Introduction

Cellulose is one of the most abundant organic substances on the planet, being the main constituent of vegetable fiber. Cellulose is a linear hydrophilic homopolysaccharide and this structure consists of units of β-d-glucopyranose (β-glucose) linked by glycosidic type β-(1 → 4) (Fig. 1). However, cellulose is not found only in plant organisms. In addition to plants, different microorganisms are capable of producing cellulose as Vallonia, Disctystelium slime mold (fungi), Agrobacterium, Aerobacter, Pseudomonas, and Sarcina. However, Acetobacter xylinum (Glucanacetobacter xylinus) is the only known species capable of producing cellulose in commercial quantities and is therefore extensively studied [1–5].

Fig. 1
figure 1

Molecular structure of cellulose with degree of polymerization (DP) n

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Cellulose, due to its high availability and outstanding chemical and physical properties, is a polymer widely used in a variety of commercial applications in the food, textile, and pharmaceutical industries. For these and other applications, cellulose is obtained from mechanical and chemical (pulping) processing of woods to remove lignin and hemicelluloses, in this case, a pulp is obtained commercially as a percentage of α-cellulose ranging between 96 and 98%. The bacterial cellulose (BC) obtained from A. xylinum is pure with about 99% cellulose without the need to perform steps of delignification and bleaching. The material in this condition can be employed in the production of cellulosic derivatives; since for production of these derivatives, cellulose must have the highest purity possible [6].

The production of cellulose derivatives significantly broadens the applications of cellulose by modifying the polymer chemically in terms of hydrophobicity, processability, and solubility. For application as excipient in pharmaceutical industry, an important aspect is the improvement in processability of cellulose; in this sense, the most important cellulose derivative is microcrystalline cellulose (MCC). MCC, introduced in the early 1960s, is regarded as the best excipient for direct compression tableting [7]. The MCC is typically produced from wood pulp and cotton linters; recently, new lignocellulosic sources have been used in the production of MCC. However, all these sources have in common the need for removal of lignin and hemicellulose for subsequent production of MCC.

In this study, we propose the use of BC produced by G. xylinus as a source for production of MCC due to its high purity, which excludes the need of carrying out the deslignification and bleaching steps necessary for lignocellulosic materials. The MCC is obtained on an industrial scale by hydrolysis of cellulose using dilute mineral acids. Once the cellulose obtained from different sources differs in properties (crystallinity, moisture, surface and pore structure, and molecular mass), different properties of MCC are expected. Considering this aspect, in this study, MCC produced from BC was compared with vegetal MCC and commercial MCC. The samples were characterized by degree of polymerization (DP) obtained by technique of viscosity, via changes in the crystallinity degree and the pattern of X-ray diffraction (XRD) (Wide Angle X-ray Diffraction—WAXD) and evaluated in terms of change in the thermal stability of samples followed by thermogravimetric analysis (TG). The modification of fiber structure was accompanied by Scanning electron microscopy (SEM).

Experimental

MCC preparation

Bacterial cellulose membranes were supplied by Fibrocel Produtos Biotecnológicos LTDA, Ibiporã, Brazil. Vegetal cellulose (VC) sheets produced from bleached Kraft Eucalyptus pulp were supplied by Bahia Pulp, and commercial MCC was supplied by Merck (cellulose microcrystalline for thin-layer chromatography).

The degree of polymerization (DP) of cellulose samples was determined by viscometry using an Ostwald viscometer according to Brazilian technical standard NBR7730 [8].

To achieve the synthesis of MCC, mineral acids (HCl, H2SO4, and HNO3) are commonly used [9]. In this study, the cellulose fibers (BC and Vegetal Cellulose) were hydrolyzed in 2.5 mol L−1 of H2SO4 at a reflux system under constant stirring for 120 min according to Paralikar et al. [10]. The hydrolyzed pulp was thoroughly washed with distilled water until pH 7.0, and was wetted with ethanol and dried in an oven at 37 °C until constant mass.

X-ray powder diffraction studies

Diffraction patterns were obtained using a Kristalloflex Simens X-ray diffractometer. The diffraction patterns were recorded using Cu-Kα radiation with Ni filter from 4° to 70°. The samples were pressed into pellets (25 mm in diameter) by compression of 0.25 g. These diffraction patterns were intended to identify structural changes in crystalline and amorphous phases after processing the cellulose in the derivative studied (MCC). The separation of crystalline peaks and amorphous halo was performed by deconvolution using the function Pseudo—Voigt 1, whose equation is described below:

$$ y = y_{0} + A\left[ {m_{\text{u}} {\frac{2}{\pi }}{\frac{{w_{\text{L}} }}{{4(x - x_{\text{c}} )^{2} + w_{\text{L}}^{ 2} }}} + (1 - m_{\text{u}} ){\frac{{\sqrt {4\,{\text{In 2}}} }}{{\sqrt {\pi w_{\text{G}} } }}}\left( {{{ - 4\,{\text{In }}2} \mathord{\left/ {\vphantom {{ - 4\,{\text{In }}2} {w_{\text{G}}^{ 2} }}} \right. \kern-\nulldelimiterspace} {w_{\text{G}}^{ 2} }}} \right)(x - x_{\text{c}} )^{2} } \right] $$
(1)

