A method for producing polyhydroxyalkanoate (PHA) by fermentation using food processing tail water and application thereof

Abstract

This invention discloses a method and application for producing polyhydroxyalkanoates (PHA) through fermentation of food processing wastewater, relating to the field of bio-based material manufacturing technology. The method includes the following steps: S1, after removing slag from oily food processing wastewater, it is separated into an aqueous phase and an oil-containing concentrated phase by air flotation; S2, the oil-containing concentrated phase is subjected to at least two stages of countercurrent washing with water, and the washing effluent is incorporated into the aqueous phase. The mixture is then hydrolyzed, clarified, nutrient-adjusted, and sterilized to prepare a microbial proliferation solution; the washed oil-containing concentrated phase is subjected to lipase hydrolysis, solid-liquid separation, pH adjustment, and sterilization to prepare a PHA accumulation solution with a total organic carbon to total nitrogen mass ratio of not less than 30. This invention uses the oil-water phase of the same wastewater as a starting point, using the nitrogen-containing aqueous phase for proliferation and the washed low-nitrogen oil phase for accumulation, avoiding the need for separate commercial carbon and organic nitrogen sources.

Description

Technical Field

[0001] This invention relates to the field of bio-based material manufacturing technology, specifically to a method and application for producing polyhydroxyalkanoates (PHA) by fermenting food processing wastewater. Background Technology

[0002] Slaughtering, meat processing, and seafood processing generate high-organic wastewater containing soluble protein, ammonium nitrogen, suspended particles, and animal and vegetable oils. Conventional treatment typically treats oils as a load affecting biological treatment, removing them via flotation and disposing of them as low-value waste oil; soluble nitrogen is then removed in subsequent biological treatment units. While this approach reduces the pollution load, it does not fully utilize the carbon sources from oils and the nitrogen sources from proteins.

[0003] PHA is a polyester synthesized intracellularly by various microorganisms under nutrient-imbalanced conditions. Cupriavidusnecator requires carbon, nitrogen, phosphorus, and trace elements for rapid proliferation during nutrient-sufficient stages, while directing carbon flow towards PHA accumulation during carbon-sufficient but nitrogen-limited stages. If the same oily food processing wastewater contains both nitrogen and oil, and is used directly as a culture medium without classification, the emulsification and mass transfer of the oil will hinder proliferation, while the continuous input of nitrogenous components will weaken the nitrogen restriction required for the accumulation stage.

[0004] Current wastewater-to-PHA (Probiotic Acid) solutions mostly involve acidifying the entire wastewater stream before supplying it to a mixed microbial community, or propagating it in a conventional culture medium and then adding commercial fatty acids. Simply using flotation, lipase hydrolysis, two-stage fermentation, or dissolved oxygen control cannot solve the problem of the opposite demand for carbon and nitrogen components in the same wastewater during the two physiological stages; and directly adding high doses of oil-containing phases to the tank may cause inhibition of long-chain fatty acids and fluctuations in oxygen transfer. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method and application for producing polyhydroxy fatty acid esters (PHA) through fermentation of food processing wastewater. Starting with the oil-water phase of the same wastewater stream, the nitrogen-containing aqueous phase is used for proliferation, and the washed low-nitrogen oil phase is used for accumulation, avoiding the need to set up separate commercial carbon sources and organic nitrogen sources.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for producing polyhydroxyalkanoates (PHA) through fermentation of food processing wastewater, comprising the following steps:

[0007] S1. After removing slag from the wastewater from oily food processing, the wastewater is separated into an aqueous phase and an oily concentrated phase by air flotation.

[0008] S2. The oil-containing concentrated phase is washed with water in at least two stages of countercurrent washing. The washing effluent is combined with the aqueous phase and hydrolyzed, clarified, nutrient-adjusted and sterilized to prepare a cell proliferation solution. The washed oil-containing concentrated phase is hydrolyzed with lipase, separated into solid and liquid, pH adjusted and sterilized to prepare a PHA accumulation solution with a total organic carbon to total nitrogen mass ratio of not less than 30.

[0009] S3. Inoculate Cupriavidus necator into the cell proliferation broth and carry out the first stage of aerobic culture using a fed-batch method. When the ammonium nitrogen in the fermentation broth is not higher than 50 mg / L, and the dissolved oxygen, measured by air saturation, rises by at least 10 percentage points relative to the point of suspension within 5-15 minutes after the suspension, stop feeding the cell proliferation broth.

[0010] S4. Except for the nitrogen-containing components in the tailwater of the PHA accumulation liquid, without adding any other nitrogen-containing nutrients, supply the PHA accumulation liquid in stages or continuously to maintain the free fatty acids in the fermentation broth (calculated as oleic acid) at 0.2-1.5 g / L, and carry out the second stage of aerobic culture.

[0011] S5. Isolate PHA-containing bacterial cells and extract PHA.

[0012] Furthermore, the wastewater from oily food processing originates from slaughtering, meat processing, or aquatic product processing and is collected before the main biological treatment unit; after slag removal, the chemical oxygen demand is 2000-20000 mg / L, the animal and vegetable oil content is 0.2-5.0 g / L, the total nitrogen content is 80-1500 mg / L, the pH is 5.5-8.5, and the total residual chlorine is not higher than 0.5 mg / L.

[0013] The advantage of this preferred scheme is that it limits the raw materials to before the main biological treatment, while retaining the oils and soluble nitrogen available for fermentation; at the same time, it uses COD, animal and vegetable oils, total nitrogen, pH and total residual chlorine to define the treatable window, avoiding the mistaken inclusion of terminal wastewater where organic matter has been deeply removed or where residual chlorine still has a significant antibacterial effect in the protection scope.

[0014] Furthermore, the dissolved air pressure for air flotation is 0.35-0.55 MPa, the reflux ratio is 20%-40%, and the residence time is 15-30 min; two-stage or three-stage countercurrent washing is adopted, the temperature is 45-60℃, the ratio of the total mass of water added to the mass of the oil-containing concentrated phase is 0.5-2.0:1, and fresh water is added to the final stage and then sequentially enters the preceding stage in a countercurrent manner.

[0015] The advantage of this preferred scheme is that the flotation parameters first concentrate the oil into a washable concentrated phase, followed by two or three stages of countercurrent washing, and then the entrained soluble nitrogen is carried back to the aqueous phase with a smaller amount of fresh water. This operation simultaneously improves the nitrogen nutrient recovery rate on the propagation side and the carbon-nitrogen ratio on the accumulation side, providing a material basis for the subsequent two-stage cultivation.

[0016] Further, the aqueous phase and washing effluent are hydrolyzed for 2-6 hours at 45-55℃ and pH 6.5-7.5 with the addition of neutral protease at a rate of 500-2000 U per gram of soluble protein. After clarification, no more than 10% of the final volume of homologous PHA accumulation solution is added to adjust the total organic carbon to total nitrogen mass ratio to 8-18, and phosphorus, magnesium, and trace elements are supplemented. No organic carbon or organic nitrogen sources other than those from the effluent source are added, and the effluent source liquid should not be less than 85% of the final volume.

[0017] The advantages of this preferred approach are: neutral proteases convert large protein molecules in the aqueous phase into readily available peptides and amino acids; adjusting the carbon-to-nitrogen ratio with a small amount of homologous accumulation solution can supplement the carbon source for proliferation without introducing exogenous organic carbon and nitrogen. The proportion of wastewater source liquid and the carbon-to-nitrogen ratio together ensure that the culture medium is both resource-efficient and supports stable proliferation.

[0018] Furthermore, after washing, lipase is added to the oil-containing concentrated phase at 500-3000 U per gram of oil, and hydrolyzed at 38-48℃ and pH 7.0-8.0 for 3-8 h to ensure that the oil hydrolysis rate is not less than 60%. After removing insoluble proteins and homogenizing, the free fatty acid content in the accumulated liquid is 20-80 g / L (based on oleic acid), the total organic carbon to total nitrogen mass ratio is 30-100, and the median droplet size is 5-30 μm.

[0019] The advantages of this preferred method are: external lipase hydrolysis pre-converts triglycerides into absorbable free fatty acids, and the removal of insoluble proteins and control of droplet size can reduce nitrogen entrainment and oil droplet aggregation. The free fatty acid concentration, carbon-to-nitrogen ratio, and D50 jointly limit the carbon source intensity, nitrogen limitation degree, and mass transfer state of the accumulation liquid.

[0020] Further, the strain was Cupriavidus necator DSM 545 or H16. Before fermentation, the seed culture medium containing fructose and ammonium sulfate was replaced with cell proliferation broth at volume ratios of 20%, 40%, 60%, 80%, and 100%, respectively, with each ratio being passaged 2-4 times. The lag period for this stage should not exceed 8 hours, and the culture sample should be sampled at an equal volume, centrifuged, washed, and resuspended to the original volume. If the absorbance at the endpoint of 600 nm decreases by no more than 10% compared to the previous replacement ratio, then the next stage can begin.

