CN115786408A - Method for improving low-rank coal biological gas production rate - Google Patents

Abstract

The invention aims to provide a method for improving the biological gas production rate of low-rank coal by using nitric acid and sphingosine bacillus cornutatus, which belongs to the technical field of coal bioconversion, and the method adopts nitric acid to oxidize the low-rank coal and uses culture medium supernatant for culturing the sphingosine bacillus cornutatus to clean the low-rank coal; then carrying out aerobic degradation on the cleaned nitric acid oxidized coal by using sphingosine torvum, eliminating the adverse effect of the nitric acid adsorbed in the coal, promoting the release of fatty acid and other active organic matters in the coal, and preparing degradation products; finally, degrading the aerobic degradation product by using methanogenic bacteria group to generate methane. The method reduces the using amount of the cleaning solution of the nitric acid oxidized coal, eliminates the adverse effect of residual nitric acid in the nitric acid oxidized coal on biological gas production, promotes the dissolution and release of fatty acid substances in the coal, enhances the interaction between a substrate and a methanogenic flora, and finally improves the biological gas production rate.

Description

Translated fromChinese

一种提高低阶煤生物产气量的方法A method for increasing the biogas production of low-rank coal

技术领域technical field

本发明属于煤的生物转化技术领域，具体涉及一种利用硝酸和矢野口鞘氨醇杆菌（Sphingobium yanoikuyae）提高低阶煤生物产气量的方法。The invention belongs to the technical field of coal biotransformation, and in particular relates to a method for increasing the biological gas production of low-rank coal by using nitric acid and Sphingobium yanoikuyae.

背景技术Background technique

煤炭是一种不可再生资源，节约高效地开发利用煤炭资源并解决伴生的环境问题，对实现“双碳”目标和我国经济社会的可持续发展具有重要现实意义。低阶煤是我国煤炭资源的重要组成部分，虽然其热值较低、水分和灰分较高，但随着能源资源的枯竭，其价值也逐渐被人们重视。然而，通过直接燃烧的方式会产生大量的硫氧化物、氮氧化物和二氧化碳，带来严重的环境问题；而通过化学方法气化和液化需要的反应条件高，会带来较高的能耗及环境污染。利用生物技术实现低阶煤的清洁高效利用，可以克服传统工艺对环境造成的不利影响，并具有低能耗和高效的特点。Coal is a non-renewable resource. It is of great practical significance to realize the goal of "double carbon" and the sustainable development of my country's economy and society to develop and utilize coal resources economically and efficiently and solve the associated environmental problems. Low-rank coal is an important part of my country's coal resources. Although its calorific value is low and its moisture and ash content are high, its value has gradually been valued by people with the depletion of energy resources. However, a large amount of sulfur oxides, nitrogen oxides and carbon dioxide will be produced through direct combustion, which will cause serious environmental problems; while gasification and liquefaction by chemical methods require high reaction conditions, which will bring high energy consumption and environmental pollution. Using biotechnology to achieve clean and efficient utilization of low-rank coal can overcome the adverse impact of traditional processes on the environment, and has the characteristics of low energy consumption and high efficiency.

生物气化是指通过为产甲烷菌群提供适宜的生长条件，促进其生长和繁殖，经过复杂的生物化学代谢过程，将煤中的有机物转化为甲烷的过程。当前，国内外很多高校和科研院所已经实现了实验室内的低阶煤生物气化技术，但距离工业应用尚有一定差距，主要原因是产气量较低。研究表明，通过对煤进行预处理以提高煤的生物可降解性，然后以预处理后的煤为底物进行生物产气，可以提高生物产气量。Biogasification refers to the process of converting organic matter in coal into methane through complex biochemical metabolic processes by providing suitable growth conditions for methanogenic bacteria to promote their growth and reproduction. At present, many universities and scientific research institutes at home and abroad have realized low-rank coal biogasification technology in the laboratory, but there is still a certain distance from industrial application, mainly due to the low gas production. Studies have shown that the biogas production can be increased by pretreating coal to improve its biodegradability, and then using the pretreated coal as a substrate for biogas production.