Since W L and W G are the widths at half height for the components of the Gaussian and Lorentz equation, A is the area and m u is the form factor. The crystallinity index can be evaluated from the Eq. 2:

$$ x_{\text{c}} (\% ) = {\frac{{A_{\text{c}} }}{{A_{\text{c}} + A_{A} }}} \times 100 $$
(2)

where A C and A A are the areas of crystalline peaks and amorphous halo, respectively.

Scanning electron microscopy

Scanning electron microscopy was performed using a Top Com 600 system. The samples were coated with a carbon layer with a thickness of 15 nm.

Thermogravimetric analysis (TG)

A SDT (TA Instruments) Thermogravimetric analyzer was used to study the thermal stability of the different MCC samples. The heating rate was set at 10 °C min−1 over a temperature range of 10–900 °C. Measurements were carried out in nitrogen atmosphere, with a rate of flow of 50 cm3 min−1.

Results and discussion

Degree of polymerization

The hydrolysis of BC and the VC to prepare MCC was carried out using 2.5 mol L−1 of sulfuric acid. Viscometry experiments showed that these pulp samples reached a lower degree of polymerization after their reflux with acid during 120 min. It is known that lowering the degree of polymerization is due to degradation of the glycosidic chains of cellulose. Table 1 shows the DP of the MCC prepared from the BC and VC pulps. The results are compared to a commercial Avicell-MCC.

Table 1 Degree of polymerization obtained from viscosity measurements
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The chain length of cellulose, expressed in numbers of constituents of anhydrousglucose, with DP, varies with the source and type of treatment of raw material. In the case of cellulose from wood pulp, the values are generally 300–1700. In the case of cotton and other fibrous plants the values of DP are in the range 800–1500. For the BC, the DP obtained from viscometry was 2800, which equates to an average molecular mass of 453,600 g mol−1. The MCC obtained commercially (Avicell) presents values of DP between 150 and 300 [11]. For the MCC produced in this study, it was obtained a value of degree of polymerization of 250 for the pulp produced with BC, 170 for the MCC produced with plant cellulose, and for the commercial MCC, it was obtained a value of degree of polymerization of 225. These results are within the range reported in the literature.

X-ray diffraction studies

X-ray diffraction is a useful technique to follow the change in cellulose crystallinity as the sequence of reactions progressed [12]. The XRD pattern of BC, VC, and MCC samples is shown in Fig. 2. The calculated crystallinity indexes of the different samples are given in Table 2. As shown in Fig. 2, all samples of MCC have a typical crystal lattice for cellulose I [13]. Also, all samples had similar CrI values with slightly higher values for MCC derived from vegetal cellulose. One possible explanation for this may be related to the morphological differences between VC and BC. Bacterial cellulose has a higher purity and degree of polymerization than vegetal cellulose, moreover, the fibers are arranged to form a hollow space and network structure like a membrane. This allows the BC to absorb large amounts of water when ground and exposed to acidic or basic aqueous solutions. In plants, cellulose is organized in a cellular hierarchical structure linked to other components such as hemicellulose, lignin, and pectin. After delignification and bleaching, cellulose pulp absorbs less water than the BC. These structural differences significantly influence the accessibility and reactivity of cellulose, as well as the final properties of MCC produced. This fact can be observed in the patterns of XRD and degree of crystallinity of the samples of bacterial MCC and BC. The reduction of crystallinity of MCC-BC after the hydrolysis can be explained due to the extensive swelling during hydrolysis possibly leading to disruption of crystalline regions, with the increase of the amorphous sample in comparison with BC and the samples produced from plant cellulose.

Fig. 2
figure 2

X-ray diffraction patterns of BC, VC, MCC-BC, MCC-VC, and Avicell-MCC

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Table 2 Crystallinity index (CrI) of all samples
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The XRD of BC shows three diffraction peaks at 2θ = 15°, 17°, and 22.5°, which are attributed to the interplanar distance characteristic of the phases Iα and Iβ of the elementary cellulose crystalline structure (100 Iα, 110 Iβ, and 010 Iβ refer to the angle 15° and 110 Iα and 200 Iβ to 22.5°) [14]. The hydrolysis modifies the pattern of X-rays with change of relative intensity of the peaks and the appearance of a shoulder around 2θ equals to 21.50. The etching occurs preferentially in the amorphous regions of cellulose. However, changes in the profile of MCC diffractograms indicate change in crystalline structure with a possible contribution of the polymorphic form of cellulose II.

