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Brazilian Archives of Biology and Technology

Print version ISSN 1516-8913On-line version ISSN 1678-4324

Braz. arch. biol. technol. vol.61  Curitiba  2018  Epub June 25, 2018

http://dx.doi.org/10.1590/1678-4324-2016160696 

Biological and Applied Sciences

Production of Cellulose and Profile Metabolites by Fermentation of Glycerol by Gluconacetobacter Xylinus

Francielle Lina Vidotto 1  

Geovana Piveta Ribeiro 1  

Cesar Augusto Tischer 1   *  

1Universidade Estadual de Londrina - UEL - Departamento de Bioquímica e Biotecnologia - CCE, Londrina, Paraná, Brasil.

ABSTRACT

Because of the widespread occurrence of cellulose in nature, many organisms use glycerol as a source of carbon and energy, so these organisms have drawn attention to the potential use of glycerol bioconversion. The bacteria Gluconacetobacter xylinus, a strictly aerobic strain that performing incomplete oxidation of various sugars and alcohols to cellulose biosynthesis. For this reason, we modify the Hestrim-Schram medium, associating glycerol, glucose and sucrose varying their concentration. The fermentations were performed statically at a temperature of 28˚C for a period of 10 days. The pH, membrane formation, crystallinity and the production of some metabolites of the 4th, 7th and 10th days was evaluated. The results showed a higher yield of membrane in the medium containing glucose, gly 1 + glu2 on 10 fermentation of 3.5 g %. Through solid-state NMR gave the crystallinity of the membranes, where there was a clear trend toward higher crystallinity membranes with 7 days of fermentation. Metabolic products found in the media by analysis of NMR spectroscopy in liquid were similar, especially for the production of alanine and lactate that were present in all media. The leucine and threonine were present in various media, although in small quantities has been found glutamate.

Key Words: Bacterial cellulose; Glycerol; Metabolites; Gluconacetobacter xylinus; Solid state nuclear magnetic resonance; 1H nuclear magnetic ressonance

INTRODUCTION

Cellulose β-(1-4)-D-glucan, is the unbranched polymer composed of dozens of linear glucose units arranged in parallel, forming strongly hydrogen bonds intra and inter regular chain. It is widely distributed in nature, and mainly produced by vascular plants, much of its biosynthesis occurs in plant cell walls, being associated with lignin, hemicellulose and other components1,2. Besides plants, many biological organisms distributed in the realms are able to produce this exopolymer as bacteria, fungi, protists, and tunicates. The bacterial cellulose produced by bacteria is chemically pure, free of components such as lignin and hemicellulose, which increase industrial processes due to chemical treatment to remove these components. It is extremely hydrophilic with greater capacity to absorb water compared with the cellulose originated from plants and have a significantly higher degree of polymerization, and can be produced in different substrates and have different shapes. It is endowed with unique physical properties such as ultrafine fibers highly crystalline meshes and high purity, thus, it is a biological material with potential industrial use. These properties combined with a three-dimensional nanostructure provides a wide range of applications ranging from paper and textile industry to the food on the polymer industry3-5. Among the bacteria producing it the Gluconacetobacter xylinus species is known as the largest producer6, is a gram-negative, able to grow and produce cellulose from various substrates7. A distinct advantage in the study is that its cellulose fibrils are a metabolically inert product, highly pure and of extracellular deposition8,9. To produce cellulose many carbon sources may be used, some substrates have been studied such as mannitol, glycerol, fructose, sucrose and galactose seeking to obtain better yields and characteristics of the cellulose fibers. In pursuit of the use of low cost substrates, glycerol is an option, which is the main by-product of biodiesel production and available in plenty currently. Glycerol is a good source of carbon and energy for the growth of various microorganisms, being suitable for biotechnological production of a number of chemicals in fermentative processes2,10-12. Beyond the production of cellulose, bioconversion of substrates also generates various metabolites such as dihydroxyacetone, propanediol, succinate, citrate, butanediol, lactate, amino acids, among others13,14. Therefore, studies have been devoted to improve the biotechnological production of cellulose through tests of selected strains, different ways of fermentation and different formulations. The objective of the study was to test the association of glycerol per annum with sources of glucose and sucrose, in order to obtain a cellulose yield and analysis of metabolites formed during fermentation. For the characterization of the products obtained during fermentation techniques such as NMR 13C CP-MAS in liquid and solid state.

MATERIAL AND METHODS

Bacterial strain and culture medium

The microorganism used was Gluconacetobacter hansenii ATCC 23769 (former Acetobacter xylinus Yamada) obtained from the André Tosello Foundation of Campinas, São Paulo-Brazil. The medium used was modified Hestrin-Schramm15, wherein the carbon source matched to associations of sources glucose, sucrose and glycerol as follows: glycerol 2% + glucose 4%, glycerol 1% + glucose 2%, glycerol 2% + 8% sucrose and glucose 8%.

