Ralstonia eutropha ATCC 17699 (Cupriavidus necator) was purchased from ATCC (Manassas, VA, USA) through CES University. The bacterium was cryopreserved at -20 °C in tryptic soy and broth medium (TSB) in 30% glycerol, inside a 1.5 mL vial, and subcultured in TSB under standard monthly conditions.

The inoculum used for culture in the reactor was carried out in 1L Erlenmeyer flasks containing 300 mL of TSB medium, at 30°C and 150 rpm for 12 hours, using a vial with the bacterial strain cryopreserved.

The culture was carried out in a 5L bioreactor (New Brunswick) with automatic control of agitation at 200 rpm, a temperature of 30^oC, air flow of 5 L/min and pH 7.0, controlled with 1N HCL and NaOH solutions.

The vinasse for experimentation was donated by a sugar-alcohol company in Colombia. It was centrifuged at 8,000 rpm for 7 min to remove particulate matter. The supernatant was then characterized to determine total sugar, Ca, Mg and K content. On the other hand, commercial molasses employed (high viscosity sugar syrup) contained 60-63 % (w/w) sucrose and 3-5 % (w/w) reducing sugars. Due to the high sugar content, this was diluted to obtain the ratio and concentration of sugar required for experimentation.

Mineral salt medium (MSM) employed in the culture as a reference medium contained (per liter): Glucose 20 g; Na₂HPO₄.7H₂O 6,7 g; KH₂PO₄ 1,5 g; (NH₄)₂SO₄ 1,0 g; MgSO₄.7H₂O 0,2 g; iron and ammonium citrate 60 mg; CaCl₂.2H₂O 10 mg; element trace solution 1 ml. Element trace solution (per liter): H₃BO₃ 0,3 g; CoCl₂.6H₂O 0,2 g; ZnSO₄.7H₂O 0,1 g; MnCL₂.4H₂O 30 mg; NaMoO₄.2H₂O 30 mg; NiCl₂.6H₂O 20 mg; CuSO₄.5H₂O 10 mg [14].

Biomass concentration was determined by gravimetry, using dry weight determination with 5mL samples of culture broth. The samples were passed through membrane filters (Millipore, 0.45 µm filters) in a filtration equipment with a vacuum pump, to be then dried at 70°C for 24 hours and weighed in an analytical balance to constant weight.

A high-performance liquid chromatography (HPLC) Agilent Technologies 1200, model 61362A, with a column of separation ionic interchange Animex HPX-87H, 300x7.8mm was employed in sugars determination. The samples were centrifuged at 7,000 rpm for 5 min and aliquots of the supernatant were taken, which were diluted in mobile phase according to the concentration range of the standard curve employed. Each diluted sample was run through 0.2 µm regenerated cellulose filters and injected under the following conditions: 20 µL sample, 0.6 mL/min flow, 35^oC and 12 min run time. All samples were analyzed in triplicate.

The extraction of the PHA produced was carried out following Yu & Chen methodology, where the sample was subjected to a sterilization process at 120^oC for 30 min, to be then is recovered by centrifugation at 5,000 g for 12 min and washed with distilled water; then, 200 mL of 0.1 M H₂SO₄ are suspended in solution and subjected to digestion at 100 ^oC for 2 hours, adjusted to pH 10 with 5N NaOH and centrifuged at 4,000 rpm for 20 min to recover the biopolymer. This is resuspended in a 6% NaCl solution to be bleached and is again recovered by centrifugation and washed with distilled water. Determination of the PHA production was performed using dry basis quantification [15].

For the characterization of the PHA produced, Fourier Transform Infrared Spectroscopy (FTIR) technique was used, with a Perkin-Elmer Spectrum BX infrared spectrophotometer and ART module from 4,000 to 400 cm^-1 and a Sigma-Aldrich branched PHB (Polyhidroxibutyrate) standard with 99% purity.

Three one-way experimental designs were performed. The first factor was evaluated with different relations of molasses/vinasse (M/V: 100/0, 75/25, 50/50, 25/75); the second factor was the nitrogen source (with and without nitrogen source); the third factor was the salts supplement (with and without salts supplement). The nitrogen and salts from the MSM medium were used as a reference for the second and third factor. All experiments were carried out in triplicate, using biomass, sugars, and biopolymer as response variables.

A one-way variance analysis (ANOVA) and a Tukey HSD multiple comparisons test were performed to evaluate the data. The results that presented values of p-value < 0.05 were interpreted as statistically significant variables using the software StatGraphics Centurion 17.1 Demo.

Vinasse was characterized according to available sugar content, which was quantified by HPLC; this, together with other components that can be seen in Table 1, were analyzed at the Laboratory of Physicochemical Analysis, Institute of Chemistry (Laboratorio de Análisis Fisicoquímico del Instituto de Química - LAFQ) , University of Antioquia.

