EP3969602A1 - Process for the production of bacterial nanocellulose from lignocellulosic biomass of eucalyptus globulus and culture media and nanocellulose produced according to the process - Google Patents

Abstract

The present invention relates to a process for producing bacterial nanocellulose using sugar-rich liquor from Eucalyptus globulus lignocellulosic biomass. The process consists on cooking lignocellulosic biomass from Eucalyptus globulus, hydrolyzing the resulting pulp, constituting a culture media which includes the resulting sugar-rich liquor, inoculating the culture media, fermenting the culture media and washing the nanocellulose bacterial obtained. The described process and associated culture media allow the production of bacterial cellulose in yields comparable or even higher than with other common culture media, without requiring any detoxification step and without potentiating any inhibitory effect on the production of nanocellulose. Additionally, it allows for a greater sustainability of the pulp and paper industry and of the production of bacterial cellulose, by enabling the use of residual biomass.

Description

DESCRIPTION

Title of Invention

PROCESS FOR THE PRODUCTION OF BACTERIAL NANOCELLULOSE FROM LIGNOCELLULOSIC BIOMASS OF EUCALYPTUS GLOBULUS AND CULTURE MEDIA AND NANOCELLULOSE PRODUCED ACCORDING TO THE PROCESS

Technical Field

The present invention relates to a process for producing bacterial nanocellulose production involving the use of Eucalyptus globulus lignocellulosic biomass, to a culture media that includes Eucalyptus globulus lignocellulosic biomass sugar- rich liquor and bacterial nanocellulose produced with the described process and culture media. The invention further relates to a culture media and nanocellulose produced according to the process.

Background Art

Bacterial nanocellulose (BNC) is a form of cellulose naturally synthesized by different species of bacteria. This biopolymer has an enormous potential for application in different areas, such as in the medical and cosmetic industries, in composites and in the food and electronic segments (Jozala et al, 2016).

This application potential related to the unique characteristics of bacterial nanocellulose, such as, for example, a high degree of purity, porosity, crystallinity, high degree of polymerization, high mechanical resistance in the wet state, high water holding capacity, low density, biocompatibility, biodegradability and non-toxicity (Campano et al, 2016; Hong et al., 2011; Jozala et al, 2016; Tsouko et al, 2015; Yamanaka et al., 1989). Different strains of bacteria have been studied for the production of this biopolymer. Komagataeibacter strains have been intensively studied due to the high nanocellulose production capacity. These studies consider the optimization of the culture media and of the entire fermentation process, in order to obtain higher yields than those usually obtained.

Despite BNC unique properties and applications potential, there are still limitations associated with its industrial production due to high operating costs and low yields on bacterial nanocellulose. Multiple efforts have been directed to promote greater productivity and yields, using low-cost raw materials, such as rotten fruits, locust bean whey, beans, and corn liquor as a protein source, molasses as a carbon source, yeast for beer, industrial drinks residues, fruit peels, rice husks and fruit juices as industrial food residues (Bilgi et al, 2016; Fane et al, 2016; Kumbhar et al, 2015; Kurosumi et al, 2009; Lin et al, 2014). It has also been reported the use of effluents from a distillery, of the glycerol present in biodiesel, sludge with residual fibers, wheat straw, agricultural corn, and cotton-based textile waste and waste water (Jozala et al, 2015; Chen et al, 2013; Cheng G. et al, 2017; Cheng Z. et al, 2017; Dahman et al, 2010; Hong et al, 2012; Huang et al, 2016; Kongruang et al, 2007; Ruka et al, 2012; Vazquez et al, 2013; Zhao et al, 2018;).

The work of Guo and co-authors describes different methods of detoxifying fir hydrolysate for the production of bacterial nanocellulose, this detoxification being necessary for obtaining the desired high yields (Xiang et al, 2013). The use of a culture medium with wheat straw hydrolysates also requires a previous detoxification, which can be achieved by a treatment with various alkaline compounds, including calcium hydroxide, sodium hydroxide and ammonia, and their combination with activated carbon or with a laccase enzyme. (Hong et al, 2011).