Culture methods

The fermentation temperature was static at 28˚C for cooling heating incubator (Sterilifer, São Paulo, Brazil). For each 500 mL of medium culture was added 4 mL of standard bacteria, than divided into 10 plates with approximately 40 mL of medium each. All fermentations lasted for 10 days, and on day 4, 7 and 10 the membranes were removed from the petri dishes and separated from the culture medium.

Treatment of bacterial cellulose

Obtained celluloses underwent through a cleaning process in 2% NaOH solution and then were stored in 1% NaOH solution for 48 hours, after this period it were washed in distilled water until neutralization16.

Yield bacterial cellulose

Produced cellulose mass was determined by drying and weighing the membranes, dried at a temperature of 80˚C for 15-30 minutes. After drying, the membranes were weighed on an analytical balance and their yield calculated according to the formula MCB. 100 / Si, where MCB is the dry mass of cellulose and Si the initial amount of sugar present in each plate, adapted from Goelzer and cols16.

Solid state NMR and Crystallinity index

To obtain the crystallinity index it was used the analysis on NMR of solid state, where the membrane fragments were added in 4 mm rotor and subjected to analyze at CP-MAS NMR probe with rotation of 10k Hz, using Bruker standard pulse. To calculate the crystallinity index (CI) it was carried out the ratio of the integrated peaks Ic / (Ia + Ic) (x100) to crystalline regions / amorphous regions, peak fit using Bruker TopSpin 3.1 and Origin 8.1 software16.

NMR analysis of the metabolites

With the liquid medium removed from the plates, centrifuged and dried by lyophilizing, suspended on Deuterium Oxide 99.9%, lyophilized again, and finally solubilized on Deuterium Oxide 99.9% with 0,312 mM of (trimethyl)propionic-2,2,3,3-d4 acid (TSP) as chemical shift and concentration reference to metabolites analysis by NMR. Samples were performed in multinuclear 5 mm BBI probe in quartz tubes NMR. Programs used in the processing of the NMR spectra were on Chenomx NMR suite, using adapted protocol from Lee17 and cols, and examples of the metabolite profiler tool are showed on supplemental material.

RESULTS AND DISCUSSIONS

Membrane formation and Income

All fermentations occurred initially at pH 5, by the end of fermentation in medium containing glucose the pH varied between 3.69 and 5.8, and in medium containing sucrose the lowest pH was 5.03 and the highest 5.34. The membrane formation began on day 7 of fermentation, medium containing glucose on composition (glu8) was the only that showed it at 4 days of fermentation (Table 1). The membranes obtained with the medium glu8 aspect is thick, swollen and bulky, though has little cellulose mass, corresponding mainly to the water, since this can be absorbing about 100x its weight in water after produced. The water is retained during the process of biosynthesis, trapped by the fibers that develop around18,19.

Table 1 Total carbon mass ratio in the medium added with the mass of the membrane obtained for each medium. 

Yield g%
Composition 4 7 10
glu8 1.9 2.6 3
gly2+glu4 0 0.4 1.7
gly2+sac8 0 0.7 1.1
gly1+glu2 0 1.6 3.5

The medium gly1+glu2 had the highest final yield with 3,5 g%, with a very reasonable income. In a medium containing sucrose, gly2 + sac8, the presence of a thin membrane on day 7 was noticed and day 10 the membrane even thin, occupied the entire area of the petri dish. It was observed that in glu8 a progressive increase of CB, but other medium required a longer time.

Analysis by Nuclear Magnetic Resonance solid (CP MAS13C rmn) of bacterial cellulose membrane

The cellulose samples were submitted for analysis to compare the crystallinity of the material obtained in different ways (Table 2). The amount of the bacterial cellulose crystallinity index in the literature with glucose as carbon source CI vary between 71% and 79%. In media containing sucrose crystallinity the rates found were 69% and 81,2% CI20-22. Values were mostly similar to those observed in previous studies, between 67 and 79%, yet an analysis of absolute crystallinity values indicate a clear trend for a higher crystallinity in the membranes obtained with 7 days of fermentation in medium that had glycerol associated, as gly2+glu4 with 73% CI and gly2+sac8 with 79,4% CI (Table 2).

Table 2 Crystallinity index of the membranes in culture media containing glycerol. 