煤的预处理方法主要包括化学（硝酸、双氧水、高锰酸钾等）法和生物法。硝酸预处理被普遍认为是一种高效的化学预处理方法，但由于硝酸具有强酸性和氧化性，需要对预处理后的煤进行反复的清洗，以消除对产甲烷菌的生长造成的不利影响，这实际上提高了工艺的复杂性和成本。而且，一些与煤大分子结构紧密结合的硝酸很难清洗干净，这些硝酸分子会随着生物产气过程中煤的降解作用而缓慢释放，造成微生物生长环境氧化还原电位升高、pH值降低，从而对产甲烷过程造成不利影响。生物预处理主要是利用一些好氧微生物对煤进行降解，以提高煤的产气效率。在生物预处理过程中，微生物通过产生一些胞外酶、碱、螯合剂、表面活性剂等对煤产生降解作用，但总体来看生物预处理的效果有限。Coal pretreatment methods mainly include chemical (nitric acid, hydrogen peroxide, potassium permanganate, etc.) and biological methods. Nitric acid pretreatment is generally considered to be an efficient chemical pretreatment method, but due to the strong acidity and oxidizing properties of nitric acid, repeated cleaning of the pretreated coal is required to eliminate the adverse effects on the growth of methanogens , which actually increases the complexity and cost of the process. Moreover, it is difficult to clean some nitric acid tightly combined with the macromolecular structure of coal. These nitric acid molecules will be released slowly with the degradation of coal during the biogas production process, resulting in an increase in the oxidation-reduction potential and a decrease in the pH value of the microbial growth environment. This will adversely affect the methane production process. Biological pretreatment mainly uses some aerobic microorganisms to degrade coal to improve the gas production efficiency of coal. In the process of biological pretreatment, microorganisms degrade coal by producing some extracellular enzymes, alkalis, chelating agents, surfactants, etc., but overall the effect of biological pretreatment is limited.

此外，在生物产气阶段，由于煤颗粒中孔裂隙大小的限制，微生物很难进入煤体中发挥作用；一些在厌氧降解产物也会由于煤的吸附作用而残留在其孔隙中，难以释放到环境中被微生物进一步转化成甲烷。这些原因也影响了生物产气量的提高。In addition, in the stage of biological gas production, due to the limitation of the size of pores and fissures in coal particles, it is difficult for microorganisms to enter the coal body to play a role; some anaerobic degradation products will also remain in the pores due to the adsorption of coal, making it difficult to release In the environment, it is further converted into methane by microorganisms. These reasons also affect the improvement of biogas production.

综上，硝酸预处理方法清洗难度大，其残留的氧化性和强酸性会对对生物产气过程造成不利影响；生物预处理虽然条件温和、操作简单，但是预处理效果有限，对产气量的提高并不明显；生物产气过程中直接使用煤粉使得产气效果不理想。因此，需要改进生物产气前煤的预处理方法并提高产气阶段的产气量。In summary, the nitric acid pretreatment method is difficult to clean, and its residual oxidation and strong acidity will adversely affect the biological gas production process; although the biological pretreatment conditions are mild and the operation is simple, the pretreatment effect is limited, and the gas production rate is limited. The improvement is not obvious; the direct use of pulverized coal in the biological gas production process makes the gas production effect unsatisfactory. Therefore, it is necessary to improve the pretreatment method of coal before biogas production and increase the gas production in the gas production stage.

