Method for enhancing caproic acid production from lactic acid by coupling nano ferroferric oxide and cytochrome C

Abstract

The invention relates to the technical field of environmental protection, in particular to a method for enhancing caproic acid production from lactic acid by coupling nano ferroferric oxide and cytochrome C. Adding nano ferroferric oxide and cytochrome C into the lactic acid wastewater to be treated inoculated with the caproic acid functional bacterial liquid at the same time, so that the nano ferroferric oxide and the cytochrome C are coupled; according to the system, the synergistic effect of lactic acid and hexanoic acid in the electron transfer process is fully exerted, conversion from lactic acid to hexanoic acid is promoted, and the yield of 55% hexanoic acid is increased; besides, a coupling system formed by nano ferroferric oxide and cytochrome C can still maintain good performance under long-time operation or severe conditions, shows excellent durability and adaptability, and provides a new solution for improving water treatment efficiency and reliability.

Description

Technical Field

[0001] The invention relates to the technical field of environmental protection, and in particular to a method for coupling nano-ferroferric oxide and cytochrome C to enhance the production of caproic acid from lactic acid. Background Art

[0002] In recent years, anaerobic fermentation technology has been widely used to treat organic waste to generate high-value medium-chain carboxylic acids. Through carbon chain growth reactions, substances such as lactic acid, ethanol and water-soluble short-chain fatty acids can be converted into more hydrophobic medium-chain fatty acids, such as caproic acid. Caproic acid has been widely used in food, biofuel, rubber, resin synthesis and pharmaceutical fields due to its low solubility, high hydrophobicity, low O / C ratio and high energy density. Its use value is much higher than conventional biofuels such as ethanol, acetic acid and butyric acid.

[0003] In the biosynthesis of caproic acid, substances such as lactic acid act as electron donors and eventually produce caproic acid through a series of enzymatic reactions. In this process, electron transfer efficiency is a key factor affecting caproic acid production. In recent years, conductive materials and electrofermentation technology have been widely used to promote the efficiency of electron transfer in microbial metabolism. Conductive materials such as biochar, zero-valent iron, and ferroferric oxide have played an important role in enhancing anaerobic fermentation production due to their wide sources and relatively low prices.

[0004] Cytochrome C (Cyt C) is an electron transfer protein widely present in the inner membrane of eukaryotic mitochondria and bacteria, and plays a key role in the biological electron transfer chain. Through the cyclic transformation of oxidation and reduction states, cytochrome C plays an important role in the extracellular electron transfer process of microorganisms. It can not only directly participate in electron transfer, but also mediate electron transfer and indirectly assist electron transfer, thereby promoting the metabolic process of microorganisms and improving the generation efficiency of target products. It has good application potential in many fields such as energy production, wastewater treatment, and bioremediation.

[0005] In summary, both conductive materials and cytochrome C show great potential in electron transfer. If conductive materials and cytochrome C can be combined and applied in the process of lactic acid fermentation to produce caproic acid, whether it can promote the improvement of caproic acid conversion efficiency to a certain extent, or overcome other difficulties in the prior art, the inventor has conducted research on this technical solution. Summary of the invention

[0006] The purpose of the invention is to provide a method for coupling ferrosiric oxide and cytochrome C to enhance the production of caproic acid from lactic acid.

[0007] To achieve the purpose of the present invention, the technical solution adopted is:

[0008] Based on the current research status, in this application, a conductive material is coupled with cytochrome C (Cyt C) to promote the efficiency of lactic acid to caproic acid fermentation conversion and increase caproic acid production by regulating the efficiency of electron transfer.

[0009] When selecting conductive materials, it was found that nano-iron tetroxide (Fe3O4 NPs) is a ferromagnetic nanomaterial with good biocompatibility in the biomedical field. Its transfer efficiency and conductivity in the electron transfer process are better than those of ferrous chloride, ferric oxide and other materials. This is due to the fact that Fe 2+ and Fe 3+ Rapid electron transfer between; at the same time, the ferromagnetism of nano-iron tetroxide enables it to accelerate electron transfer under an external magnetic field, and it can also show high activity and strong stability in catalytic reactions. Therefore, nano-iron tetroxide is selected as the conductive material for coupling with cytochrome C.

