Biochar as well as preparation method and application thereof in petroleum degradation

Abstract

The invention relates to the technical field of biological environmental protection, and particularly discloses biochar, a preparation method thereof and application of the biochar in petroleum degradation, and the biochar is used for improving the petroleum degradation rate in combination with petroleum degrading bacteria. The biochar is prepared by drying and crushing biomass, carbonizing at a heating rate of 10 DEG C / min to 12 DEG C / min, carbonizing at a constant temperature of 400 DEG C to 800 DEG C for 10 min to 12 min, and naturally cooling; and the petroleum degrading bacterium is pseudomonas (Pseudomonas). The invention also discloses a preparation method of the petroleum degrading bacterium. According to the present invention, the prepared biochar can effectively adsorb petroleum, and the degradation efficiency of the biochar on the petroleum is significantly improved after the Pseudomonadaceae is immobilized.

Description

Technical Field

[0001] The invention relates to the technical field of biological environmental protection, and in particular to a biochar and a preparation method thereof and an application thereof in petroleum degradation. Background Art

[0002] Oil is an important raw material for many products in life. This non-renewable resource brings convenience to humans, but it also threatens the ecological environment and human health due to many improper operations or accidents. The main components of oil are a mixture of alkanes, cycloalkanes, aromatic hydrocarbons, etc., many of which are harmful to humans. After polluting the environment, they will also enter the biological body through material circulation. As the city of oil, Daqing is characterized by a large wetland area, sufficient surface water, high salinity and alkali content in the water, and an average annual temperature of 4°C, which weakens the self-cleaning ability of the water body. When faced with the threat of oil leakage, it will directly lead to a decline in the health level of aquatic plants and animals and humans. At present, the methods for dealing with oil pollution in surface water can be roughly divided into physical salvage, chemical decomposition, biodegradation, or a combination of the above methods. The biodegradation method stands out for its characteristics of low secondary pollution to the environment and low capital cost. For example, a large number of researchers have isolated microorganisms that degrade oil from the environment and used them in the degradation of oil, but oil-degrading bacteria are difficult to perform their effective functions due to their mutual influence with indigenous bacteria or the influence of environmental uncertainties, resulting in a low degradation rate. Therefore, how to reduce the mutual influence between oil-degrading bacteria and indigenous bacteria to improve the efficiency of oil degradation is an urgent problem to be solved. Summary of the invention

[0003] In order to reduce the mutual influence between oil-degrading bacteria and indigenous bacteria, the present invention provides a biochar and a preparation method thereof and an application thereof in oil degradation. The present invention prepares biochar and uses biochar to adsorb and fix Pseudomonas, thereby reducing the direct contact between oil-degrading bacteria and indigenous bacteria, reducing the influence of indigenous bacteria on oil-degrading bacteria, and thus significantly improving the degradation efficiency of oil-degrading bacteria on oil.

[0004] The present invention provides an application of biochar in oil degradation, using biochar to adsorb and fix Pseudomonas (Pseudomonadaceae) to prepare an immobilized bacterial agent, which is added to a sample to be treated containing oil;

[0005] The biochar is prepared by drying and crushing the biomass and then carbonizing it at a heating rate of 10°C / min to 12°C / min, reaching a charcoal burning temperature of 400°C to 800°C, carbonizing it at a constant temperature for 10min to 12min, and then naturally cooling it down.

[0006] The present invention utilizes the prepared biochar with rich pore structure and large surface area to provide a stable habitat, match the adsorption and fixation of Pseudomonas to obtain an immobilized bacterial agent, and thus fix the Pseudomonas on the biochar. However, due to the competitive relationship and the special biochar preparation method, indigenous microorganisms in the environment (such as Arthrobacter, Alcaligenes, Flavobacterium, Achromobacter and Nocardia, etc.) are difficult to be adsorbed into the biochar prepared by the present invention, which reduces the direct contact between Pseudomonas and indigenous microorganisms in the environment, and reduces the interaction and influence between indigenous microorganisms and Pseudomonas, so that the Pseudomonas fixed on the biochar can efficiently degrade organic matter in petroleum, especially the hydrocarbon components therein, and the biochar simultaneously adsorbs petroleum and coordinates the Pseudomonas to jointly reduce the petroleum content in the environment.

[0007] Furthermore, 10 ml to 20 ml of the immobilized bacterial agent is added to every 1 L of the sample to be processed.

[0008] Furthermore, the preparation process of the immobilized bacterial agent is as follows: 100 g to 150 g of biochar is added to every 1 L of Pseudomonas bacterial liquid for adsorption and fixation to obtain the immobilized bacterial agent.

[0009] Furthermore, 100 g of biochar was added to every 1 L of Pseudomonas bacterial solution for adsorption and fixation.