发明内容Contents of the invention

本发明针对现有技术的不足，提供一种提高低阶煤生物产气量的方法。该方法首先以硝酸对低阶煤进行氧化，用培养过矢野口鞘氨醇杆菌（Sphingobium yanoikuyae）的培养基上清液清洗；然后以矢野口鞘氨醇杆菌对清洗后的硝酸氧化煤进行好氧降解，最后以降解产物为底物进行生物产气。The invention aims at the deficiencies of the prior art and provides a method for increasing the biological gas production of low-rank coal. The method first oxidizes low-rank coal with nitric acid, washes it with the medium supernatant cultured with Sphingobium yanoikuyae; then uses Sphingobium yanoikuyae to aerobically degrade the cleaned oxidized coal with nitric acid , and finally use the degradation products as substrates for biogas production.

本发明采用如下技术方案：The present invention adopts following technical scheme:

第一步，取低阶煤粉碎至10-20目（粒径约1-2mm），用6mol/L的硝酸进行氧化，再用培养过矢野口鞘氨醇杆菌的培养基的上清液对硝酸氧化后的煤粉进行清洗，收集清洗好的煤粉；The first step is to take low-rank coal and crush it to 10-20 mesh (particle size is about 1-2mm), oxidize it with 6mol/L nitric acid, and then use the supernatant of the culture medium of Sphingobacter yanoguchi to treat the nitric acid The oxidized coal powder is cleaned, and the cleaned coal powder is collected;

第二步，用矢野口鞘氨醇杆菌对步骤一中清洗好的硝酸氧化煤进行降解，收集降解产物；In the second step, degrade the cleaned nitric acid oxidized coal in step 1 with Sphingobacter yanoguchi, and collect the degradation products;

第三步，用产甲烷菌群对步骤二中的降解产物进行生物产气。In the third step, the degradation product in the second step is biogas-produced by the methanogenic flora.

本发明的有益效果如下：The beneficial effects of the present invention are as follows:

1.本发明利用硝酸对煤进行高效预处理，而无需反复清洗。1. The present invention uses nitric acid to efficiently pretreat coal without repeated cleaning.

2. 本发明利用矢野口鞘氨醇杆菌对硝酸氧化煤进行降解并消除硝酸造成的不利影响，促进了煤中脂肪酸类物质的溶解，进一步提高处理效果。2. The present invention utilizes Sphingobacterium yanoguchi to degrade nitric acid oxidized coal and eliminate the adverse effects caused by nitric acid, which promotes the dissolution of fatty acid substances in coal and further improves the treatment effect.

3. 本发明利用好氧降解产物进行生物产气，促进了产甲烷菌群与底物的相互作用，提高了生物产气量。3. The present invention utilizes aerobic degradation products for biological gas production, which promotes the interaction between methanogenic bacteria and substrates and increases the amount of biological gas production.

附图说明Description of drawings

图1为发酵液pH值的变化图。Figure 1 is a graph showing the change in pH value of the fermentation broth.

图2为好氧降解吸光度随时间的变化图。Figure 2 is a graph showing the change of absorbance with time in aerobic degradation.

图3为降解产物的气质联用分析图，其中，a为上清液，b为沉淀。Figure 3 is the GC analysis diagram of the degradation product, where a is the supernatant and b is the precipitate.

具体实施方式Detailed ways

本发明所用的好氧降解细菌为矢野口鞘氨醇杆菌（Sphingobium yanoikuyae）；本发明所用的产甲烷菌群为从山西蓝焰煤层气有限公司SH-121煤层气井出水口中富集的原位产甲烷菌群。The aerobic degradation bacteria used in the present invention is Sphingobium yanoikuyae; Methanogens.

