Evaluation of the addition of glycerol to Cupriavidus necator culture medium over Poly ( 3-hydroxybutyrate ) production

Glycerol was used as a source of additional carbon in the production of Poly(3-hydroxybutyrate) (P(3HB)). The inverted sugar and glycerol concentrations and the temperature of the Cupriavidus necator culture medium were evaluated using a Central Composite Rotational Design (CCRD). The results showed that the increase in temperature and sugar concentration led to an increase in production and P(3HB) accumulation and when 15 g L -1 of glycerol was added better results were obtained, however these were not considered statistically significant. The best results were obtained at 38 °C and with 30 g L -1 of inverted sugar. Although not considered statistically significant, the addition of 15 g L -1 of glycerol increased the P(3HB) accumulation percentage by 15 %, thus in kinetic terms, greater productivity was obtained in 0.32 g L -1 h -1 polymer. Larger scale assays are being conducted to verify if the addition of glycerol improves the thermal and mechanical properties of the synthesized polymer.


INTRODUCION
Poly(3-hydroxybutyrate) -P(3HB) -is a natural and biodegradable polyester from the polyhydroxyalkanoates (PHAs) family, synthesized and accumulated internally in diverse micro-organisms [1].It is a photopolymer and is highly crystalline, rigid and brittle, which reduces its application, however, the addition of nucleating agents, plasticizers or other additives have been used to give new applications to P(3HB) [2].According to Gumel et al. (2012) [3] the type of carbon source used as a substrate does not only affect the polymer produced in terms of physical-chemical and mechanical properties but also its yield and monomeric composition.
The P(3HB) applications are the most diverse, being used for biodegradable packaging, agricultural product packaging for transporting young plants [4], tissue engineering, due to their biocompatibility, for manufacturing prostheses and other medical components as it does not generate toxic substances during degredation [5,6], for toothbrushes [7], in the formulation of capsules for the controlled release of pharmaceuticals and pesticides [8].
Most of the technologies so far implemented for the microbial production of PHA are not economically competitive with the production of synthetic plastics, although they are already commercially available.Therefore, efforts have been made to achieve more cost-effective and sustainable PHA production processes [9,10].Many researchers have been focusing on the use of low cost sugars as a source of carbon for an efficient PHAs [11] production.On the other hand, with the growing increase of biodiesel production, the cost of glycerin originating from the production process has been decreasing due to an increase in supply, often making its refinement process economically unfeasible.Silva et al. (2009) [12] have already indicated crude glycerol as being a promising source of abundant carbon for industrial microbiology.Within this context, the aim of this study was to evaluate the use of glycerol as a source of additional carbon in the Cupriavidus necator medium culture in the production and accumulation of P(3HB).

Experimental planning:
The C. necator cells were cultivated in baffled 1.000 mL Erlenmeyer flasks containing 300 mL of Mineral Medium (MM).The concentrations of the inverted sugar carbon sources (glucose and fructose) (varying from 0 to 30 g L -1 ) and/or glycerol (varying from 0 to 30 g L -1 ) and culture medium temperature (varying from 28 to 38 °C) according to central composite rotational design (CCRD) 2 3 with 3 central points.The culture media were maintained under stirring at 150 min -1 , for 24 h.The statistical analyses were conducted with the help of Statistica 7.0 software.

CCRD validation:
Assays were conducted, in triplicate, under conditions indicated by CCRD as the highest producer of P(3HB).

Cell growth:
Dry weigh was determined by using pre-weighed 2 mL Eppendorf tubes. 2 mL of culture broth was centrifuged at 9,000 rpm for 10 min.The supernatant was discarded and the cell pellet was washed twice with distilled water.The cell pellet was dried 24 h at 60 °C and cooled down to room temperature in a desiccator.The weight difference was used to determine the dry biomass.

Substrate consumption:
Substrate consumption (glucose/fructose and glycerol) was verified by high performance liquid chromatography (HPLC) (Merck Hitachi model D-7000IF) with refractive index detector model RI-71 (Merck), with Transgenomic, model ICSep ICE-ION Column (protons exchange).The mobile phase was 8.5 mmol.L -1 H 2 SO 4 and the flow rate was 0.4 mL.min -1 .The column temperature was maintained at 70 °C and the volume injection was 10 µL.

P3(HB) determination:
2 mL of the medium was centrifuged and the cells were washed twice with distilled water, then submitted to methanolysis, according to the method based on Braunegg et al. (1978) [13], with modifications proposed by Brandl et al. (1995) [14].The P(3HB) was then dosed by gas chromatography.