[0010] Furthermore, the concentration of the Pseudomonas bacterial solution is 0.1 g / ml to 0.2 g / ml.

[0011] Furthermore, the concentration of the Pseudomonas bacterial solution is 0.2 g / ml. Adding 100 g of biochar to every 1 L of 0.2 g / ml Pseudomonas bacterial solution is just enough to achieve a saturated adsorption and fixation state of Pseudomonas, thereby achieving the best degradation state and lowering the cost.

[0012] Furthermore, the Pseudomonas bacterial liquid is prepared by inoculating Pseudomonas in a liquid culture medium and culturing the culture medium to a logarithmic growth phase, then collecting the bacterial cells by centrifugation and preparing a bacterial liquid with a concentration of 0.1 g / ml to 0.2 g / ml.

[0013] Furthermore, after the bacterial cells are collected by centrifugation, a Pseudomonas bacterial solution with a concentration of 0.1 g / ml to 0.2 g / ml is prepared using sterile water, PBS or liquid culture medium.

[0014] Furthermore, the biomass is reed stalks.

[0015] The present invention also provides a biochar, the preparation process of which is as follows: after washing and removing impurities from reed stems, drying at 80°C to 90°C, crushing, and then carbonizing at a heating rate of 10°C / min to 12°C / min, carbonizing at a constant temperature for 10min to 12min after reaching a charcoal burning temperature of 400°C to 800°C, and then naturally cooling to obtain biochar. The yield of biochar obtained by the preparation process of biochar provided by the present invention is high. The prepared biochar can effectively adsorb petroleum, and after adsorbing petroleum, the buoyancy of biochar can be improved while reducing the surface viscosity of petroleum. When the present invention uses reed stem biomass raw material and the charcoal burning temperature is 800°C, the floating rate of the prepared biochar is significantly higher than the floating rate of biochar prepared using other biomass as raw materials and at other charcoal burning temperatures, and the ash content of the biochar prepared using reed stems is significantly higher than that of biochar prepared using other biomass as raw materials and at other charcoal burning temperatures, and the ash content of the biochar prepared using reed stems is significantly higher than that of the biochar prepared using other biomass.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The floating capacity of the biochar prepared by the present invention is 25.7% to 62.2%, the biochar yield is 23.0% to 41.1%, and the ash content is 3.0% to 5.0%, among which the reed stem biochar has the highest floating capacity: 62.2%; the ash content is 5.0%, and the oil degradation rate of the combined Pseudomonadaceae is 45.3%.

[0018] The present invention uses reed stalks, soybean husks, corn stalks and pine needles as biomass materials to prepare biochar at different temperatures. It is found that the yield of the obtained biochar decreases with the increase of temperature, but the type of biomass has no significant effect on the yield and ash content of the biochar. For surface water oil pollution, biochar with a high floating rate can more efficiently adsorb oil before it precipitates itself. After adsorbing oil, its buoyancy can be improved while reducing the surface viscosity of the oil. Experimental analysis shows that the floating rate of 800℃ reed stalk biochar with a particle size greater than 100 mesh is as high as 62.2% and has strong stability.

[0019] The present invention utilizes the prepared biochar with rich pore structure and large surface area, provides a stable habitat, matches the adsorption and fixation of Pseudomonas to obtain an immobilized bacterial agent, thereby fixing Pseudomonas on the biochar, and due to the competitive relationship and the special biochar preparation method, the indigenous microorganisms in the environment (such as arthroblasts, alcaligenes, Flavobacterium, colorless bacteria and Nocardia, etc.) are difficult to adsorb into the biochar prepared by the present invention, reducing the direct contact of Pseudomonas with the indigenous microorganisms in the environment, reducing the interaction and influence between the indigenous microorganisms and Pseudomonas, so that the Pseudomonas fixed on the biochar efficiently degrades oil, and the biochar adsorbs oil at the same time, and coordinates the Pseudomonas to jointly reduce the oil content in the environment. In the present invention, adding 1g of biochar to every 10ml of Pseudomonas bacterial liquid with a concentration of 0.2g / ml can just reach the state of saturated adsorption and fixation of Pseudomonas. Achieve the best degradation state, and the cost is low. Biochar not only improves the activity of microorganisms, but also adsorbs oil pollutants, reduces its toxicity to the environment, thereby improving the efficiency of oil degradation. The present invention can improve the petroleum degradation rate of Pseudomonas LD23 by 12.1% after fixing the Pseudomonas LD23 with biochar. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The effects of different oscillation times and particle sizes on the buoyancy of the reed stem biochar prepared in Example 1 of the present invention;

[0022] In the figure, A shows the effect of reed stem biochar oscillation treatment at different time on the floating rate;

[0023] B is the effect of particle size of reed stem biochar on the floating rate.