The patent application document US20170283842A1 describes the production of bacterial nanocellulose using a cassava bagasse hydrolysate. However, as described in the patent document, this hydrolysate additionally needs to be subjected to a detoxification step using, for example, sodium hydroxide and an enzyme. US20170283764A1 takes into account the description of the processing of plant material into raw material for the production of bacterial cellulose. Once again, there is the need to carry out a detoxification step with sodium hydroxide and activated carbon.

Summary of Invention

In a first aspect, the present invention provides a process for producing bacterial nanocellulose using sugar-rich liquor from lignocellulosic biomass of Eucalyptus globulus according to claim 1, which consists on cooking lignocellulosic biomass from Eucalyptus globulus, hydrolyzing the resulting pulp, constituting a culture media including the resulting sugar-rich liquor, inoculating the culture media, fermenting the culture media and washing the bacterial nanocellulose obtained. Several Eucalyptus globulus lignocellulosic biomasses can be used as sugar sources, from Eucalyptus globulus bark to liqueurs resulting from biomass cooking processes, such as sulfate (Kraft) and sulfite processes. In fact, the cooking procedure involved in one of the steps of the invention presented in this document can be carried out by one of these delignification processes.

Preferred embodiments of this invention consider Eucalyptus globulus bark as the selected lignocellulosic biomass, sulfate cooking and an enzymatic hydrolysis. Another aspect of the invention is the culture media including the sugar-rich liquor from the lignocellulosic biomass of Eucalyptus globulus. A further aspect of the current invention is the nanocellulose obtained by the process described. It has a series of textural properties of interest, and tailored according to the desired application, with respect to, for example, compressive strength, cohesion, resilience and water holding capacity.

Looking in more detail at the process described in this invention when considering the use of Eucalyptus globulus bark, in the pulp and paper industry, about 35% of the material that enters the pulp and paper mills becomes waste, of which, approximately 56% are used for energy recovery and the remaining 44% do not have a sustainable environmental solution (Kamali et al, 2015, Scott et al, 1995). The disposal of waste in landfills is an expensive and harmful solution for the environment. Therefore, finding new alternatives for the use of the waste generated represents an emerging priority for the pulp and paper industry.

The strategy of using waste as a carbon or protein source for the culture medium allows providing a more sustainable destination for the generated waste. These residues, which have no value as a product, originate an excess of operating costs associated to the needed additional treatments so that they can be disposed of. Additionally, the disposal solution will still be harmful to the environment. Therefore, the use of these residues allows a greater sustainability of the entire process, while being beneficial for the environment.

It is possible to verify that the Eucalyptus globulus lignocellulosic biomass sugar-rich liquor allows the production of bacterial cellulose with yields comparable, or even superior, to other common culture media, and not requiring any detoxification step for the production of bacterial cellulose, unlike to what is described at the available literature on the need for steps to detoxify culture media derived from other lignocellulosic materials. Without potentiating any inhibitory effect on the production of biomass and bacterial cellulose, the use of Eucalyptus globulus biomass as a carbon source promotes a greater sustainability to the entire process, both in the pulp and paper industry and in the bacterial cellulose fermentation process.

Brief Description of Drawings

Figure 1 - Yield of BNC (g/L) produced with K.xylinus ATCC 700178 in different culture media.

Figure 2 - Yield of BNC (g/L) produced with K.hansenii ATCC 53582 in different culture media.

Detailed Description and Description of Preferred Embodiments

The following description considers practical cases of application of the invention described in this document, in detail, but not limited to, according to preferred embodiments of realizing the invention.