矢野口鞘氨醇杆菌（Sphingobium yanoikuyae）所用的培养基为LB培养基，其组成为：蛋白胨10g，酵母粉5g，NaCl 10g，(固体培养基中含有琼脂15g)，蒸馏水1000mL，pH为7.4～7.6。先将甘油保藏的矢野口鞘氨醇杆菌（Sphingobium yanoikuyae）恢复至室温后，用接种环挑取保藏液在LB固体培养基平板上划线分离，在30℃下培养至长出单菌落。用接种环在固体培养基平板上挑取单菌落，接种至LB液体培养基中，在恒温摇床（30℃，150rpm）中培养24-36h，制成种子液。The medium used by Sphingobium yanoikuyae is LB medium, which consists of: 10g peptone, 5g yeast powder, 10g NaCl (solid medium contains 15g agar), 1000mL distilled water, pH 7.4-7.6 . After the Glycerol-preserved Sphingobium yanoikuyae (Sphingobium yanoikuyae) was returned to room temperature, the preservation solution was picked up with an inoculation loop, streaked and separated on the LB solid medium plate, and cultured at 30°C until a single colony grew. Use an inoculation loop to pick a single colony on a solid medium plate, inoculate it into LB liquid medium, and culture it in a constant temperature shaker (30°C, 150rpm) for 24-36h to make a seed solution.

产甲烷菌群所用的培养基组成为：K₂HPO₄ 2.9g，KH₂PO₄ 1.5g，NH₄Cl 1.8g，MgCl₂0.4g，酵母萃取物0.2g，L-半胱氨酸盐酸盐0.5g，去离子水1000mL。取产甲烷菌群保藏液5mL接种到120mL培养基中厌氧培养15天，作为种子液。The medium composition used for the methanogenic bacteria group is: K₂ HPO₄ 2.9g, KH₂ PO₄ 1.5g, NH₄ Cl 1.8g, MgCl₂ 0.4g, yeast extract 0.2g, L-cysteine hydrochloride Salt 0.5g, deionized water 1000mL. Take 5 mL of the methanogenic flora preservation solution and inoculate it into 120 mL medium for anaerobic culture for 15 days as the seed solution.

本实施例所用的低阶煤采自河南义马千秋矿，粉碎至10-20目（粒径约1-2mm），在鼓风干燥箱中70℃烘干。The low-rank coal used in this example was collected from Qianqiu Mine in Yima, Henan, crushed to 10-20 mesh (particle size about 1-2mm), and dried in a blast drying oven at 70°C.

实施例1 煤的硝酸氧化及清洗方法的改进Example 1 Nitric acid oxidation of coal and improvement of cleaning method

取100g低阶煤煤粉，缓慢加入6mol/L的硝酸100mL并不断搅拌，直至反应结束，静置24小时。Take 100g of low-rank coal powder, slowly add 100mL of 6mol/L nitric acid and keep stirring until the reaction is over, and let stand for 24 hours.

在250mL三角瓶加入体积为150 mL LB培养基，高温（121℃，20min）灭菌。冷却后加入1mL矢野口鞘氨醇杆菌种子液，放入恒温摇床（30℃，150rpm）中培养14天，测量发酵液的pH变化，结果如图1所示。可以看出，矢野口鞘氨醇杆菌培养过程中产生了一些碱性物质造成发酵液pH升高，第9天发酵液pH值达到了9.01，随后保持稳定。Add a volume of 150 mL LB medium to a 250 mL Erlenmeyer flask, and sterilize at high temperature (121 °C, 20 min). After cooling, 1 mL of Sphingobacter yanoguchi seed liquid was added, and cultured in a constant temperature shaker (30°C, 150 rpm) for 14 days, and the pH changes of the fermentation broth were measured, and the results are shown in Figure 1. It can be seen that some alkaline substances were produced during the cultivation of Sphingobacter yanoguchi, which caused the pH of the fermentation broth to rise, and the pH value of the fermentation broth reached 9.01 on the 9th day, and then remained stable.