[0024] Figure 2 The effect of the ratio of the amount of biochar to the amount of oil used in the reed stem biochar prepared in Example 1 on the adsorption of oil;

[0025] In the figure, A is the adsorption of oil when the ratio of biochar to oil is 0.050g:0.2g;

[0026] B is the adsorption of oil when the ratio of biochar to oil is 0.075g:0.2g;

[0027] C is the adsorption of oil when the ratio of biochar to oil is 0.1g:0.2g;

[0028] D is the adsorption of petroleum when the ratio of biochar to petroleum is 0.125g:0.2g.

[0029] Figure 3 This is the growth curve of strain LD23.

[0030] Figure 4 This is the bacterial solution concentration-absorbance curve. In the figure, bacterial concentration is the bacterial solution concentration.

[0031] Figure 5 This is the saturation time test for biochar adsorption bacteria.

[0032] Figure 6 This is the petroleum standard curve. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0034] The experimental materials provided by the present invention are as follows:

[0035] 1. Materials and instruments

[0036] (1) Materials: Pseudomonas (Pseudomonadaceae), currently deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCC No.11679.

[0037] (2) Biomass: reed stalks, soybean husks, corn stalks, and pine needles, all provided by Heilongjiang Bayi Agricultural University.

[0038] (3) Reagents: agar, yeast powder, calcium chloride, sodium hydroxide, peptone, ammonium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium carbonate, ferrous sulfate, copper sulfate, manganese sulfate, magnesium sulfate, petroleum ether, anhydrous sodium sulfate.

[0039] (4) Instruments: Vacuum tube furnace SK3-2-10-8 (Hangzhou Zhuochi Instrument Co., Ltd.); electric constant temperature incubator DRP-9162 (Shanghai Senxin Experimental Instrument Co., Ltd.); portable pressure steam sterilizer XFS-280A (Zhejiang Xinfeng Medical Instrument Co., Ltd.); high-speed refrigerated centrifuge CR-21G (Hitachi, Japan); visible spectrophotometer VIS-723G (Beijing Rayleigh Analytical Instrument Co., Ltd.); electronic balance JA2003N (Shanghai Qinghai Instrument Co., Ltd.); pH meter PB-10 (Beijing Sartorius Scientific Instrument Co., Ltd.); full temperature oscillator HZQ-Q (Donglian Electronic Technology Development Co., Ltd.); sterile laboratory bench DL-CJ-2N (Harbin Donglian Co., Ltd.); electric constant temperature blast drying oven DGG-9070BD (Shanghai Senxin Experimental Instrument Co., Ltd.).

[0040] (5) Culture medium:

[0041] LB medium: NaCl 10g / L, peptone 10g / L, yeast powder 5g / L, agar 20g / L, distilled water to 1L, PH = 7.0 [5] .

[0042] Improved petroleum fermentation medium: crude oil 2g / L, NH4NO3 3.0g / L, KH2PO4 0.5g / L, K2HPO4 0.5g / L, trace element solution 3.0ml / L, distilled water to 1L, pH adjusted to 8.5 with Na2Co3 [6] .

[0043] Trace element solution: NaCl 1.0g / L, FeSO4·7H2O 1.0g / L, CaCl2 1.0g / L, CuSO4 1.0g / L, MnSO4 1.0g / L, MgSO4·3H2O 1.0g / L, distilled water to 1L. CaCl2 and MgSO4·3H2O need to be sterilized separately.

[0044] Example 1: A biochar and a method for preparing the same.

[0045] The reed stems were cleaned with deionized water, cut into short stems of 1-3 cm, and the debris was removed for later use.

[0046] The treated material was placed in an electric constant temperature drying oven at 80°C and dried to constant weight, then taken out and crushed in a grinder to obtain reed stem powder, which was then placed in an electric constant temperature drying oven again for use.

[0047] The reed stem powder was added to a porcelain boat with a length of 10 cm, a height of 2 cm, and a width of 5 cm, and compacted, and then evacuated to a vacuum of -0.1 Pa. It was placed in a vacuum tube furnace and carbonized at a heating rate of 10°C / min. After reaching the predetermined charcoal burning temperature of 800°C, it was carbonized at a constant temperature for 10 minutes, and then naturally cooled to 25°C and taken out to obtain reed stem biochar.

[0048] Example 2: A biochar and a method for preparing the same.

[0049] Wash the soybean hulls with deionized water, cut them into short poles of 1-3 cm, remove debris, and set aside.

[0050] The treated material was placed in an electric constant temperature drying oven at 80°C and dried to constant weight, then taken out and crushed in a grinder to obtain soybean hull powder, which was then put back into the electric constant temperature drying oven for use.