The following examples take into account: as an example of lignocellulosic biomass the use of a residual biomass, the bark of Eucalyptus globulus; the inclusion, or not, of pre-treatments of this biomass such as, for example, extraction with water and alcohol; culture media and strains bacteria; biomass sulfate cooking, the also called Kraft process; exemplifying the suitability of biomass sugar-rich liquor from Eucalyptus globulus for the production of bacterial nanocellulose, with yields comparable or even higher than other common culture media and without the need for detoxification steps characteristic of the use of culture media derived from lignocellulosic material. Examples

Preparation of an hydrolysate from the bark of Eucalyptus globulus

Two hydrolysates were obtained from the bark of Eucalyptus globulus. The difference resides on the previous extraction carried out of the Eucalyptus globulus bark. Hydrolysates obtained with an ethanol: water extraction (Eucalyptus globulus lignocellulosic biomass sugar-rich liquor, EBH1) and without any prior bark extraction (Eucalyptus globulus lignocellulosic biomass sugar-rich liquor, EBH2).

Regarding EBH1, the Eucalyptus globulus bark extraction with ethanol and water was carried out in a rotary digester, with 52% ethanol and 48% water, v/v, for 264 minutes at a maximum temperature of 83 °C. Subsequently, the extracted Eucalyptus globulus bark was subjected to Kraft cooking in a rotary digester, at 170 °C for 60 minutes, with an alkaline charge of 0.22 (ratio between the mass of active alkali, in Na₂₀ equivalents, and the biomass mass) and a liquid-solid ratio of 8: 1.

The pulp produced after the Kraft cooking, at a consistency of 5% (mass percentage of pulp in relation to the total volume of hydrolysis), was subjected to an enzymatic hydrolysis in a buffered medium (0.05M sodium citrate buffer), using a commercial enzymatic cocktail (enzymatic load of 20 FPU/g carbohydrates), at an optimum temperature of 50 °C, with mechanical stirring, for 24 h.

Preparation of the inoculum and static fermentation of cultures Two strains of bacteria were used for the production of bacterial cellulose, Komagataebacter hansenii ATCC 53582 and Komagataebacter xylinus ATCC 700178. Both strains were maintained in a Hestrin-Schramm (HS) culture medium (Hestrin et al, 1954), in the solid state with 20.0 (g / L) agar. The cells were cultured in 1L conical flasks containing 100 mL of HS with the following composition (in g/L): glucose 20.0 (Fischer chemical), peptone 5.0 (Liofilchem), yeast extract (Liofilchem) 5.0, disodium phosphate 2.27 (Labkem) and citric acid 1.26 (Panreac). HS culture medium is the most commonly used synthetic culture medium for the production of BNC. Before inoculation, the HS medium was autoclaved at 121 °C for 20 minutes. After inoculation under sterile conditions, cultures were incubated for 48 h at 30 °C. Then, the formed cellulose membranes were agitated in order to release the bacteria trapped inside the cellulose matrix, into the residual medium. This residual medium was used as an inoculum, corresponding to 10% (v/v) of the final volume to ferment. The inoculated media were incubated at 30 °C for 9 days (at a fixed depth of the culture media of 1 cm in 100 ml flasks).

Tables 1 and 2 describe the composition of the different culture media tested for K. xylinus 700178 and K. hansenii 53582, respectively. In addition to Eucalyptus globulus (EBH) lignocellulosic biomass sugar-rich liquor, culture media having molasses in their composition were also tested. The amount of sugars (total and differentiated) was quantified by HPLC (described in analytical methods), at the beginning and at end of fermentation (table 3). To the media tested with K. xylinus, 1.5% (v/v) of ethanol was added (table 1).

Like ethanol, the addition of ammonium sulfate provides an increase in the BNC yield for the K.xylinus strain (Rodrigues et al, 2019). Therefore, ammonia sulfate was also added to the media with lignocellulosic biomass sugar-rich liquor from Eucalyptus globulus (table 1). However, for the K. hansenii strain, no ethanol or ammonium sulphate was added, since the influence of these components for the K. hansenii strain is not known (table 2). The objective is to enhance the production of bacterial cellulose with the sugar syrups sugar-rich liquor of lignocellulosic biomass of Eucalyptus globulus (EBH). Table 1 - Compositions of the culture media used for the strain K. xylinus 700178.

strain 53582. BNC volumetric yield

After 9 days of fermentation, the bacterial cellulose produced was washed with 0.1 M NaOH at room temperature to remove residues from the culture medium and any trapped cells. Subsequently, the membranes were washed with distilled water, also at room temperature, until the pH of the extract was equal to that of the distilled water. After drying at 37 °C, the dry membranes were weighed to calculate the BNC volumetric yield (g/L) according to the following equation: Analytical methods

