Structural Properties of Bacterial Cellulose Film Obtained on a Substrate Containing Sweet Potato Waste

Abstract

The paper presents the results of research on the microstructure of bacterial cellulose (BC-SP) obtained on a medium containing sweet potato peel, which was compared to cellulose obtained on a synthetic medium containing sucrose and peptone (BC-N). The properties of cellulose were analyzed using the methods: size exclusion chromatography (SEC), X-ray diffraction (XRD), scanning electron microscope (SEM), and computer microtomograph (X-ray micro-CT). BC-SP was characterized by a higher degree of polymerization (5680) and a lower porosity (1.45%) than BC-N (4879, 3.27%). These properties give great opportunities to cellulose for various applications, e.g., the production of paper or pulp. At the same time, for BC-SP, a low value of relative crystallinity was found, which is an important feature from the point of view of the mechanical properties of the polymer. Nevertheless, these studies are important and constitute an important source of knowledge on the possibility of using cheap waste plant materials as potential microbiological substrates for the cultivation of cellulose-synthesizing micro-organisms with specific properties.

1. Introduction

Bacterial cellulose (BC) is a polymer with great application potential, synthesized by aerobic micro-organisms. Due to its high mechanical strength, high crystallinity, and a much greater degree of polymerization than plant cellulose, it has become a promising polymer for use in various technical fields, and even in medicine.

The main quality parameters of cellulose, determining its desired properties, is the crystallinity and the degree of polymerization. Allomorph Iα is dominant in the bacterial polymer. Aleshina et al. [1] indicate that it may constitute from 70 to 100% of the morphological composition, and additionally the quality and composition of the culture medium on which cellulose is synthesized affects its level in cellulose. Skiba et al. [2] reported that the synthesis of cellulose on unconventional substrates from plant materials causes a reduction in crystallinity and a decrease in the content of Iα in the polymer. The same authors, referring to the works of other authors, indicated that cellulose synthesized on a substrate from agricultural waste in the form of grape bagasse is characterized by a content of allomorph Iα from 70 to 56%. Another important morphological parameter influencing the high tensile strength of cellulose is the degree of crystallinity. In addition, this parameter may vary depending on the method of culturing cellulose-synthesizing micro-organisms [3], the types of carbon source and other components of the medium [4], or the procedure and method of drying [5]. 6. Illa et al. [6] showed that in the case of conventional drying, the degree of crystallinity of bacterial cellulose was slightly higher than during drying by lyophilization. Particular attention is paid to the influence of the composition of the culture medium on the degree of crystallinity of the cellulose. Because the development of low-cost culture media, on which it will be possible to obtain high-quality polymer, additionally with high efficiency, can guarantee its commercial application. Xu et al. [7], using a substrate of sweet potatoes, obtained cellulose with a crystallinity ranging from 83 to 87%. Other authors report that cellulose obtained on substrates containing agricultural waste in the form of oil palm leaf juice [8] or sweet sorghum leaves [9] was characterized by much lower crystallinity.

The properties of bacterial cellulose are also inextricably linked to its degree of polymerization, which is much higher than that of its plant-based counterpart and can be up to 20,000 [10]. Like crystallinity, the degree of polymerization of cellulose can be influenced by various external factors accompanying the synthesis process by micro-organisms. Surma-Ślusarska et al. [11] obtained cellulose on a substrate with glucose and mannitol with a degree of polymerization of approximately 1700, while Betlej et al. [12] obtained a cellulose polymerization degree of 6080 on a substrate with sucrose and peptone.

The conditions for culturing cellulose-synthesizing micro-organisms, including the composition of the culture medium, have a significant impact on the structural features of cellulose, which will reflect its properties. One of the key features of a bacterial polymer, determining its potential utility, is tensile strength and porosity. Porosity seems to be of particular importance in the case of the use of cellulose in the form of medical dressings, being gas-permeable and thus preventing the growth of anaerobic bacteria in places protected by it [13].