[0021] The advantage of this preferred approach is that incremental replacement culture allows the bacteria to gradually adapt to the salt, organic acids, and trace inhibitors in the tailwater; using the lag phase and the OD change after washing and resuspending as dual conditions for entering the next stage can avoid false adaptation judgments caused by the turbidity of the original solution.

[0022] Furthermore, in the first stage, the inoculum size is 5%-15%, the temperature is 28-33℃, the pH is 6.8-7.2, and the dissolved oxygen is 30%-50%, with initial loading combined with fed-batch culture for 8-24 h; preferably, the culture is transferred when the ammonium nitrogen is not higher than 30 mg / L and the dissolved oxygen recovers by at least 15 percentage points within 10 min of pausing feeding. In the second stage, the temperature is 28-33℃, the pH is 6.8-7.2, and the dissolved oxygen is 20%-40%, with culture for 18-48 h; with the PHA accumulation solution stopped, feeding is started or resumed when the free fatty acid is below 0.3 g / L and the dissolved oxygen recovers by at least 8 percentage points within 5 min, and feeding is stopped when the free fatty acid reaches 0.8-1.0 g / L or the dissolved oxygen is below 20%.

[0023] The purpose of this preferred scheme is as follows: In the first stage, the ammonium nitrogen threshold is used to characterize the near depletion of the nitrogen source, and the recovery of DO after pausing nitrogen-containing feeding is used to characterize the decline in the uptake of readily available substrates; in the second stage, the response of free fatty acids and DO under the stopped feeding state determines the resumption of feeding, and the upper limit of fatty acids or low DO under the feeding state determines the pause. The two sets of signals constrain the transition and substrate inhibition, respectively, avoiding control based solely on culture time or cumulative feed amount.

[0024] Further, after cultivation, a cross-flow membrane with a pore size of 0.1-0.2 μm is used to retain the bacterial cells. The permeate is heat-treated and used as the first-stage wash replenishment for the next batch, accounting for 10%-30% of the total wash volume in this stage. The concentrated bacterial cells are then subjected to high-pressure homogenization to disrupt cell walls, enzymatic removal of non-PHA cell material, washing with water, and drying to obtain PHA. Unused fermentation broth enters the main biological treatment unit, where PHA is used to manufacture biodegradable packaging films, packaging inserts, or agricultural covering materials.

[0025] The advantage of this preferred design is that the cross-flow membrane first separates the bacterial cells from the fermentation broth, and the permeate after heat treatment is only returned to the next batch of first-stage washing, so that its residual nitrogen ultimately enters the proliferation side with the first-stage washing effluent, without directly contaminating the oil phase that has completed the final washing. This reuse location takes into account both water conservation and the low nitrogen stability of the accumulated broth.

[0026] Compared with existing technologies, this invention provides a method and application for producing polyhydroxyalkanoates (PHA) through fermentation of food processing wastewater, which has the following beneficial effects: This invention starts with the oil-water phase separation of the same wastewater stream, using the nitrogen-containing aqueous phase for propagation and the washed low-nitrogen oil phase for accumulation, avoiding the need for additional, independent commercial carbon and organic nitrogen sources. Countercurrent washing is not simply purifying the oil phase: on the one hand, it transfers soluble nitrogen entrained in the oil phase to the propagation liquid, improving the recovery of usable nitrogen in the front stage; on the other hand, it increases the carbon-nitrogen ratio of the accumulation liquid, strengthening the nitrogen constraint in the later stage, thus producing a synergistic effect in both stages.

[0027] Outside the tank, lipase is used to pre-convert triglycerides into free fatty acids, reducing dependence on the strain's extracellular lipolysis capacity. Combined with droplet size control, this improves the availability of hydrophobic substrates and batch reproducibility. The ammonium nitrogen threshold confirms near nitrogen depletion, and the recovery of dissolved oxygen after feeding pauses reflects a decrease in the uptake rate of readily available substrates. Switching stages only after both conditions are met reduces premature or delayed switching caused by relying solely on time or a single nitrogen index. The second stage uses a combined start-stop system based on residual free fatty acids and dissolved oxygen to maintain an excess of carbon source without excessive accumulation, reducing the impact of high-concentration long-chain fatty acids on membrane structure, oxygen transfer, and cell activity. A portion of the permeate after cross-flow retention is returned to the next batch of first-stage washes, with its residual nitrogen entering the proliferation medium with the first-stage wash effluent, avoiding direct contact with the oil phase after the final wash, thus balancing water reuse and the stability of the low-nitrogen-accumulation solution. Attached Figure Description

[0028] Figure 1 This is a laser confocal microscope image of the oil-containing concentrated phase obtained after air flotation separation of the wastewater from oily food processing in Example 2 of the present invention.

[0029] Figure 2 This is a diagram showing the total nitrogen mass distribution in the aqueous phase, the washing effluent from each stage, and the oil-containing concentrated phase after washing during the three-stage countercurrent washing process in Example 2 of the present invention.

[0030] Figure 3 The image shows the laser diffraction particle size distribution curve of the PHA accumulation solution obtained in Example 2 of this invention after lipase hydrolysis, removal of insoluble proteins, and homogenization.

[0031] Figure 4 This is a transmission electron microscope image of an ultrathin section of Cupriavidus necator bacterial cells after the second stage of culture in Example 2 of the present invention. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Please see Figures 1-4 This invention provides a method and application for producing polyhydroxyalkanoates (PHA) through fermentation of food processing wastewater:

[0034] Unless otherwise specified, percentages are by mass, liquid ratios are by volume, and temperatures are in degrees Celsius; at least three parallel samples must be prepared for each test. The listed instrument brands are not necessarily limited; any equipment with equivalent accuracy and functionality may be used.

[0035] Process wastewater preceding the main biological treatment unit refers to the liquid generated and collected during the cleaning, raw material processing, or cutting of food production lines, but which has not yet entered the main biochemical units such as anaerobic, aerobic, or membrane bioreactors; it does not include the final effluent that has already undergone deep treatment.

[0036] Both the first-stage and final-stage washes are named according to the flow direction of the oil-concentrated phase; the oil-concentrated phase first contacts the first-stage wash solution and last contacts the final-stage fresh water. The aqueous phase separated in the final stage flows counter-currently to the previous stage.

[0037] The total organic carbon to total nitrogen mass ratio is expressed as the ratio of the TOC and TN mass concentrations measured at the same homogeneous liquid sample and the same sampling time point. Oil-containing samples are first thoroughly mixed at 40°C and then immediately separated to reduce sampling deviations caused by oil leaching.

[0038] The hydrolysis rate of fats and oils was calculated based on the reduction in triglycerides before and after hydrolysis, and cross-validated by the amount of free fatty acids generated; free fatty acids were uniformly converted into oleic acid equivalents.

[0039] OD 600 Do not directly read the turbid tailwater stock solution. Take an equal volume of culture sample, centrifuge, wash twice with 0.85% sodium chloride and resuspend to the original volume, read the absorbance at 600 nm, and subtract the sterile blank under the same conditions. The lag period is determined by the time it takes for the corrected OD to continuously increase.

[0040] Dissolved oxygen recovery is expressed as percentage points of air saturation. During the judgment period, the corresponding feed is stopped, but the temperature, pH, stirring speed, aeration rate, and tank pressure settings remain unchanged; the online DO sampling interval is no longer than 10 seconds.

[0041] PHA content is the percentage by mass of methanol-hydrolyzable PHA monomers in the dry cell culture; PHA titer is the mass of PHA per unit final fermentation broth volume. The final volume includes all added liquid and is corrected for 20°C.

[0042] Cupriavidus necator DSM 545 and Cupriavidus necator H16 were obtained from publicly available bacterial culture collections. Neutral proteases and lipases were prepared using food-grade or fermentation-grade formulations, with the supplier's stated activity as the initial value, and the actual activity was verified using the corresponding substrate method before the dosage was calculated. All tanks and pipelines in contact with the bacterial culture were steam-sterilized or treated with equivalent heat intensity before use.

[0043] The standard seed culture medium contains 20 g fructose, 2.0 g (NH4)2SO4, 1.5 g KH2PO4, 3.5 g K2HPO4, 0.25 g MgSO4·7H2O, and 1.0 mL of trace element solution per liter, with a pH of 7.0. The trace element solution contains 2.0 g FeSO4·7H2O, 2.0 g CaCl2·2H2O, 0.50 g MnSO4·H2O, 0.50 g ZnSO4·7H2O, 0.05 g CuSO4·5H2O, and 0.05 g H3BO3 per liter. After filtration through 0.22 μm, it is added to the sterilized culture medium.

[0044] For washing of the oil-containing concentrated phase, the evaluation is based on the third batch after three consecutive batches have reached a stable material balance. If the first batch lacks the permeate from the previous batch, process water of the same ionic strength is used instead; from the second batch onwards, it is reused according to the specified ratio. The mass of the aqueous phase, oil phase, washing liquid, and solid residue is recorded for each batch. Batches with a material balance closure rate of less than 95% are not included in the statistics.