Total quantification of sugar and proteins High Performance Liquid Chromatography (HPLC) with the Aminex HPX-87H IEX column, PU-2080 Plus pump (JASCO), DG-2080-53 degasser (JASCO), AS12057-Plus 191 (JASCO) automatic sample injector and a 2031 Plus The RI detector (JASCO) was used to identify and quantify the concentrations of cellobiose, glucose and xylose in the EBH sugar hydrolysate (and EBH media). It was also used to identify and quantify the sucrose, fructose and glucose concentrations of the molasses and Mel CSL medium. For the HS culture medium, glucose was quantified. The conditions used to quantify the initial and residual sugars in the culture media were as follows: flow rate of the mobile phase (H2S045 mM) at 0.05 ml / min; column temperature 35 ° C. The injected volume was 20 pL. The concentration of cellobiose, glucose, xylose, sucrose and fructose were determined based on the calibration curves obtained using the pure compounds with concentrations ranging from 0.01 g/L to 30 g/L. The sugar composition in the EBH1 hydrolysate was: glucose 131 g/L, xylose 13.2 g/L and cellobiose 3.70 g/L. The composition of the EBH2 hydrolysate was: glucose 70.0, xylose 14.5 and cellobiose 4.1. The composition of molasses (g/L) determined was: sucrose 687.7, glucose 20.6 and fructose 12.8. The protein in the CSL substrate was determined by the Kjedhal method (Bradstreet RB, 1954). The sample digestion was performed in an eight-tube Digester Foss Tecator/Labtec. The distillation was carried out in a Foss distiller, Model Kjeltec 8400 Analyzer Unit. The total protein present in the CSL was 177.1 g/L.

Figures 1 and 2 represent the results in terms of BNC yield with different culture media. For the K. xylinus strain, the synthetic media HS 1 (glucose levels of 15.6 g/L) and HS 2 (with 45.6 g/L of glucose present in the culture medium) were tested. Both synthetic media showed low yields of bacterial nanocellulose (2.3-2.4 g/L). The production of BNC with the strain K. xylinus 700178 reached a yield of BNC of 7.6 g/L after 9 days of fermentation (figure 1). This yield, in turn, shows the productive capacity of K. xylinus. Therefore, this test was done for comparison purposes. The media containing the hydrolysate, with a previous extraction with ethanol: water (EBH1 HS, EBH1 CSL) and without previous extraction (EBH2 HS, EBH2 CSL) presented yields lower than with Mel CSL, and higher than with a synthetic media (figure 1). The use of the hydrolysate without previous extraction of the bark of Eucalyptus globulus (EBH2), allowed yields slightly higher than with the hydrolysate that had been previously extracted with ethanol: water (EBH1), although it is not a significant difference (figure 1). It is also verified that the interaction of the substrate EBH2 with the corn liquor (CSL) yielded a maximum production of 4.3 g/L (EBH2 CSL) (figure 1). Molasses and corn liquor (CSL) are very economical sources of carbon and protein, commonly used in industrial fermentations. As already mentioned, molasses is composed of sucrose, glucose and fructose, where sucrose is the predominant carbon source. Through table 3, a high consumption of sucrose (93%) and a lower consumption of glucose (34%) are observed in K. xylinus (table 3). The fact that the K. xylinus strain gives preference to sucrose, may explain the lower yield obtained with EBH hydrolysates, where there is no sucrose in its composition (only glucose, xylose and cellobiose). Another factor that enhances greater yields with the K. xylinus strain is the addition of CSL. This is a supplement rich in amino acids, vitamins and minerals, which offer a buffering capacity to the culture medium. It is reported in the literature that the production of BNC decreases during fermentation, due to the production of gluconic acid, a by-product that lowers the pH (Tsouko et al, 2015, da Silva et al, 2019). Therefore, with the addition of CSL, the culture medium not only has a buffering effect, but also has vitamins and minerals that can enhance the production of BNC. Figure 2 shows the BNC yields obtained with the strain K. hansenii 53582. The synthetic media, HS 1 (25 g/L of glucose present in the medium), HS 2 and HS CSL 2 (with 50 g/L of glucose present in the media) present BNC yields between 4.2 and 6.6 g/L, with the BNC yield being higher with the HS medium of lower glucose concentration (HS 1) (figure 2). Regarding the EBH1 and EBH2 media (with 43 and 49 g/L of total sugars, respectively) the BNC yields varied between 3 to 5 g/L (figure 2). Higher values were obtained with the use of the EBH1 hydrolysate, instead of the addition of EBH2, although the difference is not significant. In addition, the addition of CSL to the EBH media did not promote a greater production of BNC (figure 2). Among the tests with EBH hydrolysates, the EBH1 HS medium stands out, due to the higher BNC production (5.2 g/L) (figure 2). Similar yields were obtained with the HS 2 and HS CSL 2 media (figure 2).

