vinegars (100 µl) were diluted 1/5 and 1/10 and spread on HS plates. After incubation at 30°C for 5 days, individual isolates were transferred to new HS agar plates and further incubated at 30°C. The strains were also stored in 20% (w/w) glycerol at -80°C in the Seoul National University Fungal Collection (SFC, Korea) for further use.

Selection and Identification of BNC-Producing Strains

To investigate BNC-producing ability, all strains isolated from ripened fruits and traditional vinegars were individually inoculated in 20 ml HS media. The formation of pellicles on the surface of the culture media was visually verified and the BNC- producing ability was compared after cultivation at 30°C for 7 days [16]. Then, the strain that produced the highest amount of cellulose was selected for further experiments.

The selected bacterial strain was identified based on 16S rRNA sequence analysis. Bacterial 16S rDNA was amplified using a Maxime PCR PreMix Kit (iNtRON Biotechnology, Korea) with 27F forward and 1492R reverse primers [17]. Colony PCR amplification was performed with a PCR mixture containing 1.0 µl of each primer, 23.0 µl sterilized distilled water, and one colony of a bacterial strain. PCR conditions were as follows: denaturation at 95°C for 10 min, 35 cycles of elongation at 95°C for 40 sec, at 55°C for 40 sec, and at 72°C for 60 sec, and final extension at 72°C for 5 min. PCR products were separated and visualized via 1% agarose gel electrophoresis, and purified using an Expin PCR Purification Kit (GeneAll, Korea). Sequencing was performed using an automated DNA analyzer system (PRISM 3730XL DNA Analyzer, Applied Biosystems, USA) from Macrogen (Korea). The sequences were checked and edited using MEGA v.5 [18]. Identification was conducted using both BLAST, against the EzBioCloud database [19], and phylogenetic analysis based on the neighbor-joining method with Kimura-2 parameter and 1,000 bootstrap replicates. The sequence was deposited into GenBank under accession number MH045739.

Optimization of Culture Conditions

Growth of the selected strain was examined by inoculating the strain in HS medium in 250-ml flasks and incubating at 30°C for 7 days with shaking at 200 rpm. Culture viability was examined by plating daily on HS agar after vigorous shaking and filtering through sterile four-layered gauze to detach cells from the cellulose, and counting the number of colony forming units (CFU) of viable bacterial cells. For BNC production, a colony of the selected strain was inoculated in 100 ml HS medium in a 250-ml flask and incubated at 30°C for 3 days with shaking at 200 rpm. Five percent (v/v) of the culture broth supernatant was then inoculated in 100 ml of medium in 250-ml flasks for the optimization of culture conditions.

To optimize culture conditions, the effect of various temperatures (20°C, 25°C, 30°C, and 37°C) and agitation on BNC production for 7 days was compared. BNC amounts were also compared to that of Komagataeibacter xylinus (ATCC10245), which is a well-known cellulose-producing bacteria [20]. In addition, the composition of the growth medium was optimized by adding various carbon sources (glucose, mannitol, fructose, sucrose, maltose, and lactose; concentrations 1%–5%), nitrogen sources (yeast extract, corn steep liquor, beef extract, malt extract, and peptone; 0.5%–3%), acids (acetic acid, lactic acid, and succinic acid; 0%–0.3%), phosphate sources (KH2PO4, K2HPO4, NaH2PO4, and (NH4)2HPO4; 0.27%) and ethanol 0%–2.5%.

To determine statistical significance, a one-way analysis of variance with post hoc testing and a least significant difference test were performed using Statistica software (version 7.1, StatSoft, Tulsa, OK).

Bacterial Nanocellulose Purification

BNC pellicles produced at the air-liquid interface of the medium were immersed in 0.3 N NaOH at 90°C for 20 min, placed on filter paper (No. 2; Advantec, Japan), and rinsed with distilled water until the pH of filtrate became neutral to ensure the complete removal of sodium hydroxide. The total solids content of the filtered pellicles was obtained by drying at 80°C for 48 h in triplicate.