[0010] After coupling nano-iron tetroxide with cytochrome C as a conductive material, cytochrome C is used as an electron mediator to assist Fe 2+ and Fe 3+ The electron transfer between them is enhanced, and the promotion effect of nano-ferroferric oxide on cytochrome C is utilized to increase the production of caproic acid.

[0011] The present application specifically provides a method for coupling nano-ferroferric oxide and cytochrome C to enhance lactic acid-type caproic acid production, wherein Fe3O4 NPs and Cyt C are simultaneously added to the wastewater to be treated inoculated with a caproic acid functional bacterial solution to couple Fe3O4 NPs and Cyt C, and finally a caproic acid-producing working solution consisting of a caproic acid functional bacterial solution, wastewater to be treated, Fe3O4 NPs and Cyt C is obtained.

[0012] Further, the concentration of Fe3O4 NPs in the working solution is 0.2-5 g / L, and the concentration of Cyt C in the working solution is 20-100 mg / L;

[0013] Furthermore, the concentration of Fe3O4 NPs in the working solution is 0.2-1 g / L, and the concentration of Cyt C in the working solution is 20-50 mg / L.

[0014] Furthermore, the method for coupling nano-ferroferric oxide and cytochrome C to enhance the production of caproic acid from lactic acid specifically comprises the following steps:

[0015] (1) Acclimation and enrichment of caproic acid functional bacteria: The caproic acid bacterial solution was cultivated and acclimated, and a solution containing lactic acid was continuously added, and then the microbial flora was enriched and screened to obtain a caproic acid functional bacterial solution that can utilize lactic acid to produce caproic acid under anaerobic fermentation conditions. OD 600 The value is 0.8±0.2.

[0016] Furthermore, the conditions of the acclimation and enrichment process are as follows: the lactic acid concentration in the added lactic acid solution is controlled at 10-15 gCOD / L (1 g lactic acid is equivalent to 1.067 gCOD); after adding the lactic acid solution, the initial pH value of the solution is adjusted to 5.5±0.5, the temperature is maintained at 35±2°C during the acclimation and enrichment process, and the culture is carried out under anaerobic conditions. The lactic acid solution is supplemented once every 7 days to keep the lactic acid concentration at 10-15 gCOD / L; after 3-5 times of lactic acid solution feeding, the caproic acid concentration is measured. When the caproic acid concentration no longer increases significantly, the acclimation and enrichment process is completed, and a caproic acid functional bacterial solution is obtained.

[0017] (2) Multi-source coupling to enhance the production of caproic acid from lactic acid: the caproic acid functional bacterial solution obtained in step (1) is inoculated into the wastewater to be treated containing lactic acid, and Fe3O4 NPs and Cyt C are added at the same time to obtain a caproic acid-producing working solution.

[0018] Furthermore, the purity of the Fe3O4 NPs is 97%, and the diameter ranges from 50 to 100 nm; the concentration of the Fe3O4 NPs in the working solution is 0.2 to 5 g / L;

[0019] Furthermore, the selected cytochrome C is derived from horse heart (95%, SDS-PAGE); the concentration of Cyt C in the working solution is 20-100 mg / L;

[0020] Furthermore, the concentration of Fe3O4 NPs in the working solution is 0.2-1 g / L, and the concentration of Cyt C in the working solution is 20-50 mg / L.

[0021] The conditions for multi-source coupling to enhance the production of caproic acid from lactic acid are as follows: the pH of the wastewater containing lactic acid to be treated is adjusted to between 6.0±0.5, and then the caproic acid functional bacterial solution obtained in the above step (1) is inoculated into the wastewater containing lactic acid to be treated at 60%-70% of the total volume of the working solution, and Fe3O4 NPs and Cyt C are added to obtain a working solution, the initial pH of the working solution is adjusted to 5.5±0.5, and then nitrogen is filled for 10 minutes to ensure that the reaction is carried out under anaerobic conditions.

