The In Silico Optimization of a Batch Reactor for D-Fructose Production Using the Cetus Process with In Situ Cofactor Quick Regeneration
Abstract
1. Introduction
2. The Experimental Enzymatic Reactor
3. Kinetic Model of Biocatalytic Process
4. Dynamic Model of Enzymatic Batch Reactor
5. BR Simulation and Optimization
5.1. Nominal BR Simulation and Selection of Control Variables
5.2. Single-Objective Function Optimization (NLP) of the BR
Max Ω, where Ω = [P(t)]
5.3. Optimization Problem Constraints
- (a)
- The BR model in Equation (1);
- (b)
- To limit the excessive consumption of raw materials (especially the costly enzymes), feasible searching limits are imposed on the control/decision variables (Table 2), based on the previous trials of Maria et al. [47,73], and on literature information [74,75,76]. In mathematical terms, the constraints (b) translate to
6. Optimization Results and Discussion
- -
- -
- -
- A comparison of all BR operating alternatives in terms of P production and consumption of raw materials (based on the initial load) is presented in Table 7.
- (1)
- The non-optimal DS1–DS4 BR experimental runs defined in Table 2 perform much better if the NADPH is regenerated in situ. Thus, the realized yields (4.9/35, 11/35, 7.8/15 in Table 7) are very low if NADPH is not regenerated, though the yields are 100% if NADPH is regenerated. This is a major reason to use the in situ cofactor regeneration for this process.
- (2)
- The non-optimal BR operation (DS1–DS3) using in situ NADPH regeneration resulted in the high consumption of enzymes as a result of the operating alternatives in Table 7. This sub-optimal operation can be improved by applying an NLP procedure using the optimization objective in Equation (2), subjected to the constraints in Section 5.3. Thus, one obtains the optimal BR operation in Table 7 (last row), with the species dynamics plotted in Figure 3. Compared to the experimental nominal, non-optimal BR operation (DS1–DS4), with or without cofactor regeneration, the optimized BR with cofactor regeneration resulted in a 25% lower consumption of NADPH, though the amount of the processed substrate is approximately 3× higher. Moreover, the consumption of costly enzymes (ALR, FDH) is roughly half.
- (3)
- By analyzing the NLP optimal operating policy of the BR, shown in Table 7 and Figure 3, the following conclusion can be derived: the P-productivity increases with the initial substrate [kDG, NADPH] concentrations if enough enzymes (ALR, FDH) are present and if ALR (and FDH) is not deactivated too fast. To better fulfill such a condition, the best alternative could be to use more stable enzymes, that is, immobilized on suitable porous supports [86,87,88] (not investigated here).
- (4)
- For sufficiently stable (immobilized) enzymes (ALR, FDH), DF production maximization clearly depends on the available amount of substrate (kDG) and cofactor (NADPH). As the kDG results from Step 1 of the Cetus process [73], a more realistic optimization must concomitantly consider both linked Cetus processes. Some trials have already been conducted [89].
7. Conclusions
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
Abbreviations and Notations
Cj, | Species j concentration |
Kj, kj, y, kc2, KM2 | Kinetic model constants |
k | Rate constants vector |
Species j reaction rate | |
Temperature | |
Time | |
Batch time | |
Ω | Optimization objective function |
[x] | Concentration of species “x” |
Index | |
0,o | Initial |
Abbreviations | |