IC% membrane
Composition 4 7 10
glu8 73 67.6 71.4
gly2+glu4 73 68.5
gly2+sac8 79.4 70.4
gly1+glu2 69.9 67.6

Relating the crystallinity with the income it can be considered that these characteristics are inversely proportional, as on the 7th day of fermentation crystallinity indexes were higher, membranes yields were low. Considering that the pairing of chains occurs as a phenomenon of post synthesis, from a certain chain length this becomes stationary, pressing the movement of bacteria in the opposite direction to the film formation described by Hesse23 with the acronym NOC (nematic ordered cellulose), and results in a less effective pairing of cellulose chains from the CeSa (cellulose synthase)24,25. Besides the crystalline regions, cellulose comprises amorphous regions, which depending on the treatment and the cellulose source its degree of crystallinity varies, and its composition determines the type cellulose membrane forming. The literature describes two types of cellulose produced naturally by G. xylinus, the Cel I under normal conditions, and Cel II when subjected to heat stress or mechanical26.

Analyses of solid NMR spectra were very similar among themselves, showing characteristic signs for mostly crystalline cellulose type Cel I. The chemical signals obtained exhibit a mixture of Cel Iα and Iβ as well as those obtained by Faria-Tischer26, where observed anomeric signal (C1) in the 103 ppm region, the signals for crystalline and amorphous C4 (82-92 ppm) and to C6 between 60 and 65 ppm (Figure 1). Cellulose I is composed of two distinct crystalline phases known as Iα and Iβ which differ by different diffraction patterns, cellulose I (alpha) triclinic and cellulose I (beta) monoclinic. The difference between them is the hydrogen bonds and the cellulose chains frame5,27,28.

Figure 1 CP MAS 13C nmr spectrum of bacterial cellulose produced in medium gly2 + sac8 of 10 days fermentation. 

Metabolite Analysis by liquid state nuclear magnetic resonance (1H nmr)

For studies of metabolites and metabolic profiles, analysis by Nuclear Magnetic Resonance (NMR) is an important and advantageous tool due to the ease of preparation of the samples to be analyzed. In the analysis by 1 H nmr in liquid state, it was carried out the quantification of the metabolites present in the media during the 10-day period, where samples were taken on days 4, 7 and 10 of fermentation, and all results for metabolites profile are summarized on table 1 of the supplemental material. During fermentation by specific bacteria, G. xylinus, microbial synthesis of cellulose microfibrils is the major metabolite produced, but during this synthesis other metabolites are produced due to microbial metabolism. In addition to identifying the metabolites, 1 H nmr was also used to check the consumption of the carbon sources glucose and sucrose during the fermentation. It was possible to observe a decrease in glucose concentrations on media gly2+glu4 and gly1 + glu2 on the 4˚, 7˚ and 10˚ day of fermentation. Among these, the media glu2 + glu4 had the highest consumption of glucose, due to the participation of glucose in the beginning of biosynthetic pathway to the production of CB and secondary metabolites. Since the media containing sucrose had the presence of glucose, it is due to the breakdown of the disaccharide sucrose into monosaccharides glucose, wherein the media with a higher concentration of glucose, gly2 + sac8, showed high conversion to glucose during the first days of fermentation. In the media gly2 + sac8 sucrose concentration was kept high even on the 10th day of fermentation with 92.5%, this is due to high concentration added in the medium, wherein the microorganism is not able to convert this concentration on the period of 10 days. Most of the identified metabolites were amino acids, and these are synthesized during glycolysis or the citric acid cycle. Alanine is a nonessential amino acid found in all living organisms, synthesized from pyruvate and amino acids with branched side chains such as valine, leucine and isoleucine, are commonly produced by reduction of the pyruvate amination. Alanine was present in all media but in those containing glycerol, concentration of this metabolite was higher and in medium containing only glucose, glu8, its concentration decreased with each passing day. The presence of aspartate and the amino acid lysine and tronina are fully connected as the aspartate, in bacteria, is the precursor of the production of these amino acids. Aspartate was identified only in the medium gly2 + glu4 on day 7 and in the gly1+ glu2 on day 4, with small concentrations. Leucine was detected by the method in the medium gly2 + glu4 where its concentration was higher at day 10 with 4.8%, to the medium gly1+ glu2 its highest concentration of 4.2% was detected on day 4. To the mediums glu2 + sac8 and glu8, it was not possible to detect the presence of the metabolite. Valine was detected only in the media containing the combination of glycerol and glucose with small concentrations. Threonine was detected in small amounts in almost all media as observed. Its highest concentration was observed in gly2 + glu4 with 2.6% and other media ranged from 0,4 to 0,7%. Glutamate was identified only on the 7th day in the medium gly2 + glu4. Lysine was also identified only in the medium gly2 + glu4 on day 7 and 10, with concentrations of 1.1% and 3.4%. The low presence of this amino acid and aspartate in the media is due to its low concentration, being difficult to identify it. Liu and colleagues29 conducted a study analyzing the profile of metabolites produced in agitated and static cultures by G. xylinus by GC-MS analysis, as in this research, they also identified the presence of amino acids alanine, valine and leucine, due to its precursors participate in the metabolic pathway of the microorganism. DHA is a physiological product of the cells involved in the metabolic pathway. The glycolytic pathway uses DHA for ATP generation. The diidroxicetona metabolite is produced by microbial fermentation of glycerol from species of the Acetobacter family30-32. The diidroxicetona is one of the metabolites most commonly found in fermentations with glycerol, but found in small quantities and only in the medium gly2 + sac8 on day 7 with 1.2% and on day 10 with 1.3%. The presence of lactate in the culture medium was identified by 1H NMR analysis on all media with higher concentration at day 10 in the medium gly2 + sac8 with 14.6%. Studies report the presence of lactate in the fermentation by Enterobacteriaceae and the accumulation of primary metabolites such as acetate and secondary as lactate33,34. Traces of propylene glycol were identified among gly2 + sac8 on day 7 with 0.2%. It is described the presence of propylene glycol in studies of gram-negative bacteria using glycerol as substrate35.