Scanning electron microscopy (SEM)

Figure 3 shows SEM micrographs of the different samples. Through the images we observed that BC (Fig. 3a) presents a three-dimensional structure formed by nanometric fibers. Figure 3b shows the structure of the vegetal cellulose, with thicker fibers than those seen in BC.

Fig. 3
figure 3

SEM graphs of BC (a), VC (b), MCC-Avicell (c), MCC-VC (d), and MCC-BC (e and f)

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According to the images of the samples of MCC, it can be inferred that, by acid hydrolysis, cellulose particles were obtained, similar in appearance and size to those observed for commercial MCC (Fig. 3c) and produced from other sources such as lignocellulosic materials [15]. The images also show that the particles formed are smaller than 30 μm, and also that for the MCC obtained from BC showed more uniformity compared with other samples.

Thermogravimetric analysis

The thermal behaviors of BC and plant cellulose have been reported [16, 17].Thermal stability of the different MCC samples was studied using TG. Figure 4 shows the TG curves of these samples and the Table 3 gives the data obtained from these curves.

Fig. 4
figure 4

TG curves and differential TG curves of BC (a), VC (b), MCC-BC (c), MCC-VC (d), and MCC-Avicell (e)

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Table 3 TG data obtained for cellulose and MCC samples
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The TG curves for all the samples show two stages of mass loss within the temperature range 25–600 °C. The first degradation stage for the samples occurs at a temperature range 25–100 approximately. This process is assigned to water evolution. For BC and VC (Fig. 4a, b), between 270 and 398 °C, we observe a mass loss due to degradation processes of cellulose, such as depolymerization, dehydration, and decomposition of glycosyl units followed by the formation of a charred residue [18]. The same event is observed for the MCC commercial and MCC prepared from BC (MCC-BC) and cellulose plants MCC (VC). For MCC (Fig. 4c (MCC-BC), d (MCC-VC)), we observed a decrease in decomposition temperature compared to original celluloses and commercial MCC. This difference can be attributed to several aspects like the difference between the sources, considering the use of bacterial and plant cellulose and processing differences, since the process for obtaining the commercial MCC is not known in all its stages [19].

The difference between thermal stability of some cellulosic materials showed in Fig. 4 and Table 3 can be attributed to crystallinity, moisture content, the porous structure, and the polymerization degree of materials before and after chemical and/or mechanical treatment. Roman and Winter [20] showed that for samples hydrolyzed with sulfuric acid, the increase of sulfate ions concentration leads to a decrease in degradation temperature of cellulose prepared. The presence of these ions can influence the thermal stability of MCC in different ways according to the porosity of the structure and the ability to absorb water and promote the swelling of bacterial and plant cellulose. In this case, the decrease in thermal stability of MCC-BC in relation to BC can be explained in terms of reduced structural order during the hydrolysis due to swelling of the cellulose, caused by sulfuric acid solution, a good swelling agent for cellulose. This swelling can change crystalline regions on cellulose and increase amorphous fraction. Because of increased accessibility in this case, the degradation temperature of MCC-BC was decreased. The reduction of crystallinity can be confirmed by the results obtained from analysis of X-ray patterns of the samples. For the sample of vegetal MCC (MCC-VC), a decrease in the polymerization degree is one of the aspects that reduce its thermal stability catalyzed by the presence of sulfate ions leading to degradation temperatures of MCC-VC to a value lower than that found for MCC-BC and commercial MCC.

A more detailed observation of the samples TGS MCC-BC (Fig. 4c) and MCC-VC (Fig. 4d) shows a decrease in the slope of the event which occurs between 200 and 450 °C, moreover, the peak in the curves DTG has a shoulder. These aspects are indicative of the presence of a heterogeneous structure [21], which could lead to production of fractions of material with different crystallinities and polymerization degree. In this case, the thermal stability of MCC also depends on the heterogeneity of the sample produced. This fact is not unusual, since the acid hydrolysis proceeds in heterogeneous phase. The existence of heterogeneity can generate fractions of material with greater or lesser resistance to hydrolysis, in this case, the fraction of material less resistant to hydrolysis is responsible for the decrease in thermal stability. The hydrolysis time is one of the essential parameters in reducing the heterogeneity of the samples produced. This is one of the key parameters that have been optimized and will be presented in future study.

In this first study, we are concerned to show the feasibility of producing MCC from a cellulose source was little explored in the production of cellulose derivatives. The results show that the material produced has the characteristics of MCC. The system shows an ivory-white aspect when a viscous suspension is produced; this aspect has been reported in the literature [22].

Conclusions

In this study, we report the viability of producing MCC from the BC from the acid hydrolysis using sulfuric acid. The material was produced as a powder with a DP 250 within the expected range for a commercial MCC. The standard X-ray indicates preferential attack of the amorphous regions with small changes in crystal structure that can be attributed to a contribution of cellulose II.