[0045] Example 1

[0046] This embodiment uses wastewater from slaughtering and processing, with each batch consisting of 5.0 m³. After removing hair, meat scraps, scales, and other visible solids through a 1.0 mm sieve, a homogeneous sample is taken for analysis. The results show a chemical oxygen demand (COD) of 2000 mg / L, animal and vegetable oil concentrations of 0.20 g / L, total nitrogen of 80 mg / L, pH of 5.5, and total residual chlorine of 0.50 mg / L. All liquid volumes are converted to 20°C. Three parallel samples are taken from each testing point.

[0047] The slag removal effluent was fed into a dissolved air flotation (DAF) unit with a dissolved air pressure of 0.35 MPa, a reflux ratio of 20%, and a hydraulic retention time of 15 min. The clarified flotation solution was collected as the aqueous phase, and the emulsified oily residue after dewatering of the slag was collected as the oil-containing concentrated phase. The equipment and pipelines were pre-circulated with 80°C hot water for 30 min.

[0048] Two-stage countercurrent washing was performed based on the flow direction of the oil-concentrated phase. The washing temperature was maintained at 45℃, and the ratio of total water mass to oil-concentrated phase mass was 0.5:1. Fresh water was added only to the final stage, and the aqueous phase separated from the final stage was used in the reverse direction for the previous stage. After stirring for 10 minutes in each stage, the phases were separated using a disc centrifuge at an equivalent centrifugal force of 5000×g, and the aqueous layer was used as the washing effluent. After entering the steady-state batch, the heat-treated permeate from the previous batch of fermentation was used as the first-stage supplementary washing liquid, accounting for 10% of the total washing liquid volume for that stage. All washing effluent was combined with the aqueous phase.

[0049] In preparing the cell proliferation broth, the aqueous phase and washing effluent were mixed thoroughly. Neutral protease was added at 500 U / g per gram of soluble protein at 45℃ and pH 6.5, and hydrolysis was carried out for 2 h. The temperature was then raised to 85℃ and maintained for 15 min to inactivate the enzyme. The mixture was centrifuged at 8000×g for 10 min, and the supernatant was collected. PHA accumulation solution from the same batch, comprising 10% of the final volume of the cell proliferation broth, was added to adjust the total organic carbon to total nitrogen mass ratio to 8. Then, 1.0 g of KH₂PO₄, 1.0 g of K₂HPO₄, 0.25 g of MgSO₄·7H₂O, and 1.0 mL of trace element solution were added per liter. No organic carbon or nitrogen sources other than those from the wastewater were added; the wastewater source liquid comprised 85% of the final volume. The mixture was sterilized at 121℃ for 20 min and cooled for later use.

[0050] To prepare the PHA accumulation solution, sterile water was added to the washed oil-containing concentrated phase, and lipase was added at a rate of 500 U / g per gram of oil. Hydrolysis was carried out at 38℃ and pH 7.0 for 3 h with stirring. After hydrolysis, the enzyme was inactivated at 85℃ for 15 min, and insoluble proteins were removed by centrifugation at 8000×g for 10 min. The fatty acid-containing liquid phase was then homogenized using a high-shear homogenizer at 12000 r / min for 3 min. After adjustment with water or the same batch of washed oil phase, the oil hydrolysis rate of the accumulation solution was 62.0%, the free fatty acid content (based on oleic acid) was 20 g / L, the total organic carbon to total nitrogen mass ratio was 30, and the median droplet size was 5.0 μm. The pH was adjusted back to 7.0, and the solution was sterilized at 115℃ for 20 min and cooled under nitrogen protection.

[0051] The bacterial strain was *Cupriavidus necator* DSM 545. During acclimatization, the prepared bacterial cell proliferation broth was used to successively replace the conventional seed culture medium containing 20 g / L fructose and 2 g / L ammonium sulfate at volume ratios of 20%, 40%, 60%, 80%, and 100%, with each subculture repeated twice. Each subculture was performed with a 5% inoculum at 30°C and 180 r / min. After centrifugation, washing with 0.85% sodium chloride water, and resuspending the sample, OD values ​​were measured. 600 This is to eliminate background turbidity in the effluent. Only when the lag period does not exceed 8 hours and the final OD value is... 600 When the rate decreases by no more than 10% from the previous level, it will move to the next replacement ratio.

[0052] Fermentation was carried out in a 20 L mechanically stirred tank. An initial 3.0 L of cell proliferation solution was added, followed by 5% (or equivalent) of the initial working volume) of adaptation seed culture. The temperature was 28°C, and the pH was 6.8. Dissolved oxygen was maintained at 30% through cascaded regulation of agitation and aeration. No additional feed was added for the first 2 hours. Subsequently, 2.0 L of cell proliferation solution was added uniformly, and cultivation continued until the total first-stage duration of 8 hours. When ammonium nitrogen dropped to 30 mg / L, the temperature, pH, agitation, aeration, and back pressure settings were kept constant, except for pausing the cell proliferation solution. Within 5 minutes, a 15% increase in dissolved oxygen was observed compared to the point of pause. Based on this, the nitrogen-containing feed solution was stopped, and the second stage commenced.

[0053] In the second stage, apart from the trace nitrogenous components from the tailwater accompanying the PHA accumulation solution, no additional nitrogenous nutrients were added. The temperature and pH remained at 28℃ and 6.8, respectively, and the dissolved oxygen was set at 20%. The accumulation solution was controlled by rapid alkaline titration every 30 minutes: with the PHA accumulation solution supply stopped, the peristaltic pump was started when the free fatty acid (based on oleic acid) dropped to 0.20 g / L and the dissolved oxygen recovered by at least 8 percentage points within 5 minutes; when the free fatty acid reached 0.80 g / L or the dissolved oxygen was below 20%, the process was paused, with continuous or segmented feeding and incubation for 18 hours. A total of 10.0 L of PHA accumulation solution was supplied in the second stage. At the end of the incubation, sterile water was used to compensate for sampling and evaporation losses, and the final working volume was corrected to 15.0 L. Sampling was immediately centrifuged at 4℃. The aqueous phase was used for the determination of free fatty acids, ammonium nitrogen, and total organic carbon, while the bacterial cells were used for dry weight and PHA determination.

[0054] After cultivation, the bacterial cells were retained using a ceramic cross-flow membrane with a pore size of 0.10 μm. The permeate was kept at 80℃ for 30 min and used as replenishment for the first-stage wash of the next batch at the aforementioned ratio of 10%; the unused portion was sent to the main biological treatment unit. The concentrated bacterial solution was homogenized twice under high pressure at 80 MPa, and then treated with 100 U / g dry bacterial cells of protease and nuclease at pH 8.0 and 50℃ for 2 h. After washing three times with sterile water, the solution was vacuum dried at 60℃ to constant weight to obtain PHA sample E1.

[0055] Example 2

[0056] This embodiment uses wastewater from meat processing, with each batch consisting of 1.0 m³. After removing hair, meat scraps, scales, and other visible solids through a 1.0 mm sieve, a homogeneous sample is taken for analysis. The results show a chemical oxygen demand (COD) of 11000 mg / L, animal and vegetable oil concentrations of 2.60 g / L, total nitrogen of 790 mg / L, pH of 7.0, and total residual chlorine of 0.25 mg / L. All liquid volumes are converted to 20°C. Three parallel samples are taken from each testing point.

[0057] The slag removal effluent was fed into a dissolved air flotation (DAF) unit with a dissolved air pressure of 0.45 MPa, a reflux ratio of 30%, and a hydraulic retention time of 22.5 min. The clarified flotation solution was collected as the aqueous phase, and the emulsified oily residue after dewatering of the slag was collected as the oil-containing concentrated phase. The equipment and pipelines were pre-circulated with 80°C hot water for 30 min.

[0058] A three-stage countercurrent washing process was performed, with the direction of the oil-concentrated phase flow as the baseline. The washing temperature was maintained at 52.5℃, and the ratio of total water mass to oil-concentrated phase mass was 1.25:1. Fresh water was added only to the final stage, and the aqueous phase separated from the final stage was used in the reverse direction for the previous stage. After stirring for 10 minutes in each stage, the mixture was separated into layers using a disc centrifuge at an equivalent centrifugal force of 5000×g, and the aqueous layer was used as the washing effluent. After entering the steady-state batch, the heat-treated permeate from the previous batch of fermentation was used as the first-stage supplementary washing liquid, accounting for 20% of the total washing liquid volume for that stage. All washing effluent was combined with the aqueous phase.

[0059] In preparing the cell proliferation broth, the aqueous phase and washing effluent were mixed thoroughly. Neutral protease was added at 1250 U / g of soluble protein per gram at 50℃ and pH 7.0, and hydrolysis was carried out for 4 h. The temperature was then raised to 85℃ and held for 15 min to inactivate the enzyme. The mixture was centrifuged at 8000×g for 10 min, and the supernatant was collected. PHA accumulation solution from the same batch, comprising 6% of the final volume of the cell proliferation broth, was added to adjust the total organic carbon to total nitrogen mass ratio to 13. Then, 1.0 g of KH₂PO₄, 1.0 g of K₂HPO₄, 0.25 g of MgSO₄·7H₂O, and 1.0 mL of trace element solution were added per liter. No organic carbon or nitrogen sources other than those from the wastewater were added; the wastewater source liquid comprised 92% of the final volume. The mixture was sterilized at 121℃ for 20 min and cooled for later use.

