A simplified processing line from vinasse to succinate crystal is designed in this chapter based on previous work, experts’ suggestions as well as our own system in order to explain the industrialization potential and economic value of our project.
The processing line is mainly composed of four parts: pretreatment, degradation & production, purification and crystallization. Pretreatment aims at adjusting the vinasse’s pH to proper pH for our engineering bacteria. Fermentation contains two subunits, where engineered T. reesei and P. putida are inoculated in sequence. Purification with reference to experts from JOYOU CHEMICAL AND ENGINEERING CO., LTD utilizes the rising extraction technology which has been verified to save 3000–4000 yuan/batch. Crystallization is where succinate crystals are finally produced which is a rather mature technology.
In the best-case scenario, for each 500 kg vinasse, about 34.3 kg succinate can be produced in 10–12 days.
Generally, about 4 % of vinasse is composed of organic acid (Table 1), and according to our detection, the pH of our vinasse sample is around 4.36–4.45. Although we have cultivated acid-fast microbes surviving at pH 4, the acid produced with the growth may result in lower pH. Therefore, a pretreatment to adjust the pH to 5 is designed here[1][2].
Table 1. Major Components of Vinasse
| Component | Content % |
|---|---|
| Water | 60–65.3 |
| Cellulose | 10.1–37.7 |
| Hemicellulose | 12.6–19.6 |
| Lignin | 11.2–21.3 |
| Organic Acid | ~4 % |
Tip: Following calculations are conducted with the average of the bounds for each component.
According to literature research, common kinds of organic acid have an average pKa around 4.0 and an average molecular weight around 130 g/mol (Table 2). As it has been mentioned above, the original pH takes 4.4.
Table 2. Major Organic Acid in Vinasse
| Component | pKa | Molecular Weight (g/mol) |
|---|---|---|
| Quinic Acid | 3.46 | 192.17 |
| Vitamin C | 4.17 | 176.12 |
| Lactic Acid | 3.86 | 90.08 |
| Citric Acid | 4.76 | 192.12 |
| Fumaric Acid | 3.03; 4.44 | 116.07 |
| Malic Acid | 2.83 | 134.09 |
| Propionic Acid | 4.86 | 74.08 |
| Glutaric Acid | 4.34; 5.42 | 132.11 |
| Oxalic Acid | 1.71; 3.67 | 90.03 |
| Succinate | 3.9 | 118.09 |
| Butyric Acid | 4.83 | 88.11 |
| Phenylmalonate | – | 180.16 |
| Isovaleric acid | 4.77 | 102.13 |
Tip: The data all come from CAS SciFinder.
Calculate with the formula:
$$ \mathrm{pH} = \mathrm{p}K_a + \lg\frac{[\mathrm{A}^-]}{[\mathrm{HA}]} $$
Therefore, for each 500 kg vinasse, only 39 mL 1 mol/L NaOH aq is needed in this unit.
The moisture content of the vinasse is usually between 60 % and 65 % , and even after pH adjustment it doesn’t contain much water. Therefore, in order to better degrade insoluble plant fibers in vinasse, solid-state fermentation (SSF) is chosen.
Requirements for a proper reactor:
Rostary drum reactor is a well-acknowledged type of reactor for SSF meeting the standards above. The reactor contains cylindrical containers usually installed on a roller system that serves both as a support and for rotation a clock-shaped driving mechanism. The rotational speed is typically 1, 6, or 16 revolutions per minute. The reactor is equipped with inlet and outlet ports. The inlet pipe can reach the bottom of the drum or branch off at different parts of the pipe. The end part is a nozzle. Generally, a blower or an air compressor is used for ventilation. The air is sterilized with sulfuric acid and then moistened with sterile distilled water. Operations such as mixing, steam sterilization, cooling, inoculation, cultivation, and even drying can all be carried out in the reactor.[3]
The technology is industrially available. For example, in China, rotary drum reactors from 30 L to 2000 L (custom-tailored) are provided by Changzhou Sungod Biotechnology[4] and EastBio[5].
T. Reesei is inoculated to the pretreated vinasse first for primary degration of cellulose, hemicellulose, and lignin to provide better nutritional conditions for P.Putida. Both cellulose and hemicellulose can be transfered to glucose and lignin can be transfered to Aromatic monomer which can be used by our wngineered P.Putida. Based on existing technology, the potential degration rate of cellulose, hemicellulose and lignin are as follows[6][7]:
Table 3. Potential Degradation Rate in 8–10 Days
| Component | Content % |
|---|---|
| Cellulose | 39.1 |
| Hemicellulose | 13.1 |
| Lignin | 31.5 |
Table 4. Target Components 8–10 Days after First Inoculation
| Component | Content % | Amount (kg) |
|---|---|---|
| Cellulose | 25.7 | 72 |
| Hemicellulose | 25.0 | 70 |
| Lignin | 19.6 | 55 |
| Glucose | 20.2 | 56.5 |
| Aromatic Monomer | 9.5 | 26.5 |
P. Putida is dierctly inoculated to the broth after the first fermentation, since we’ve constructed a mutualistic system for the two microbes. In this process, T.Reesei continues to contribute on the degration of cellulose, hemicellulose, and lignin, while P.Putida is cultivated and transfers aromatic monomer into succinate.