[0051] The soybean hull powder was added to a porcelain boat with a length of 10 cm, a height of 2 cm, and a width of 5 cm, and compacted, and then evacuated to a vacuum of -0.1 Pa. It was placed in a vacuum tube furnace and carbonized at a heating rate of 10°C / min. After reaching the predetermined charcoal burning temperature of 800°C, it was carbonized at a constant temperature for 10 minutes, and then naturally cooled to 25°C and taken out to obtain soybean hull biochar.

[0052] Example 3: A biochar and a method for preparing the same.

[0053] The corn stalks were washed with deionized water, cut into short stalks of 1-3 cm, and the debris was removed for later use.

[0054] The treated material was placed in an electric constant temperature drying oven at 80°C and dried to constant weight, then taken out and crushed in a grinder to obtain corn stalk powder, which was then put back into the electric constant temperature drying oven for use.

[0055] The corn stalk powder was added to a porcelain boat with a length of 10 cm, a height of 2 cm, and a width of 5 cm, and compacted, and then evacuated to a vacuum of -0.1 Pa. It was placed in a vacuum tube furnace and carbonized at a heating rate of 10°C / min. After reaching the predetermined charcoal burning temperature of 800°C, it was carbonized at a constant temperature for 10 minutes, and then naturally cooled to room temperature and taken out to obtain corn stalk biochar.

[0056] Example 4: A biochar and a method for preparing the same.

[0057] Wash the pine needles with deionized water, cut them into short poles of 1-3 cm, remove debris, and set aside.

[0058] The treated material was placed in an electric constant temperature drying oven at 80°C and dried to constant weight, then taken out and crushed in a grinder to obtain pine needle powder, which was then put back into the electric constant temperature drying oven for use.

[0059] Pine needle powder was added to a porcelain boat with a length of 10 cm, a height of 2 cm, and a width of 5 cm, and compacted, and then evacuated to a vacuum of -0.1 Pa. It was placed in a vacuum tube furnace and carbonized at a heating rate of 10°C / min. After reaching the predetermined charcoal burning temperature of 800°C, it was carbonized at a constant temperature for 10 minutes, and then naturally cooled to room temperature and taken out to obtain pine needle biochar.

[0060] Example 5: A biochar and a method for preparing the same.

[0061] The preparation steps are basically the same as those in Example 1, except that the predetermined charcoal burning temperature is set to 600°C.

[0062] Example 6: A biochar and a method for preparing the same.

[0063] The preparation steps are basically the same as those in Example 1, except that the predetermined charcoal burning temperature is set to 400°C.

[0064] Example 7: A biochar and a method for preparing the same.

[0065] The preparation steps are basically the same as those in Example 2, except that the predetermined charcoal burning temperature is set to 600°C.

[0066] Example 8: A biochar and a method for preparing the same.

[0067] The preparation steps are basically the same as those in Example 2, except that the predetermined charcoal burning temperature is set to 400°C.

[0068] Example 9: A biochar and a method for preparing the same.

[0069] The preparation steps are basically the same as those in Example 3, except that the predetermined charcoal burning temperature is set to 600°C.

[0070] Example 10: A biochar and a method for preparing the same.

[0071] The preparation steps are basically the same as those in Example 3, except that the predetermined charcoal burning temperature is set to 400°C.

[0072] Example 11: A biochar and a method for preparing the same.

[0073] The preparation steps are basically the same as those in Example 4, except that the predetermined charcoal burning temperature is set to 600°C.

[0074] Example 12: A biochar and a method for preparing the same.

[0075] The preparation steps are basically the same as those in Example 4, except that the predetermined charcoal burning temperature is set to 400°C.

[0076] The biochar prepared in Examples 1-4 was used as the research object, the floating rate of the biochar was detected, the yield and ash content of the reed stem biochar with the best floating rate were selected, the effect of different biochar amounts on the adsorption of oil was studied, the strain growth curve was determined, the bacterial concentration-absorbance curve and dry weight and wet weight were determined, the saturation time of biochar adsorption of bacterial liquid was determined, and the oil degradation rate of biochar was determined. The specific methods and test results are as follows:

[0077] 1. Methods

[0078] 1. Determination of floating rate

[0079] The biochar prepared in Examples 1-4 was placed in an electric constant temperature drying oven at 80°C and dried to constant weight. Take about 0.005g of biochar sample cooled to room temperature in a 40ml test tube (the mass of biochar is recorded as m1), add 30ml of distilled water and mix well, place in a full temperature oscillator, set to 15°C, 150r / min and oscillate for 24h. Mark the culture dish, and the mark is consistent with the mark of each group of biochar. Cut the filter paper into a fan shape to scoop out the sample floating on the water surface, rinse the sample with distilled water onto the filter paper in the funnel, and repeat several times until all the water surface samples in the test tube are scooped out. Put the filter paper with biochar collected in the corresponding culture dish, dry it in an electric constant temperature drying oven to constant weight, cool it to room temperature and weigh the mass as m2. The floating rate can be calculated by the following formula:

[0080] Floating rate (%) = m2 / m1×100%.