[0060] To prepare the PHA accumulation solution, sterile water was added to the washed oil-containing concentrated phase, and lipase was added at a rate of 1750 U / g per gram of oil. Hydrolysis was carried out at 43℃ and pH 7.5 with stirring for 5.5 h. After hydrolysis, the enzyme was inactivated by holding the solution at 85℃ for 15 min, and insoluble proteins were removed by centrifugation at 8000×g for 10 min. The fatty acid-containing liquid phase was then homogenized using a high-shear homogenizer at 12000 r / min for 3 min. After adjustment with water or the same batch of washed oil phase, the oil hydrolysis rate of the accumulation solution was 80.2%, the free fatty acid content (based on oleic acid) was 50 g / L, the total organic carbon to total nitrogen mass ratio was 65, and the median droplet size was 17.5 μm. The pH was adjusted back to 7.5, and the solution was sterilized at 115℃ for 20 min and cooled under nitrogen protection.

[0061] The bacterial strain was *Cupriavidus necator* DSM 545. During acclimatization, the prepared bacterial cell proliferation broth was used to successively replace the conventional seed culture medium containing 20 g / L fructose and 2 g / L ammonium sulfate at volume ratios of 20%, 40%, 60%, 80%, and 100%, with each subculture repeated three times. Each subculture was performed with a 5% inoculum at 30°C and 180 r / min. After centrifugation, washing with 0.85% sodium chloride water, and resuspending, the OD value was measured. 600 This is to eliminate background turbidity in the effluent. Only when the lag period does not exceed 8 hours and the final OD value is... 600 When the rate decreases by no more than 10% from the previous level, it will move to the next replacement ratio.

[0062] Fermentation was carried out in a 20 L mechanically stirred tank. An initial 3.0 L of cell proliferation solution was added, followed by 10% (or equivalent) of the initial working volume of adaptation seed culture. The temperature was 30.5 °C, and the pH was 7.0. Dissolved oxygen was maintained at 40% through cascaded regulation of agitation and aeration. No additional feed was added for the first 2 hours. Subsequently, 2.0 L of cell proliferation solution was added uniformly, and cultivation continued until the total first-stage duration of 16 hours. When ammonium nitrogen dropped to 20 mg / L, the temperature, pH, agitation, aeration, and back pressure settings were kept constant, except for pausing the cell proliferation solution. Within 10 minutes, a 18% increase in dissolved oxygen was observed compared to the point of pause. Based on this, the nitrogen-containing feed solution was stopped, and the second stage commenced.

[0063] In the second stage, apart from the trace nitrogenous components from the tailwater accompanying the PHA accumulation solution, no additional nitrogenous nutrients were added. The temperature and pH remained at 30.5℃ and 7.0, respectively, and the dissolved oxygen was set at 30%. The accumulation solution was controlled by rapid alkaline titration every 30 minutes: with the PHA accumulation solution supply stopped, the peristaltic pump was started when the free fatty acid (based on oleic acid) dropped to 0.28 g / L and the dissolved oxygen recovered by at least 8 percentage points within 5 minutes; when the free fatty acid reached 0.90 g / L or the dissolved oxygen was below 20%, the process was paused, with continuous or segmented feeding and incubation for 33 hours. A total of 8.0 L of PHA accumulation solution was supplied in the second stage. At the end of the incubation, sterile water was used to compensate for sampling and evaporation losses, and the final working volume was corrected to 13.0 L. Sampling was immediately centrifuged at 4℃. The aqueous phase was used for the determination of free fatty acids, ammonium nitrogen, and total organic carbon, while the bacterial cells were used for dry weight and PHA determination.

[0064] After cultivation, the bacterial cells were retained using a ceramic cross-flow membrane with a pore size of 0.15 μm. The permeate was kept at 80℃ for 30 min and used as replenishment for the first-stage wash of the next batch at the aforementioned ratio of 20%; the unused portion was sent to the main biological treatment unit. The concentrated bacterial solution was homogenized twice under high pressure at 80 MPa, and then treated with 100 U / g dry bacterial cells of protease and nuclease at pH 8.0 and 50℃ for 2 h. After washing three times with sterile water, the solution was vacuum dried at 60℃ to constant weight to obtain PHA sample E2.

[0065] Example 3

[0066] This embodiment uses wastewater from aquatic product processing, with each batch consisting of 0.50 m³. After removing hair, meat scraps, scales, and other visible solids through a 1.0 mm sieve, a homogeneous sample is taken for analysis. The results show a chemical oxygen demand (COD) of 20,000 mg / L, animal and vegetable oil concentrations of 5.00 g / L, total nitrogen of 1,500 mg / L, pH of 8.5, and total residual chlorine of 0.05 mg / L. All liquid volumes are converted to 20°C. Three parallel samples are taken from each testing point.

[0067] The slag removal effluent is fed into a dissolved air flotation (DAF) unit with a dissolved air pressure of 0.55 MPa, a reflux ratio of 40%, and a hydraulic retention time of 30 min. The clarified flotation solution is collected as the aqueous phase, and the emulsified oily residue after dewatering the slag is collected as the oil-containing concentrated phase. The equipment and pipelines are pre-circulated with 80°C hot water for 30 min.

[0068] A three-stage countercurrent washing process was performed, with the direction of the oil-concentrated phase flow as the baseline. The washing temperature was maintained at 60°C, and the ratio of total water mass to oil-concentrated phase mass was 2.0:1. Fresh water was added only to the final stage, and the aqueous phase separated from the final stage was used in the reverse direction for the previous stage. After stirring for 10 minutes in each stage, the mixture was separated into layers using a disc centrifuge at an equivalent centrifugal force of 5000×g, and the aqueous layer was used as the washing effluent. After entering the steady-state batch, the heat-treated permeate from the previous batch of fermentation was used as the first-stage supplementary washing liquid, accounting for 30% of the total washing liquid volume for that stage. All washing effluent was combined with the aqueous phase.

[0069] In preparing the cell proliferation broth, the aqueous phase and washing effluent were mixed thoroughly. Neutral protease was added at 2000 U / g per gram of soluble protein at 55℃ and pH 7.5, and hydrolysis was carried out for 6 h. The temperature was then raised to 85℃ and held for 15 min to inactivate the enzyme. The mixture was centrifuged at 8000×g for 10 min, and the supernatant was collected. 2% of the final volume of the cell proliferation broth containing the same batch of PHA accumulator was added to adjust the total organic carbon to total nitrogen mass ratio to 18. Then, 1.0 g of KH₂PO₄, 1.0 g of K₂HPO₄, 0.25 g of MgSO₄·7H₂O, and 1.0 mL of trace element solution were added per liter. No organic carbon or nitrogen sources other than those from the wastewater were added; the wastewater source liquid comprised 98% of the final volume. The mixture was sterilized at 121℃ for 20 min and cooled for later use.

[0070] To prepare the PHA accumulation solution, sterile water was added to the washed oil-containing concentrated phase, and lipase was added at a rate of 3000 U / g per gram of oil. Hydrolysis was carried out at 48℃ and pH 8.0 for 8 h with stirring. After hydrolysis, the enzyme was inactivated by holding the solution at 85℃ for 15 min, and insoluble proteins were removed by centrifugation at 8000×g for 10 min. The fatty acid-containing liquid phase was then homogenized using a high-shear homogenizer at 12000 r / min for 3 min. After adjustment with water or the same batch of washed oil phase, the oil hydrolysis rate of the accumulation solution was 92.1%, the free fatty acid content (based on oleic acid) was 80 g / L, the total organic carbon to total nitrogen mass ratio was 100, and the median droplet size was 30.0 μm. The pH was adjusted back to 8.0, and the solution was sterilized at 115℃ for 20 min and cooled under nitrogen protection.

[0071] The strain was *Cupriavidus necator* H16, obtained from a publicly available strain depository and revived according to the supplier's instructions. During acclimatization, the prepared cell proliferation broth was used to sequentially replace the conventional seed culture medium containing 20 g / L fructose and 2 g / L ammonium sulfate at volume ratios of 20%, 40%, 60%, 80%, and 100%, with each subculture repeated four times. Each subculture was performed with a 5% inoculum at 30°C and 180 r / min. After centrifugation, washing with 0.85% sodium chloride water, and resuspending, the OD was measured. 600 This is to eliminate background turbidity in the effluent. Only when the lag period does not exceed 8 hours and the final OD value is... 600 When the rate decreases by no more than 10% from the previous level, it will move to the next replacement ratio.

[0072] Fermentation was carried out in a 20 L mechanically stirred tank. An initial 3.0 L of cell proliferation solution was added, followed by 15% (or equivalent) of the initial working volume) of adaptation seed culture. The temperature was 33°C, and the pH was 7.2. Dissolved oxygen was maintained at 50% through cascaded regulation of agitation and aeration. No additional feed was added for the first 2 hours. Subsequently, 2.0 L of cell proliferation solution was added uniformly, and cultivation continued until the total first-stage duration of 24 hours. When ammonium nitrogen dropped to 5 mg / L, the temperature, pH, agitation, aeration, and back pressure settings were kept constant, except for pausing the cell proliferation solution. Within 10 minutes, a 20% increase in dissolved oxygen was observed compared to the point of pause. Based on this, the nitrogen-containing feed solution was stopped, and the second stage commenced.