[0022] The reaction was then placed in a shaker at 35±2°C and cultured at 120-150 rpm for 10-20 days.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. By coupling Fe3O4 NPs and Cyt C, the interaction between Fe3O4 NPs and Cyt C is utilized to improve the efficiency of electron transfer. At the same time, Cyt C is used as a mediating electron in the electron transfer process to promote the cyclic conversion of oxidized Fe(III) and reduced Fe(II), significantly improve the charge and discharge capacity of Fe3O4 NPs, enhance the efficiency of lactic acid oxidation and carbon chain extension, and increase the hexanoic acid yield by 55%.

[0025] 2. OD of bacterial solution after coupling of Fe3O4 NPs with Cyt C 600 The value has been significantly improved, and it has the potential to replace traditional immobilized membrane fillers to enrich bacterial liquid, providing a more efficient and economical method for bacterial liquid enrichment.

[0026] 3. The Fe3O4 NPs and Cyt C coupling system exhibits good stability and durability. Fe3O4 NPs can maintain the stability of its structure and performance under a variety of environments. The introduction of Cyt C helps to protect Fe3O4 NPs from interference from the external environment, extending the service life of the coupling system, so that the system can still maintain good performance under long-term operation or harsh conditions. It is suitable for environmental fields such as sewage treatment and provides a new solution for improving the efficiency and reliability of water quality treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Cyclic voltammetry curves of Fe3O4 NPs and Cyt C before and after coupling in the present invention;

[0028] Figure 2 The bacterial solution images are shown in the present invention without adding exogenous substances (a) and after adding Fe3O4 NPs coupled with Cyt C (b);

[0029] Figure 3 The three-dimensional fluorescence spectrum of the extracellular polymers after adding single Fe3O4 NPs or Cyt C and coupling the two in the present invention; DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0032] in,

[0033] The increase in caproic acid was the caproic acid accumulation minus the initial background value of caproic acid. During the data processing, the average of the caproic acid accumulation on days 12 to 16 was uniformly used as the caproic acid accumulation.

[0034] Example 1 Experiment on promoting lactic acid fermentation broth to produce caproic acid by using nano-ferroferric oxide as a single exogenous source

[0035] The caproic acid bacteria liquid obtained from a Shandong Biotechnology Co., Ltd. was inoculated into synthetic wastewater containing 15gCOD / L lactic acid and inorganic nutrients at an inoculation ratio of VS:COD=1:3, and then the pH of the reaction system was adjusted to 5.5±0.5 with NaOH solution and HCl solution. At the same time, 50mmol / L of sodium 2-bromoethylsulfonate (BES) was added to inhibit the activity of methanogens. N2 was then introduced for 5min to discharge the headspace, and the system was sealed and placed in a constant temperature shaker at 35±2℃ and 150rmp for acclimation and culture.

[0036] Feed is added once every 7 days to keep the lactic acid concentration in the solution at 10-15 gCOD / L. After fermentation for about 15-20 days, when the caproic acid production in the acclimated solution is stabilized at 2.5±0.5 g / L, acclimation is completed to obtain caproic acid functional bacterial solution.

[0037] The volume of the hexanoic acid functional bacterial solution to be inoculated can be calculated according to the volume of the wastewater to be treated, and the mass fraction of Fe3O4 NPs to be added can be calculated according to the volume of the wastewater to be treated and the volume of the hexanoic acid functional bacterial solution to be inoculated.

[0038] 30 mL of lactic acid synthetic wastewater was taken into a reaction bottle, the pH of the lactic acid synthetic wastewater was adjusted to between 6.0±0.5, and a certain mass of Fe3O4 NPs was added thereto to obtain a mixed solution. The mixed solution was first ultrasonically pretreated for 8 minutes, and then 70 mL of caproic acid functional bacterial liquid was inoculated into the lactic acid synthetic wastewater mixed solution containing Fe3O4 NPs to obtain caproic acid-producing working solution. The final total volume of the working solution was 100 mL.

[0039] The concentration of lactic acid in 100 mL of working solution was 20 gCOD / L, and the concentration of Fe3O4 NPs in 100 mL of working solution was 0.2 g / L.

[0040] The initial pH of the working solution was adjusted to 5.5±0.5, and then N2 was introduced for 5 min to exhaust the headspace. The solution was sealed and placed in a constant temperature shaker at 35°C and 150 rpm for culture. The pH was adjusted every two days to stabilize it at 5.5±0.5.