[0081] The test results show that the reed stem biochar prepared in Example 1 has the highest floating rate. Therefore, it was used in subsequent experiments.

[0082] Biochar with high floating rate was selected to determine the effect of oscillation time on the floating rate, and the floating rate on the 1st and 7th days of oscillation was specifically tested.

[0083] The reed stem biochar prepared in the steps of Example 1 was passed through a 100-mesh particle size after burning, and the remaining steps were the same as in Example 1. The floating rate of the reed stem biochar with a particle size of 100 mesh was tested and compared with the floating rate of the reed stem biochar obtained in Example 1.

[0084] 2. Determination of yield and ash content

[0085] Yield determination: The yield of biochar refers to the percentage of biochar produced by pyrolysis at various temperatures to the original biomass. First, an empty porcelain boat is calcined at high temperature in a muffle furnace for about 1 hour, and after cooling, it is weighed and recorded as m1. The dry biomass sample is weighed and placed in a porcelain boat that has been calcined to a constant weight. The weight of the porcelain boat filled with biomass powder is recorded as m2, and the total weight of the biochar and porcelain boat after carbonization is recorded as m3. The yield can be calculated using the following formula:

[0086] Yield (%) = (m3-m1) / (m2-m1) × 100%

[0087] Ash content determination: Ash content refers to the percentage of the remaining amount of biomass material after complete combustion under aerobic conditions and sufficient oxygen in the original biomass. First, burn the empty porcelain boat at high temperature in a muffle furnace for about 1 hour, weigh it after cooling and record it as m1, weigh the dry biomass sample, place it in the porcelain boat that has been burned to constant weight, and the weight of the porcelain boat filled with biomass powder is recorded as m2. Finally, burn it completely to constant weight under 800℃ with sufficient oxygen, weigh it after cooling and record it as m4. The ash content can be calculated using the following formula:

[0088] Ash content (%) = (m4-m1) / (m2-m1)×100%.

[0089] 3. Effect of different biochar amounts on oil adsorption

[0090] Prepare 4 triangular flasks, add 0.2g petroleum and 100ml distilled water respectively, sterilize at 121℃ for 20 minutes, cool to room temperature, add 0.05, 0.075, 0.1, 0.125g of biochar with the best floating effect (reed stem biochar prepared in Example 1), shake for 7 days, take photos to observe the dynamic adsorption status and roughly analyze the adsorption ratio.

[0091] 4. Determination of strain growth curve

[0092] Low-temperature petroleum-degrading bacteria LD23 were inoculated into 100ml LB liquid culture medium, and the culture conditions were 15℃, 150r / min, and cultured for 12h. Take the culture solution and inoculate it into three 100ml LB liquid culture media at 5% inoculation volume. The mixed LB liquid culture medium was stored in sterile test tubes with a specification of 5ml on the sterile operating table, and 0, 1.5, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26h were used as measurement time nodes. Three groups of parallel controls were measured each time, and a total of 45 test tubes were labeled a, b, and c according to the corresponding time. The uninoculated LB liquid culture medium was used as the blank control, and all culture conditions were 15℃ and 150r / min. Take the inoculated and uninoculated LB liquid culture media at the corresponding time nodes, and immediately store them in a 4℃ refrigerator. At the last time, measure the absorbance values ​​of the obtained bacterial solution at 600nm (using OD 600 With time as the abscissa and absorbance as the ordinate, the growth curve of LD23 low-temperature petroleum-degrading bacteria was obtained.

[0093] 5. Bacterial concentration-absorbance curve and dry weight and wet weight

[0094] Take LD23 in the logarithmic growth phase, centrifuge at 4000r / min for 10min, rinse with sterile water twice, centrifuge for 10min each time. Discard the supernatant and prepare the gradient bacterial solution with sterile water, and measure the absorbance value of each at 600nm (OD 600 With bacterial concentration (mg / ml) as the horizontal axis and absorbance as the vertical axis, the bacterial-absorbance standard curve of LD23 was obtained:

[0095] Y=3.6515OD 600 -2.5419(R 2 =0.9968)

[0096] Weighing method to measure the dry weight and wet weight of the strain: Take LD23 in the logarithmic growth phase, centrifuge at 4000r / min for 10min, rinse twice with sterile water, centrifuge for 10min each time. Discard the supernatant, weigh the wet weight of the bacteria m1, dry at 80℃ to constant weight, and weigh the dry weight m2. The ratio of dry weight to wet weight can be calculated using the following formula:

[0097] Dry-wet weight ratio (%) = m2 / m1×100%.

