CN106978346B - Method for improving strain discharge amount of PHA (polyhydroxyalkanoate) synthesis strain output section - Google Patents
Method for improving strain discharge amount of PHA (polyhydroxyalkanoate) synthesis strain output section Download PDFInfo
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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Abstract
The invention discloses a method for improving the strain discharge amount of PHA synthesis bacteria in the output section, which takes PHA synthesis bacteria discharged from an SBR reactor in the output section of the PHA synthesis bacteria as a bacteria source, carries out substrate segmented supplementation to realize the expanded culture of the PHA synthesis bacteria, and then enters the third stage to complete the synthesis of PHA so as to greatly improve the integral PHA output and volumetric productivity of the process. The 'carbon source storage/endogenous growth' expanded culture mode introduced by the invention bears higher process load, so that the screening section of the PHA-producing flora can realize stable and controllable operation in a lower substrate load range, the contradiction between stable process operation and high PHA yield is solved, and the large-scale application of mixed PHA synthesis can be effectively promoted. After the 'carbon source storage/endogenous growth' expanded culture mode provided by the invention is embedded into the traditional three-stage process, the overall PHA yield of the process can be obviously improved, and the maximum PHA yield is about 80 times of that of the original process.
Description
Technical Field
The invention belongs to the technical field of biodegradable plastic synthesis and waste resource recovery, and relates to a high-yield polyhydroxyalkanoate process based on mixed flora expansion culture.
Background
Polyhydroxyalkanoate (PHA) is a biopolyester, which is an energy-storing substance synthesized intracellularly by microorganisms to resist unbalanced external environmental stress. PHA is similar in physical properties to traditional thermoplastics, so it can replace traditional chemically synthesized plastics, alleviating the problem of increasingly severe "white contamination". At present, the commercial promotion of the biosynthesis of degradable Plastics (PHA) is mainly pure bacterial fermentation, but the relatively high raw material cost, disinfection cost and microbial separation and purification cost limit the large-scale application of PHA. The PHA production process by the mixed flora is a completely open fermentation process, does not need substrate sterilization and strict prevention of mixed bacteria pollution, can utilize waste carbon sources, and is a hotspot of technical research on the basis of a wastewater acid production regulation and control technology.
The three-stage PHA synthesis process of mixed flora is generally divided into the following three stages:
1) substrate acid production stage: fermenting the complex organic substrate into small molecular organic acid;
2) PHA synthesis bacteria output section: domesticating PHA synthesizing bacteria in a Sequencing Batch Reactor (SBR) by taking small molecular organic acid as a carbon source, and regularly discharging the domesticated PHA synthesizing bacteria after the SBR stably runs, wherein the discharged PHA synthesizing bacteria have PHA synthesizing capacity but low PHA content in vivo and can accumulate PHA in vivo under high organic load and aerobic conditions;
3) a PHA synthesis section: and (3) taking micromolecular organic acid as a substrate, taking PHA (polyhydroxyalkanoate) synthesis bacteria discharged in the second stage as bacterial sludge, and adding the organic acid substrate in batches under aerobic conditions to complete PHA synthesis in the PHA synthesis bacteria.
The second stage is particularly critical among the three stages, because only SBR finishes domestication of PHA synthesis bacteria and can stably discharge the PHA synthesis bacteria as much as possible, and the third stage can synthesize more PHA by utilizing an organic acid substrate. The second stage of domestication generally adopts a 'satiation-hungry' (FF) mode under an aerobic condition, namely, a 'nutrient enrichment-nutrient deficiency' growth condition is created for mixed flora along with the reaction process under the aerobic reaction condition. The FF-based three-stage mixed flora process can realize the screening of PHA synthesis bacteria, the PHA synthesis bacteria have higher PHA content in the third stage and have achieved the level close to the PHA content fermented by pure bacteria, but in order to maintain the stable discharge of the PHA synthesis bacteria in the second stage, the organic load of the flora domestication stage (the second stage) needs to be controlled in a lower range, which directly results in the too low yield of mixed flora for PHA synthesis, thereby limiting the improvement of the total PHA yield of the process. Compared with the pure bacteria PHA synthesis process which is generally put into commercial operation, the large-scale application of the mixed bacteria process with resource benefit and environmental benefit is delayed, and the lower PHA yield is one of the key reasons. Therefore, the operation mode of the second stage, namely the domestication stage of the PHA synthesis bacteria is improved, the stability of SBR is maintained, the discharge amount of the PHA synthesis bacteria is improved, the total PHA yield is obviously improved, and the important propulsion effect is generated on the large-scale application of the mixed flora PHA synthesis.
