Production and Characterization of Bacterial Cellulose ... - Springer Link

Biotechnology and Bioprocess Engineering 20: 410-416 (2015) DOI 10.1007/s12257-014-0793-y

RESEARCH PAPER

Production and Characterization of Bacterial Cellulose by Leifsonia sp. CBNU-EW3 Isolated from the Earthworm, Eisenia fetida Palanivel Velmurugan, Hyun Myung, Muthusamy Govarthanan, Young-Joo Yi, Sang-Ki Seo, Kwang-Min Cho, Nanh Lovanh, and Byung-Taek Oh

Received: 14 November 2014 / Revised: 12 February 2015 / Accepted: 1 March 2015 © The Korean Society for Biotechnology and Bioengineering and Springer 2015

Abstract A total of five bacterial strains were isolated from earthworm, Eisenia fetida and examined for bacterial cellulose (BC) production in Hestrin–Schramm medium (HS). Among the five strains tested, CBNU-EW3 exhibited excellent BC production and was identified as Leifsonia sp. by 16S rDNA sequence analysis. BC production by Leifsonia sp. CBNU-EW3 was optimum at pH 5, 30°C, and with glucose and yeast extract as carbon and nitrogen sources, respectively, according to 15 day-long experiments. (XRD) analysis of the dried pellicle indicated that the BC was partially crystalline type I. Fourier transform infrared spectroscopy (FT-IR) analysis showed that the obtained pellicle contained the same functional groups as typical Palanivel Velmurugan†, Muthusamy Govarthanan, Young-Joo Yi, ByungTaek Oh Division of Biotechnology, Advanced Institute of Environment and Bioscience, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan 570-752, Korea Hyun Myung† Department of Ecology Landscape Architecture – Design, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan 570 752, Korea Sang-Ki Seo Korea Rural Community Corp., Naju 520-350, Korea Kwang-Min Cho National Institute of Crop Science, RDA, Iksan 570-080, Korea Nanh Lovanh USDA-ARS, AWMRU, 230 Bennett Lane, Bowling Green, KY 42104, USA Byung-Taek Oh* Plant Medical Research Center, College of Agricultural and Life Sciences, Chonbuk National University, Jeonju 561-756, Korea Tel: +82-63-850-0838; Fax: +82-63-850-0834 E-mail: [email protected] †

These authors contributed equally to this work.

BC. Field emission scanning electron microscopy (FESEM) images showed that the BC micro-fibril matrix consisted of a flat surface with large pore size and cellulose aggregation. Keywords: bacterial cellulose, Leifsonia sp., optimization, characterization

1. Introduction Cellulose is a natural biopolymer composed of glucose monomers with β-1,4 glycosidic linkages. Cellulose represents one of the major groups of industrially important polysaccharides and is widely used in several industries, such as food, textiles, medicine, environment, and paper [1]. Cellulose is commonly extracted from plants as well as some animals and microorganisms. Bacterial celluloses are universally desired for many industrial applications because of their high crystalline content, degree of polymerization, tensile strength, and degree of purity [2]. Several Grampositive and Gram-negative bacterial isolates belonging to Aerobacter, Acetobacter, Achromobacter, Agrobacterium, Alacaligenes, Azotobacter, Gluconacetobacter, Pseudomonas, Rhizobium, Sarcinaa, and Rhodococcus genera have been reported to be efficient producers of bacterial cellulose (BC), an extracellular polysaccharide [3-5]. Indeed, BC is more crystalline and free from other cellular entities compared to plant cellulose. Furthermore, BC has the potential to be a good alternative for several industries that currently rely on plant cellulose, such as those working on creating artificial skin, composite reinforcements, and electronic paper applications [6,7]. Earthworms are the most important soil invertebrates

Production and Characterization of Bacterial Cellulose by Leifsonia sp. CBNU-EW3 Isolated from the Earthworm, Eisenia fetida