However, it should be remembered that the guarantee of the production volume of bacterial cellulose and its global demand is the reduction of production costs, while maintaining excellent physical and mechanical properties. According to Rivas et al. [14], the cost of cultivation on standard microbiological media may account for approximately 30% of the total cost of the process, therefore, efforts should be made to search for alternative sources of nutrients in the processes of microbial cultivation. It seems that a good alternative to synthetic substrates may be waste from plant production, which are rich in sugars, proteins, vitamins, and microelements necessary for the development of cellulose-synthesizing micro-organisms. At the same time, the management and reuse of plant waste can bring many benefits, including by reducing the costs of exportation and disposal or the production of new products.

The aim of the study was to investigate the structural features of bacterial cellulose, such as crystallinity, degree of polymerization and porosity, obtained on the culture medium from sweet potato peel and to compare them to the characteristics of cellulose obtained on a semi-synthetic medium containing sucrose and peptone. The indirect goal of the study was therefore to determine the suitability of plant waste materials, grown in many countries on a large scale, as a low-cost substrate for the production of high-quality polymer for various applications. In this way, we indicate environmentally friendly methods of bacterial cellulose production, which can be used in many industrial areas.

2. Materials and Methods

Bacterial cellulose (BC) was synthesized by micro-organisms known as Symbiotic Culture of Bacteria and Yeast (SCOBY) grown on two types of media. SCOBY were obtained from the organic farm Wolanin (Wolanin, Szczawnik, Poland). According to literature data, the dominant bacterial cultures are the species Acetobacter xylium, A.pasteurianus, A. aceti, and Gluconobacter oxydans [15], among the fungi yeasts belonging to Saccharomyces, Saccharomycodes, Schizosaccharomyces, or Zygosaccharomyces [16] are the those that are dominant. The test cultures were stored on agar slants containing 0.03% peptone (Biomaxima SA, Lublin, Poland), 0.05% yeast extract (Biomaxima SA, Lublin, Poland), 2.5% glucose (PPF HASCO-LEK S.A., Wrocław, Poland), and 2.5% agar (AphaVit, Biała Podlaska, Poland). Before starting the experiment, an inoculum of micro-organisms was taken and introduced into 100 cm³ of a liquid medium containing peptone, yeast extract, and glucose and cultured for 14 days in a heat incubator. During this time, the formation of bacterial cellulose on the surface of the medium was checked. Cultures were carried out in glass beakers with a diameter of 5 cm. The test culture was homogenized and used for inoculation of the media used in the test.

The reference medium contained 10% sucrose (Krajowa Spółka Cukrowa SA, Toruń, Poland) and 0.03% peptone (Biomaxima SA, Lublin, Poland). The second type of medium was based on ingredients of vegetable origin (sweet potato peel), treated as waste. The sweet potato tubers were stored at 4 °C before the start of the study. To prepare a broth medium based on plant material: 200 g of sweet potato peel, varieties ‘Carmen Rubin’, ‘Purple’ and ‘Beauregard’, grown in the field in Żyznów (49°49′ N 21°50′ E, Poland) on the soil of the defective wheat complex, with a slightly acidic reaction (pH = 6.1, in 1N KCl), suspended in 500 cm³ of water and ground with a blender, model MMBM401W (Bosch, Gerlingen, Germany). Thus, a homogeneous homogenate was prepared. The individual sweet potato homogenates were combined and then mixed. The homogenate was then filtered through the filter paper using a water pump, separating the clear solution from the solids. A clear solution was used as a microbiological broth medium, divided into equal portions, and sterilized in a steam autoclave (Spółdzielnia Mechaników SMS, Warsaw, Poland) for 20 min at 121 °C. A total of 1 cm³ of the inoculum was sterile added to both types of media. Cultures were incubated in a heat incubator (J.P. Selecta Laboratory Equipment Manufacturer, Barcelona, Spain) for a period of 14 days. The incubation temperature was 26 ± 2 °C. After the end of the cultivation time, the cellulose was purified according to the procedure described by Betlej et al. [17]. Both the cellulose obtained on the standard medium (BC-N) and the cellulose obtained on the sweet potato peel medium (BC-SP) were washed several times with distilled water, then rinsed in 0.1% NaOH solutions (Avantor Performance materials Poland SA, Gliwice, Poland) and 0.1% citric acid (Avantor Performance Materials Poland SA, Gliwice, Poland). Distilled water was always used between uses of the individual alkali and acid solutions and at the end of the rinsing process. The polymer thus prepared was dried at a temperature of 24 ± 2 °C in a laboratory dryer (J.P. Selecta Laboratory Equipment Manufacturer, Barcelona, Spain) until obtaining the constant mass of the polymer. The total sugar content in individual sweet potato varieties was presented and described by Krochmal-Marczak et al. [18] in earlier studies (