[0041] The caproic acid concentration was measured by gas chromatography every 2 days, and the final measured caproic acid accumulation was 4.26 g / L, and the relative increase rate of caproic acid was 15%.

[0042] Example 2

[0043] The experimental process of this embodiment is the same as that of embodiment 1, except that the concentration of Fe3O4 NPs in 100 mL of working solution is 1 g / L.

[0044] The final hexanoic acid accumulation was 4.49 g / L, and the relative increase rate of hexanoic acid was 28%.

[0045] Example 3

[0046] The experimental process of this embodiment is the same as that of embodiment 1, except that the concentration of Fe3O4 NPs in 100 mL of working solution is 5 g / L.

[0047] The final hexanoic acid accumulation was 4.36 g / L, and the relative increase rate of hexanoic acid was 21%.

[0048] Example 4

[0049] The experimental process of this embodiment is the same as that of embodiment 1, except that the concentration of Fe3O4 NPs in 100 mL of working solution is 10 g / L.

[0050] The final hexanoic acid accumulation was 3.83 g / L, and the relative increase rate of hexanoic acid was -10%.

[0051] Example 5 Experiment on promoting caproic acid production in lactic acid fermentation broth using cytochrome C as a single exogenous source

[0052] The experimental process of this embodiment is the same as that of embodiment 1, except that the exogenous additive Fe3O4 NPs is replaced by Cyt C, and the concentration of Cyt C in 100 mL of working solution is 20 mg / L; and no ultrasonic pretreatment is required, and only shaking is required to mix it in the reaction bottle.

[0053] The final hexanoic acid accumulation was 4.22 g / L, and the relative increase rate of hexanoic acid was 13%.

[0054] Example 6

[0055] The experimental process of this embodiment is the same as that of embodiment 5, except that the concentration of Cyt C in 100 mL of working solution is 50 mg / L.

[0056] The final hexanoic acid accumulation was 4.26 g / L, and the relative increase rate of hexanoic acid was 15%.

[0057] Example 7

[0058] The experimental process of this embodiment is the same as that of embodiment 5, except that the concentration of Cyt C in 100 mL of working solution is 100 mg / L.

[0059] The final hexanoic acid accumulation was 3.91 g / L, and the average relative increase rate of hexanoic acid was -5%.

[0060] Example 8 An experiment on promoting the production of caproic acid by lactic acid fermentation broth by coupling ferric oxide with cytochrome C

[0061] (1) Acclimation and enrichment of caproic acid functional bacteria:

[0062] The caproic acid bacteria liquid obtained from a Shandong Biotechnology Co., Ltd. was inoculated into synthetic wastewater containing 15gCOD / L lactic acid and inorganic nutrients at an inoculation ratio of VS:COD=1:3, and then the pH of the reaction system was adjusted to 5.5±0.5 with NaOH solution and HCl solution. At the same time, 50mmol / L of sodium 2-bromoethylsulfonate (BES) was added to inhibit the activity of methanogens. N2 was then introduced for 5min to discharge the headspace, and the system was sealed and placed in a constant temperature shaker at 35±2℃ and 150rmp for acclimation and culture.

[0063] Feed is added once every 7 days to keep the lactic acid concentration in the solution at 10-15gCOD / L. After fermentation for about 15-20 days, when the caproic acid production in the acclimated liquid stabilizes at 2.5g / L, acclimation is completed to obtain caproic acid functional bacterial liquid.

[0064] (2) Screening of single exogenous reinforcement conditions: The data in Examples 1-7 were comprehensively analyzed to screen out the best single exogenous reinforcement conditions.

[0065] (3) Multi-source coupling to enhance the production of caproic acid from lactic acid: 30 mL of 20 gCOD / L lactic acid synthetic wastewater was taken into a reaction bottle, the pH of the lactic acid synthetic wastewater was adjusted to between 6.0±0.5, and a certain mass of Fe3O4 NPs and Cyt C were added thereto to obtain a mixed solution. The mixed solution was first subjected to ultrasonic pretreatment for 8 min, and then 70 mL of caproic acid functional bacterial liquid was inoculated into the 20 gCOD / L lactic acid synthetic wastewater mixed solution containing Fe3O4 NPs and Cyt C to obtain a working solution. The total volume of the working solution was 100 mL.

