Method for rapidly degrading petroleum hydrocarbons in high-salinity oily wastewater
By using a mixed bacterial agent of Pseudomonas and denitrifying achromobacterium to treat high-salt and oily wastewater, the problem of slow degradation by a single bacterial species was solved, and the dominant bacterial community was quickly established, thereby improving the degradation efficiency of petroleum hydrocarbons and the wastewater treatment effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUANGDONG GANGHANG ENVIRONMENTAL PROTECTION TECH CO LTD
- Filing Date
- 2024-09-24
- Publication Date
- 2026-08-04
AI Technical Summary
In existing technologies, single bacterial species degrade slowly in the treatment of high-salt and oily wastewater, making it difficult to establish a dominant bacterial community in a short period of time, and thus failing to effectively meet the treatment requirements of wastewater with high petroleum hydrocarbon content.
A mixed bacterial agent of Pseudomonas and denitrifying achromobacter is used. After fermentation and concentration, it is mixed with an auxiliary liquid and added to high-salt and oily wastewater. The wastewater is degraded in an aerobic environment. Hydrophilic polyurethane filler and nutrient source are used to regulate and optimize the hydraulic retention time and environmental conditions.
It enabled the rapid establishment of a dominant microbial community in a short period of time, improved the degradation rate of petroleum hydrocarbons and COD load tolerance, significantly improved the wastewater treatment effect, and achieved the best petroleum hydrocarbon degradation effect.
Smart Images
Figure CN119707093B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of oily wastewater treatment, and in particular relates to a method for rapidly degrading petroleum hydrocarbons in high-salt oily wastewater. Background Technology
[0002] With the rapid development of the petroleum industry, a large amount of oily wastewater is inevitably generated during the production, storage, transportation, processing, and use of petroleum, especially high-salt oily wastewater generated in petroleum processing and port transportation. This wastewater is characterized by high crude oil content, high salinity, and large batch-to-batch fluctuations, which significantly impacts the continuous operation of wastewater treatment processes, particularly the biochemical treatment section, ultimately resulting in unsatisfactory wastewater treatment effects that fail to meet treatment requirements.
[0003] Currently, methods for treating high-salt, oily wastewater fall into three main categories: physical, chemical, and biological methods. Biological treatment methods are characterized by high treatment efficiency, mild conditions, low energy consumption, and no secondary pollution. They are typically used in the third stage of oily wastewater treatment, after multiple stages of oil removal including static oil separation, flotation oil separation, and filtration. At this stage, the COD of the wastewater is... Cr The concentration is 1000-1500 mg / L, and the organic matter is mainly long-chain alkanes and other recalcitrant hydrocarbons in the heavy oil. It is in an emulsified state in water, with oil droplet size less than 50 μm and salinity of 0.5-1.5%. It is necessary to cultivate special functional microorganisms that are salt-tolerant and have a high efficiency in degrading long-chain alkanes, and introduce them into the biochemical system to improve the degradation efficiency of organic matter.
[0004] The high efficiency of biological treatment methods mainly depends on the dominance and abundance of highly efficient alkane-degrading bacteria. In wastewater treatment, low efficiency is typically due to long hydraulic retention times and short microbial retention times.
[0005] In related research and development, patent CN118389348A discloses a strain of Pseudomonas sp. Z1 that can produce co-surfactants. This strain can transport petroleum hydrocarbons into the cell for degradation by producing co-surfactants, achieving a degradation rate of 85% for alkanes under suitable salinity and pH conditions. Patent CN118406583A discloses a strain of Achromobacter denitrifying chloroforms M1 suitable for the degradation of medium- and long-chain alkanes in saline crude oil wastewater. This strain can produce lipopeptide protein surfactants, achieving a degradation rate of 90% for crude oil wastewater after 3 days under suitable salinity and pH conditions. The above strains, under aerobic conditions, alter the microemulsion state of petroleum hydrocarbons in water by secreting surfactants or co-surfactants extracellularly, thereby adsorbing petroleum hydrocarbons around the cells and transporting them into the cells. Through fatty acid metabolism, petroleum hydrocarbons are converted into CO2. Some petroleum hydrocarbons are also adsorbed on the cell surface, and due to the density difference between the bacterial cell / petroleum hydrocarbon mixture, they may either remain suspended in the aqueous phase or settle into the sludge.
