5. RESULTS AND DISCUSSION
5.1. Two-Stage Continuous Production of poly-3-hydroxybutyrate
5.1.1. Effect of the dilution rate in the first-stage
Different dilution rates were tested and for each one the experimental data analyzed in order to find a steady state. Figure 13 shows an example of that for a dilution rate in the first bioreactor, D1, of 0.31 h-1.
Table 9 – Experimental dilution rate studied on the first-stage bioreactor.
D1 (h-1) 0.10 0.15 0.21 0.24 0.26 0.28 0.31 0.32
Figure 13 - Performance of the first-stage bioreactor over time for a dilution rate of 0.31 h-1. A steady state is reached between 37 to 51 hours of fermentation. Glucose concentration (◆), cell dry weight (▲), P(3HB) content (×), phosphate concentration (■) and P(3HB) concentration (●).
A range of dilution rates between 0.10 and 0.32 h-1 were studied under phosphate-rich conditions in the first-stage bioreactor (Table 9). Figure 14 shows the steady-state values of cell dry weight (CDW), glucose concentration, phosphate concentration and P(3HB) concentration in the first stage continuous culture, with the respective standard deviation at different dilution rates.
Figure 14 – Effect of dilution rate on residual glucose concentration (◆), CDW (▲), P(3HB) concentration (●) and phosphate concentration (■) in the first-stage bioreactor, under phosphate-rich conditions.
0.0 1.0 2.0 3.0 4.0 5.0 6.0
0 10 20 30 40 50 60 70
0 10 20 30 40 50 60 70
Phosphate concentra]on (g/L) P(3HB) concentra]on (g/L) Glucose concentra]on (g/L), CDW (g/L) PHB content (%)
Time (h)
CSTR 1
Gluc1 CDW1 % PHB Phosp1 PHB1
0.0 1.0 2.0 3.0
0 10 20 30 40 50
0.08 0.12 0.16 0.20 0.24 0.28 0.32
Phosphate concentra]on (g/L)
Glucose, CDW, P(3HB) concentra]on (g/L)
D1 (h-‐1)
CSTR 1
Glucose CDW PHB Phosphate
37h - 51h
25
Figure 14 suggests that CDW was not affected by the dilution rate in the range of 0.10-0.28 h-1, attaining a value of approximately 31 g/L. At dilution rates above 0.28 h-1, the CDW started to decrease and it dropped to 20.7 g/L at 0.32 h-1. Glucose concentration was zero until dilution rate reached 0.21 h-1 and started to increase at higher dilution rates. It increased quickly when the dilution rate approached 0.32 h-1, which could indicate that this dilution rate was close to the wash-out point of the continuous culture system.An average P(3HB) content of 8.8 % was found in the first bioreactor . With the increase of dilution rate, phosphate concentration increased, while P(3HB) concentration did not present a tendency, as shown in Figure 15. A residual concentration of P(3HB) was obtained for all the dilution rates tested even in the presence of phosphate , suggesting that B. sacchari has a mechanism which leads to a residual P(3HB) accumulation even under phosphate-rich conditions, or suggesting that B. sacchari could probably be under a limitation of other nutrient like oxygen (because the dissolved oxygen (DO) was close to zero during the whole cultivation).
Figure 15 - Effect of the dilution rate on the residual biomass (▲), phosphate (■) and P(3HB) concentration (●
), P(3HB) content (◆) in the first-stage bioreactor.
For a two-stage bioreactor system, Du G. et al., 2001 [21] obtained for the first bioreactor a maximum residual biomass concentration of 27.1 g/L at a dilution rate of 0.21 h-1 which is lower than the one obtained in this study (31 g/L).
The volumetric productivity of P(3HB) is by definition the amount of P(3HB) produced per hour per bioreactor volume. Eq.(7) was used to calculate the volumetric productivity of P(3HB) in the first-stage. Analogously, the volumetric productivity of residual biomass in the first bioreactor was calculated by Eq. (6).