According to the FBA model we’ve conducted, in the ideal case, each gram of biomass theoretically produces 5.34 g succinate each other. The consumption and production of relevant components each hour by 1 gram of biomass are as follows:
Table 5. Partial Fluxes in Second Fermentation
| Component | Production Rate (g h⁻¹ g-biomass⁻¹) |
|---|---|
| Cellulose & Hemicellulose | –2.53 |
| Lignin | –0.32 |
| Glucose | –1.01 |
| Aromatic Monomer | –0.31 |
| Succinate | +5.34 |
| Biomass | +0.60 |
According to suggestions from relavant experts, the model shows the ideal case, and when applied to reality, might be proper to take 40%. Therefore, in following calculations, it is assumed that each gram of biomass theoretically produces 2.14 g succinate each other.
Therefore, after t h:
The mass of Biomass:
$$ \begin{align} m_{bt} = (1 + v_b)^t \times m_{b0} = (1+0.6)^t \times 13.5 \end{align} $$
The mass of glucose consumed:
$$ \begin{align} \int_0^t m_{bt} \times v_g dt \end{align} $$
It is assumed that the propotion of each nutrient in the rate of their consumption is in line with the one in medium. We might consider only one nutrient and glucose is taken as example. Glucose’s final concentration should not be lower than its flux set in the medium.
$$ \begin{align} m_{g0} - \int_0^t m_{bt} \times v_g dt & \geq v_g \times m_{bt} \\ \Rightarrow 56.5 - \int_0^t 1.6^t \times 13.5 \times 1.01 / 1000 \times dt & \geq 1.01/1000 \times 1.6^t \times 13.5 \end{align} $$
So, the fermentation can at most sustain 15 h. Then the production of succinate after 15 h from 500 kg vinasse is ∫0tmbt × vsdt = ∫0151.615 × 13.5 × 2.14/1000 × dt = 70.9 kg.
Tip: vb - the biomass 1 g biomass can produce in one hour; vg - the glucose 1 g biomass can consume in one hour; vs - the succinate 1 g biomass can produce in one hour; mb0 - the primary biomass; mbt - the biomass after t h; mg0 - the primary mass of glucose.
After this unit, there might be 40.6 kg succinate recovered for each 500 kg vinasse.
Separation of liquid and solid in the broth is the first step to purify succinate. This step is composed of two filtration subunits of different purity. Now that around 40% of vinasse is solid, plate-and-frame filter can be applied first for rough separation. Later, ultrafiltration with is employed to remove tiny particles and microbe debris.
Plate-and-frame filter is one of the most commonly used filters in the early years of the chemical industry and is still widely used. A plate-and-frame filter press consists of alternating plates and hollow frames. The plates are lined on both sides with a filter cloth, while the frames create chambers for cake buildup during filtration. Frames include ports for feeding and washing, and plates have outlets for draining filtrate. This type of filter offers simplicity, low cost, and flexibility. It operates well at high pressures and capacity can be easily modified by adding or removing plates and frames. When operated correctly, it produces a denser and drier cake than most other filters with liquid recovery rate over 98%[8].
Ultrafiltration is primarily used for the retention of particles and macromolecules smaller than 0.1 μm, with membrane pore sizes ranging from 0.05 μm down to a few nanometers, generally not exceeding 1 nm[9]. The feed liquid flows at high speed inside the membrane tubes. Driven by pressure, the clarified permeate—containing small molecular components—passes outward through the membrane in a perpendicular direction, while the turbid concentrate rich in macromolecular components is retained by the membrane. A ceramic ultrafiltration membrane with a pore size of 0.01 μm can be used for this process[10].
According to literature research, distillation in a rotary vacuum evaporator (Hei-VAP Expert, heidolp) at 350 mbar and 80 °C can effectively remove volatile fatty acids, i.e. acetic and formic acid, and ethanol from the succinate mixture. In industry, the device might have to be assembled with evaporator, PHE, Steam Ejector,etc.
Amine-based extractants like TOA extract organic acids from the solution through reaction with the undissociated acid to form an acid-amine complex:
$$ \begin{align} H_{n}A + mTOA \rightleftharpoons H_{n}A · (TOA)_{m} \end{align} $$
Then, 6 N NaOH guaranteen the complete recovery of the acids from organic phase for the pH of the aqueous phase to values higher than pKa of the acids. According to literature research, when extractant used was tri-n-octylamine (TOA) with 1-octanol as a diluent for 6 hour, the process achieved 57.3 % recovery with relatively high succinate selectivity[11]. Method of the same principle has been realized in industry according to experts from JOYOU CHEMICAL AND ENGINEERING CO.,LTD, though the technical details are unaccacible to us.
This unit is rather common and mature for production of solid chemical products.
The succinate solution is concentrated first as saturated solution at 80 °C (Wt(succinate). % = 41.45), and then it is cooled down to 20 °C (Wt(succinate). % = 6.446), so that succinate can crystallize.
For solution with 40.6 kg succinate, mMax = 40.6 × (41.45 − 6.446)/41.45 = 34.4 kg.
That is to say that in the best-case scenario, for each 500 kg vinasse, about 34.3 kg succinate can be produced in 10~12 days.