[0066] According to the volume of wastewater to be treated, the volume of hexanoic acid functional bacterial solution to be inoculated and the mass fraction of Fe3O4NPs and Cyt C to be added can be calculated: the concentration of Fe3O4NPs in 100mL working solution is 0.2g / L, and the concentration of Cyt C is 20mg / L; the initial pH of the working solution is adjusted to 5.5±0.5, and then nitrogen is filled for 10 minutes to ensure that it reacts under anaerobic conditions.

[0067] The final hexanoic acid accumulation was 4.54 g / L, and the relative increase rate of hexanoic acid was 31%.

[0068] Example 9

[0069] The experimental process of this embodiment is the same as that of embodiment 8, except that in step (3), the concentration of Fe3O4NPs in 100mL working solution is 0.2g / L, and the concentration of Cyt C is 50mg / L.

[0070] The final hexanoic acid accumulation was 4.59 g / L, and the relative increase rate of hexanoic acid was 34%.

[0071] Example 10

[0072] The experimental process of this embodiment is the same as that of embodiment 8, except that in step (3), the concentration of Fe3O4NPs in 100mL of working solution is 0.2g / L, and the concentration of Cyt C is 100mg / L.

[0073] The final hexanoic acid accumulation was 4.30 g / L, and the relative increase rate of hexanoic acid was 17%.

[0074] Embodiment 11

[0075] The experimental process of this embodiment is the same as that of embodiment 8, except that the concentration of Fe3O4 NPs in 100 mL of working solution is 1 g / L, and the concentration of Cyt C is 20 mg / L.

[0076] The final hexanoic acid accumulation was 4.78 g / L, and the relative increase rate of hexanoic acid was 45%.

[0077] Example 12

[0078] The experimental process of this embodiment is the same as that of embodiment 8, except that the concentration of Fe3O4 NPs in 100 mL of working solution is 1 g / L, and the concentration of Cyt C is 50 mg / L.

[0079] The final hexanoic acid accumulation was 4.97 g / L, and the relative increase rate of hexanoic acid was 55%.

[0080] Embodiment 13

[0081] The experimental process of this embodiment is the same as that of embodiment 8, except that the concentration of Fe3O4 NPs in 100 mL of working solution is 1 g / L, and the concentration of Cyt C is 100 mg / L.

[0082] The final hexanoic acid accumulation was 4.41 g / L, and the relative increase rate of hexanoic acid was 24%.

[0083] Embodiment 14

[0084] The experimental process of this embodiment is the same as that of embodiment 8, except that the concentration of Fe3O4 NPs in 100 mL of working solution is 5 g / L, and the concentration of Cyt C is 20 mg / L.

[0085] The final caproic acid concentration was 4.47 g / L, and the relative increase rate of caproic acid was 28%.

[0086] Embodiment 15

[0087] The experimental process of this embodiment is the same as that of embodiment 8, except that the concentration of Fe3O4 NPs in 100 mL of working solution is 5 g / L, and the concentration of Cyt C is 50 mg / L.

[0088] The final hexanoic acid accumulation was 4.56 g / L, and the relative increase rate of hexanoic acid was 33%.

[0089] Comparative Example 1:

[0090] In this example, based on Example 1, no additional exogenous substances were added, and the initial background value of hexanoic acid was 2.26 g / L, which was used as a blank control (CK group), and the data obtained from Comparative Example 1 and Examples 1-15 were analyzed.

[0091] The increase in caproic acid is the accumulation of caproic acid minus the initial background value of caproic acid. In the data processing process, the average of the accumulation of caproic acid from the 12th to the 16th day is used as the accumulation of caproic acid. The specific data are as follows:

[0092] Table 1 Effects of different concentrations of Fe3O4NPs and CytC and their coupling on hexanoic acid production

[0093]

[0094]

[0095] Through the data analysis in Table 1, it can be found that in Examples 1-4, a single Fe3O4 NPs is added as an exogenous substance. After adding 1g / LFe3O4 NPs, the accumulation of caproic acid reaches 4.49g / L, and the relative increase rate is 28%. When the addition amount is 5g / LFe3O4 NPs, although the caproic acid production increases, the accumulation of caproic acid shows a downward trend. When the addition amount of Fe3O4NPs in Example 4 is increased to 10g / L, both the caproic acid production and the caproic acid accumulation show a significant decrease, and the relative increase rate of caproic acid is negative.