将第9天的发酵液离心（6000rpm，15min），收集上清液。取10g硝酸氧化后的煤粉，分别用上清液和去离子水作为清洗液对其进行清洗直至pH为7.0，计算清洗液的消耗量，结果如表1所示。可以看出，由于上清液中含有的碱性物质可以快速中和煤粉中的硝酸，其用量比去离子水降低了58%，显著减少了清洗液的用量，降低了清洗难度。The fermented liquid onday 9 was centrifuged (6000 rpm, 15 min), and the supernatant was collected. Take 10g of pulverized coal oxidized by nitric acid, wash it with supernatant and deionized water as cleaning solution until the pH is 7.0, calculate the consumption of cleaning solution, and the results are shown in Table 1. It can be seen that since the alkaline substances contained in the supernatant can quickly neutralize the nitric acid in the coal powder, its dosage is 58% lower than that of deionized water, which significantly reduces the dosage of cleaning fluid and reduces the difficulty of cleaning.

表1 清洗液用量对比Table 1 Comparison of the amount of cleaning solution

实施例2 好氧降解时间的确定Embodiment 2 Determination of aerobic degradation time

在250mL三角瓶加入体积为150 mL LB培养基和1g硝酸氧化煤，高温（121℃，20min）灭菌。冷却后加入1mL矢野口鞘氨醇杆菌种子液，放入恒温摇床（30℃，150rpm）中培养。Add 150 mL of LB medium and 1 g of oxidized coal nitric acid into a 250 mL Erlenmeyer flask, and sterilize at high temperature (121 °C, 20 min). After cooling, add 1 mL of Sphingobacter yanoguchi seed solution, and put it in a constant temperature shaker (30°C, 150rpm) for cultivation.

研究表明，450nm处的吸收与腐殖酸的生成有关，降解液在450nm处吸光度的大小可以表征煤的降解效果。因此，在培养的第1、2、3、6、8、9、14天取降解液离心，收集上清液样品，采用紫外可见分光光度计（尤尼柯UV4802），以去离子水作为空白，测定其450nm处吸光度值，结果如图2所示。从图2可以看出，发酵液的在450nm处的吸光度值在第9天即可达到稳定，因此将矢野口鞘氨醇杆菌对煤的好氧降解时间确定为9天。Studies have shown that the absorption at 450nm is related to the generation of humic acid, and the absorbance of the degradation solution at 450nm can characterize the degradation effect of coal. Therefore, on the 1st, 2nd, 3rd, 6th, 8th, 9th, and 14th days of culture, the degradation solution was taken and centrifuged, and the supernatant sample was collected, and a UV-visible spectrophotometer (Unico UV4802) was used, and deionized water was used as a blank , Determination of its absorbance value at 450nm, the results are shown in Figure 2. It can be seen from Figure 2 that the absorbance value of the fermentation broth at 450 nm can reach stability on the 9th day, so the aerobic degradation time of coal by Sphingobacter yanoguchi is determined to be 9 days.

实施例3 好氧降解产物的制备方法The preparation method of embodiment 3 aerobic degradation products

在250mL三角瓶加入体积为150 mL 的LB培养基和加入1g硝酸氧化煤，高温（121℃，20min）灭菌。冷却后加入1mL矢野口鞘氨醇杆菌种子液，放入恒温摇床（30℃，150rpm）中培养9天，收集上清液，作为好氧降解产物。Add LB medium with a volume of 150 mL and 1 g of oxidized coal nitric acid to a 250 mL Erlenmeyer flask, and sterilize at high temperature (121 °C, 20 min). After cooling, add 1 mL of Sphingobacterium yanoguchi seed solution, put it in a constant temperature shaker (30°C, 150rpm) and cultivate it for 9 days, and collect the supernatant as an aerobic degradation product.

实施例4 好氧降解率的测定The mensuration ofembodiment 4 aerobic degradation rate

收集实施例3中矢野口鞘氨醇杆菌好氧降解后的煤样，用去离子水20mL清洗3次以去除吸附的微生物，在70℃烘干后测定其质量。降解率的计算方法为：P=（m₀-m₁）/m₀·100%，其中，P为降解率，%；m₀为煤样初始质量，g；m₁为经生物降解残余煤样质量，g。Collect the coal samples aerobically degraded by Sphingobacter yanoguchi in Example 3, wash them three times with 20 mL of deionized water to remove adsorbed microorganisms, and measure their quality after drying at 70°C. The calculation method of the degradation rate is: P=(m₀ -m₁ )/m₀ ·100%, where P is the degradation rate, %; m₀ is the initial mass of the coal sample, g; m₁ is the residual coal after biodegradation Sample mass, g.