Table 1. Total sugars in sweet potato with peel based on studies by Krochmal-Marczak et al. [16].

Cultivars	Total Sugars (Average) g kg−1 FM *
‘Purple’	4.90
‘Beauregard’	7.42
‘Carmen Rubin’	9.43

* FM—fresh matter.

). Krochmal-Marczak et al. [19] in other studies reported that the average protein content in dry matter in the raw material used is 1.35 g 100 g⁻¹, the average content of vitamin C is 22.86 mg 100 g⁻¹, and macroelements (P, K, Ca, Mg, Na) are 0.26, 2.12, 0.51, 0.13, and 0.19 mg 100 g⁻¹, respectively

2.1. Polymerization Degree and Crystallinity of Bacterial Cellulose

The degree of polymerization of bacterial cellulose was determined by the size exclusion chromatography (SEC) method [20]. The degree of polymerization of bacterial cellulose were determined according to the methodology described by Antczak et al. [21] and Waliszewska et al. [22], with changes described by Betlej et al. [12]

The crystallinity of polymer was analyzed using a TUR M-62 X-ray diffractometer (Carl Zeiss AG, Jena, Germany) with the method described by Betlej et al. [12]. On the basis of XRD tests, the structural parameters of cellulose were determined:

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Crystallite size was calculated using the Scherrer equation (Equation (1)):

$$\mathrm{D}=\frac{\mathrm{k}·\mathrm{\lambda }}{\mathrm{\beta }·\cos \mathrm{\theta }}$$

(1)

where D is the crystallite size perpendicular to the plane; k-Scherrer constant; λ is the X-ray wavelength; β is the full-width at half-maximum in radians; and θ is the Bragg angle.

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The crystallinity of bacterial cellulose by comparison of the areas under crystalline peaks and the amorphous curve was determined. Deconvolution of peaks was performed by the method proposed by Hindeleh and Johnson [23].

After the separation of X-ray diffraction lines, the relative crystallinity was determined by comparing the areas under crystalline peaks and the amorphous curve. Relative crystallinity (%) was calculated using Equation (2).

$$\mathrm{Relative} \mathrm{crystallinity}=\frac{\mathrm{crystalline} \mathrm{area}}{(\mathrm{crystalline}+\mathrm{amorphous})\mathrm{area}}$$

(2)

2.2. Microstructure of Bacterial Cellulose

The microstructure of bacterial cellulose was examined using a Hitachi scanning electron microscope, (TM-3000, Hitachi Ltd., Tokyo, Japan). Gold was used as a sputter (Cressington 108 auto sputter coater, Netherlands). The cross-section was observed. The photos of the samples at accelerating voltages equal to 15 kV were taken with 500 and 1000 magnification, and the record was saved using SEM software (TM3000, Hitachi Ltd., Tokyo, Japan).

2.3. Porosity Analysis

To examine the porosity of bacterial cellulose, samples were analyzed using X-ray micro-CT Skyscan 1272 system (Bruker, Kontich, Belgium). The parameters of the process carried out were as follows: X-ray source, voltage at 40 kV, and 193 μA current. Scans were done with a rotation step of 0.3° and a resolution of 25 μm. NRecon software (Bruker, Kontich, Belgium) was used to reconstruct cross-section images from μCT projection into 3D images. The determination of porosity was done with the application of CTAnn software (Bruker). Raw images were binarized at a threshold value of 25–255, and custom processing with internal plugins (despeckle, ROI shrink-wrap, 3D analysis) were applied for the selected volume of interest. The images were binarized by means of assigning pixels with lower intensity as background (air, pores) and pixels with higher intensity as matter. Two samples of each experimental variant were scanned.