A, A* | NADPH, NADP+ |
ALR | Aldose reductase |
BR | Batch reactor |
DG | D-glucose |
DF | D-fructose |
DS1–DS4 | The data sets obtained by Maria and Ene [2013] in batch experiments aimed at investigating the kDG conversion to D-fructose |
E, ENZ | ALR enzyme |
Ein, E*Ay | Inactive forms of the enzyme E |
FBR | Fed-batch reactor |
FDH | Formate dehydrogenase |
GMO | Genetically modified organisms |
HFCS | High-fructose/glucose syrup |
HFS | High-fructose syrup |
kDG | Keto D-glucose |
Max | Maximum |
Min | Minimum |
NADPH | Nicotinamide adenine dinucleotide phosphate—reduced form |
NLP | Nonlinear programming |
P | Product (D-fructose) |
P2Ox | Pyranose 2-oxidase |
R1, R2 | Main reactions of the second step of the Cetus process (Figure 1) |
S | Substrate (kDG) |
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Characteristics | Glucose Isomerization [a,d] | Cetus Two-Step Process [b] | Inulin Hydrolysis [c] |
---|---|---|---|
Number of steps | 1 | 2 | 1 |
Conversion (%) | 50 (limited by the equilibrium) [d] | 99 | 99.5 |
Raw material availability | Glucose from the starch of crops, molasses, cellulose, and food processing byproducts [42,43] | Genetically modified chicory crop; cultures of Aspergillus sp. | |
Impurities in the product | Yes | Traces | Negligible amounts |
Reaction type | Enzymatic isomerization | Enzymatic oxidation (step 1), followed by enzymatic reduction (step 2) | Enzymatic hydrolysis |
Enzyme mobility | Immobilized [d] | Free (suspended) | Immobilized |
Enzyme stability and other additives | Intracellular glucose-isomerase (e.g., Streptomyces murinus) of low stability; metal (Al) salts | Pyranose 2-oxidase (P2Ox) and catalase (step 1); aldose reductase and NAD(P)H (step 2); enzymes are costly | Inulinase |
Temperature | 50–60 °C | 25–30 °C/ 25–30 °C | 55 °C (40–60 °C) |
Reaction time (h) | 7 | 3–20 (step 1); 25 (step 2) | 13 |
pH | 7–8.5 | 6.5–7(–8.5); 7–8.5 | 5.5 |
Reaction steps | 1 isomerization | 2 oxidation (step 1), reduction (step 2) | 1 hydrolysis |
Coenzyme necessary? | No | Yes, catalase for (step 1) to prevent P2Ox quick inactivation; NAD(P)H for step 2. NAD(P)H is continuously regenerated in situ. | No |
Product purification | Difficult [d] | Simple (due to high selectivity) | Simple (due to high selectivity) |
Product purity | 2–5% impurities [d] | High (99.9%) | High (99.9%) |
Parameter | Nominal Initial Value | Remarks | |
---|---|---|---|
Data set # 1 (DS1) | [S]o = [kDG]o | 35 mM | Other species initial conc. [P]o = 0; [A(+)]o = [NADP(+)]o = 0; [EA]o = 0 |
[A]o = [NADPH]o | 35 mM | ||
[E]o = [ALR]o | 0.0048 U/mL | ||
Data set # 2 (DS2) | [S]o = [kDG]o | 35 mM | |
[A]o = [NADPH]o | 35 mM | ||
[E]o = [ALR]o | 0.00257 U/mL | ||
Data set # 3 (DS3) | [S]o = [kDG]o | 35 mM | |
[A]o = [NADPH]o | 6 mM | ||
[E]o = [ALR]o | 0.0055 U/mL | ||
Data set # 4 (DS4) | [S]o = [kDG]o | 15 mM | |
[A]o = [NADPH]o | 35 mM | ||
[E]o = [ALR]o | 0.006 U/mL | ||
Temperature, pH | 25 °C, 7 [47,51] (optimal) | pH buffer | |
Optimization limits of initial loads | [S]o ∈ [5–100], mM [47,73] [NADPH]o ∈ [5–80], mM [74,75] | [E]o ∈ [0.003–0.1] U/mL [47,76] [FDH] ∈ [100–2000] (U/L) [12,51] | |
NADPH regeneration | [HCOO]o = [kDG]o | Similar to Maria [55]; Slatner et al. [51] | |
[CO2]o = 0; [FDH]o = 1000 U/L (adopted as an average) | [FDH]o should be determined using optimization | ||
Reactor volume (L) | 1 | Up to 3 L capacity | |
Batch time (tf) (h) | 24 | For DS1–DS4 | |
Solubility in water | DG (kDG) | 5–7 M | (25–30 °C) [77] |