[0006] While the strains disclosed in the aforementioned patents exhibit high degradation rates for petroleum hydrocarbons, their effectiveness in actual wastewater biochemical treatment processes is limited by the tolerance and degradation pathways of single strains. Typically, a prolonged period and continuously increasing dosages are required after addition to establish a dominant bacterial community and achieve a high petroleum hydrocarbon degradation rate. However, the hydraulic retention time in actual wastewater treatment is usually short, and the relatively slow degradation rate of the strains cannot fully meet the requirements. This problem is particularly prominent in the treatment of wastewater with high petroleum hydrocarbon content (2000 ppm). Considering the actual wastewater treatment capacity and economic costs, it is necessary to develop a method for the rapid and efficient degradation of petroleum hydrocarbons in high-salt, oily wastewater, building upon existing methods. Summary of the Invention
[0007] The specific technical solution of this invention is as follows:
[0008] A method for rapidly degrading petroleum hydrocarbons in high-salt, oily wastewater, characterized by comprising the following steps:
[0009] 1) Pseudomonas and denitrifying achromobacter were cultured separately in fermentation medium; wherein:
[0010] Culture conditions for Pseudomonas: culture medium salinity 1.5%; initial carbon source concentration 5 g / L, fed-batch concentration 0.5 g / L; initial pH 7.5; dissolved oxygen 50%; culture temperature 30℃.
[0011] Culture conditions for denitrifying achromobacterium: culture medium salinity 1.0%; initial carbon source concentration 5 g / L, fed-batch concentration 0.5 g / L; initial pH 7.5; dissolved oxygen 50%; culture temperature 30℃; among which:
[0012] The Pseudomonas species described is disclosed in patent CN118389348A, strain number Z1, and is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No.30070;
[0013] The denitrifying achromobacterium is disclosed in patent CN118406583A, strain number M1, and is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No.29698;
[0014] 2) Concentrate the bacterial cultures of the two strains to a cell concentration of not less than 8 × 10⁻⁶. 13 CFU; then the concentrated solutions of the two strains were mixed at a live bacteria ratio of 1:1, and then an auxiliary solution of equal volume was added to the mixed bacterial solution to obtain the bacterial agent; the components of the auxiliary solution are as follows: Na2HPO4 1.0g / L, KH2PO4 0.5g / L, NH4Cl 1.1g / L, MgSO4 0.2g / L, CaCl2 0.02g / L, Fe2(SO4)3 0.02g / L, NaCl 30g / L, hexadecane 2000ppm, and the balance being water;
[0015] 3) Add bacterial agents and hydrophilic polyurethane fillers to high-salt, oily wastewater to degrade petroleum hydrocarbons in an aerobic environment.
[0016] Furthermore, the degradation of petroleum hydrocarbons in the wastewater in step 3) above is carried out in an aerobic tank, with a daily wastewater inflow of 8-10 m³. 3 The appropriate dosage of the microbial agent is as follows: the initial dosage is 1-2 kg, and then 0.5-1 kg is added daily until the COD of the wastewater is not higher than 300 mg / L.
[0017] Furthermore, during the degradation of petroleum hydrocarbons in the wastewater in step 3) above, the pH of the wastewater is maintained at 6.5 to 7.5, and the dissolved oxygen in the wastewater is maintained at 2.5 to 4.5 ppm.
[0018] Furthermore, in step 3) above, after the initial addition of the bacterial agent, an appropriate amount of C, N, and P nutrients are added to the wastewater, so that the molar ratio of C, N, and P in the wastewater after addition is 100:5:1.
[0019] Furthermore, in step 3) above, the amount of hydrophilic polyurethane filler added is 0.1 to 0.2 times the total volume of wastewater by volume ratio.
[0020] Furthermore, the initial COD of the high-salt oily wastewater is 1000–3000 mg / L, and the salinity is 0.8–2.0%.
[0021] Furthermore, in step 1), the culture time for Pseudomonas is 40 hours; the culture time for Denitrifying Achromobacterium is 42 hours.