Disclosure of Invention
In order to overcome the defect of low output of strains at the output section of PHA synthesis bacteria, the invention provides a method for improving the discharge amount of strains at the output section of PHA synthesis bacteria.
The purpose of the invention is realized by the following technical scheme:
a method for improving the strain discharge amount of a PHA synthesis strain output section comprises the following steps:
firstly, establishing a bacterial sludge expanding culture system: the bacterial sludge spread culture system consists of N bacterial sludge spread culture reactors, the operation of each bacterial sludge spread culture reactor is divided into N repeated batches along time, and each batch is divided into N-1 and N-2 sections;
secondly, when each bacterial sludge expanding culture reactor in the step one is started, a certain amount of bacterial sludge taken from the PHA synthetic bacteria output section of the three-section type mixed bacteria PHA process is placed in the bacterial sludge expanding culture reactor, and an expanding culture mode of carbon source storage/endogenous growth is adopted, namely: adding a proper amount of substrate A in the n-1 section, controlling the mass ratio of the substrate A to the microorganisms to be not higher than 6.4 g COD/g VSS/d, then aerating the mixed solution, and monitoring the dissolved oxygen level of the reaction system to be not lower than 3 mg/L; when the dissolved oxygen level in the system reaches a saturated state, stopping aeration, entering a static settling stage, discharging a certain volume of supernatant, and entering an n-2 section of the batch; adding a substrate B with the same volume as that of the discharged supernatant into the original bacterial sludge expanding culture reactor, aerating the mixed solution, and monitoring the dissolved oxygen level of the reaction system to ensure that the dissolved oxygen level is not lower than 3 mg/L; after the ammonium ions in the system are completely consumed, the reaction is stopped and enters a static precipitation stage, and the supernatant is discharged and enters the next batch of operation: substrate feeding A → aeration → static sinking → water discharging → substrate feeding B → aeration → static sinking → water discharging; and circulating and increasing the carbon source supplement amount in a gradient manner until the total biomass/the initial biological biomass for propagation is between 40 and 60 g/g.
Compared with the existing mixed PHA, the invention has the following advantages:
1. the 'carbon source storage/endogenous growth' expanded culture mode provided by the invention can realize large proliferation of PHA-producing biomass and effectively maintain the PHA synthesis capacity of the flora (figure 3);
2. after the 'carbon source storage/endogenous growth' expanded culture mode provided by the invention is embedded into the traditional three-stage process, the PHA yield of the whole process can be obviously improved, and the maximum PHA yield is about 80 times of that of the original process (figure 4);
3. from the whole process, the 'carbon source storage/endogenous growth' expanded culture mode introduced by the invention bears higher process load, so that the screening section of PHA-producing flora can realize stable and controllable operation in a lower substrate load range, the contradiction between stable process operation and high PHA yield is solved, and the large-scale application of mixed PHA synthesis can be effectively promoted;
4. the carbon source used in the invention can be a nitrogen-deficient or nitrogen-free high-concentration organic waste carbon source, such as molasses wastewater acidification liquid, papermaking wastewater acidification liquid and crude glycerol (biodiesel byproduct), and can bring certain environmental benefits by reducing biochemical oxygen demand while realizing resource recovery by using the waste carbon source.