2.4. Statistical Analysis

TIBCO company software (STATISTICA program, version 13, Palo Alto, CA, USA) was used to conduct the ANOVA analysis. The samples of bacterial cellulose film were divided into homogenous groups with the use of Tukey’s test (α = 0.05).

3. Results

3.1. Characteristics of the Crystallinity and Degree of Polymerization of Bacterial Cellulose

Bacterial cellulose is a polymer characterized by high crystallinity, which is a decisive feature influencing the mechanical and physical properties of the polymer. XRD analysis is a key method for imaging crystallinity to verify the effect of various nutrient media on the crystallization properties of BC. X-ray patterns of the BC-N and BC-SP polymers presented in Figure 1 show significant differences in the heights as well as the widths of the diffraction peaks, which proves some changes in the supermolecular structure. In the case of BC-N obtained on a standard medium, typical diffraction maxima originating from the polymorphic variety of cellulose I were observed (Figure 1). The recorded diffraction peaks at the diffraction angles of 2θ corresponded to the crystal planes (100), (010), (110) of cellulose type I_α [1]. On the basis of the performed calculations, it has been shown that for bacterial cellulose from standard medium the value of the degree of crystallinity is 65%, which is close to the crystallinity value obtained on Hestrin–Schramm substrates, so far considered as reference substrates for cellulose-synthesizing micro-organisms [24]. The crystallinity of the cellulose obtained on the sweet potato medium was relatively low at 27%. Fan et al. [25] also observed lower crystallinity of cellulose obtained on media containing plant components.

The conducted research also showed significant differences in the determined sizes of crystallites in individual types of cellulose. It can be noted (

Table 2. Based structural properties of bacterial cellulose obtained from different medium broth.

Parameter	BC-N	BC-SP
Crystallite size (D) of (100) plane (Ǻ)	44.4	70.3
Crystallite size (D) of (010) plane (Ǻ)	56.4	94.1
Crystallite size (D) of (110) plane (Ǻ)	50.2	77.6
Relative crystallinity (%)	65	27
Parameter	BC-N (SD) *	BC-SP (SD) *
Molar mass:
Number average Mn (kg/mol)	266 a (29)	336 b (11)
Weight average Mw (kg/mol)	791 a (14)	920 a (55)
Molar mass dispersity Đ	3.01 a (0.41)	2.75 a (0.28)
Polymerization degree DPw	4879 a (98)	5680 b (383)

* SD—standard deviations in parentheses. The different lowercase letters in row show different homogeneous groups with the use of Tukey’s HSD test with α = 0.05.

) that bacterial cellulose from sweet potato peel medium is characterized by a much larger crystallite size (70–94 Ǻ depending on the plane) compared to BC-N cellulose, where the crystallite size is in the range of approximately 44–56 Ǻ) (Table 2). The reason for this phenomenon can also be seen as BC-SP is not a pure cellulose. On the subject, information can be found that bacterial cellulose contains up to 90–95% pure cellulose, the remaining components may be fractions of other polysaccharides, such as levane [26].

Despite its low crystallinity, BC-SP is characterized by a higher degree of polymerization compared to BC-N (Table 2). The reason for this can be seen in the greater availability of saccharides in the sweet potato medium than in the standard medium containing only sucrose. Sweet potatoes are a rich source of sugars, both mono and polysaccharides [27], and the latter can be broken down by enzyme into simple sugars, which are then used by micro-organisms not only for energy purposes but also in the process of polymer synthesis. In addition, the medium based on plant ingredients is rich in compounds such as vitamins, minerals, and enzymes, which can additionally regulate cellular processes or affect complex enzyme complexes involved in the biosynthesis of the polymer [28].