[0096] Combined with the analysis of the production of caproic acid after adding Cyt C, the relative increase rate of caproic acid in the reaction bottle with 50 mg / L Cyt C added in Example 6 was 15%. When the addition amount of Cyt C was 100 mg / L, the relative increase rate and calculated yield of caproic acid showed a downward trend.

[0097] Through a preliminary analysis of the effect of single exogenous addition on the caproic acid yield, it can be concluded that when the Fe3O4 NPs addition concentration is in the range of 0.2-5.0 g / L and the Cyt C addition concentration is in the range of 20-100 mg / L, the caproic acid output of lactic acid wastewater can be effectively increased.

[0098] After coupling Fe3O4 NPs with Cyt C, the production of caproic acid was further improved: in Example 8, after 0.2 g / L Fe3O4 NPs was coupled with 50 mg / L Cyt C, the final production of caproic acid was 4.54 g / L, and the relative increase rate of caproic acid was 31%; when the concentration of Fe3O4 NPs added was kept constant at 0.2 g / L, the relative increase rate of caproic acid was not obvious with the increase of Cyt C concentration, and the relative increase rate of caproic acid in Example 9 was only 34%. In Example 10, the concentration of Cyt C was further increased to 100 mg / L, and the production of caproic acid was lower than that of 0.2 g / L Fe3O4 NPs coupled with 20 mg / L Cyt C (Example 8);

[0099] When the concentration of Fe3O4NPs is increased to 1g / L, the maximum relative increase rate of hexanoic acid in the reaction bottle (Example 12) of 1g / LFe3O4NPs coupled with 50mg / L Cyt C can reach 55%, while the sum of the relative increase rates of adding 1g / LFe3O4NPs or 50mg / LCyt C is only 43%;

[0100] When the concentration of Fe3O4NPs was increased to 5g / L, after coupling with different concentrations of Cyt C, it had no obvious effect on the change in the yield of lactic acid fermentation converted into caproic acid. In addition, in the single external aid enhancement experiment, when the concentration of Fe3O4NPs was 5g / L, the relative increase rate of caproic acid showed a downward trend. It can be reasonably inferred that when the concentration of Fe3O4NPs was 5g / L and coupled with different concentrations of Cyt C, it had a certain promoting effect on the conversion of lactic acid fermentation into caproic acid, but compared with when the concentration of Fe3O4NPs was 0.2g / L or 1g / L and coupled with different concentrations of Cyt C, the promoting effect on the production of caproic acid from lactic acid was lower.

[0101] In addition, the addition of Fe3O4NPs and Cyt can increase the content of electron shuttles in microbial extracellular polymers. Figure 1 The integral area of ​​the mesocycle closed curve represents the capacitance of the material. The charge and discharge properties of the sample can be determined by cyclic voltammetry: the capacitance of Fe3O4NPs is 2.85553×10 -5 F, the capacitance of Fe3O4 NPs coupled with Cyt C is 5.75522×10 -5F. It can be seen that at the same scan rate, the cyclic voltammetry curve area in the reactor with Fe3O4 NPs coupled to Cyt C is the largest, which is 2 times higher than that of the group with only Fe3O4 NPs added. The charge and discharge capacity is stronger and the extracellular electron transfer between microorganisms is accelerated, indicating that Fe3O4 NPs coupled to Cyt C can significantly enhance the electron transfer process between microorganisms and the electrode surface.

[0102] Figure 2 The results show that the bacterial population of the control group without adding exogenous substances is more evenly distributed and has less sediment, indicating that the enrichment of the bacterial population is weaker. In contrast, the bacterial solution under the action of Fe3O4 NPs and coupled Cyt C has cotton-like sediment, indicating that the enrichment of the bacterial solution has been significantly improved. On the one hand, the coupling effect makes the bacterial solution increase faster, and on the other hand, Fe3O4 NPs can prevent the loss of bacterial solution.