Drawings
FIG. 1 is a schematic view of the operation of a bacterial sludge propagation reactor according to the present invention;
FIG. 2 is a schematic view of the insertion of an expanded culture mode into a conventional three-stage PHA synthesis process;
FIG. 3 is a specific example of the process of biomass amplification and PHA synthesis capacity of mixed flora in the enlarged cultivation process, wherein the black solid line represents the effective working volume of the enlarged cultivation batch reactor, the black solid square represents the intracellular maximum PHA synthesis ratio of the mixed flora, and the black open circle represents the biomass of the mixed flora;
FIG. 4 is a schematic diagram of the mode of operation of the expanding culture process for continuous production of PHA according to the present invention and a comparison of the PHA yield in the actual operation, wherein C1 represents the first bacterial sludge expanding culture batch reactor, and so on, taking the production cycle of 11 days as an example;
in the figure, the PHA yield value on the ordinate was calculated from the actual running results.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings, but not limited thereto, and any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.
The invention provides a method for improving the strain discharge amount of a PHA (polyhydroxyalkanoate) synthesis strain output section, which comprises the following specific steps of:
firstly, establishing a bacterial sludge expanding culture system: the bacterial sludge spread culture system consists of N bacterial sludge spread culture reactors, as shown in FIG. 1, the operation of each bacterial sludge spread culture reactor is divided into N repeated batches (batch 1, batch 2 … …, batch N) along the time, each batch is divided into two sections (N-1 and N-2) along the time, and two substrates, substrate A: mainly comprises a carbon source which does not contain nitrogen elements or limits nitrogen and takes small molecular organic acid (acetic acid, propionic acid, butyric acid and valeric acid) or glycerol as a main component; substrate B: for the strain nutrient solution without carbon source, the relative proportion of N, P element in the strain nutrient solution and the carbon source in the substrate A should satisfy the following conditions: COD (carbon source)/N/P = 100/(6-10)/(1-1.5) (mass ratio), and magnesium, calcium and trace elements are added to meet the growth requirement of the microorganism.
Secondly, when each bacterial sludge expanding culture reactor in the step one is started, a certain amount of bacterial sludge taken from the PHA synthesis bacterial output section of the three-section type mixed bacterial PHA process is placed in the reactor, and an expanding culture mode of carbon source storage/endogenous growth is adopted, namely: adding a proper amount of substrate A in the first batch 1-1 stage, controlling the mass ratio of the substrate A to the bacterial sludge to be not higher than 6.4 g COD/gVSS/d, aerating the mixed solution, monitoring the dissolved oxygen level of the reaction system to be not lower than 3 mg/L, and converting the extracellular carbon source into PHA particles to be stored in cells (a carbon source storage stage) by the PHA production mixed flora at the stage. When the dissolved oxygen level in the system reaches a saturated state, stopping aeration, entering a static settling stage, and discharging supernatant with a certain volume into the 1-2 stages of the batch. Adding substrate B with the same volume as that of the discharged supernatant into the original bacterial sludge reactor in the section 1-2 of the batch, aerating the mixed solution and monitoring the dissolved oxygen level of the reaction system to ensure that the dissolved oxygen level is not lower than 3 mg/L, and carrying out cell growth by using a carbon source stored in cells and free nutrient elements by the PHA-producing flora in the stage (an endogenous growth section). After the ammonium ions in the system are completely consumed, the reaction stops entering a static settling stage, the supernatant is discharged to enter a next batch 2-1, and the complete batch operation of the bacterial sludge culture expanding reactor comprises the actions of 'substrate feeding A → aeration → static settling → water discharging → substrate feeding B → aeration → static settling → water discharging', and the steps are circulated (as shown in figure 1). And when the biomass in the system is increased to a certain degree and the sludge load of the reaction system is lower than 1.6 g of COD/g of VSS/d, adding the substrate A in the carbon source storage section to enable the sludge load to be close to 6.4 g of COD/g of VSS/d, and synchronously increasing the concentration of the nutrient elements in the endogenous growth section according to the mass ratio of the nutrient elements to the carbon source in the step one. In order to prevent the accumulation of inert biomass, a certain amount of mixed bacteria and sludge can be discharged before the second stage of precipitation of each batch during the culture expanding reaction, and the mixed liquor (accounting for the total volume ratio) is preferably discharged from 1/10-1/20 every day. The number of batches included in a complete propagation cycle is preferably 40-60, which satisfies the biomass amplification multiple (total biomass at the end of propagation/total biomass at the beginning of propagation, g/g).