结果显示，经过矢野口鞘氨醇杆菌9天的降解后，煤粉的降解率达到了32.14±2.73%，这表明矢野口鞘氨醇杆菌对低阶煤具有较好的降解效果。The results showed that after 9 days of degradation by Sphingobacter yanoguchi, the degradation rate of coal powder reached 32.14±2.73%, which indicated that Sphingobacter yanoguchi had a good degradation effect on low-rank coal.

实施例5 好氧降解产物的有机组分分析Example 5 Organic Component Analysis of Aerobic Degradation Products

用盐酸将实施例3中降解产物pH值调节至2.0后静置过夜，离心（6000rpm，10min）收集沉淀并在70℃烘干。取烘干后的降解产物1 g加入5 mL甲醇，在50℃浸泡萃取72 h。萃取完成后，将上清液用氮吹仪45 ℃浓缩至1mL待测。The pH value of the degradation product in Example 3 was adjusted to 2.0 with hydrochloric acid and allowed to stand overnight, and the precipitate was collected by centrifugation (6000 rpm, 10 min) and dried at 70°C. Take 1 g of the dried degradation product and add 5 mL of methanol, soak and extract at 50 °C for 72 h. After the extraction was completed, the supernatant was concentrated to 1 mL at 45 °C with a nitrogen blower for testing.

用固相萃取方法对沉淀上清液中有机物进行提取，萃取流程为：（1）在萃取柱（Agilent Bond Elut C18，500 mg，120 µm，6 mL）中依次加入5 mL甲醇、5 mL去离子水进行活化。（2）加入上清液10 mL，以流速约2 mL/min过柱。（3）过完柱后，先用10 mL去离子水冲洗，再抽真空去除残留水分。（4）用2 mL甲醇洗脱，收集洗脱液，在氮吹仪中45 ℃浓缩至1.0mL待测。The organic matter in the precipitation supernatant was extracted by solid phase extraction. The extraction process was as follows: (1) Add 5 mL of methanol and 5 mL of Ionized water for activation. (2) Add 10 mL of supernatant and pass through the column at a flow rate of about 2 mL/min. (3) After passing through the column, rinse with 10 mL of deionized water, and then vacuum to remove residual water. (4) Elute with 2 mL of methanol, collect the eluate, and concentrate it to 1.0 mL in a nitrogen blower at 45 °C for testing.

采用气质联用仪（Agilent 7890A-5795C）进行组分分析。色谱柱采用Agilent VF-WAXms（30 m×250 μm×0.25 μm），载气为高纯度的氦气，进样量0.8 μL，柱流速1.0 mL/min，初始温度60℃，保持2 min，以10℃/ min 的速率升温至250℃，保持20 min。分析完成后用NIST08数据库进行有机物鉴定。Component analysis was performed using a gas chromatography-mass spectrometer (Agilent 7890A-5795C). The chromatographic column used Agilent VF-WAXms (30 m×250 μm×0.25 μm), the carrier gas was high-purity helium, the injection volume was 0.8 μL, the column flow rate was 1.0 mL/min, the initial temperature was 60°C, and it was kept for 2 min, then The temperature was raised to 250 °C at a rate of 10 °C/min and held for 20 min. After the analysis was completed, the NIST08 database was used for organic identification.