[0098] 6. Determination of saturation time of biochar adsorption bacterial solution

[0099] Centrifuge the LD23 bacterial solution in the logarithmic growth phase, add sterile water to make 100 ml of 10 mg / ml (1 g is added to 100 ml), then add 0.1 g of biochar and dynamically adsorb in a 15°C shaker. Take a small amount of the bacterial solution and filter it every hour. Measure the absorbance value at 600 nm (OD 600 The saturation time of biochar adsorption bacterial solution was obtained according to the standard curve, with sterile water treated in the same way as the control.

[0100] When making the immobilized bacterial agent, in order to ensure the consistency of the inoculation amount in the petroleum fermentation medium, this experiment selected 0.1g of the selected biochar and 1ml of LD23 bacterial solution with a concentration of 0.2g / ml for full adsorption and mixing.

[0101] 7. Determination of oil degradation rate

[0102] Petroleum standard curve determination: Using UV spectrophotometry, use 50ml volumetric flasks to prepare the petroleum standard curve gradient solution with concentrations of 0.01g / ml, 0.02g / ml, 0.03g / ml, and 0.04g / ml, and dilute it 10 times. Use a UV spectrophotometer to measure the absorbance value of each at 350nm (using OD 350 With oil concentration as the horizontal axis and absorbance as the vertical axis, the oil standard curve is obtained.

[0103] In the experimental group with bacterial solution, the inoculation amount in each 100 ml petroleum fermentation medium is 1 ml of LD23 bacterial solution with a concentration of 0.2 g / ml. To reduce the error, 0.1 g of the biochar prepared in Example 1 is fully adsorbed and mixed with 1 ml of LD23 bacterial solution with a concentration of 0.2 g / ml, and then the mixed solution is placed in the corresponding petroleum fermentation medium (three parallels are made for each group). The control without bacterial solution and the blank petroleum fermentation medium are used as CK, and they are cultured in a shaker at 150 r / min for 7 days; 10 ml of petroleum ether is added to the culture solution and fully shaken for 2 minutes, and it is poured into a separating funnel and left to stand for stratification for 2 minutes; the petroleum ether layer is repeatedly extracted 3 times, and the extract is filtered with a funnel plugged with a little absorbent cotton and anhydrous sodium sulfate on it.

[0104] Using ultraviolet spectrophotometry, collect the extract in a 50 ml volumetric flask, dilute to volume with petroleum ether, and dilute 10 times. Use an ultraviolet spectrophotometer to measure the absorbance value at 350 nm (using OD 350 express).

[0105] Degradation rate (%) = (AB) / A × 100%;

[0106] Where: A is the absorbance of the control, and B is the absorbance of the sample.

[0107] 2. Experimental Results

[0108] 1. Determination of the floating rate of different biochars

[0109] The results are shown in Table 1. A horizontal comparison of the same biomass at different temperatures shows that the floating rate of biochar has a positive correlation with temperature, and the firing temperature of 800°C has the most significant effect on the floating rate of biochar (p<0.05). This is because as the firing temperature increases, the porosity and surface area of ​​the biochar increase, resulting in an increase in the floating rate.

[0110] The type of biomass is also one of the main factors affecting the floating rate of biochar. A longitudinal comparison of different biomasses at the same temperature shows that the overall floating rate of the four biochars is corn stalks < soybean husks < pine needles < reed stalks. Among the four biochars, the floating rate of reed stalk biochar is the most significant (p < 0.05).

[0111] Table 1 Effect of different biomass and different temperatures on biochar floating rate (%)

[0112] Floating rate (%) 400℃ 600℃ 800℃ Reed Example 6: 34.2±1.4A c Example 5: 50.9±4.7B b Example 1: 62.2±1.3C d Soybean Hulls Example 8: 30.0±0.8A b Example 7: 33.2±1.7B a Example 2: 35.4±0.5B b Corn stalks Example 10: 26.7±1.6A a Example 9: 29.5±1.6A a Example 3: 25.7±1.9Aa Pine Needles Example 12: 36.5±0.1Ac Example 11: 31.7±5.2Aa Example 4: 51.4±5.2B c

[0113] Note: Capital letters indicate significant differences among different treatment groups with the same biomass at different temperatures (P<0.05). Lowercase letters indicate significant differences among different treatment groups with the same temperature (P<0.05).