Principal Component Analysis (PCA) of metabolites

In PCA chart metabolites are represented by vectors, which when suffer decomposition, will be indicated by other vectors in realtion to a main axis component (AC). The vectors that show high correlation with the axis of CP, whether horizontal or vertical, are those considered longer and also are those that best explain the variability between samples presented in this component36,37.

Figure 2 Projections of production of metabolites through glu8 on day 4, 7 and 10. 

Based on the projection of 8% glucose medium (Figure 5) is possible to observe through the vectors a correlation between days of fermentation and metabolites produced. Only three metabolites have been identified among them being threonine, alanine and lactate. The presence of threonine was favored at day 10 while lactate fermentation on day 4, but alanine was favored in both fermentation periods.

Metabolites sucrose, alanine, lactate , leucine, lysine and threonine, had their production favoured on day 10 of fermentation on medium gly 2 + glu4. While glutamate and aspartate were favoured on day 7. On the 4th day of fermentation it was not possible to observe the projections of the vectors due to small concentrations of metabolites produced (Figure 3). Projection of medium gly 1 + glu2 is shown in Figure 4, where vectors show metabolites favouring production on day 10, thus identifying the presence of sucrose, alanine, lactate, leucine and valine. On day 4 metabolites are also produced, but in low concentrations, and it is favoured the production of threonine and aspartate.

Figure 3 Projections of production of metabolites through gly 2 + glu4 on days 4, 7 and 10.  

Figura 4 Projections of production of metabolites through gly 1 + glu2 on days 4, 7 and 10. 

In the only medium containing glycerol and sucrose (gly2 + sac8) (Figure 5) it is shown that on day 4 of fermentation, small concentrations of metabolites fermentation were produced and the production of lactate and DHA were not favored at this time, but it was positive at day 10 of fermentation. On the 7th day occurred the production of propylene glycol that did not favor the production of alanine, present in small concentrations.

Figure 5 Projections of production of metabolites through glu2 + sac8 on days 4, 7 and 10. 

CONCLUSION

Glycerol could be used as carbon source for bacterial cellulose production with no loses for yield, crystalline index and type of crystalline arrangement, and gives metabolites with the same profile obtained for the usual glucose medium.

During the production of bacterial cellulose membranes, metabolites formed from the carbon sources glycerol and sucrose gives yield with the medium gly1 + glu2 production rate similar to usual medium.

The bacterial cellulose membranes analyzed by NMR spectroscopy in solid state showed characteristic signals for crystalline celluloses type Cel I. There was a clear tendency for greater crystallinity membranes with 7 days of fermentation with the exceptionally high value for medium gly2 + sac8 with a value of 79,4% CI. The metabolic products found in the media by analysis of NMR spectroscopy in liquid state were similar, especially the production of alanine and lactate that were present in all media. This way, a research is important in seeking ways to obtain cellulose membranes with low production cost.

ACKNOWLEDGMENTS

The authors wish to thank the Spectroscopy Facility Laboratory (SPEC-UEL-CT INFRA 2009-01.10.0534.01) at the Universidade Estadual de Londrina. The authors would also like to acknowledge financial support from CNPq-National Counsel of Technological and Scientific Development (447861/2014-0, and 479992/2013-4).

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Received: February 03, 2016; Accepted: July 14, 2016

*Author for correspondence: cesar.tischer@me.com, cesar.tischer@uel.br

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