[0103] OD 600 The value can be used to reflect the cell concentration in the fermentation broth. From Table 2, it can be seen that in the initial stage, that is, when no exogenous additives are added (Comparative Example 1), the OD 600 The value is 0.56. After adding 1g / LFe3O4 NPs (Example 2) on day 16, the OD 600 The value can reach 1.25. After 1g / L Fe3O4 NPs is coupled with 50mg / L Cyt C (Example 12), the OD 600 The value can reach 1.47, while the OD of comparative example 1 600 The value was only 0.89, indicating that the microorganisms were rapidly enriched after the addition of Fe3O4NPs, and the enrichment effect of microorganisms was more obvious after coupling with CytC.

[0104] Table 2 OD of different experimental groups at different culture times 600 value

[0105]

[0106] Figure 3 This is the three-dimensional fluorescence spectrum of microbial extracellular polymers after adding Fe3O4 NPs and Cyt C. As shown in the figure, (a) the fluorophore with an Ex / Em of 275–280 / 300–350 at the peak represents humic acid-like substances; (b) the fluorophore with an Ex / Em of 220–230 / 290–325 at the peak represents tryptophan-like substances.

[0107] It can be seen that the group with 1g / LFe3O4 NPs coupled with 50mg / LCyt C (Example 12) has the highest content of extracellular polymeric substances (EPS). Fe3O4 NPs not only act as electron transfer carriers themselves, but also increase the concentration of electron shuttles (humic acid-like) in EPS. After coupling with Cyt C, the rate of extracellular electron transfer can be further accelerated.

[0108] In summary, in the entire working system, the coupling system obtained by coupling Fe3O4 NPs and Cyt C has good stability and durability. During the experiment, it was found that Fe3O4 NPs not only performed well in improving the efficiency of electron transfer, but also showed excellent performance in enriching the bacterial community. The addition of Cyt C further improved the enrichment effect on the bacterial community. The treatment environment during sewage treatment is relatively harsh and time-consuming, so the stability and durability of the Fe3O4 NPs and Cyt C coupling system are particularly important. The coupling of Fe3O4 NPs and Cyt C enhances the system's ability to resist interference from the external environment, thereby extending the service life of the coupling system, so that the system can still maintain good performance under long-term operation or harsh conditions.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for coupling nano-ferroferric oxide and cytochrome C to enhance the production of caproic acid from lactic acid, characterized in that: Fe3O4 NPs and Cyt C are simultaneously added to the wastewater to be treated inoculated with caproic acid functional bacteria liquid to obtain a caproic acid-producing working solution, wherein the concentration of Fe3O4 NPs in the working solution is 0.2-5 g / L, and the concentration of Cyt C in the working solution is 20-100 mg / L.

2. A method for coupling nano-ferroferric oxide and cytochrome C to enhance the production of caproic acid from lactic acid, characterized in that: Fe3O4 NPs and Cyt C are simultaneously added to the wastewater to be treated inoculated with caproic acid functional bacteria liquid to obtain a caproic acid-producing working solution, wherein the concentration of Fe3O4 NPs in the working solution is 0.2-1 g / L, and the concentration of Cyt C in the working solution is 20-50 mg / L.

3. A method for coupling nano-ferroferric oxide and cytochrome C to enhance the production of caproic acid from lactic acid, characterized in that: Specifically include the following steps: (1) Acclimation and enrichment of caproic acid functional bacteria: The caproic acid bacteria liquid is cultivated and acclimated by continuously adding lactic acid solution to achieve the purpose of enriching and screening the microbial flora, and finally the caproic acid functional bacteria liquid containing lactic acid capable of producing caproic acid under anaerobic fermentation environment is obtained; (2) Multi-source coupling to enhance the production of caproic acid from lactic acid: the caproic acid functional bacterial solution obtained in step (1) is inoculated into the wastewater to be treated containing lactic acid, and Fe3O4 NPs and Cyt C are added simultaneously to obtain a caproic acid-producing working solution.