[0073] In the second stage, apart from the trace nitrogenous components from the tailwater accompanying the PHA accumulation solution, no additional nitrogenous nutrients were added. The temperature and pH remained at 33℃ and 7.2, respectively, and the dissolved oxygen was set at 40%. The accumulation solution was controlled by rapid alkaline titration every 30 minutes: with the PHA accumulation solution supply stopped, the peristaltic pump was started when the free fatty acid (based on oleic acid) dropped to 0.29 g / L and the dissolved oxygen recovered by at least 8 percentage points within 5 minutes; when the free fatty acid reached 1.00 g / L or the dissolved oxygen was below 20%, the process was paused, with continuous or segmented feeding and incubation for 48 hours. A total of 7.0 L of PHA accumulation solution was supplied in the second stage. At the end of the incubation, sterile water was used to compensate for sampling and evaporation losses, and the final working volume was corrected to 12.0 L. Sampling was immediately centrifuged at 4℃. The aqueous phase was used for the determination of free fatty acids, ammonium nitrogen, and total organic carbon, while the bacterial cells were used for dry weight and PHA determination.

[0074] After cultivation, the bacterial cells were retained using a ceramic cross-flow membrane with a pore size of 0.20 μm. The permeate was kept at 80℃ for 30 min and used as replenishment for the first-stage wash of the next batch at the aforementioned ratio of 30%; the unused portion was sent to the main biological treatment unit. The concentrated bacterial solution was homogenized twice under high pressure at 80 MPa, and then treated with 100 U / g dry bacterial cells of protease and nuclease at pH 8.0 and 50℃ for 2 h. After washing three times with sterile water, the solution was vacuum dried at 60℃ to constant weight to obtain PHA sample E3.

[0075] Example 4

[0076] This embodiment uses wastewater from slaughtering and processing, with each batch consisting of 1.2 m³. After removing hair, meat scraps, scales, and other visible solids through a 1.0 mm sieve, a homogeneous sample is taken for analysis. The results show a chemical oxygen demand (COD) of 9800 mg / L, animal and vegetable oil concentrations of 2.40 g / L, total nitrogen of 720 mg / L, pH of 6.9, and total residual chlorine of 0.18 mg / L. All liquid volumes are converted to 20°C. Three parallel samples are taken from each testing point.

[0077] The slag removal effluent was fed into a dissolved air flotation (DAF) unit with a dissolved air pressure of 0.45 MPa, a reflux ratio of 30%, and a hydraulic retention time of 22 min. The clarified flotation solution was collected as the aqueous phase, and the emulsified oily residue after dewatering of the slag was collected as the oil-containing concentrated phase. The equipment and pipelines were pre-circulated with 80°C hot water for 30 min.

[0078] A three-stage countercurrent washing process was performed based on the flow direction of the oil-concentrated phase. The washing temperature was maintained at 52°C, and the ratio of total water mass to oil-concentrated phase mass was 1.2:1. Fresh water was added only to the final stage, and the aqueous phase separated from the final stage was used in the reverse flow to the previous stage. After stirring for 10 minutes in each stage, the mixture was separated into layers using a disc centrifuge at an equivalent centrifugal force of 5000×g, and the aqueous layer was used as the washing effluent. After entering the steady-state batch, the heat-treated permeate from the previous batch of fermentation was used as the first-stage supplementary washing liquid, accounting for 20% of the total washing liquid volume for that stage. All washing effluent was combined with the aqueous phase.

[0079] In preparing the bacterial cell proliferation broth, the aqueous phase and washing effluent were mixed thoroughly. Neutral protease was added at 1200 U / g per gram of soluble protein at 50℃ and pH 7.0, and hydrolysis was carried out for 4 h. The temperature was then raised to 85℃ and held for 15 min to inactivate the enzyme. The mixture was centrifuged at 8000×g for 10 min, and the supernatant was collected. PHA accumulation solution from the same batch, comprising 6% of the final volume of the bacterial cell proliferation broth, was added to adjust the total organic carbon to total nitrogen mass ratio to 1:3. Then, 1.0 g of KH₂PO₄, 1.0 g of K₂HPO₄, 0.25 g of MgSO₄·7H₂O, and 1.0 mL of trace element solution were added per liter. No organic carbon or nitrogen sources other than those from the wastewater were added; the wastewater source liquid comprised 92% of the final volume. The mixture was sterilized at 121℃ for 20 min and cooled for later use.

[0080] To prepare the PHA accumulation solution, sterile water was added to the washed oil-containing concentrated phase, and lipase was added at a rate of 1700 U / g per gram of oil. Hydrolysis was carried out at 43℃ and pH 7.5 with stirring for 5.5 h. After hydrolysis, the enzyme was inactivated by holding the solution at 85℃ for 15 min, and insoluble proteins were removed by centrifugation at 8000×g for 10 min. The fatty acid-containing liquid phase was then homogenized using a high-shear homogenizer at 12000 r / min for 3 min. After adjustment with water or the same batch of washed oil phase, the oil hydrolysis rate of the accumulation solution was 79.8%, the free fatty acid content (based on oleic acid) was 50 g / L, the total organic carbon to total nitrogen mass ratio was 65, and the median droplet size was 17.2 μm. The pH was adjusted back to 7.5, and the solution was sterilized at 115℃ for 20 min and cooled under nitrogen protection.

[0081] The bacterial strain was *Cupriavidus necator* H16. During acclimatization, the prepared bacterial cell proliferation broth was used to successively replace the conventional seed culture medium containing 20 g / L fructose and 2 g / L ammonium sulfate at volume ratios of 20%, 40%, 60%, 80%, and 100%, with each subculture repeated three times. Each subculture was performed with a 5% inoculum at 30°C and 180 r / min. After centrifugation, washing with 0.85% sodium chloride water, and resuspending the sample, OD values ​​were measured. 600 This is to eliminate background turbidity in the effluent. Only when the lag period does not exceed 8 hours and the final OD value is... 600 When the rate decreases by no more than 10% from the previous level, it will move to the next replacement ratio.

[0082] Fermentation was carried out in a 20 L mechanically stirred tank. An initial 3.0 L of cell proliferation solution was added, followed by 10% (or equivalent) of the initial working volume of adaptation seed culture. The temperature was 30.5 °C, and the pH was 7.0. Dissolved oxygen was maintained at 40% through cascaded regulation of agitation and aeration. No additional feed was added for the first 2 hours. Subsequently, 2.0 L of cell proliferation solution was added uniformly, and cultivation continued until the total first-stage duration of 16 hours. When ammonium nitrogen dropped to 45 mg / L, the temperature, pH, agitation, aeration, and back pressure settings were kept constant, except for pausing the cell proliferation solution. Within 15 minutes, a 10 percentage point increase in dissolved oxygen was observed compared to the point of pause. Based on this, the nitrogen-containing feed solution was stopped, and the second stage commenced.

[0083] In the second stage, apart from the trace nitrogenous components from the tailwater accompanying the PHA accumulation solution, no additional nitrogenous nutrients were added. The temperature and pH remained at 30.5℃ and 7.0, respectively, and the dissolved oxygen was set at 30%. The accumulation solution was controlled by rapid alkaline titration every 30 minutes: with the PHA accumulation solution supply stopped, the peristaltic pump was started when the free fatty acid (based on oleic acid) dropped to 1.20 g / L and the dissolved oxygen recovered by at least 8 percentage points within 5 minutes; when the free fatty acid reached 1.50 g / L or the dissolved oxygen was below 20%, the process was paused, with continuous or segmented feeding and incubation for 33 hours. A total of 8.0 L of PHA accumulation solution was supplied in the second stage. At the end of the incubation, sterile water was used to compensate for sampling and evaporation losses, and the final working volume was corrected to 13.0 L. Sampling was immediately centrifuged at 4℃. The aqueous phase was used for the determination of free fatty acids, ammonium nitrogen, and total organic carbon, while the bacterial cells were used for dry weight and PHA determination.

[0084] After cultivation, the bacterial cells were retained using a ceramic cross-flow membrane with a pore size of 0.15 μm. The permeate was kept at 80℃ for 30 min and used as replenishment for the first-stage wash of the next batch at the aforementioned ratio of 20%; the unused portion was sent to the main biological treatment unit. The concentrated bacterial solution was homogenized twice under high pressure at 80 MPa, and then treated with 100 U / g dry bacterial cells of protease and nuclease at pH 8.0 and 50℃ for 2 h. After washing three times with sterile water, the solution was vacuum dried at 60℃ to constant weight to obtain PHA sample E4.

[0085] Unless otherwise specified in the instructions, the following comparative examples are the same as those in Example 2.

[0086] Comparative Example 1

[0087] Except for the elimination of the three-stage countercurrent washing and the direct lipase hydrolysis of the oil-containing concentrated phase from the air flotation, all other raw material batches, water addition amounts, enzymatic hydrolysis, nutrient adjustment, strains, culture, and recovery conditions were the same as in Example 2. The resulting sample was numbered C1.