[0022] This invention can rapidly degrade petroleum hydrocarbons in high-salt, oily wastewater. The degradation rate of petroleum hydrocarbons by the bacterial agent and its tolerance to COD load in the wastewater are significantly higher than those of single bacterial species, while also exhibiting good salinity adaptability. In practical wastewater biochemical treatment, this demonstrates that a dominant microbial community can be rapidly established with a relatively small dosage and within a short time, effectively improving the abundance and diversity of microorganisms in the system. Continuous operation can achieve optimal petroleum hydrocarbon degradation within a limited hydraulic retention time. Attached image description:
[0023] Figure 1 Results of growth antagonism analysis between the two strains.
[0024] Figure 2 Comparison of short-term degradation efficiency of single strains and mixed strains with different ratios.
[0025] Figure 3 Comparison of alkane degradation efficiency of mixed bacteria under different salinity conditions.
[0026] Figure 4 The effect of different initial alkane concentrations on the degradation capacity of mixed bacteria.
[0027] Figure 5 Growth curve of Pseudomonas Z1 during fermentation.
[0028] Figure 6 Growth curve of denitrifying achromobacter M1 during fermentation.
[0029] Figure 7 The trend of dissolved oxygen changes in the system when using microbial agents to treat oily wastewater from ports in an A / O wastewater treatment system.
[0030] Figure 8 The pH change trend of the system when the bacterial agent is used in the A / O wastewater treatment system for the treatment of oily wastewater in ports.
[0031] Figure 9 COD variation trend when using microbial agents to treat oily wastewater from ports in an A / O wastewater treatment system. Detailed Implementation
[0032] The technical solution of the present invention will be further described below with reference to specific embodiments. The materials and methods used in the present invention all belong to the conventional technical field.
[0033] Example 1
[0034] Antagonistic Analysis of Strains: The antagonistic effects of different bacterial strains were analyzed using the Oxford cup diffusion method. The specific procedure was as follows: LB solid plates with a salinity of 1% were prepared according to standard methods. The components (g / L) of the solid LB medium were tryptone 10, yeast extract 5, NaCl 10, and agar 20. Bacterial suspensions of *Achromobacterium denitrifyingis* M1 and *Pseudomonas* Z1 were spread onto the solid plates, face up, and left to stand for 5 minutes to allow absorption. Then, two sterile Oxford cups were gently placed vertically on the left and right sides of the solid plates containing the bacterial suspensions, and gently pressed with forceps to ensure close contact with the medium surface. 200 μL of the corresponding bacterial inoculum was added to the left Oxford cup, and the same volume of sterile water was added to the right Oxford cup as a control. Finally, after standing at room temperature for 1 hour, the plates were placed upright in an incubator and incubated at 30°C for 12–18 hours, after which antagonistic zones were observed.
[0035] Experimental results showed that there was no antagonistic growth between denitrifying achromobacter M1 and alkali-producing pseudomonads Z1. Figure 1 As shown in Figure A, no antagonistic zone was formed between the plate coated with denitrifying achromobacter M1 and the Oxford cup on the right side containing the Pseudomonas aeruginosa inoculum. Similarly, no antagonistic zone was formed between the plate coated with Pseudomonas aeruginosa Z1 and the Oxford cup on the right side containing the denitrifying achromobacter M1 inoculum. Figure 1 B).
[0036] Example 2
[0037] Comparison of short-term degradation efficiency of single strains and mixed strains with different ratios: Two strains, *Achromobacterium denitrifyingis* M1 and *Pseudomonas* Z1, were activated using hexadecane as the sole carbon source and cultured to the logarithmic growth phase. The bacterial culture was then adjusted to OD using the dilution method. 600 nmWith the same values, mixed inoculum solutions were prepared according to the ratio of viable counts of denitrifying achromobacterium M1 and Pseudomonas Z1 at 1:1, 1:2, and 2:1, respectively. These solutions were inoculated into a culture medium containing 1000 ppm alkane at an inoculation rate of 8% (v / v). After incubation at 30℃ and 160 r / min for 24 h in a shaker, samples were taken to determine the alkane degradation rate. A single-strain control group was also set up for comparison. Both the control and experimental groups had three replicates. The components of the alkane culture medium were as follows (g / L): Na2HPO4 0.6, KH2PO4 0.3, NH4Cl 0.66, MgSO4 0.12, CaCl2 0.012, Fe2(SO4)3 0.012, hexadecane 145 μL, pH adjusted to 7.5, salinity 1%, and diluted to 1 L with distilled water. The solution was sterilized at 121℃ for 20 min. For details on the alkane extraction procedure and the calculation method of alkane degradation rate, please refer to "Determination of Petroleum by Water Quality - Ultraviolet Spectrophotometry (Trial)" (HJ 970–2018). First, the supernatant of different bacterial solutions was extracted using the n-hexane extraction method, and the absorbance value of the extract at 225 nm was measured. Then, the alkane degradation rate was calculated.