due to their capacity for modifying the physical properties of soil and facilitating the decomposition of organic matter. The beneficial effects of earthworms on soil properties are attributed to their feeding activities and interactions with soil microorganisms. Specifically, their pattern of consumption involves the breakdown and incorporation of large amounts of mineral soil and organic matter, both of which contain a variety of microorganisms. In this way, earthworms act as vectors for the dispersal of soil microorganisms. However, there are several key differences between the gut conditions of earthworms and the soil environment [8]. In the present study, we investigated the production of BC by Leifsonia CBNU-EW3 (Chonbuk National University-earthworm isolate 3), which was isolated from a single earthworm species (Eisenia fetida). To date, the majority of similar studies have utilized both Gram-positive and -negative cellulose-producing bacterial isolates, although there has been no previous report on BC production by Leifsonia sp. Gram-positive aerobic bacteria. With respect to BC production, each cellulose-producing bacterial strain has its own optimum cultural parameters for maximum cellulose production. Thus, medium optimization, primarily through selecting an appropriate formulation, is often used to achieve higher BC yields. Indeed, numerous studies have explored the use of different media formulations with various carbon and nitrogen sources in order to optimize BC production [9,10]. In the present study, BC production was optimized according to different sources of carbon and nitrogen, as well as pH, temperature, and incubation time. Accordingly, the objectives of this study were to (i) isolate, identify and screen BC-producing bacteria from earthworms, (ii) identify an optimal media formulation for enhanced BC production, and (iii) characterize BC production using field emission-scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR).

2. Materials and Methods 2.1. Materials and reagents HS media ingredients (peptone, yeast extract and other

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nitrogen source) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glucose, additional carbon sources, disodium hydrogen phosphate, sodium hydroxide and citric acid were purchased from DaeJung Chemicals, Seoul, South Korea. 2.2. Isolation of BC-producing bacteria from earthworms Three earthworms (E. fetida) were collected from grassy soil at Chonbuk National University, Iksan, South Korea. Bacteria from earthworms were isolated as described previously by Kim et al. [8] with minor modifications. Briefly, the surface of each earthworm was washed three times with 70% ethanol, and the whole earthworm was pulverized by mortar and pestle under aseptic conditions. A serially diluted suspension was then plated using the pour plate technique onto Hestrin–Schramm (HS) agar medium composed of an aqueous solution of 2% (w/v) glucose, 0.5% (w/v) peptone, 0.5% (w/v) yeast extract, 0.27% (w/v) Na2HPO4, and 1.15 g/L citric acid. Plates were incubated at 30°C for 7 days and observed for bacterial growth. After incubation for 7 days, white creamy bacterial colonies were isolated according to colony morphology (Fig. 1). To prepare pre-inoculums, individual isolates were inoculated into HS liquid medium and incubated at 30°C for 48 h under static conditions. Next, each pre-inoculum was inoculated (10% v/v) into 50 mL of liquid production medium in 500 mL Erlenmeyer flasks, followed by incubation at 30°C for 12 days to screen for BC production. Among a total of five isolates, one was identified as having potent BC pellicle production and was stored at 4°C for further use. 2.3. Bacteria strain identification DNA extraction and 16S rDNA sequencing was used to identify strain CBNU-EW3. Chromosomal DNA extraction was performed according to the methods developed by Kim et al. [11] and Rainey et al. [12]. Briefly, 16S rRNA bacterial universal primers 27f (5’-AGAGTTTGATCA TGGCTCAG-3’) and 1492R s (5’-TACGGTTACCTTGTT ACGACTT-3’) were used for PCR amplification. Purified amplicons were sequenced using ABI PRISM (Model 3700, CA, USA). The most closely related species to the strain were identified using BLAST from the NCBI database,

Fig. 1. Earthworm bacterial colony morphology on HS agar medium and the BC produced by Leifsonia sp. CBNU-EW3.