4. The method for enhancing the production of caproic acid from lactic acid by coupling nano-ferroferric oxide and cytochrome C as claimed in claim 3, characterized in that: Specifically include the following steps: (1) Acclimation and enrichment of caproic acid functional bacteria: the caproic acid bacterial solution is cultivated and acclimated, a lactic acid solution with a concentration of 10 to 15 gCOD / L is continuously added, and the mixed microbial flora is enriched and screened to obtain a caproic acid functional bacterial solution that can utilize lactic acid to produce caproic acid under an anaerobic fermentation environment; (2) Multi-source coupling to enhance the production of caproic acid from lactic acid: the caproic acid functional bacterial solution obtained in step (1) is inoculated into the wastewater to be treated containing lactic acid according to 60%-70% of the total volume of the working solution, and Fe3O4 NPs and Cyt C are added to obtain a caproic acid-producing working solution; the purity of the Fe3O4 NPs is 97%, the diameter is 50-100 nm, the concentration of the Fe3O4 NPs in the working solution is 0.2-5 g / L; the concentration of Cyt C in the working solution is 20-100 mg / L.

5. The method for enhancing the production of caproic acid from lactic acid by coupling nano-ferroferric oxide and cytochrome C as claimed in claim 4, characterized in that: Specifically include the following steps: (1) Acclimation and enrichment of caproic acid functional bacteria: culturing and acclimating the caproic acid bacterial solution, continuously adding a lactic acid solution with a concentration of 10 to 15 gCOD / L, adjusting the initial pH value of the solution to 5.5±0.5, maintaining the temperature at 35±2°C during the acclimation and enrichment process, culturing under anaerobic conditions, replenishing the lactic acid solution once every 7 days to maintain the lactic acid concentration at 10 to 15 gCOD / L, and obtaining the caproic acid functional bacterial solution capable of utilizing lactic acid to produce caproic acid under an anaerobic fermentation environment; (2) Multi-source coupling to enhance the production of caproic acid from lactic acid: the pH of the wastewater containing lactic acid to be treated is adjusted to between 6.0±0.5, and then the caproic acid functional bacterial solution obtained in step (1) is inoculated into the wastewater containing lactic acid to be treated according to 60%-70% of the total volume of the working solution, and Fe3O4 NPs and Cyt C are added to obtain a caproic acid-producing working solution; the purity of the Fe3O4 NPs is 97%, the diameter is 50-100nm, the concentration of the Fe3O4 NPs in the working solution is 0.2-5g / L; the concentration of the Cyt C in the working solution is 20-100mg / L; the initial pH of the working solution is adjusted to 5.5±0.5, and then nitrogen is filled for 10 minutes to ensure that it reacts under anaerobic conditions.

6. The method for enhancing the production of caproic acid from lactic acid by coupling nano-ferroferric oxide and cytochrome C as claimed in claim 5, characterized in that: Specifically include the following steps: (1) Acclimation and enrichment of caproic acid functional bacteria: the caproic acid bacterial solution is cultivated and acclimated, and a lactic acid solution with a concentration of 10 to 15 gCOD / L is continuously added, and the initial pH value of the solution is adjusted to 5.5±0.

5. During the acclimation and enrichment process, the temperature is maintained at 35±2°C, and the culture is carried out under anaerobic conditions. The lactic acid solution is supplemented once every 7 days to keep the lactic acid concentration at 10 to 15 gCOD / L; after 3 to 5 times of lactic acid solution feeding, the caproic acid concentration is measured. When the caproic acid concentration no longer increases significantly, the acclimation and enrichment process is completed, and the caproic acid functional bacterial solution capable of producing caproic acid by using lactic acid under an anaerobic fermentation environment is obtained; (2) Multi-source coupling to enhance the production of caproic acid from lactic acid: the pH of the wastewater containing lactic acid to be treated is adjusted to between 6.0±0.5, and then the caproic acid functional bacterial solution obtained in step (1) is inoculated into the wastewater containing lactic acid to be treated according to 60%-70% of the total volume of the working solution, and Fe3O4 NPs and Cyt C are added to obtain a caproic acid-producing working solution; the purity of the Fe3O4 NPs is 97%, the diameter is 50-100nm, the concentration of the Fe3O4 NPs in the working solution is 0.2-5g / L; the concentration of the Cyt C in the working solution is 20-100mg / L; the initial pH of the working solution is adjusted to 5.5±0.5, and then nitrogen is filled for 10 minutes to ensure that it reacts under anaerobic conditions, and the working solution is placed in a shaker at 35±2°C and 120-150rpm for batch shaking culture for 10-20 days.

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