Analysis of BNC Properties by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR)

To evaluate the conformational characteristics of the BNC fibrils obtained from the medium, the BNC pellicles were analyzed by FTIR (Nicolet iS5 FTIR Spectrometer, Thermo Scientific, USA) and SEM (SU8220; Hitachi, Japan). For FTIR analysis, the lyophilized sample was cut into pieces of a predetermined size and spectra in the range of 500 to 4,000 cm-1 were obtained at a resolution of 4 cm-1 , with 16 scans for each sample in the transmission mode. For SEM imaging, the lyophilized sample was coated with a thin platinum film and all images were taken at an accelerating voltage of 15 kV.

Results and Discussion

Screening of Nanocellulose-Producing Bacteria and Identification of the Isolate

A total of 147 bacterial strains were isolated from various fruits (citrus fruits-11 strains, persimmons-92, and tomatoes- 9) and traditional vinegars (apricot-4, banana-1, Chinese matrimony vine-2, lespedeza-10, peach-3, persimmon-6, Sanghwang mushroom-2, and rice-7) using HS medium. Among them, 24 strains isolated from ripened persimmons only produced extracellular compounds, which could be cellulose polymer. Particularly, SFCB22-18 produced the greatest amount of polymer (1.70 g/l) (Fig. 1).

Figure 1. Comparison of bacterial nanocellulose production by strains isolated from ripened persimmons. Box with asterisk indicates significantly different at p < 0.05.

Strain SFCB22-18 was identified based on its 16S rRNA sequence. Primary identification using BLAST against the EzBioCloud database showed similarities of 99.7%, 99.6%, and 99.6% to K. intermedius TF2T, K. oboediens DSM 11826T, and K. medellinensis NBRC 3288T, respectively. Phylogenetic analysis based on the neighbor-joining method showed that strain SFCB22-18 was close to K. medellinensis NBRC 3288T with a high support value (80%), while it was clustered with several Komagataeibacter spp. (Fig. 2). Thus, the isolated strain remained as Komagataeibacter sp. SFCB22- 18 because of its unclear phylogenetic relationship with closely related species.

Figure 2. Neighbor-joining tree inferred from 16S rRNA showing the relationship of strain SFCB22-18 with the most closely related members of the genus Komagataeibacter. Bootstrap scores of >70 are presented at the nodes. The scale bar indicates the number of nucleotide substitutions per site.

So far, either acetic acid-producing species or poly- saccharide-producing species have been isolated from persimmons and persimmon vinegars. For example, Aceto- bacter syzygii, K. intermedius (= Gluconacetobacter intermedius) and K. europaeus were observed during the acetification of persimmon [21]. In addition, Gluconacetobacter sp. RKY5, K. xylinus TJU-S8, K. xylinus KJ-1 (= A. xylinum), and K. intermedius TF2 were observed in persimmon vinegars [22, 23]. Mostly, they are producing insoluble cellulose polymers and soluble extracellular polysaccharides. In this study, we have found that some of the bacterial stains isolated from ripened persimmons were able to produce insoluble extracellular polysaccharide. The extracellular polysaccharide which would be assumed as cellulose was examined with FTIR (Fig. 3). A typical absorption spectrum of BNC was observed, such as OH stretching at 3,400 cm-1, CH stretching at 2,897 cm-1, COC stretching at 1,030 cm-1, etc. [24, 25]. Thus, we assumed that the produced extracellular pellicles from Komagataeibacter sp. SFCB22-18 are mainly composed of cellulose.