[0088] Comparative Example 2

[0089] The three-stage countercurrent washing was performed as usual, but the washing effluent was not combined with the aqueous phase but sent to wastewater treatment separately; the volume of the bacterial proliferation solution that was missing was made up with deionized water, and all other conditions were the same as in Example 2. The resulting sample was numbered C2.

[0090] Comparative Example 3

[0091] Except for the absence of lipase and stirring for only 5.5 h at the same temperature and pH, all other conditions were the same as in Example 2. The resulting sample was designated C3.

[0092] Comparative Example 4

[0093] Except for the first stage, where the process immediately transitioned to the second stage when ammonium nitrogen dropped to 30 mg / L, and the pause in feeding and confirmation of dissolved oxygen recovery were not performed, all other conditions were the same as in Example 2. The resulting sample was designated C4.

[0094] Comparative Example 5

[0095] At the start of the second stage, the same amount of PHA accumulation solution as the cumulative supply in the second stage of Example 2 was added at once, without starting or stopping based on free fatty acids and dissolved oxygen; all other conditions were the same as in Example 2. The resulting sample was numbered C5.

[0096] Comparative Example 6

[0097] Cupriavidus necator DSM 545 cultured on conventional fructose seed culture medium was directly inoculated into the bacterial proliferation solution without 20%-100% incremental subculturing; all other conditions were the same as in Example 2. The resulting sample was numbered C6.

[0098] Comparative Example 7: Permeate used for final stage washing

[0099] The permeate after heat treatment was added to the final washing stage at 20% of the total volume of the final washing liquid, instead of being used as supplementary water for the first washing stage; all other conditions were the same as in Example 2. The resulting sample was numbered C7.

[0100] Performance testing methods

[0101] Wastewater and process liquid water quality. Chemical oxygen demand (COD) was determined according to HJ 828-2017 "Determination of Chemical Oxygen Demand in Water - Dichromate Method"; total nitrogen was determined according to HJ 636-2012 "Determination of Total Nitrogen in Water - Alkaline Potassium Persulfate Digestion Ultraviolet Spectrophotometric Method"; ammonium nitrogen was determined according to HJ 535-2009 "Determination of Ammonia Nitrogen in Water - Nessler's Reagent Spectrophotometric Method"; total organic carbon was determined according to HJ501-2009 "Determination of Total Organic Carbon in Water - Combustion Oxidation-Non-Dispersive Infrared Absorption Method"; animal and vegetable oils were determined according to HJ 637-2018 "Determination of Petroleum and Animal / Vegetable Oils in Water - Infrared Spectrophotometric Method"; pH was determined according to HJ 1147-2020 Electrode Method; and total residual chlorine was determined according to HJ 586-2010 DPD Spectrophotometric Method. High-oil samples were first thoroughly homogenized and diluted within the allowable range of each standard.

[0102] Flotation and washing effects. Oil recovery rate is calculated using a closed-loop accounting method, combining the total oil mass before flotation with the oil mass in the oil-containing concentrated phase, the flotation aqueous phase, and the residue. Nitrogen transfer to the propagation side is calculated as the percentage of the sum of the total nitrogen (TN) mass in the aqueous phase and all washing effluent to the total TN mass of the effluent after sludge removal. Each flow is weighed independently or measured using a calibrated flow meter.

[0103] Free fatty acids and lipid hydrolysis rate. Free fatty acids were determined using the validated potassium hydroxide ethanol solution titration method, calculated as oleic acid. Sampling and endpoint determination followed the acid value procedure of Method I in GB 5009.229-2025. Triglycerides were determined by gas chromatography or high performance liquid chromatography. The hydrolysis rate results from the two methods differed by more than 5 percentage points, and the determination was repeated.

[0104] Droplet size. The PHA accumulation solution was gently inverted 10 times before measurement without additional sonication; the median particle size D50 of the volume distribution was determined according to GB / T 19077-2024 "Particle size analysis by laser diffraction".

[0105] Cell dry weight and viability. Take 10.0 mL of fermentation broth, centrifuge at 10000×g for 10 min, wash twice with an equal volume of deionized water, and vacuum dry at 60℃ until the difference between two consecutive weighings does not exceed 0.5 mg. Calculate the dry cell concentration. Cell viability was determined by fluorescent staining and counting on a flow cytometer. A control of live bacteria not exposed to the culture medium and a control of dead bacteria treated with 70% isopropanol were included.

[0106] Intracellular PHA content and titer. Accurately weigh 10-20 mg of lyophilized bacterial cells, add acidified methanol and chloroform containing an internal standard, and hydrolyze in methanol at 100℃ for 3 h in a sealed environment. After cooling, add water to separate the layers, and quantify 3-hydroxy fatty acid methyl esters using gas chromatography-flame ionization detector (GC-FID). Prepare a calibration curve with at least five points using certified purity PHA, with a correlation coefficient not less than 0.995, and a spiked recovery rate controlled at 90%-110%. The PHA titer is obtained by multiplying the dry bacterial cell concentration by the intracellular PHA mass fraction.

[0107] Carbon utilization and pollution reduction effects. The cumulative input organic carbon is calculated by summing the TOC mass of the initial liquid and each feed liquid. Unutilized carbon is calculated using a closed-loop accounting method based on the dissolved TOC of the final fermentation broth, tail gas carbon dioxide, and sampling losses. PHA carbon conversion is used for cross-sectional comparisons within the same batch. COD before and after entering the main biological treatment is determined according to HJ 828-2017, and the COD reduction rate of the unreused portion is reported.

[0108] PHA resins and products. The appearance, volatile matter, ash content, and purity of recycled PHA shall be in accordance with GB / T 30293-2013 "Biomanufacturing of Polyhydroxyalkanoates". The sample preparation and requirements for blown films shall refer to GB / T 33897-2017 "Biopolyester Polyhydroxyalkanoate (PHA) Blown Films", and the tensile properties shall be determined in accordance with GB / T 1040.3-2006, which is still valid as of the date of this manuscript; the final aerobic biodegradability under controlled composting conditions shall be determined in accordance with GB / T 19277.1-2025.

[0109] Table 1 shows the data for phase separation, washing, and the two types of culture media.

[0110] sample Oil recovery rate / % Nitrogen transfer rate to the proliferation side / % C / N ratio of proliferation solution Accumulation solution C / N Oil hydrolysis rate / % D50 / μm E1 82.4 77.8 8 30 62 5 E2 91.3 89.1 13 65 80.2 17.5 E3 95.6 93.4 18 100 92.1 30 E4 90.8 88.7 13 65 79.8 17.2 C1 91 62.3 13.1 12 80 17.8 C2 91.2 55.6 17 65 80.1 17.6 C3 91.1 88.9 13 64 8.6 44.8 C7 91.2 82.5 13 44 79.9 18

[0111] Table 2 shows the data on fermentation and pollution reduction performance.

[0112] sample Dry bacterial cells / g·L⁻¹ PHA content / % PHA titer / g·L⁻¹ Volumetric productivity / g·L⁻¹·h⁻¹ Organic carbon utilization rate / % COD reduction rate of unused liquid / % Cell viability / % E1 3.1 80.6 2.5 0.1 42 55 91 E2 15.8 80.4 12.7 0.26 75.1 80.5 95 E3 24.6 74.4 18.3 0.25 78 86 93 E4 12.6 69.8 8.8 0.18 60 71 82 C1 16 45 7.2 0.15 58 70 88 C2 11.5 79.1 9.1 0.19 65 72 94 C3 11 55.5 6.1 0.12 44 60 89 C4 13.5 67.4 9.1 0.19 62 70 91 C5 8.7 43.7 3.8 0.08 33 48 69 C6 10.7 55.1 5.9 0.12 47 62 84 C7 13.8 69.6 9.6 0.2 64 72 90

[0113] Table 3 shows the transition stability data.

[0114] plan Transition time / h <![CDATA[Ammonium nitrogen after phase transition / mg·L⁻¹]]> Pause for 10 minutes, DO rebounds by percentage points Transition time CV / % PHA content / % E2 Dual Signal 16.0±0.4 19.6±1.3 18.4±1.1 2.5 80.4±1.6 C4 Single Nitrogen Signal 13.8±1.7 28.7±2.0 5.2±2.4 12.3 67.4±3.8

[0115] PHA Products and Application Examples

[0116] Application Example 1

[0117] The PHA obtained in Example 2 was mixed with 40 parts by weight of PHA, 35 parts by weight of poly(butylene adipate / terephthalate), 20 parts by weight of polylactic acid, 3 parts by weight of tributyl acetylacetonate, 1 part by weight of talc, and 1 part by weight of epoxy chain extender. The temperatures of each zone of the twin-screw extruder from the feed end to the die head were 145°C, 155°C, 165°C, 170°C, and 165°C, respectively, with a screw speed of 180 r / min. After water cooling and pelletizing, the pellets were vacuum dried at 60°C for 8 h. A packaging film with a thickness of 30±3 μm was obtained by blowing the film at a temperature of 155-165°C, a blow-up ratio of 2.5, and a draw ratio of 3.0.