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which led to the identification of Leifsonia sp. [13]. 2.4. Optimization of BC production HS medium was modified to investigate the effects of different carbon and nitrogen sources, as well as pH, temperature and incubation time on the production of BC. Glucose was substituted with 11 carbon sources, namely, sucrose, mannitol, fructose, glycerol, xylose, cellobiose, galactose, arabinose, sorbitol, starch and ethanol, in HS medium at a final concentration of 20 g/L. With respect to the nitrogen source, yeast extract was replaced with several organic and inorganic sources, namely, peptone, potassium nitrate, ammonium nitrate, di-ammonium phosphate, ammonium chloride, ammonium sulphate, sodium nitrate, urea, ammonia, ammonium ferrous sulfate, and monosodium glutamate, all at a final concentration of 5 g/L. Different pH (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0), temperature (20, 25, 30, 35, and 40°C) and incubation times (1 ~ 15 days) were also studied. 2.5. Purification of BC pellicles After production of BC at optimum conditions for 9 days at 30°C, BC pellicles were harvested by discarding the media and washing with copious amounts of nanopure water (conductivity = 18 µΩ/m, TOC < 3 ppb, Barnstead, Waltham, MA, USA). Next, the pellicles were rinsed three times in a 2 wt. % NaOH solution at 90°C for 60 min to eliminate adherent bacteria. Finally, samples were repeatedly washed with copious amounts of distilled water until the pH was less than 7.0. The wet cellulose pellicles were then dried at 105°C until a constant weight was reached, after which the concentration was determined as g/L (mass (g) of BC/volume (L) of culture medium). The isolated pellicles were used for further instrumental analysis. 2.6. Characterization of BC A portable digital pH meter (Hanna, HI 9124, USA) was used to evaluate pH changes. FT-IR spectra of the BC pellicle were obtained with a Perkin-Elmer FT-IR spectrophotometer (Norwalk, USA) in diffuse reflectance mode at a resolution of 4 particles/cm in KBr pellets. XRD was used to determine the crystalline nature of the BC pellicle using a Rigaku X-ray diffractometer (Rigaku, Japan). BC morphology was studied by field emission scanning electron microscope (FESEM, AURIGA, Carl Zeiss AG, Jena, Germany) after gold coating.

3. Results and Discussion 3.1. Bacteria isolation and identification The present study evaluated the potential for using bacteria

Biotechnology and Bioprocess Engineering 20: 410-416 (2015)

isolated from earthworms to determine the production of industrially important BC. After screening several bacteria isolates for BC production, one bacterial isolate was identified as a potent producer and was designated CBNUEW3. Polymerase chain reaction amplification of the partial 16S rDNA of the CBNU-EW3 isolate resulted in the predicted 900-bp amplicon. The amplified PCR product of the isolate was then sequenced and compared with available sequences in the NCBI database. The CBNU-EW3 isolate exhibited a 97% identity with that of Leifsonia sp. (GenBank Accession No. HM58775). These results were consistent with previous studies reporting the isolation of Leifsonia sp. from whole earthworm flora [14,15]. 3.2. Production and optimization of BC Fig. 2A shows the production of BC as a function of pH. The maximum BC production was obtained at pH 5.0 after incubation for 12 days. This result was consistent with previous reports of several Gram-positive and Gram-negative bacteria strains capable of producing high yields of BC [16-19]. Specifically, two studies reported that BC production depends on pH and bacterial strain, wherein a neutral to slightly acidic pH range was found to be optimal [7]. Interestingly, BC production was high at an optimum pH range, but was relatively low to non-existent when initiated during HS medium preparation before pH adjustment. Neutral pH plays a key role in BC production by converting glucose into (keto) gluconic acid, which decreases cell viability and lowers pH [20]. Fig. 2B shows the influence of incubation time on BC production by Leifsonia sp. CBNU-EW3, which was initiated at an initial pH of 5. The initial formation of the BC pellicle was visible on top of the media by the fifth day and continued through the 15th day of incubation at 30°C. In this experiment, BC production was initiated by utilization of dissolved oxygen in the medium, which stimulated bacteria close to the surface of the liquid medium to produce cellulose in pellicle form. In addition, compared with other BC-producing strains, Leifsonia sp. CBNU-EW3 required a slightly longer time for BC production. Temperature is a crucial parameter that affects both growth and cellulose production. Fig. 2C shows that optimal production of BC (up to 3.5 g/L) by Leifsonia sp. CBNU-EW3 was achieved at 30°C and an initial pH of 5. This result was consistent with several other previous reports showing that 30°C is optimal for BC production in several bacteria strains [19,21-23]. BC production by Leifsonia sp. CBNU-EW3 was also investigated using culture media enriched with different carbon sources, as shown in Fig. 2D. The mass of BC produced per liter of medium was determined after incubation for 15 days in static culture at 30°C. The glucose-rich source yielded the highest amount (4 g/L) of BC, whereas


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Fig. 2. BC yield at various optimized parameters of (A) pH, (B) incubation time, (C) temperature, (D) carbon source, and (E) nitrogen source.

sucrose yielded 3.8 g/L, mannitol 3.8 g/L, fructose 2.7 g/L and glycerol 2.5 g/L. Maltose, cellobiose, xylose and galactose appeared to be less suitable carbon sources for BC production by Leifsonia sp. CBNU-EW3. Specifically, xylose, cellobiose, galactose, arabinose, starch and ethanol produced a smaller BC pellicle, the appearance of which indicated that these were not suitable carbon sources for BC production (Fig. 2E). Nitrogen-rich yeast extract yielded

3.8 g/L BC, followed by peptone (3.4 g/L), potassium nitrate (3.2 g/L), ammonium nitrate (3.0 g/L) and to a lesser extent di-ammonium phosphate, ammonium chloride, ammonium sulphate, sodium nitrate and urea. Ammonia, ammonium ferrous sulfate and monosodium glutamate resulted in an inadequate BC yield and were deemed as inappropriate nitrogen sources for BC production by Leifsonia sp. CBNU-EW3.