In the case of synthetic media, it is also found that the addition of CSL did not enhance the production of BNC. However, it was with the HS 1 medium that the highest BNC yield (6.7 g/L) was observed (figure 2). This can be explained by the fact that this medium has only 25 g/L of glucose, while the remaining media (synthetic and with the hydrolysate EBH1 and EBH2) have 40-50 g/L total sugars. As already mentioned in the literature (Tsouko et al, 2015, da Silva et al, 2019), there may be an inhibition of BNC production, due to the fact that there is an excess of carbon source in the culture medium. This excess can increase the production of gluconic acid, which in turn, inhibits the production of BNC, due to the decrease in the pH.

In addition to the BNC yields obtained, it is important to note the percentage of sugars consumed during fermentation (table 3). These data allow, above all, verifying whether the entire carbon source was consumed and which carbon source was most consumed by the strain. With the K.xylinus 700178 strain, as already mentioned, a higher consumption of sucrose than glucose is observed (table 3; Mel CSL). However, as seen in other media, in the absence of sucrose, the K.xylinus strain also consumes glucose. In the case of synthetic media, only 60% to 63% of glucose was consumed, while in the media with EBH hydrolysates, glucose consumption varied between 82 and 97% at the end of 9 days of fermentation. It is important to note that this higher consumption of glucose in the medium with EBH did not reflect in much higher BNC yields (figure 1; table 3). In the metabolism of K. xylinus the greater consumption of glucose may have led to a greater production of by-products such as gluconic acid, as already mentioned, limiting the production of BNC. Regarding sugar consumption with K. hansenii 53582, there was a high consumption of glucose in synthetic media, especially in the HS medium with 25 g/L (94% of glucose consumed) (table 3). In the synthetic media with higher initial glucose concentrations (50 g/L), consumption was observed between 74 and 84%. Regarding the consumption of sugars in the media with EBH, there was a higher consumption of glucose (EBH1 HS-93%; EBH2 HS-86%) when the CSL was not added to the culture medium (table 3). EBH media with CSL show a reduction in glucose consumption to 64 and 76% (table 3). Observing the effect of the addition of CSL and the yields of BNC obtained, it is also verified that the CSL did not enhance the production of BNC, for K. hansenii 53582 (figure 2; table 3). In both strains, in the EBH media, similar glucose consumption was observed, but it was in the K. hansenii strain that higher BNC yields were observed, as a result of this glucose consumption (figures 1 and 2; table 3). Also in both strains, it was found that xylose was consumed in the media with the EBH hydrolysates (table 3). This consumption was around 50% in the two strains with all media, except for EBH1 CSL medium, with K. hansenii 53582 which was 90% (table 3). However, this consumption is residual compared to glucose consumption, since the initial concentration of xylose in the various media, varied between 1.3 and 8.5 g/L. Regarding the cellobiose present in the EBH media, it is observed that these represent 3-7% of the total sugars in the EBH media (table 3). Therefore, a residual amount of sugars were not available for consumption by the Komagaebacter strains. When carrying out these tests, it was noted that the lignocellulosic biomass sugar-rich liquor of Eucalyptus globulus (EBH) do not have an inhibitory effect on the production of biomass and BNC. Comparing strains, higher BNC yields were observed with K. hansenii 53582. It should be noted that the tested strains are different and therefore the components used, such as the hydrolysates EBH1 and EBH2 and CSL end up influencing the production of BNC, in different ways. On the one hand, CSL enhanced the production of BNC in the K. xylinus strain but did not have a significant influence on BNC yields, for the K.hansenii 53582 strain. Comparing the tests with the EBH media, there is a greater use of sugar consumption for the production of BNC, with the K. hansenii strain than with K. xylinus. Lower results were obtained with the strain K. xylinus since this strain preferably consumes sucrose for the production of BNC.