降解产物气质联用分析结果如图3所示。从降解产物沉淀的甲醇萃取物中检测到了大量的羧酸类物质。羧酸主要包括庚烷酸、十二酸、十三烷酸、十四酸、13-甲基十四酸、十六酸、十八酸等中长链饱和脂肪酸，9-十六烯酸、9-十八烯酸、顺-10-庚烯二酸等长链不饱和脂肪酸，乙二酸、丁二酸、戊二酸等小分子二羧酸和4-氧代戊酸、3-羟基-2-甲基戊二酸等氧取代羧酸，详细的组分见表2。从上清液中主要检测到一些的小分子的醛、醇、酚和杂环化合物，主要包括4-甲基-2-戊醇、吡咯、苯乙醛、苯乙酸、1,4-苯二甲酸、吲哚、3-甲基-1-氢-吲哚等。沉淀上清液中的有机物被称为黄腐酸，其分子量比较小，但具有更高的水溶性和生物活性。总之，降解产物中含有丰富的脂肪酸和其它活性有机物，是对产甲烷菌群有利的营养物质。The analysis results of degradation products by gas chromatography-mass spectrometry are shown in Figure 3. A large number of carboxylic acids were detected from the methanol extract of the degradation product precipitation. Carboxylic acids mainly include medium and long-chain saturated fatty acids such as heptanoic acid, dodecanoic acid, tridecanoic acid, myristic acid, 13-methyl tetradecanoic acid, palmitic acid, octadecanoic acid, 9-hexadecenoic acid, Long-chain unsaturated fatty acids such as 9-octadecenoic acid and cis-10-heptenedioic acid, small molecule dicarboxylic acids such as oxalic acid, succinic acid, glutaric acid, and 4-oxopentanoic acid, 3-hydroxy Oxygen-substituted carboxylic acids such as -2-methylglutaric acid, see Table 2 for detailed components. Some small molecule aldehydes, alcohols, phenols and heterocyclic compounds were mainly detected from the supernatant, mainly including 4-methyl-2-pentanol, pyrrole, phenylacetaldehyde, phenylacetic acid, 1,4-benzenedi Formic acid, indole, 3-methyl-1-hydro-indole, etc. The organic matter in the precipitation supernatant is called fulvic acid, which has a relatively small molecular weight, but has higher water solubility and biological activity. In short, the degradation products are rich in fatty acids and other active organic matter, which are beneficial nutrients for methanogenic bacteria.

表2 降解产物中有机酸组分Table 2 Organic acid components in degradation products

实施例6生物产气中好氧降解产物添加量的确定Determination of the amount of aerobic degradation product added in the biological gas production in Example 6

在250mL厌氧瓶中加入体积为150 mL 的产甲烷菌群培养基，分别加入2.5mL、5.0mL、7.5mL、10mL、12.5mL实施例3中获得的降解产物作为实验组，以不添加降解产物作为空白组，高温（121℃，20min）灭菌。冷却后加入8mL产甲烷菌群种子液，放入恒温箱（30℃）。研究表明，煤在产甲烷菌群的作用下会产生氢气、甲烷和二氧化碳等气体，产气量一般在30天左右达到稳定。因此，根据第32天的产气量来评估好氧降解产物的添加量。In a 250mL anaerobic bottle, add a volume of 150mL methanogenic flora culture medium, add 2.5mL, 5.0mL, 7.5mL, 10mL, 12.5mL of the degradation products obtained in Example 3 as the experimental group, without adding degradation products The product was used as a blank group and sterilized at high temperature (121°C, 20min). After cooling, add 8mL of methanogenic flora seed solution, and put it in an incubator (30°C). Studies have shown that under the action of methanogenic bacteria, coal will produce gases such as hydrogen, methane and carbon dioxide, and the gas production will generally stabilize in about 30 days. Therefore, the addition of aerobic degradation products was evaluated based on the gas production on day 32.