Table 1

Source: The authors.

According to Table1, the sugar content in the vinasses reached 11.64 g/L, so it was required to complement the medium with an additional carbon source such as molasses, according to the carbon requirements of the MSM. The pH also needed to be adjusted but Ca, Mg and K contributions exceeded those required by the reference medium [14]. However, Zuñiga et al. (2013) report pH values in vinasse of 4.6-4.7 and 7.4 - 8.4 g/L in total solids [11], in agreement with the values in this work.

Table 2 shows the concentration of sugars consumed by R. eutropha under different molasses/vinasse ratios. The growth of bacteria in all cultures was evidenced by microscopy. It was also observed that the highest yield of total sugars was reached in the proportion 25/75 molasses/vinasse, presenting a statistically significant difference between the other proportions (p-value <0.05). Similarly, Dos Santos et al. reported a yield of 0.79 or 79 % of sugars consumed in an R. eutropha culture, using vinasse obtained from cane ethanol production [16].

Fig. 1 shows a profile of time-quantified sugars for the cultivation of R. eutropha at an M/V ratio of 25/75 with supplementation of nitrogen and salt sources (1.0 g/L of (NH₄)₂SO₄) and according to the MSM culture medium. It also shows the consumption of total sugars (sucrose, glucose, and fructose), the gradual consumption of sucrose, and how at 18 hours post-culture the total glucose had been consumed.

Table 2

Source: The authors.

Figure 1 Source: The authors.

The results in Table 3 correspond to the evaluation of the culture supplemented with nitrogen and salt sources, with M/V of 25/75. Although there were no statistically significant differences between treatments, PHA productivity, Y_p/s and consumption of sugars were higher in the mixture supplemented with nitrogen and salt sources.

Table 3

Source: The authors.

Finally, in Fig. 2, a comparison was made between the MSM reference culture and the culture using an M/V ratio of 25/75 supplemented with nitrogen and salt sources. In both cultures, the consumption of total sugars with values of 18.60 and 15.56 g / L is appreciated, which represents an efficiency in the consumption of sugars of 83.1 and 71.3%, respectively.

Figure 2 Source: The authors.

In the culture with molasses/vinasse, a lag phase of 9 hours could also be observed, unlike the culture with the MSM medium. This difference in the lag phase may be related to the low concentration of glucose in the medium forcing R. eutropha to hydrolyze the available sucrose.

Table 4 shows the results of the parameters determined for each crop; no statistically significant differences were found between the parameters evaluated except the total of sugars consumed. In this way, the M/V medium can be considered suitable for the cultivation of R. eutropha, with the capacity to accumulate up to 97.8 % of PHA with respect to the produced biomass and a yield of 97.8 % in total sugars consumed. However, Nonato et al. cultivated R. eutropha with a medium enriched with cane molasses with a productivity of 1.44 g PHA/L.h, but with a biopolymer content of 65-70% [17].

Table 4

Source: The authors.

Other researchers, such as El-Sayed et al., also carried out cultures with mixtures of glucose and sucrose obtaining 0.8 g/L PHB, but with a change in the culture, strategy reached 7.31 g/L PHB [18]. On the other hand, Pramanik A et al. also used vinasse as a culture medium for the production of PHAs but using the strain Haloarcula marismortui, with which a content of 23-30% was obtained in the dry biomass with a productivity between 0.015 and 0.020 g / L.h of polymer [19].

By comparing the spectra of the polymers obtained in Fig. 3 with the R. eutropha strain versus the polyhydroxy butyrate standard (PHB), an equal pattern is observed in the formed bands. The peaks corresponding to the region of the spectrum of 1,740 cm^-1 and 2,970 cm^-1 are characteristic of C = O and CH₃ functional groups of the monomeric PHB [20].

Figure 3 Source: The authors.

The spectra obtained from R. eutropha showed a correlation of 99.25% with the pattern of bands in the PHB commercial polymer employed, indicating thus that the polymer obtained from R. eutropha is of the PHB type [21] [22]. According to reports, this polymer is highly crystalline (> 50%). The melting and glass transition temperature is about 180 ° C and 4 ° C, respectively. It has some mechanical properties comparable to degradable synthetic polyesters, such as PLA. During the storage time at room temperature, it undergoes a secondary crystallization of the amorphous phase, as a result, the tensile and elongation modulus increase while the polymer becomes more brittle and harder [23] [24].

This biopolymer is an attractive material for tissue engineering due to its inherent biocompatibility. It has been shown to enhance the proliferation of different cell types and since PHB is susceptible to various processing methods, it can be mixed with other types of biocompatible molecules such as the hydroxyapatite mineral to create films, fibrous mats or non-crystalline chains, adaptable to a specific use [25] [26].