[0114] As can be seen from Table 1, the reed stem biochar prepared in Example 1 has the strongest floating ability, which is significantly higher than that of other examples. Therefore, further experiments were conducted on the relationship between the floating rate of the reed stem biochar prepared in Example 1 and time and the biochar particle size.

[0115] The results are as follows Figure 1 As shown in Figure 2, the increase in oscillation time has little effect on the floating rate of 800℃ reed stem biochar, thus confirming its stability ( Figure 1 A). The floating rate of biochar with a particle size greater than 100 mesh is significantly higher than that of biochar with a particle size less than 100 mesh. Therefore, 800℃ reed stem biochar with a particle size greater than 100 mesh was selected as the immobilization carrier ( Figure 1 B).

[0116] 2. Determination of yield and ash content of different biochars

[0117] As shown in Table 2, the yield of biochar is related to the consumption of organic matter during the firing process. The yield decreases with the increase of temperature. The yield relationship corresponding to the three groups of temperatures is 800℃<600℃<400℃(p<0.05). This is because the firing is more complete with the increase of temperature, and the quality of organic matter decreases. The yield of biochar has no significant effect on the type of biomass, that is, the consumption of organic matter during the firing process is similar.

[0118] Table 2 Effect of different biomass and different temperatures on biochar yield (%)

[0119] Yield (%) 400℃ 600℃ 800℃ Reed 36.0±2.4B a 30.6±0.7Aa 29.5±0.2Ac Soybean Hulls 35.0±2.7B a 27.8±1.2Aa 23.0±0.4Aa Corn stalks 41.1±5.5B a 31.0±0.2Aa 25.3±1.0Ab Pine Needles 32.7±0.9B a 30.2±1.7AB a 28.2±0.3Ac

[0120] Note: Capital letters indicate significant differences among different treatment groups with the same biomass at different temperatures (P<0.05). Lowercase letters indicate significant differences among different treatment groups with the same temperature (P<0.05).

[0121] Table 3 Effect of different biomass on ash content

[0122] Biomass Ash content (%) Reed 5.0±1.0a Soybean Hulls 4.8±0.4a Corn stalks 3.0±0.5a Pine Needles 3.9±0.8a

[0123] Note: The same letters indicate significant differences among different biomass and treatment groups (P<0.05).

[0124] As shown in Table 3, from the effects of different biomasses on ash content, it can be seen that the relationship between the ash content measured after complete oxidation and the type of biomass is corn stalks < pine needles < soybean husks < reed stalks, but there is no significant difference between them.

[0125] 3. Effect of different biochar amounts on oil adsorption

[0126] like Figure 2As shown in the figure, after 7 days of dynamic adsorption, it was observed that after adding 0.1g of selected biochar to adsorb 0.2g of oil, the adsorption saturation was roughly reached. When the adsorption capacity is too high, the oil concentration is too high, which may lead to reduced activity or even death of the strain.

[0127] 4. Determination of strain growth curve

[0128] like Figure 3 As shown in the figure, from the growth curve of strain LD23, 0-3h is the growth delay phase, 3-16h is the logarithmic phase, and 16h and later is the stable phase. Since the bacteria in the logarithmic phase have the highest activity, this experiment selected the logarithmic growth phase of 14h for subsequent experiments.

[0129] 5. Bacterial concentration-absorbance curve and dry weight and wet weight

[0130] like Figure 5 As shown, the trend equation of the bacterial concentration-absorbance curve is: y = 0.1418x + 0.0195 (R 2 =0.9978), the approximate bacterial concentration can be calculated by the absorbance of bacterial concentration according to the equation. The bacterial concentration is referred to as bacterial concentration.

[0131] Table 4 Relationship between dry weight and wet weight of bacteria

[0132] Wet weight(g) Dry weight (g) Dry to wet weight ratio (%) 0.53±0.05 0.10±0.01 19.1±0.8

[0133] From the relationship between dry weight and wet weight in Table 4, it can be seen that the ratio of dry weight to wet weight of the strain is 19.1±0.8%.

[0134] 6. Determination of saturation time of biochar adsorption bacterial solution

[0135] like Figure 5 As shown, the adsorption time of the biochar prepared in Example 1 reached saturation at 5 h, providing a time reference for the subsequent immobilization treatment.