[0038] The results show that Figure 2 As shown, the alkane degradation efficiency in the culture system was optimal when the ratio of the two strains was 1:1, reaching over 80% in 24 hours, significantly higher than that of single strains and mixed strains with other ratios. In subsequent examples, the mixed strains or inoculants were all prepared using a 1:1 ratio of two strains.
[0039] Example 3
[0040] Comparison of alkane degradation efficiency of mixed bacteria under different salinities: Alkane-selective culture media (containing 1000 ppm n-hexadecane) with salinities of 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% were prepared. The mixed bacteria were inoculated at an 8% inoculum (v / v) into the sterilized alkane-selective culture media at the above different salinities. The initial bacterial concentration (OD) after inoculation was... 600 The value was 0.24. Single strains M1 and Z1 were used as blank control groups.
[0041] After incubation at 30℃ with shaking for 24 h, the supernatants of different bacterial cultures were extracted using the n-hexane extraction method. The absorbance of the extract at 225 nm was measured, and the alkane degradation rate was calculated. The extraction steps and the method for calculating the alkane degradation rate are the same as in Example 2.
[0042] The results are as follows Figure 3 As shown, the mixed bacteria achieved the highest alkane degradation rate at a salinity of 1–1.5%, exceeding 80% in all cases. When the salinity reached 2.5%, the alkane degradation rate remained at around 55% after 24 hours, and the alkane degradation rate in the salinity range of 1–2.5 was significantly higher than that of the single strains M1 and Z1.
[0043] Example 4
[0044] Effect of different initial alkane concentrations on the degradation ability of mixed bacteria: Alkane-selective culture media with initial alkane concentrations (ppm) of 3000, 2000, 1000, and 500 were prepared, with a salinity of 1% and a pH of 7.5. The mixed bacteria were inoculated into the sterilized selective culture media with different initial alkane concentrations at an inoculation rate of 8% (v / v). The initial concentration of the bacterial culture after inoculation was measured. 600 The value was 0.24. Single strains M1 and Z1 were used as blank control groups.
[0045] After incubation at 30℃ with shaking for 24 h, the supernatants of different bacterial cultures were extracted using the n-hexane extraction method. The absorbance of the extract at 225 nm was measured, and the alkane content was calculated. The extraction procedure and the method for calculating the alkane content were based on the "Determination of Petroleum Hydrocarbons in Water - Ultraviolet Spectrophotometry (Trial)" (HJ 970–2018).
[0046] The results are as follows Figure 4 As shown, when the alkane concentration was <1000, there was no significant difference in alkane degradation between the control and experimental groups. When the initial alkane concentration was >1000, the alkane degradation amount in the experimental group was significantly higher than that in the control group as the alkane concentration increased, indicating that the mixed bacteria had a much higher tolerance to high concentrations of alkane than the single strain. Under the same conditions, after 24 hours of cultivation, the amount of residual alkane in the mixed bacterial solution was much lower than that in the single strain control group.
[0047] Example 5
[0048] Application of microbial agents in A / O wastewater treatment processes:
[0049] 1) Pseudomonas Z1 and denitrifying achromobacter M1 were cultured separately in a 5L fermenter:
[0050] Culture conditions for strain Z1: culture medium salinity 1.5%; initial carbon source concentration 5 g / L, fed-batch concentration 0.5 g / L; initial pH 7.5, adjusted using fed-batch ammonia; dissolved oxygen 50%, adjusted by stirring speed; culture temperature 30℃, adjusted using cooling water.