The EBH hydrolysates show their great potential for the production of BNC, since they are produced using low-cost raw materials. The use of Eucalyptus globulus bark as a carbon source, promotes a greater sustainability of the entire process, both in the pulp industry and in the BNC fermentation process.

Table 3 - Results obtained by HPLC, regarding the sugar consumption of the strains tested in the different culture media.

*Fructose formed during the consumption of sucrose, during fermentation;

**Cellobiose is not consumed during fermentation;

References

Bilgi, E., et al., Optimization of bacterial cellulose production by Gluconacetobacter 97 xylinus using carob and haricot bean. International journal of biological 98 macromolecules, 2016. 90: p. 2-10.

Bradstreet RB (1954) Kjeldahl method for organic nitrogen. Anal Chem 26(1):185-187

Campano, C., et al., Enhancement of the fermentation process and properties of 76 bacterial cellulose: a review. Cellulose, 2016. 23(1): p. 57-91.

Chen, L., et al., Biotransformation of wheat straw to bacterial cellulose and its 118 mechanism. Bioresource technology, 2013. 135: p. 464-468. 119

Chen, G., et al., Bioconversion of waste fiber sludge to bacterial nanocellulose and use 120 for reinforcement of CTMP paper sheets. Polymers, 2017. 9(9): p. 458. 121

Cheng, Z., et al., Green synthesis of bacterial cellulose via acetic acid pre-hydrolysis 115 liquor of agricultural corn stalk used as carbon source. Bioresource technology, 2017. 116234: p. 8-14.

Dahman, Y., K.E. Jayasuriya, and M. Kalis, Potential of biocellulose nanofibers 109 production from agricultural renewable resources: preliminary study. Applied 110 biochemistry and biotechnology, 2010. 162(6): p. 1647-1659. da Silva, F. A. S., et al., Optimization of bacterial nanocellulose fermentation using recycled paper sludge and development of novel composites. Applied Microbiology and Biotechnology, 2019. 103 (21-22): p. 9143-9154. Fan, X., et al., Production of nano bacterial cellulose from beverage industrial waste 85 of citrus peel and pomace using Komagataeibacter xylinus. Carbohydrate Polymers, 862016. 151: p. 1068-1072.

Hong, F., et al., Wheat straw acid hydrolysate as a potential cost-effective feedstock for 82 production of bacterial cellulose. Journal of Chemical Technology & Biotechnology, 83 2011. 86(5): p. 675-680.

Hong, F., et al., Bacterial cellulose production from cotton- based waste textiles: enzymatic saccharification enhanced by ionic liquid pretreatment. Bioresource 101 Technology, 2012. 104: p. 503-508.

Hestrin, S., et al., Synthesis of cellulose by Acetobacter xylinum. 2. Preparation of freeze-dried cells capable of polymerizing glucose to celulose. Biochem Journal, 1954. 58(2): p. 345-352.

Huang, C., et al., Using wastewater after lipid fermentation as substrate for bacterial 103 cellulose production by Gluconacetobacter xylinus. Carbohydrate polymers, 2016. 136: 104 p. 198-202.

Jozala, A.F., et al., Bacterial cellulose production by Gluconacetobacter xylinus by 128 employing alternative culture media. Applied microbiology and biotechnology, 2015. 12999(3): p. 1181-1190.