[0118] Application Example 2

[0119] Take 60 parts by weight of PHA obtained in Example 2, 30 parts by weight of polylactic acid, 5 parts by weight of poly(butylene adipate / terephthalate), 3 parts by weight of tributyl acetyl citrate, and 2 parts by weight of nucleating talc powder, and granulate them after vacuum drying at 60°C for 8 hours. Set the temperature of the injection molding machine barrel from the feed end to the nozzle to 150°C, 160°C, 170°C, and 170°C, the mold temperature to 55°C, the injection pressure to 75 MPa, and the holding pressure to 20 s to produce a food packaging inner tray.

[0120] Application Example 3

[0121] Take 30 parts by weight of PHA obtained in Example 2, 50 parts by weight of poly(butylene adipate / terephthalate), 15 parts by weight of polylactic acid, 3 parts by weight of tributyl acetylacetonate, 1 part by weight of carbon black masterbatch, and 1 part by weight of chain extender, granulate them according to the extrusion conditions of Application Example 1, and blow-dry them at 150-165℃ to form an agricultural cover film with a thickness of 12±2 μm. Before laying, test the film thickness, longitudinal and transverse tensile strength, and elongation at break, and evaluate the final aerobic biodegradability according to the controlled composting method.

[0122] Table 4

[0123] sample index Test value PHA resin E2 PHA purity / % 97.2 PHA resin E2 Volatile matter / % 0.3 PHA resin E2 Ash content / % 0.7 Packaging film Longitudinal / Transverse Tensile Strength (MPa) 31 / 27 Packaging film Longitudinal / Transverse Elongation at Break (%) 370 / 410 agricultural mulch film Longitudinal / Transverse Tensile Strength (MPa) 28 / 24 agricultural mulch film 180-day relative biodegradation rate (%) 91

[0124] As shown in Tables 1 and 2, this invention utilizes a nutrient-directed allocation method of "air flotation phase separation - countercurrent washing of the oil-containing phase - aqueous phase proliferation - low-nitrogen oil phase accumulation" to ensure that the soluble nitrogen-containing components and oil carbon sources in the same food processing wastewater are used for the cell proliferation stage and the PHA accumulation stage, respectively. In Example 2, the oil recovery rate and nitrogen transfer rate to the proliferation side reached 91.3% and 89.1%, respectively. The total organic carbon to total nitrogen mass ratio of the prepared PHA accumulation solution reached 65, and the final PHA content and PHA titer reached 80.4% and 12.7 g / L, respectively. In Comparative Example 1, after eliminating countercurrent washing, the oil recovery rate remained at 91.0%, but the nitrogen transfer rate to the proliferation side decreased to 62.3%, the total organic carbon to total nitrogen mass ratio of the PHA accumulation solution decreased to 12, and the PHA content and PHA titer decreased to 45.0% and 7.2 g / L, respectively. This indicates that countercurrent washing is not a typical oil-phase purification step, but rather a nutrient redistribution process that simultaneously achieves "washing out nitrogen entrained in the oil phase" and "recovering nitrogen-containing washing liquid," thus creating suitable nutrient conditions for both the initial bacterial proliferation and the subsequent nitrogen-limited accumulation.

[0125] The only difference between Example 2 and Comparative Example 2 is whether the washing effluent is incorporated into the aqueous phase. In Comparative Example 2, the nitrogen transfer rate to the propagation side decreased from 89.1% to 55.6%, the dry cell concentration decreased from 15.8 g / L to 11.5 g / L, and the PHA titer decreased from 12.7 g / L to 9.1 g / L; however, its PHA content still reached 79.1%. The above results indicate that incorporating the washing effluent into the aqueous phase mainly increases the recovery of available nitrogen and the cell count during the propagation stage, while having a relatively small impact on the low-nitrogen state of the accumulation liquid. Therefore, washing the soluble nitrogen entrained in the oil phase into the aqueous phase for use in the upstream propagation stage, and using the washed low-nitrogen oil phase for downstream PHA accumulation, are two complementary technical effects produced by the same material distribution measure.

[0126] By controlling the extracellular lipase hydrolysis and droplet size, the bioavailability of carbon sources in the oil-concentrated phase can be further improved. In Example 2, the oil hydrolysis rate reached 80.2%, the median droplet size in the accumulated liquid was 17.5 μm, and the PHA titer reached 12.7 g / L. In Comparative Example 3, after eliminating lipase hydrolysis, the oil hydrolysis rate was only 8.6%, the median droplet size increased to 44.8 μm, and the PHA content and titer decreased to 55.5% and 6.1 g / L, respectively. This indicates that large, unhydrolyzed oil droplets are difficult for the cells to stably take up in a short time, and easily cause uneven mass transfer and localized oil aggregation. This invention first converts triglycerides into free fatty acids outside the tank, and then controls the feed according to the residual free fatty acid concentration inside the tank. This reduces the dependence of the PHA accumulation process on the extracellular lipolysis capacity of the strain and improves the fermentation stability between different batches of tailwater.

[0127] As shown in Table 3, when both the ammonium nitrogen threshold and the dissolved oxygen recovery signal after feeding pause are used for stage transition, the transition time in Example 2 is 16.0 ± 0.4 h, with a coefficient of variation of only 2.5%, and the PHA content reaches 80.4 ± 1.6%. In Comparative Example 4, when stage transition is based solely on ammonium nitrogen concentration, the coefficient of variation of transition time increases to 12.3%, and the PHA content decreases to 67.4 ± 3.8%. This is because background turbidity of the effluent, sample dilution, and differences in nitrogen speciation may affect the ammonium nitrogen detection results, while the dissolved oxygen recovery after feeding pause can immediately reflect changes in the uptake of readily available substrates by the bacteria. Transitioning only after both signals are satisfied can reduce premature and delayed switching caused by transitioning solely based on culture time or a single nitrogen concentration.

[0128] In the second stage, the combined start-stop control of residual free fatty acids and dissolved oxygen response can prevent excessive accumulation of fatty acids. In Example 2, the PHA content, PHA titer, volumetric productivity, and cell viability were 80.4%, 12.7 g / L, 0.26 g / (L·h), and 95.0%, respectively. In Comparative Example 5, after adding an equal volume of PHA accumulation solution all at once, these indicators decreased to 43.7%, 3.8 g / L, 0.08 g / (L·h), and 69.0%, respectively. Meanwhile, the organic carbon utilization rate of Comparative Example 5 was only 33.0%, significantly lower than the 75.1% of Example 2. This indicates that adding an accumulation solution containing free fatty acids all at once easily leads to excessively high local substrate concentrations, insufficient oxygen transfer, and cell membrane damage. This invention restores feeding based on the residual free fatty acids and the recovery of dissolved oxygen during the feed stoppage state, and suspends feeding when free fatty acids reach their upper limit or dissolved oxygen decreases, thus maintaining the carbon source within a range that is "sufficient to accumulate PHA but insufficient to significantly inhibit cell growth."

[0129] Incremental substitution acclimation also significantly affected bacterial activity under real effluent conditions. In Example 2, after culturing with incremental substitutions of 20%, 40%, 60%, 80%, and 100%, the dry bacterial concentration, PHA content, and cell viability reached 15.8 g / L, 80.4%, and 95.0%, respectively; in Comparative Example 6, without incremental substitution acclimation, the corresponding indicators decreased to 10.7 g / L, 55.1%, and 84.0%. This indicates that the incremental substitution process can reduce the impact of sudden changes in salinity, organic acids, and trace inhibitors in the effluent on the bacterial cells; using the lag period and the endpoint absorbance after washing and resuspending as dual entry conditions can also eliminate the interference of background turbidity in the effluent on the judgment of bacterial adaptation status.

[0130] The location of permeate reuse also affects the low-nitrogen state of the PHA accumulation broth. In Example 2, the heat-treated fermentation permeate was used for the next batch of first-stage washing, resulting in a total organic carbon to total nitrogen mass ratio of 65, and PHA content and titer of 80.4% and 12.7 g / L, respectively. In Comparative Example 7, after using an equal amount of permeate for the final washing, the total organic carbon to total nitrogen mass ratio of the accumulation broth decreased to 44, and the PHA content and titer decreased to 69.6% and 9.6 g / L, respectively. This indicates that reusing the permeate for the first-stage washing allows residual nitrogenous components to enter the proliferation side with the first-stage washing effluent; if it is used directly for the final washing, residual nitrogen will re-enter the oil phase that has already undergone denitrification washing, thereby reducing the nitrogen constraint required for the second stage.

[0131] As shown in Table 4, after recovery and purification, the purity of the PHA resin obtained in Example 2 reached 97.2%, with volatile matter and ash content of 0.30% and 0.70%, respectively. The packaging film made from this PHA achieved longitudinal and transverse tensile strengths of 31 MPa and 27 MPa, respectively, and elongation at break of 370% and 410%, respectively. The agricultural covering film obtained from this PHA achieved longitudinal and transverse tensile strengths of 28 MPa and 24 MPa, respectively, and a relative biodegradation rate of 91% after controlled composting for 180 days. These results demonstrate that the PHA obtained in this invention can not only be used as a fermentation product for the resource recovery of food processing wastewater, but can also be further used to prepare biodegradable packaging films, packaging inserts, and agricultural covering materials, achieving synergistic effects of wastewater pollution reduction, waste oil carbon source utilization, and bio-based material manufacturing.