[0051] The growth of strain Z1 during fermentation is as follows: Figure 5 As shown: the strain has a short lag phase (0–16 h), a long exponential growth phase (20 h), and enters the stationary phase after 36 h. Biomass accumulation is rapid, with an OD600 value as high as 18. During fermentation, the strain produces a large amount of foam due to the production of co-surfactants, requiring the addition of an appropriate amount of defoamer. The fermentation time of *Pseudomonas alcaligenes* Z1 is 40 h, and the fermentation broth is used for subsequent inoculum preparation.
[0052] Culture conditions for strain M1: culture medium salinity 1.0%; initial carbon source concentration 5 g / L, fed-batch concentration 0.5 g / L; initial pH 7.5, adjusted by fed-batch ammonia; dissolved oxygen 50%, adjusted by stirring speed; culture temperature 30℃, adjusted by cooling water.
[0053] The growth of strain M1 during fermentation is as follows: Figure 6 As shown: the strain exhibits a relatively long lag phase (0–24 h), an exponential growth phase of 12 h, and enters the stationary phase after 36 h. Biomass accumulation is rapid, with an OD600 value as high as 22. Minimal foam production occurs during fermentation. The fermentation time for *Achromobacterium denitrifyingii* M1 is 42 h, and the fermentation broth is used for subsequent inoculum preparation.
[0054] 2) Incubate the fermentation broths of the two strains at 4℃ for 8–10 hours, collect the bottom cells, and discard approximately 4 / 3 of the supernatant to achieve a concentrated cell concentration of 8 × 10⁻⁶ for each strain. 13 CFU; Mix the concentrated solutions of the two strains at a 1:1 ratio of live bacteria, and then add an auxiliary solution of equal volume to the mixed bacterial solution to obtain the bacterial agent.
[0055] The composition of the above auxiliary solution is as follows: Na2HPO4 1.0g / L, KH2PO4 0.5g / L, NH4Cl 1.1g / L, MgSO4 0.2g / L, CaCl2 0.02g / L, Fe2(SO4)3 0.02g / L, NaCl 30g / L, hexadecane 2000ppm, and the balance is water.
[0056] 3) The microbial agent was applied to the A / O wastewater treatment process system, which has a total volume of 20m³. 3 A special bacteria continuous propagation biochemical system with a water treatment capacity of 10m³ 3 The system can achieve real-time intelligent monitoring of dissolved oxygen, pH, and COD. The expansion culture system is designed with port oily wastewater (COD 1000-1500 mg / L) as the influent. Hydrophilic polyurethane packing is added to the aerobic tank, and low-concentration sequential batch operation is implemented. The process mainly includes three stages: enrichment of the microbial population within the system, establishment of alkane-degrading bacteria, and treatment of oily wastewater. The enrichment of the microbial population involves pumping wastewater and sludge from the aerobic tank of the biochemical system into the aerobic zone of the special bacteria expansion culture equipment, allowing aerobic bacteria to proliferate fully under saturated aeration. Then, a small amount of bacterial agent is added daily to the raw water in the aerobic tank to establish the alkane-degrading bacteria population. In the oily wastewater treatment stage, bacterial agents are added in multiple batches, and after multiple cycles of operation, while efficiently degrading petroleum hydrocarbons, an aerobic tank biochemical community environment with alkane-degrading bacteria as the dominant bacteria is formed.
[0057] Taking the first operating cycle (3 days) in the oily wastewater treatment stage as an example, the specific operation is as follows:
[0058] On the first day, the aerobic tank was filled with 10m of water. 3 Add 1.5 kg of bacterial agent, followed by appropriate amounts of C, N, and P nutrients; the molar ratio of C, N, and P in the wastewater after addition should be 100:5:1; the amount of C nutrient source is the sum of COD in the wastewater and the added carbon source. On the second and third days thereafter, add 0.5 kg of bacterial agent daily, and measure the dissolved oxygen, pH, and COD of the aerobic tank water at 9:00, 14:00, and 19:00 daily.