And thirdly, embedding the expanding culture mode into the traditional three-stage PHA synthesis process, namely collecting sludge discharged from the enrichment reactor for expanding culture reaction, and after the period is finished, completely using bacterial sludge in the bacterial sludge expanding culture system for the subsequent third-stage PHA synthesis (figure 2). The production cycle of PHA (in days) should include a complete propagation cycle and subsequent PHA production time. And (3) sequentially operating a plurality of expanding batch reactors according to the process mode in the second step, wherein the number of the expanding batch reactors is equal to the number of days of a complete production cycle, and if the number of the expanding batch reactors is small, rounding up (as shown in figure 4). After the time of one expanding culture period, the whole process can realize continuous output of PHA by taking days as the minimum unit.
Claims (5)
1. A method for improving the strain discharge amount of a PHA synthesis strain output section is characterized by comprising the following steps:
firstly, establishing a bacterial sludge expanding culture system: the bacterial sludge spread culture system consists of N bacterial sludge spread culture reactors, the operation of each bacterial sludge spread culture reactor is divided into N repeated batches along time, and each batch is divided into N-1 and N-2 sections;
secondly, when each bacterial sludge expanding culture reactor in the step one is started, bacterial sludge taken from the PHA synthetic bacteria output section of the three-section type mixed bacteria PHA process is placed in the bacterial sludge expanding culture reactor, and an expanding culture mode of carbon source storage/endogenous growth is adopted, namely: adding a substrate A in the n-1 section, controlling the mass ratio of the substrate A to the bacterial sludge to be not higher than 6.4 g of COD/gVSS/d, then aerating the mixed solution, and monitoring the dissolved oxygen level of the reaction system to be not lower than 3 mg/L; when the dissolved oxygen level in the system reaches a saturated state, stopping aeration, entering a static settling stage, discharging a certain volume of supernatant, and entering an n-2 section of the batch; adding a substrate B with the same volume as that of the discharged supernatant into the original bacterial sludge expanding culture reactor, aerating the mixed solution, and monitoring the dissolved oxygen level of the reaction system to ensure that the dissolved oxygen level is not lower than 3 mg/L; after the ammonium ions in the system are completely consumed, the reaction is stopped and enters a static precipitation stage, and the supernatant is discharged and enters the next batch of operation: substrate feeding A → aeration → static sinking → water discharging → substrate feeding B → aeration → static sinking → water discharging; and circulating and increasing the carbon source supplement amount in a gradient manner until the total biomass/the initial biological biomass for propagation is between 40 and 60 g/g.
2. The method as recited in claim 1, wherein the substrate A is a carbon source containing no nitrogen or limited nitrogen and mainly consisting of small organic acids or glycerol.
3. The method as recited in claim 2, wherein said small organic acid is acetic acid, propionic acid, butyric acid, valeric acid.
4. The method as claimed in claim 1, 2 or 3, wherein the substrate B is a nutrient solution of the strain containing no carbon source, and the mass ratio of N, P element in the nutrient solution to the carbon source in the substrate A satisfies the following conditions: COD/N/P = 100/(6-10)/(1-1.5).
5. The method as recited in claim 1, wherein in step two, a certain amount of the mixed bacterial sludge is discharged before the second stage of the static sedimentation of each batch, wherein the daily discharge amount is 1/10-1/20 of the total volume of the mixed bacterial sludge.
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