Jozala, A.F., et al., Bacterial nanocellulose production and application: a 10-year 74 overview. Applied Microbiology and Biotechnology, 2016. 100(5): p. 2063-2072.

Kamali, M., et al., Review on recent developments on pulp and paper mill wastewater treatment. Ecotoxicol Environ Saf., 2015. 114: p. 326-42.

Kongruang, S., Bacterial cellulose production by Acetobacter xylinum strains from 106 agricultural waste products, in Biotechnology for Fuels and Chemicals. 2007, 107 Springer, p. 763-774.

Kumbhar, J.V., J.M. Rajwade, and K.M. Paknikar, Fruit peels support higher yield and 88 superior quality bacterial cellulose production. Applied microbiology and biotechnology, 2015. 99(16): p. 6677-6691

Kurosumi, A., et al., Utilization of various fruit juices as carbon source for production 91 of bacterial cellulose by Acetobacter xylinum NBRC 13693. Carbohydrate Polymers, 922009. 76(2): p. 333-335.

Lin, D., et al., Production of bacterial cellulose by Gluconacetobacter hansenii 94 CGMCC 3917 using only waste beer yeast as nutrient source. Bioresource technology, 952014. 151: p. 113-119.

Mota, I., et al., Extraction of Polyphenolic Compounds from Eucalyptus globulus Bark: Process Optimization and Screening for Biological Activity. Industrial & Engineering Chemistry Research, 51(20) 2012: p. 6991-7000.

Rodrigues, A.C., et al, Response surface statistical optimization of bacterial nanocellulose fermentation in static culture using a low-cost medium. New biotechnology, 49, 2019: p. 19-27.

Ruka, D.R., G.P. Simon, and K.M. Dean, Altering the growth conditions of 125 Gluconacetobacter xylinus to maximize the yield of bacterial cellulose. Carbohydrate polymers, 2012. 89(2): p. 613-622.

Scott G.M., et al., Sludge characteristics and disposal alternatives for the pulp and paper industry. Tappi Intern. Environ. Conf., Tappi Press, 1995, pp 269-279.

Tsouko E., et al., Bacterial cellulose production from industrial waste and by-product streams. International Journal of Molecular Sciences, 2015. 16(7): p. 14832-14849.

Vazquez, A., et al., Bacterial cellulose from simple and low cost production media by Gluconacetobacter xylinus. Journal of Polymers and the Environment, 2013. 21(2): p. 545-554.

Yamanaka, S., et al., The structure and mechanical properties of sheets prepared from 80 bacterial cellulose. Journal of Materials Science, 1989. 24(9): p. 3141-3145. Xiang, G., et al., Comparison of methods for detoxification of spruce hydrolysate for bacterial cellulose production. Microbial Cell Factories, 2013. 12(93).

Zhao, H., et al., Production of bacterial cellulose using polysaccharide fermentation 112 wastewater as inexpensive nutrient sources. Biotechnology & Biotechnological Equipment, 2018. 32(2): p. 350-356.

Claims

Claim 1. Process for producing bacterial nanocellulose using a sugar-rich liquor from lignocellulosic biomass of Eucalyptus globulus characterized by comprising the following steps: a) cooking lignocellulosic biomass from Eucalyptus globulus; b) hydrolyzing the resulting pulp; c) constituting a culture media including the resulting sugar-rich liquor; d) inoculating the culture media; e) fermenting the culture media; f) washing the obtained bacterial nanocellulose.

Claim 2. The process according to the previous claim characterized in that the lignocellulosic biomass is Eucalyptus globulus bark.

Claim 3. The process according to any of the preceding claims characterized in that the cooking of step a) is a Kraft cooking.

Claim 4. The process according to any of the preceding claims characterized in that the hydrolysis of step b) is an enzymatic hydrolysis.

Claim 5. Culture media characterized by comprising the sugar-rich liquor from lignocellulosic biomass of Eucalyptus globulus obtained by the process described in claims 1 to 4.

Claim 6. Nanocellulose obtainable by the process according to any one of claims 1 to 4.