[0059] Wastewater treatment and monitoring results are as follows Figures 7-9 As shown in the figure. The changes in dissolved oxygen are as follows: Figure 7 As shown: During this cycle, dissolved oxygen rapidly decreased to 0.9 after the addition of carbon source and bacterial agent, and further decreased to 1.6 after a second addition of bacterial agent. This indicates that the abundance of the bacterial community increased after the addition of carbon source or bacterial agent, leading to increased aerobic metabolism and a rapid decrease in dissolved oxygen. Dissolved oxygen remained between 3 and 4.5 during other time periods. pH changes are shown below. Figure 8 As shown: Except for the point where dissolved oxygen levels were low (pH dropped to 6.8) after carbon source supplementation, the pH remained stable at around 7.3 during other time periods. The COD trend is as follows: Figure 9 As shown, under the condition that the dissolved oxygen and pH of the whole system are basically stable, the COD of the wastewater also decreases rapidly. On the third day, it has decreased from the initial 730 to 289, and the COD removal rate has reached 60.41%.
Claims
1. A method for rapidly degrading petroleum hydrocarbons in high-salt, oily wastewater, characterized in that... Includes the following steps: 1) *Pseudomonas* and *Achromobacterium denitrification* were cultured separately in fermentation medium; wherein: the culture conditions for *Pseudomonas* were: medium salinity 1.5%; initial carbon source concentration 5 g / L, fed-batch concentration 0.5 g / L; initial pH 7.5; dissolved oxygen 50%; culture temperature 30℃; the culture conditions for *Achromobacterium denitrification* were: medium salinity 1.0%; initial carbon source concentration 5 g / L, fed-batch concentration 0.5 g / L; initial pH 7.5; dissolved oxygen 50%; culture temperature 30℃; wherein: the *Pseudomonas* strain disclosed in patent CN118389348A, strain number Z1, is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC. No. 30070; The denitrifying achromobacterium is disclosed in patent CN118406583A, strain number M1, and is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCCNo. 29698; 2) Concentrate the bacterial cultures of the two strains to a cell concentration of not less than 8 × 10⁻⁶. 13 CFU; then the concentrated solutions of the two strains were mixed at a live bacteria ratio of 1:1, and then an auxiliary solution of equal volume was added to the mixed bacterial solution to obtain the bacterial agent; the composition of the auxiliary solution is as follows: Na2HPO4 1.0g / L, KH2PO4 0.5g / L, NH4Cl 1.1g / L, MgSO4 0.2g / L, CaCl2 0.02g / L, Fe2(SO4)3 0.02g / L, NaCl 30g / L, hexadecane 2000ppm, and the balance being water; 3) Add bacterial agents and hydrophilic polyurethane fillers to high-salt oily wastewater and degrade petroleum hydrocarbons in the wastewater under an aerobic environment. The initial COD of the high-salt oily wastewater is 1000-3000 mg / L and the salinity is 1-1.5%.
2. The method for rapidly degrading petroleum hydrocarbons in high-salt, oily wastewater according to claim 1, characterized in that: The degradation of petroleum hydrocarbons in the wastewater in step 3) is carried out in an aerobic tank, with a daily wastewater inflow of 8-10 m³. 3 The appropriate dosage of the microbial agent is as follows: the initial dosage is 1-2 kg, and then 0.5-1 kg is added daily until the COD of the wastewater is not higher than 300 mg / L.
3. The method for rapidly degrading petroleum hydrocarbons in high-salt, oily wastewater according to claim 1, characterized in that: During the degradation of petroleum hydrocarbons in the wastewater in step 3), the pH of the wastewater is maintained at 6.5–7.5, and the dissolved oxygen in the wastewater is maintained at 2.5–4.5 ppm.
4. The method for rapidly degrading petroleum hydrocarbons in high-salt, oily wastewater according to claim 2, characterized in that: In step 3), after the initial addition of the bacterial agent, an appropriate amount of C, N, and P nutrients are added to the wastewater, so that the molar ratio of C, N, and P in the wastewater after addition is 100:5:
1.
5. The method for rapidly degrading petroleum hydrocarbons in high-salt, oily wastewater according to claim 1, characterized in that: The amount of the hydrophilic polyurethane filler added is 0.1 to 0.2 times the total volume of wastewater by volume.
6. The method for rapidly degrading petroleum hydrocarbons in high-salt, oily wastewater according to claim 1, characterized in that: In step 1), the culture time for Pseudomonas is 40 h; the culture time for Denitrifying Achromobacterium is 42 h.