3.2. Microstructure Identification Using SEM

Figure 2 illustrates the surface cross-sections of bacterial cellulose. The cross-section of the polymer obtained on the sweet potato peel medium differs significantly from that obtained on the standard substrate. BC-N has a clearly layered structure in which the individual layers are significantly folded and clearly visibly separated from each other. Void spaces between the layers are observed. The cross-section of the BC-SP is completely different. The individual layers of the polymer clearly adhere to each other, creating a uniform structure. The cross-section structure is not folded, and the individual layers are flat and firmly integrated with each other.

3.3. Porosity of Bacterial Cellulose

Porosity is one of the most important morphological parameters of materials. It is particularly important for the application of bacterial cellulose in papermaking [29] or as a medical product [30]. Tang et al. [31] showed that the porosity of cellulose depends not only on the conditions and method of cultivation but also on the polymer drying process. BC-SP cellulose was characterized by a smaller number of pores than BC-N cellulose, which may correlate with a greater degree of polymerization and thus a greater amount of microfibers and a more compact structure, which was confirmed by SEM tests. The tested polymers were characterized by exceptionally low porosity (

Table 3. Porosity of bacterial cellulose.

Parameter	BC-N (SD) *	BC-SP (SD) *
Total volume of pore space	0.43 a (0.09)	0.18 b (0.08)
Total porosity (%)	3.27 a (0.11)	1.45 b (0.22)

* SD—standard deviations in parentheses. The different lowercase letters in row show different homogeneous groups with the use of Tukey’s HSD test with α = 0.05.

). The low porosity of the two types of polymers obtained may also be due to the mild drying conditions. Moreover, as reported by Tang et al. [31], the carbon sources in the medium also have an effect on the porous structure of cellulose. The observed morphological changes may be a consequence of the use of microbiological media with a specific composition. Molina-Ramírez et al. [32] by examining the different composition of the substrate on the morphology of the synthesized cellulose using SEM scanning microscopy, showed that the nutrients contained in the microbiological substrate affect the degree of porosity, which results from the density of the cellulose nanofiber network. Studies by other authors have shown that the synthesis of bacterial cellulose on various types of substrates does not affect the size of the produced nanofibers, but with some types of substrates a polymer is obtained with a larger amount of micro- and nanofibrils [33]. The same authors also report that crystallinity is inversely related to porosity. The larger the crystal size, the smaller the number of pores, which is consistent with the results of this study.

In this study, the authors showed that cellulose obtained on the sucrose medium broth was characterized by a greater porosity than the polymer synthesized on the medium with sweet potato medium.

4. Conclusions

Culture media play a key role in the economic viability of bacterial cellulose synthesis. Striving to lower the costs of cellulose production on a large scale, readily available and cheap sources of carbon and nitrogen are sought. It seems that waste plant raw materials can successfully replace commercial microbiological substrates, while significantly reducing the costs of cellulose production for various applications. The sweet potato peel medium has proven to be suitable for the synthesis of cellulose with specific quality features. The research results presented in the paper show that the use of a microbiological medium broth based on plant-based ingredients as a medium for the synthesis of bacterial cellulose has an impact on the structural parameters of the polymer. In terms of polymer characteristics, such as degree of polymerization or porosity, it seems that this type of support is better than the standard, which is based solely on sucrose and peptone. The obtained polymer was characterized by a higher degree of polymerization, lower porosity, and a more compact structure. The degree of polymerization of SP-BC was over 14% higher than BC-N, and the percentage of porosity of cellulose obtained on the sweet potato substrate was over two times lower than BC-N. At the same time, from the point of view of crystallinity, the use of a microbiological medium based on sweet-potato peel gives worse results than on a sucrose and peptone based microbiological medium, which was only 27%. It can be concluded that the usefulness of the microbiological medium based on sweet potatoes is desirable, especially for applications of cellulose that should be characterized by a high degree of polymerization, and in this direction, it should intensify the process of polymer synthesis.