DF | ca. 22.2 M | 25 °C, pH = 7 [https://en.wikipedia.org/wiki/Fructose, accessed on 14 August 2025] | |
CO2 solubility [CO2] * | 31.3 (mM) at (25 °C) | [78,79] | |
DG (kDG) water solution viscosity | 1–3 cps (for <0.3 M) 1000 cps (4.5 M, 30 °C) vs. 1094 cps (molasses, 38 °C) | [57,80] |
The Overall Reaction R1 Shown in Figure 1 and Its Associated Side Reactions |
Rate expressions of the reactions displayed in Figure 1—right, corresponding to the mechanism of the overall reaction R1 |
, (successive Bi-Bi mechanism) ; ; ; ; ; |
Rate Constant | Value | Rate Constant | Value |
---|---|---|---|
, mM/min/(U/mL) | 3.9 106 | , 1/(mM min) | 2.07·106 |
, mM | 65.41 | , 1/min | 858.23 |
, mM | 1.24 | , mM/(U/mL) | 1.48·104 |
, mM | 1427 | , 1/min | 7.01·10−2 |
, mM | 0.886 |
, (mM/min) |
kc2 = 0.1387, 1/min/(U/L); KM2 = 8.8047 × 10−2 mM; KHC = 5.0061 × 10−2; KNADP = 90.181 |
Key Species Mass Balances in the BR (Corresponding to Equation (1)) | The Main Experimental Conditions in Table 2 |
---|---|
; ; | Liquid volume = 1 L Phosphate buffer, pH = 7; 25 °C Initial concentrations are in the following ranges: [kDG] = 15–35 mM; [NADPH] = 6–35 mM; Initial [ALR] = 2.6–6 U/L; [HCOO]o = [kDG]o [56]; [FDH] = 100–2000 U/L. If [CO2] ≥ [CO2] *, then [CO2] ≈ [CO2] *, and the excess leaves the system. FDH inactivation is neglected. Notations: S = substrate (kDG); P = product (fructose); A = NADPH; A(+) = NADP(+); E = ALR. The units are in mM, min, and U/L. (*) denotes the saturation concentration of Table 2. |
Bioreactor Operation | Raw Material Consumption (a,b,c) | DF Prod, mmol | |||||||
---|---|---|---|---|---|---|---|---|---|
kDG, mmol | NADPH, mmol | Final NADPH, mmol | ALR, (U) | FDH, (U) | |||||
BR Non-optimal experiments [47] | Without NADPH regeneration, Figure 2 (d) (very poor) | DS1 | 35 | 35 | 0.18 | 4.8 | - | 11 | |
DS2 | 35 | 35 | 0.18 | 2.57 | - | 11.1 | |||
DS3 | 35 | 6 | 0.03 | 5.5 | - | 4.9 | |||
DS4 | 15 | 35 | 0.29 | 6 | - | 7.8 | |||
With NADPH regeneration, Figure 2 (d) (good) | DS1 | 35 | 35 | 1.25 | 4.8 | 1000 | 35 | ||
DS2 | 35 | 35 | 1.06 | 2.57 | 1000 | 35 | |||
DS3 | 35 | 6 | 0.5 | 5.5 | 1000 | 35 | |||
DS4 | 15 | 35 | 1.19 | 6 | 1000 | 15 | |||
BR optimal initial load, within limits in Table 2 | With NADPH regeneration Figure 3 (e,f) (best) | kDG | 100 | 100 | 26 | 1.17 | 3.38 | 440 | 100 |
NADPH | 26 | ||||||||
ALR | 3.38 | ||||||||
FDH | 440 |
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Maria, G.; Gheorghe, D.; Muscalu, C.; Scoban, A. The In Silico Optimization of a Batch Reactor for D-Fructose Production Using the Cetus Process with In Situ Cofactor Quick Regeneration. Dynamics 2025, 5, 35. https://doi.org/10.3390/dynamics5030035
Maria G, Gheorghe D, Muscalu C, Scoban A. The In Silico Optimization of a Batch Reactor for D-Fructose Production Using the Cetus Process with In Situ Cofactor Quick Regeneration. Dynamics. 2025; 5(3):35. https://doi.org/10.3390/dynamics5030035
Chicago/Turabian StyleMaria, Gheorghe, Daniela Gheorghe, Crina Muscalu, and Andreea Scoban. 2025. "The In Silico Optimization of a Batch Reactor for D-Fructose Production Using the Cetus Process with In Situ Cofactor Quick Regeneration" Dynamics 5, no. 3: 35. https://doi.org/10.3390/dynamics5030035
APA StyleMaria, G., Gheorghe, D., Muscalu, C., & Scoban, A. (2025). The In Silico Optimization of a Batch Reactor for D-Fructose Production Using the Cetus Process with In Situ Cofactor Quick Regeneration. Dynamics, 5(3), 35. https://doi.org/10.3390/dynamics5030035