[0132] Depend on Figure 1 As can be seen, hydrophobic droplets are significantly enriched in the field of view, indicating that the flotation treatment can transfer emulsified and suspended oils in the original effluent to the oil-containing concentrated phase, providing a high concentration of oils for subsequent countercurrent washing and lipase hydrolysis. This proves that the flotation step does not only reduce the turbidity of the effluent, but also plays a role in the directional separation of oil carbon sources from the aqueous phase.

[0133] Depend on Figure 4 As can be seen, multiple complete rod-shaped or nearly circular bacterial cell cross-sections are observed, with a continuous high-electron-density envelope structure around the cell, and gray granular cytoplasm. Inside the cytoplasm, there are multiple nearly circular or elliptical low-electron-density bright areas, with some low-electron-density particles occupying a large area inside the cell, while the particles still retain a continuous cytoplasmic layer around them. No widespread breakage or large-scale dissolution of the bacterial cell envelope is observed. This indicates that the culture method of segmented supply of PHA accumulation solution and control of free fatty acids and dissolved oxygen in Example 2 can promote the formation of intracellular carbon storage particles while maintaining the basic structural integrity of the bacterial cells.

[0134] In summary, this invention, through the combination of technologies such as nutrient-directed distribution, pre-hydrolysis with lipase outside the tank, incremental strain replacement and domestication, ammonium nitrogen-dissolved oxygen dual-signal transduction, combined feeding of free fatty acids and dissolved oxygen, and first-stage recycling of permeate, prioritizes the use of soluble nitrogen in food processing wastewater for microbial proliferation, directs the use of carbon sources from washed low-nitrogen oils for PHA accumulation, and reduces substrate inhibition and batch fluctuations. When any one step in each of the single-factor comparative proportions was eliminated or changed, a decrease in nutrient distribution efficiency, microbial concentration, PHA content, PHA titer, productivity, or cell viability was observed. This indicates that the above technologies are not simply parallel but rather mutually reinforcing along a continuous process chain of "raw material distribution - microbial proliferation - stage switching - controlled accumulation - aqueous phase recycling".

[0135] The above are merely specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions, or modifications made based on the present invention to solve essentially the same technical problems and achieve essentially the same technical effects are all covered within the protection scope of the present invention.

Claims

1. A method for producing polyhydroxyalkanoates (PHA) by fermentation using food processing effluent, characterized in that, Includes the following steps: S1. After removing slag from the wastewater from oily food processing, the wastewater is separated into an aqueous phase and an oily concentrated phase by air flotation. S2. The oil-containing concentrated phase is washed with water in at least two stages of countercurrent washing. The washing effluent is incorporated into the aqueous phase liquid and then hydrolyzed, clarified, nutrient-adjusted and sterilized to prepare a cell proliferation solution. The washed oil-containing concentrated phase was hydrolyzed by lipase, separated into solid and liquid, pH adjusted and sterilized to prepare a PHA accumulation solution with a total organic carbon to total nitrogen mass ratio of not less than 30. S3. Inoculate Cupriavidus necator into the cell proliferation broth and carry out the first stage of aerobic culture by feeding. When the ammonium nitrogen in the fermentation broth is not higher than 50 mg / L, and the dissolved oxygen, measured by air saturation, rises by at least 10 percentage points relative to the point of suspension within 5-15 min after the cell proliferation broth is suspended, stop feeding the cell proliferation broth. S4. Without supplementing nitrogenous nutrients, the PHA accumulation solution is supplied in stages or continuously to maintain the free fatty acid content in the fermentation broth (calculated as oleic acid) at 0.2-1.5 g / L, and the second stage of aerobic culture is carried out. S5. Isolate PHA-containing bacterial cells and extract PHA.

2. The method of claim 1, wherein, The wastewater from oily food processing is the process wastewater generated during slaughtering, meat processing, or aquatic product processing and collected before the main biological treatment unit; after slag removal, the chemical oxygen demand of the wastewater is 2000-20000 mg / L, the content of animal and vegetable oil is 0.2-5.0 g / L, the total nitrogen content is 80-1500 mg / L, the pH is 5.5-8.5, and the total residual chlorine is not higher than 0.5 mg / L.

3. The method of claim 1, wherein, The dissolved air pressure of the air flotation is 0.35-0.55 MPa, the reflux ratio is 20%-40%, and the residence time is 15-30 min. The countercurrent washing is a two-stage or three-stage countercurrent washing, the washing temperature is 45-60℃, and the ratio of the total mass of water added to the mass of the oil-containing concentrated phase is 0.5-2.0:

1. The water added in the last stage is used in the countercurrent washing of the previous stages, and after each stage of washing, centrifugation or static stratification is performed to collect the washing effluent.

4. The method according to claim 3, characterized in that, When preparing the bacterial cell proliferation solution, the mixture of the aqueous phase and the washing effluent is used as the base liquid. At 45-55℃ and pH 6.5-7.5, 500-2000 activity units of neutral protease are added per gram of soluble protein, and hydrolysis is carried out for 2-6 hours. After clarification, the total organic carbon to total nitrogen mass ratio is adjusted to 8-18 by adding no more than 10% of the bacterial cell proliferation solution volume of PHA accumulation solution, and phosphorus, magnesium, and trace elements are supplemented. Except for the components from the tailwater source, no other organic carbon or organic nitrogen sources from non-tailwater sources are added. The proportion of tailwater source liquid in the total volume of the bacterial cell proliferation solution is not less than 85%.

5. The method according to claim 3, characterized in that, In preparing the PHA accumulation solution, water is added to the washed oil-containing concentrated phase, and 500-3000 activity units of lipase are added per gram of oil. The solution is hydrolyzed at 38-48℃ and pH 7.0-8.0 for 3-8 hours to ensure that the oil hydrolysis rate is not less than 60%. After removing insoluble proteins, the solution is homogenized to obtain a PHA accumulation solution with a free fatty acid content of 20-80 g / L, a total organic carbon to total nitrogen mass ratio of 30-100, and a median droplet size of 5-30 μm.

6. The method according to claim 1, characterized in that, The Cupriavidus necator is Cupriavidusnecator DSM 545 or Cupriavidus necator H16. Before the first stage of aerobic culture, the seed culture medium containing fructose and ammonium sulfate is replaced with the bacterial proliferation solution in sequence at volume ratios of 20%, 40%, 60%, 80%, and 100%, with each ratio being passaged 2-4 times. When the lag period of this stage of culture does not exceed 8 hours, and the absorbance at 600 nm after equal volume sampling, centrifugation, washing, and resuspending to the original volume of the culture sample decreases by no more than 10% compared to the previous replacement ratio, the next replacement ratio is entered.

7. The method according to claim 6, characterized in that, The inoculum size for the first stage of aerobic culture is 5%-15%, the culture temperature is 28-33℃, the pH is 6.8-7.2, and the dissolved oxygen is 30%-50% based on air saturation. The culture is carried out for 8-24 hours using an initial liquid loading combined with feeding. When the ammonium nitrogen is not higher than 30 mg / L, and the dissolved oxygen recovers by at least 15 percentage points within 10 minutes of pausing feeding, the culture is transferred to the second stage of aerobic culture.

8. The method according to claim 7, characterized in that, The second stage of aerobic culture is conducted at a temperature of 28-33℃, a pH of 6.8-7.2, and a dissolved oxygen level of 20%-40% (based on air saturation). The culture time is 18-48 h. When the free fatty acid content in the fermentation broth (based on oleic acid) is below 0.3 g / L, and the dissolved oxygen level recovers by at least 8 percentage points within 5 minutes after the PHA accumulation solution is stopped, the supply of PHA accumulation solution is started or resumed. When the free fatty acid content reaches 0.8-1.0 g / L or the dissolved oxygen level is below 20%, the supply of PHA accumulation solution is suspended.

9. The method according to claim 8, characterized in that, After the second stage of aerobic culture, a cross-flow membrane with a pore size of 0.1-0.2 μm is used to retain PHA-containing cells. The resulting permeate is heat-treated and used as supplementary water for the first stage washing of the next batch of oil-containing concentrated phase, accounting for 10%-30% of the total volume of the first stage washing liquid. The obtained cell concentrate is homogenized under high pressure to break the cell walls, and non-PHA cell material is removed by enzymatic hydrolysis. Then, it is washed with water and dried to obtain PHA.

10. The application of a method for producing polyhydroxyalkanoates by fermentation of food processing wastewater as described in any one of claims 1-9, characterized in that, The method is set before the main biological treatment unit of food processing wastewater. The unused portion of the fermentation broth containing PHA cells is sent to the main biological treatment unit for further treatment, and the obtained PHA is used to prepare biodegradable packaging films, packaging inserts or agricultural covering materials.

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