[0136] 7. Determination of oil degradation rate

[0137] The results are shown in Table 5. Through experimental measurements, it was found that the degradation rates corresponding to the three methods were significantly different (p<0.05), and the degradation rates of the three methods were as follows: single charcoal < single bacteria < charcoal plus bacteria, indicating that the present invention's use of charcoal plus bacteria (reed stem biochar plus LD23 bacteria prepared in Example 1) is effective in applying to petroleum pollution. Experimental analysis found that single charcoal can reduce the spread of petroleum through a certain degree of adsorption, and single bacteria face problems in terms of environmental adaptability and competition with indigenous flora in terms of degradation. In practical applications, its degradation ability will be reduced. Since biochar has the characteristics of high porosity, etc., the immobilized bacterial agent can provide efficient bacteria with early adaptation to the environment and oxygen, allowing them to survive better in unfamiliar environments, thereby improving their petroleum degradation rate. At the same time, biochar adsorbs petroleum to solve the characteristics of wide area and low concentration of surface water petroleum pollution, allowing bacteria to degrade around carbon, achieving a better effect. The petroleum standard curve is shown below. Figure 6 shown.

[0138] Table 5 Effects of different treatments on oil degradation rate

[0139] Experimental Materials Degradation rate (%) Only add charcoal 7.3±1.0a Only add bacteria 33.2±3.7b Carbon plus bacteria 45.3±4.8c

[0140] Note: The same letters indicate significant differences among different treatment groups under different experimental material treatments (P<0.05).

[0141] In summary, the present invention uses reed stalks, soybean husks, corn stalks and pine needles, which are common in Heilongjiang Province, as biomass materials to prepare biochar at different temperatures, and explores the conditions required for the biochar with the best floating rate burned by these four biomasses and the effects of different treatments on the degradation rate of the efficient strain LD23. The experimental analysis shows that the yield of biochar decreases with the increase of temperature, but the type of biomass has no significant effect on the yield and ash content of biochar. For surface water petroleum pollution, biochar with a high floating rate can more efficiently adsorb petroleum before its own precipitation. After adsorbing petroleum, its buoyancy can be improved while reducing the surface viscosity of petroleum. The experimental analysis shows that the floating rate of 800℃ reed stalk biochar with a particle size greater than 100 mesh is as high as 62.2% and has strong stability. When exploring the effects of different treatments on the degradation rate of the efficient strain LD23, it is found that the biochar can increase its petroleum degradation rate by 12.1% after fixing the efficient bacteria. This experiment uses reed stalks as the best biomass source for biochar, with low experimental cost, and provides an ideological route for the treatment of surface water petroleum pollution while effectively utilizing waste.

[0142] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0143] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An application of biochar in petroleum degradation, characterized in that: The immobilized bacteria were prepared by using biochar to adsorb and immobilize Pseudomonas (Pseudomonadaceae) and then added to the samples to be treated containing petroleum. The biochar is prepared by drying and crushing the biomass and then carbonizing it at a heating rate of 10°C / min to 12°C / min, reaching a charcoal burning temperature of 400°C to 800°C, carbonizing it at a constant temperature for 10min to 12min, and then naturally cooling it down.

2. The use of biochar in petroleum degradation according to claim 1, characterized in that: Add 10ml to 20ml of immobilized bacterial agent into every 1L of sample to be processed.

3. The use of biochar in petroleum degradation according to claim 1, characterized in that: The preparation process of the immobilized bacterial agent is as follows: 100 g to 150 g of biochar is added to every 1 L of Pseudomonas bacterial liquid for adsorption and fixation to obtain the immobilized bacterial agent.

4. The use of biochar in petroleum degradation according to claim 3, characterized in that: 100 g of biochar was added to every 1 L of Pseudomonas bacterial solution for adsorption and fixation.

5. The use of biochar in petroleum degradation according to claim 3, characterized in that: The concentration of the Pseudomonas bacterial solution is 0.1 g / ml to 0.2 g / ml.

6. The use of biochar in petroleum degradation according to claim 5, characterized in that: The concentration of the Pseudomonas bacterial solution is 0.2 g / ml.

7. The use of biochar in petroleum degradation according to claim 5, characterized in that: The Pseudomonas bacterial liquid is prepared by inoculating Pseudomonas in a liquid culture medium and culturing the culture medium to a logarithmic growth phase, then collecting the bacterial bodies by centrifugation and preparing a bacterial liquid with a concentration of 0.1 g / ml to 0.2 g / ml.

8. The use of biochar in petroleum degradation according to claim 7, characterized in that: After collecting the bacteria by centrifugation, a Pseudomonas bacterial solution with a concentration of 0.1 g / ml to 0.2 g / ml is prepared using sterile water, PBS or liquid culture medium.

9. The use of biochar in petroleum degradation according to claim 1, characterized in that: The biomass is reed stalks.

10. A biochar, characterized in that: The preparation process of the biochar is as follows: after washing and removing impurities from the reed stems, drying at 80°C to 90°C, crushing, and then carbonizing at a heating rate of 10°C / min to 12°C / min, and after reaching a charcoal burning temperature of 400°C to 800°C, carbonizing at a constant temperature for 10min to 12min, and then cooling naturally to obtain biochar.

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