Chapter 2: A closed-loop biorefinery approach for polyhydroxybutyrate (PHB)
3.3 Results and discussions
3.3.3 Performance of ABR in batch mode
Changes in the R. eutropha cells during different phases of its growth in the batch operated ABR are shown in Fig. 3.6. Turbidity of the culture was maximum during the stationary growth phase. The cells at this phase were harvested and stained with Sudan black and safranin dye, which revealed an impressive purple shading, in Fig. 3.6. It also revealed the Gram-negative nature of R. eutropha. However, blue inclusion of the cells, as shown in these images, indicates the presence of PHB inside the cells. It could be seen from the images that cells at the stationary phase showed more blue colour than at the logarithmic growth phase, signifying that PHB production was more pronounced following the exponential phase. These findings confirm that after achieving the exponential growth period, the substrate flux deviated towards PHB production by the cells (Fig. 3.6). For a clear visualization of the PHB granules, the R. eutropha cells were observed under FETEM, which revealed a moderately high number of well defined PHB granules inside the cells.
Fig. 3.6. Images showing changes observed in R. eutropha cells at a different phase of its growth in the batch-operated ABR: (a) culture broth, (b) light microscopy (100 X) magnification and (c) FETEM showing PHB granules
At the end of the 60 h batch fermentation in ABR, operated at 500 rpm and a sparging rate of 0.8 vvm, R. eutropha biomass production was observed to be exceedingly high (OD600 around 70.2). Even at 250 rpm and 0.4 vvm, the biomass produced was high (OD600 = 9.64). The high biomass titre obtained is due to the increased KLa value from 8.12 h-1 to 24.1 h-1 when sparging rate raised from 0.4 vvm to 0.8 vvm at 250 rpm (see Table 3.2). It is expected that a minimum agitation rate and air sparge rate in a fermentor yield the least biomass production owing to the oxygen limitation condition in the bioreactor, and for the same reason, deploying an unconventional reactor like ABR is favoured as it
helps in the production of R. eutropha biomass by increasing oxygen uptake by the microorganisms. However, it could be inferred from Table 3.2 that at low DO conditions, PHB accumulation by the bacterial biomass increased due to the change in substrate flux from biomass production to PHB accumulation by the bacteria. Similar observations were made by Díaz-Barrera et al. (2016), wherein an increase in agitation rate reduced PHB accumulation from 84 to 65 % (w/w). This observation on increased PHB accumulation at a low agitation rate was confirmed from the results of the gel electrophoresis analysis (Fig.
3.7). Since the bacterial cells used for the analysis were obtained at the end of the batch cultivation, protein bands were less intense. However, the band due to histone-like protein at 40 kDa is found to be intense and the intensity decreased along with an increase in the agitation rate.
Histone is a protein known to bind the DNA. In Fig. 3.7, the band at 40 kDa is a representation of histone-like proteins because these proteins bind the PHB inside the bacterial cell to form granules (Luengo et al., 2003). A schematic showing the role of histone-like proteins in R. eutropha along with TEM image of the actual R. eutropha is shown in Fig. 3.7(b-c). Thus, a decreased intensity of the band corresponding to histone- like proteins in Fig. 3.7(a) at a high agitation rate indicates reduced accumulation of PHB.
After 60 h of batch fermentation, the average PHB production values at 0.4 vvm air-sparge rate and agitation rates of 250, 350 and 500 rpm were 4.14, 8.47 and 21.18 g/L, respectively (Fig. 3.8). At the same agitation rates with an increased air sparging rate of 0.8 vvm, the PHB concentrations were increased to 12.25, 16.2 and 29.72 g/L, respectively.
Fig. 3.7. (a) Results of gel electrophoresis analysis of R. eutropha cells obtained from the batch operated ABR at varying agitation and air sparge rates, (b) Schematic showing R.
eutropha cell with PHB granules enclosed inside a histone-like protein and (c) FETEM image of an actual R. eutropha cell with PHB granules accumulated inside its cell wall
The bioreactor operated at 350 rpm, and at increased air sparge rate from 0.4 to 0.8 vvm did not yield significant enhancement in PHB accumulation. However, at the other two agitation rates, i.e., 250 and 500 rpm, an increase in air sparge rate significantly improved the PHB accumulation (Fig. 3.8). Similar results were reported in the literature, which suggests that oxygen constrained cultivation conditions result in low biomass production and PHB yields (Cavalheiro et al., 2009; Díaz-Barrera et al., 2016). Moreover, reduction in available oxygen could altogether influence native metabolic pathways in R.
eutropha, including ATP generation, which further reduces biomass and PHB production (Anusha et al., 2016; Cavalheiro et al., 2009). The results obtained from the batch operated ABR were compared with the results obtained from STBR operated at 250 rpm and 0.8 vvm air sparge rate. Increasing the STBR agitation rate resulted in lowered biomass concentration (Fig. 3.8). Whereas, ABR yielded 1.3 times higher biomass production and PHB production than the STBR operated at the optimized conditions.
Fig. 3.8. Biomass and PHB concentration at various operating conditions in the batch operated (a) ABR and (b) STBR
The results of flow cytometry analysis of R. eutropha cells in (a) positive control, (b) fermentation in ABR and (c) in STBR are depicted in Fig. 3.9 (a - c), respectively. From Fig. 3.9(b), 7% of the values fall in the upper right quadrant, which implies that ABR did not inhibit the microbial growth or impose any stress on the microbial cells. However, the mortality rate of R. eutropha cells in STBR increased to 22%, which confirms the high stress imposed by STBR even at moderate agitation and aeration rates, resulting in cell death.
Fig. 3.9. Results of flow cytometry analysis of (a) positive control (b) ABR operated at an agitation rate of 500 rpm with sparge rate of 0.8 vvm and (c) STBR operated at an agitation rate of 500 rpm with sparge rate of 0.8 vvm.