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3.3. Fourier transform infrared spectroscopy analysis of BC Fig. 3 shows the FT-IR spectra of BC produced by Leifsonia sp. CBNU-EW3. The position and intensity of peaks in the FT-IR spectra revealed that Leifsonia sp. CBNU-EW3 produced a polymeric form of BC. Characteristic absorption peaks of bacterial cellulose were absorbed at 3350/cm and also showed several bands at 1600 ~ 1250/cm, indicative of O-H stretching related to the cellulose region and CH stretching at 2930/cm. In addition, the absorption bands at 1650, 1560, 1424, and 1060/cm were attributed to C-H stretching of -CH2, O-H bending of adsorbed water, -CH2 symmetric bending and C-O-C pyranose ring skeletal vibrations, respectively [3,7,24-27]. According to studies by Nelson & Connor [28] and Bhavna et al. [29], the weak, broad band at 891/cm and strong band centered at 1424 cm-1 in the spectra of microbial cellulose correlates with CH2 scissoring, which is highly indicative of cellulose. By correlating the FT-IR spectrum described above with previous literature on BC production, we confirmed that the pellicle obtained from Leifsonia sp CBNU-EW3 was indeed bacterial cellulose.

3.4. X-ray diffraction analysis of BC Fig. 4 shows the XRD pattern of the BC pellicle obtained from Leifsonia sp. CBNU-EW3 in HS medium. There were two distinct peaks at 2θ = 15.5° and 22.5°, corresponding to crystallography planes (010) and (110), respectively. The fraction Iα and Iβ mixture of allomorphs was attributed to the cultivation condition of the bacteria and also the ratio of cellulose, which may differ according to bacterial strain. All of these results were consistent with earlier published X-ray data for BC obtained with other bacteria [7,25,26,2934]. However, compared with other bacteria, the peaks of BC produced by Leifsonia sp. CBNU-EW3 were of decreased intensity, indicating a lower crystallinity (Fig. 3).

Fig. 3. FT-IR spectrum of the BC membrane of Leifsonia sp. CBNU-EW3.

Fig. 4. XRD pattern of the BC membrane of Leifsonia sp. CBNUEW3.

3.5. Morphological identification of BC using FE-SEM The morphological, surface, and inner matrix of BC pellicles were analyzed by FE-SEM, and the results are presented in Figs. 5A and 5B. FE-SEM analysis showed that the BC pellicle consisted of an ultrafine network of cellulose microfibrils. The fibrils had a high aspect ratio and were

Fig. 5. FE-SEM micrographs of the morphology of the BC membrane of Leifsonia sp. CBNU-EW3.


assembled together to form a porous structure (Fig. 5B). Cakar et al. [35] reported that BC production in HS medium for 15 days tends to result in a light fibril structure, consistent with our findings. They also reported that BC fibrils generated in this way have a web-like structure due to the relatively longer incubation time. Fig. 5A shows the flat surface of the BC pellicle, which consisted of larger pores and cellulose fibril aggregation.

4. Conclusion We report for the first time the production of BC by the Gram-positive bacteria Leifsonia sp. CBNU-EW3 isolated from the earthworm. The production of BC was optimized using different sources of carbon and nitrogen, as well as different temperatures, pH, and incubation times. Leifsonia sp. CBNU-EW3 exhibited similar BC production compared with previous BC-producing Gram-positive strains. Microscopic evaluation of the produced BC revealed a strong network of micro fibrils. Broadly, our results suggest that Leifsonia sp. CBNU-EW3 is one of the more potent BCproducing Gram-positive bacteria strains identified to date. In conclusion, BC produced from Leifsonia sp. CBNUEW3 could play an important role in diverse industrial applications as an alternative to cellulose obtained from other sources, such as plants.

Acknowledgement This research was supported by a National Research Foundation of Korea (NRF) grant funded by the government (MEST; No. 2011-0020202).

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