Figure 3. FTIR spectra of bacterial nanocellulose. OH: O-H stretching, CH str: C-H stretching, HOH: H-O-H stretching, CH ben: symetric deformation and bending vibration of C-H, COC: C-O-C stretching

Optimization of Culture Parameters

BNC is a major form of exopolysaccharide that is produced on the surface of bacterial cultures in a slow fermentation process. In addition, BNC production is dependent on the strain type and on favorable cultivation conditions [26]. Thus, in this study, the growth and cellulose production in Komagataeibacter sp. SFCB22-18 under various fermentation conditions were investigated. The growth curve of Komagataeibacter sp. SFCB22-18 cultivated on HS media at 30 °C showed a steep increase to 3.8 × 106 CFU/ml in 3 days. Since there was no further significant increase in cell growth during the 7 days of cultivation, the inoculums cultured for 3 days were used to optimize the batch fermentation conditions, including medium components, on BNC production.

Initially, the effects of fermentation temperature and agitation on BNC production were investigated (Fig. 4). As the culture temperature increased from 20°C to 30°C, BNC concentration after 7 days, fermentation increased from 1.2 g/l to 1.6 g/l (Fig. 4A). However, a further increase in temperature to 37°C reduced BNC production, which is similar to previous results for BNC production in K. xylinus 0416 and Komagataeibacter sp. PAP1 [27, 28]. The fermentation temperature of 30°C was found to be optimal for efficient cellulose production in Komagataeibacter sp. SFCB22-18. Meanwhile, agitation during cultivation also affected the quantity and quality of the synthesized cellulose. When Komagataeibacter sp. SFCB22-18 was cultured with vigorous shaking at 200 rpm, BNC production significantly decreased by approximately 40% (Fig. 4B). These results suggested that agitation is not necessary to obtain a high amount of cellulose, probably due to a limitation in binding between newly synthesized BNC and existing BNC since cells might not have had enough time to anchor BNC at such high agitations [29]. Furthermore, a higher amount of cellulose was produced by Komagataeibacter sp. SFCB22-18 than the reference strain K. xylinus (ATCC10245). As a result, static culture condition was chosen for further experiments.

Figure 4. Effects of (A) cultivation temperature and (B) agitation on the concentration of bacterial nanocellulose produced by Komagataeibacter sp. SFCB22-18. Box with asterisk indicates significantly different at p < 0.05.

Next, the effects of different sources of carbon and nitrogen and their concentrations on BNC production were investigated (Figs. 5A-5D). Among the various types of carbon sources used (glucose, fructose, lactose, maltose, mannitol, and sucrose), glucose addition (2% w/v, standard glucose concentration in HS media) led to the highest production of BNC, followed by the addition of the same concentrations of fructose and mannitol (Fig. 5A). According to previous studies, the optimal carbon source depends on the bacterial strain since bacteria have diverse metabolic activities. For example, K. medellinensis preferred glucose over sucrose and fructose, however, the amount of cellulose produced by G. xylinus was higher in the presence of fructose or glycerol than in the presence of glucose [30, 31]. When the glucose concentration increased to 3% (w/v), BNC production improved by 160% (Fig. 5B). When glucose is readily available, bacteria do not need to convert accessible carbohydrate sources into glucose molecules before polymerization into BNC, a process that uses energy. Thus, it is likely that glucose addition to the culture medium may lead to a greater production of cellulose. However, a further increase in glucose concentration to 5% did not show any significant increase in BNC production. Thus, a glucose concentration of 3% (w/v) was selected as the optimal carbon source for efficient BNC production by Komagataeibacter sp. SFCB22-18.

Figure 5. Effects of (A) carbon source, (B) glucose concentration, (C) nitrogen source, (D) yeast extract concentration, (E) acid source, (F) acid concentration, (G) phosphate source, and (H) ethanol concentration on the concentration of bacterial nanocellulose (BNC) produced by Komagataeibacter sp. SFCB22-18. Box with patterns refers to the condition of Hestrin-Schramm (HS) medium. Bars with different letters are significantly different at p < 0.05.