𝑃𝑟𝑜𝑑!"#(𝑋𝑟)!=𝐷!∙𝑋𝑟! (6)
𝑃𝑟𝑜𝑑!"#(𝑃(3𝐻𝐵))!=𝐷!∙𝑃(3𝐻𝐵)! (7)
Figure 16 shows that the volumetric productivity of P(3HB) in the first bioreactor tends to increase with the increase of dilution rate and its value varied in a range 0.13-1.35 g/(h.L).
The volumetric productivity of residual biomass increased continuously with dilution rate from 0.10 to 0.31 h-1, and reached a maximum value of 7.88 g/(h.L). For dilution rates above 0.31 h-1, the residual biomass productivity started to decrease, as expected, since it approached wash-out conditions.
The specific P(3HB) production rate on the first bioreactor, qp1, was calculated by the Eq.
(8).
0.0 1.0 2.0 3.0 4.0 5.0
0 5 10 15 20 25 30 35
0.08 0.13 0.18 0.23 0.28 0.33 Phosphate concentra]on (g/L) P(3HB), Xr1 concentra]on (g/L) P(3HB) content (%)
D1 (h-‐1)
CSTR 1
P(3HB) % P(3HB) Xr1 Phosp1
26
𝑞!!=𝐷!∙ 𝑃(3𝐻𝐵)!𝑋𝑟!
(8)
Figure 16 – Influence of dilution rate on the volumetric productivity of residual biomass (◆) and volumetric productivity of P(3HB) (■), in the first-stage bioreactor.
The true or theoretical biomass yield on glucose is by definition the amount of biomass produced per unit glucose consumed for the purpose of cell growth. The observed biomass yield on glucose is by definition the amount of biomass produced divided by the total glucose consumed. In this work only the observed yields were calculated.
The equations used to calculate the yield of residual biomass on glucose and the yield of P(3HB) on glucose are presented in Eq. (9) and Eq. (10), respectively.
(𝑌!"/!)!= 𝐵𝑖𝑜𝑚𝑎𝑠𝑠!"#$%&'$ !
𝐺𝑙𝑢𝑐𝑜𝑠𝑒!"#$%&'( !
(9)
(𝑌!(!!")/!)! = 𝑃(3𝐻𝐵)!"#$%&'$ !
𝐺𝑙𝑢𝑐𝑜𝑠𝑒!"#$%&'( !
(10)
Where the biomass and the P(3HB) produced and the glucose consumed in the first bioreactor, (g/h), were calculated by Equations (11), (12) and (13).
𝐵𝑖𝑜𝑚𝑎𝑠𝑠!"#$%&'$ !=𝐹!"∙𝑋𝑟! (11) 𝑃(3𝐻𝐵)!"#$%&'$ !=𝐹!"∙𝑃(3𝐻𝐵)! (12) 𝐺𝑙𝑢𝑐𝑜𝑠𝑒!"#$%&'( !=𝐹𝑖!∙𝑆𝑖!−𝐹!"∙𝑆! (13) The influence of the dilution rate on the yields of residual biomass and P(3HB) on overall glucose consumption in the first bioreactor is shown in Figure 17. The yield of P(3HB) on glucose ranged from 0.02 to 0.09 gP(3HB)/gglucose. Regarding the yield of residual biomass on glucose, a plateau of approximately 0.42 gXr/gglucose from 0.10 to 0.24 h-1 can be observed and above that, it started to increase until it reached a maximum value of 0.53 g/g at 0.31 h-1. The reason it increases for higher D1 can probably be ascribed to less glucose being used for maintenance purposes at high dilution rates.
0.0 1.0 2.0 3.0 4.0 5.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
0.08 0.12 0.16 0.20 0.24 0.28 0.32
P(3HB) produc]vity (g/h.L)
Residual biomass produc]vity (g/h.L)
D1 (h-‐1)
CSTR 1
Prod (Xr1) Prod (PHB1)
27
Figure 17 - Effects of dilution rate on the observed yield of residual biomass from glucose (◆) and observed yield of P(3HB) from glucose (■), in the first-stage bioreactor.
In the study performed by Du G. et al., 2001 [43] with R. eutropha the maximum residual biomass productivity of 5.69 g/(L.h) at a dilution rate of 0.23 h-1 dilution rate and a residual biomass yield on glucose of 0.53 g/g at 0.21 h-1 dilution rate were obtained.