RESULTS AND DISCUSSION
For evaluating temperature influence on the Cupriavidus necator DSM 545 culture medium and the influence of inverted sugar and glycerol concentrations a central composite rotational design was used.
Tab. 1 shows the 17 assays conducted under the experimental conditions used and the responses obtained in the experiments.
Table 1: Experimental conditions of design assays and responses in Xt (g L -1 ), Xr (g L -1 ), P(3HB) (g L -1 ).A variance analysis (ANOVA) was conducted for P(3HB) production.Through an estimation of the pvalue supplied by ANOVA, it was verified that from the variables tested, only the inverted sugar concentration and the incubation temperature showed significant influence both on P(3HB) production as well as on the polymer accumulation percentage.The glycerol concentration and the interactions were not statistically significant.The coefficients and their interactions are part of the model to form the response surface and are presented in Equations 1 and 2, the terms in bold print are statistically significant.
As the model is predictive in the experimental region, the response surfaces for P(3HB) production are presented in Fig. 1 in relation to the inverted sugar concentration and incubation temperature within the interval tested, maintaining the glycerol concentration at 0 g L -1 (1a) and 15 g L -1 (1b).Similar profiles are observed on surfaces that represent the polymer accumulation percentage (data not presented).
As can be observed in Fig. 1a and 1b, the incubation temperature exerts significant influence on polymer production.Schneider (2006) [15] observed in his work, using the same culture medium, although with the addition of vegetable oils instead of glycerol, that an increase in temperature from 30 to 37 °C raised P(3HB) production.It can be verified that when glycerol is present in a concentration of 15 g L -1 (Fig. 1b) it reaches a production of 6.9 g L -1 as opposed to 5.3 g L -1 , under the best conditions, without the addition of glycerol, even when the glycerol concentration is not statistically significant.On adding 15 g L -1 of glycerol the P(3HB) accumulated percentage went from 50 to 65 %, a higher accumulation than that found by Cavalheiro et al. (2009) [16] who obtained 62 % accumulation using the same micro-organism in fed batch process using glycerol as a carbon source.Through the design analysis, the ideal condition for validation was reached, in triplicate, the temperature at a high axial level (38 °C) and the inverted sugar concentration also at a high axial level (30 g L -1 ) and glycerol at a low axial level (without glycerol), since the variance analysis shows that the glycerol concentration exerts no significant influence on either P(3HB) production or on the polymer accumulation percentage in cells.Even without indicating the addition of glycerol as a significant variable, it cannot be denied that a 15 % increase in polymer production is not interesting from an operating point of view in the extraction phase of this polymer.Added to this, it is not known at this time what the real contribution of adding glycerol is in terms of properties that the polymer can acquire, since the volume of the medium in the Erlenmeyer flasks is not sufficient to perform the extraction of the films for their thermal and mechanical characterization analyses.Thus it was decided to perform the model validation assays, in triplicate, not only with the predictive condition as the best but also under the conditions that in spite of not having indicated the glycerol concentration as a significant variable presented interesting responses for P(3HB) production, which were the following: 38 °C, 15 g L -1 of inverted sugar and 15 g L -1 of glycerol and 38 °C, 30 g L -1 of inverted sugar and 15 g L -1 glycerol.
In Fig. 2a, 2b and 2c the growth kinetics of Cupriavidus necator DSM 545 and P(3HB) production are presented for the above mentioned conditions and Fig 2d shows a comparison between the values predicted by the model and those obtained experimentally to production and the accumulated percentage of P(3HB) in the biomass.Table 2 presents some kinetic parameters for the three assays performed in the validation of the central composite rotational design.
The P(3HB) production reached 4.6 ± 1.0 g L -1 representing a total accumulation of 48.9 % of P(3HB) inside the cells after 24 h, without glycerol in the medium.When 15 g L -1 of inverted sugar and 15 g L -1 of glycerol were used as substrates 4.3 ± 0.8 g L -1 was reached, representing a total accumulation of 51.1 % of P(3HB) and when the culture was started with 30 g L -1 of inverted sugar and 15 g L -1 of glycerol as substrate, maximum P(3HB) concentration was (7.3 ± 1.4 g L -1 ) representing an accumulation of 68.0 % of P(3HB) in the biomass.Ganesh et al. (2015) [17] cultivated E. coli for 96 h and obtained 1.47 g L -1 of PHB (28.8% accumulation) using 20 g L -1 of crude glycerol and 4.58 g L -1 of PHB (54,5% accumulation) when 20 g L -1 of pre-treated glycerol was used.
As can be observed in Fig. 2d the values predicted for the accumulation of P(3HB) are confirmed experimentally, when taking the experimental error into account.As for P(3HB) production values, the model was confirmed experimentally for the two conditions in which the initial sugar concentration was 30 g L -1 , with and without glycerol.Only in the culture in which there was 15 g L -1 of each one of the substrates did the predicted and experimentally observed values differ by only 3 %, considering the experimental error margin.

Figure 1 :
Figure 1: Response surfaces for P(3HB) production in relation to the inverted sugar concentration (g L -1 ) and incubation temperature (°C).(a) without glycerol and (b) glycerol concentration equal to 15 g L -1 .

Figure 2 :
Figure 2:Growth kinetics of Cupriavidus necator DSM 545 and P(3HB) production at 38 °C, using 30 g L -1 of inverted sugar as substrate (a); using 15 g L -1 of inverted sugar and 15 g L -1 of glycerol as substrate (b) and using 30 g L -1 of inverted sugar and 15 g L -1 of glycerol as substrate (c).Comparison between the predictive values by the model for P(3HB) production and the polymer accumulated percentage compared to the values obtained experimentally (d).Where () represents the total biomass (Xt), () represents P(3HB) production, (---) represents the residual biomass (Xr), ( ) represents the inverted sugar concentration (Sugar) and () represents the glycerol concentration.

Table 2 :
Biomass values, P(3HB) concentration, P(3HB) accumulated percentage, cell productivity, P(3HB) productivity, substrate conversion factors in cells and substrate in P(3HB) for the different concentrations of the medium carbon source.