Among the various nitrogen sources used (yeast extract, corn steep liquor, beef extract, malt extract, and peptone), yeast extract (1% addition) led to the highest BNC production (approximate increase of 120% over BNC production in HS media) (Fig. 5C), which is in good agreement with results from previous studies for BNC production in Acetobacter sp. V6 and A. lovaniensis HBB5 [32, 33]. As the yeast extract concentration in the culture media increased to 2.5%, the amount of BNC produced increased to around 220% of that of the 0.5% yeast extract found in typical HS medium (Fig. 5D). Since further additions of yeast extract (3.0%) did not show beneficial effects in BNC production, 2.5% was chosen as the optimal yeast extract concentration for efficient cellulose production by Komagataeibacter sp. SFCB22-18.

Third, the effects of different sources of acid and the concentrations on BNC production were investigated (Figs. 5E and 5F). Various acid sources such as citrate, acetate, lactate, and succinate at concentrations of 0.115% were used individually to maintain the pH of the modified HS medium. Among them, the acetate-containing medium showed the highest BNC production, which was approxi- mately 140% of that in standard HS medium (Fig. 5E). It is probably attributable to the higher pKa value of acetate than the other acid sources; thereby indicating that acetate is the least toxic to cells during fermentation at pH 6.0. As the acetate concentration increased from 0–0.24%, the concentration of BNC pellicles continued to increase up to 149% of that produced in standard HS medium containing 0.115% citrate (Fig. 5F). Thus, 0.24% acetate concentration was selected as optimal for efficient BNC production by Komagataeibacter sp. SFCB22-18.

Fourth, the effects of the different sources of phosphate and the concentrations on BNC production were investigated (Fig. 5G). HS medium containing Na2HPO4 led to the greatest production of BNC. HS media supplemented with K2HPO4 and (NH4)2HPO4 also showed comparable BNC concentration. However, KH2PO4 and NaH2PO4 inhibited BNC production. This may be due to the fact that Na2HPO4 is more basic than NaH2PO4, which can result in greater buffering activity during fermentation.

Finally, the effect of ethanol concentration was also investigated (Fig. 5H) because some acetic acid bacteria are capable of oxidizing ethanol [34], which might be used as an energy source. When 0.5% ethanol was added, BNC concentration in the medium after 7 days of cultivation increased 1.15 fold over production in HS medium without ethanol. However, further ethanol additions did not enhance BNC production probably because of the toxicity of ethanol and thus, 0.5% ethanol concentration was considered as optimal for efficient BNC production by Komagataeibacter sp. SFCB22-18.

Time Profile of Bacterial Nanocellulose Production under Optimized Conditions

BNC production by the newly isolated Komagataeibacter sp. SFCB22-18 was explored under the optimized conditions of 3.0% glucose, 2.5% yeast extract, 0.24% acetic acid, and 0.5% ethanol with 0.27% Na2HPO4 as a phosphate source at 30°C without shaking. In comparison with BNC production in HS medium, Komagataeibacter sp. SFCB22-18 grown on the optimized medium produced about 2-fold higher BNC amounts (Fig. 6). In addition, BNC produced by Komagataeibacter sp. SFCB22-18 in the optimized medium increased continuously to up to 5 g/l during the six-week cultivation period, which is comparable to other studies showing 2-5 g/l by Gluconacetobacter sp. RKY5 and K. sucrofermentans DSM 15973 [22, 35, 36]. SEM images also showed that BC was composed of thread-like cellulosic nanofibrils whose diameter was about 20-70 nm regardless of cultured medium according to random measurement of the fibrils (Fig. 7). Therefore, the strain, Komagataeibacter sp. SFCB22-18, has high potential for the production of bacterial nanocellulose.

Figure 6. Bacterial nanocellulose concentration grown under optimized conditions during the six weeks of fermentation. Bars with asterisk indicates significantly different at p < 0.05.

Figure 7. SEM images of bacterial nanocellulose synthesized by Komagataeibacter sp. SFCB22-18 after 6 weeks of cultivation in (A) typical Hestrin-Schramm (HS) medium and (B) modified medium (HSM).

In conclusion, we isolated an extracellular compound- producing strain, Komagataeibacter sp. SFCB22-18, from ripened persimmons. The produced pellicles were characterized and identified with SEM and FTIR as nanocellulose. During the cultivation, approximately a 2- fold increase in BNC production was investigated using the optimized medium of 3.0% glucose, 2.5% yeast extract, 0.24% acetic acid, and 0.5% ethanol with 0.27% Na2HPO4 at 30°C without shaking and 7 days of cultivation. The highest BNC concentration (4.9 g/l) was obtained after 6 weeks of cultivation. Therefore, the newly isolated strain, Komagataeibacter sp. SFCB22-18, was identified as a good producer of BNC which can be used in the food and material industries.

Acknowledgments

This work (Grant No. C0004405) was supported by the Business for Cooperative R&D among Industry, Academy, and Research Institute of the Korean Small and Medium Business Administration in 2012. This research was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), the Ministry of Education (Grant No. 03030504).

Conflict of Interest


The authors have no financial conflicts of interest to declare.

Fig 1.

Figure 1.Comparison of bacterial nanocellulose production by strains isolated from ripened persimmons. Box with asterisk indicates significantly different at p < 0.05.
Journal of Microbiology and Biotechnology 2019; 29: 617-624https://doi.org/10.4014/jmb.1801.01005

Fig 2.

Figure 2.Neighbor-joining tree inferred from 16S rRNA showing the relationship of strain SFCB22-18 with the most closely related members of the genus Komagataeibacter. Bootstrap scores of >70 are presented at the nodes. The scale bar indicates the number of nucleotide substitutions per site.
Journal of Microbiology and Biotechnology 2019; 29: 617-624https://doi.org/10.4014/jmb.1801.01005

Fig 3.

Figure 3.FTIR spectra of bacterial nanocellulose. OH: O-H stretching, CH str: C-H stretching, HOH: H-O-H stretching, CH ben: symetric deformation and bending vibration of C-H, COC: C-O-C stretching
Journal of Microbiology and Biotechnology 2019; 29: 617-624https://doi.org/10.4014/jmb.1801.01005

Fig 4.

Figure 4.Effects of (A) cultivation temperature and (B) agitation on the concentration of bacterial nanocellulose produced by Komagataeibacter sp. SFCB22-18. Box with asterisk indicates significantly different at p < 0.05.
Journal of Microbiology and Biotechnology 2019; 29: 617-624https://doi.org/10.4014/jmb.1801.01005

Fig 5.

Figure 5.Effects of (A) carbon source, (B) glucose concentration, (C) nitrogen source, (D) yeast extract concentration, (E) acid source, (F) acid concentration, (G) phosphate source, and (H) ethanol concentration on the concentration of bacterial nanocellulose (BNC) produced by Komagataeibacter sp. SFCB22-18. Box with patterns refers to the condition of Hestrin-Schramm (HS) medium. Bars with different letters are significantly different at p < 0.05.
Journal of Microbiology and Biotechnology 2019; 29: 617-624https://doi.org/10.4014/jmb.1801.01005

Fig 6.

Figure 6.Bacterial nanocellulose concentration grown under optimized conditions during the six weeks of fermentation. Bars with asterisk indicates significantly different at p < 0.05.
Journal of Microbiology and Biotechnology 2019; 29: 617-624https://doi.org/10.4014/jmb.1801.01005

Fig 7.

Figure 7.SEM images of bacterial nanocellulose synthesized by Komagataeibacter sp. SFCB22-18 after 6 weeks of cultivation in (A) typical Hestrin-Schramm (HS) medium and (B) modified medium (HSM).
Journal of Microbiology and Biotechnology 2019; 29: 617-624https://doi.org/10.4014/jmb.1801.01005

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