采用气相色谱分析生物产气过程中的气体成份。仪器型号为美国安捷伦7890气相色谱，Carbonplot色谱柱（60m×320μm×1.5μm），TCD检测器，气密针进样，进样量0.5mL。进样口温度150℃，柱温箱温度30℃，检测器温度200℃。采用排水集气法测量气体体积。计算产气量时，分别从添加量为2.5mL、5.0mL、7.5mL、10mL、12.5mL的组中扣除空白组的产气量。Gas chromatography was used to analyze the gas composition in the biogas production process. The instrument model is American Agilent 7890 gas chromatograph, Carbonplot chromatographic column (60m×320μm×1.5μm), TCD detector, gas-tight needle injection, injection volume 0.5mL. The temperature of the injection port was 150°C, the temperature of the column oven was 30°C, and the temperature of the detector was 200°C. The gas volume is measured by the drainage gas collection method. When calculating the gas production, the gas production of the blank group is deducted from the groups with the addition amount of 2.5mL, 5.0mL, 7.5mL, 10mL and 12.5mL respectively.

不同好氧降解产物添加量对应的产气量如表3所示。从表3中可以看出产甲烷量随着好氧降解产物添加量的增大而增大。但在添加量大于10mL时，对产甲烷量和产二氧化碳量的提高已不明显。因此，确定选择添加10mL作为底物添加量。The gas production corresponding to different additions of aerobic degradation products is shown in Table 3. It can be seen from Table 3 that the amount of methane produced increases with the addition of aerobic degradation products. But when the addition amount is greater than 10mL, the increase of methane production and carbon dioxide production is not obvious. Therefore, it is determined to choose to add 10mL as the substrate addition amount.

表3不同好氧降解产物添加量对应的产气量Table 3 Gas production corresponding to different additions of aerobic degradation products

实施例7利用好氧降解产物产气的优势分析Example 7 Advantage Analysis of Utilizing Aerobic Degradation Products to Produce Gas

在250mL厌氧瓶中加入体积为150 mL 的产甲烷菌群培养基，分别加入10mL实施例3中获得的降解产物、5g硝酸氧化煤粉和5g未氧化的煤粉，以不添加煤粉和降解产物的作为空白组，高温（121℃，20min）灭菌。冷却后加入8mL产甲烷菌群群，放入30℃恒温箱培养36天后，按照实施例6中方法测定气体组分和产气量，结果如表4所示。计算产气量时，分别从添加降解产物和煤粉的组中扣除空白组的产气量。In a 250mL anaerobic bottle, add a volume of 150 mL of methanogenic flora medium, add 10 mL of the degradation product obtained in Example 3, 5 g of nitric acid oxidized coal powder, and 5 g of unoxidized coal powder, so as not to add coal powder and Degradation products were used as the blank group and sterilized at high temperature (121°C, 20min). After cooling, 8 mL of methanogenic flora was added, placed in a 30°C incubator for 36 days, and the gas components and gas production were measured according to the method in Example 6. The results are shown in Table 4. When calculating the gas production, the gas production of the blank group was deducted from the group added with degradation products and pulverized coal respectively.

表4不同类型底物的产气量对比Table 4 Comparison of gas production of different types of substrates

从表4可以看出相对于未氧化煤，好氧降解产物的产甲烷量提高了7倍，产二氧化碳量提高了37.3%，可见好氧降解产物具有明显的产气优势。添加硝酸氧化煤的实验组由于pH过低，未产生任何气体。It can be seen from Table 4 that compared with unoxidized coal, the methane production of aerobic degradation products has increased by 7 times, and the production of carbon dioxide has increased by 37.3%. It can be seen that aerobic degradation products have obvious advantages in gas production. The experimental group adding nitric acid to oxidize coal did not produce any gas because the pH was too low.

需要注意的是，利用本发明确定的矢野口鞘氨醇杆菌降解方法，1g硝酸氧化煤可制备至少120mL降解产物。而根据表4中数据，每10mL降解产物可产生16.1mL甲烷，120mL降解产物即可产生16.1×12=193.2mL甲烷，单位质量煤粉的产甲烷量达到了193.2÷1=193.2mL/g煤；而未氧化煤的产甲烷量为2.1÷5=0.42mL/g煤，直接利用硝酸氧化煤不可以产气。可见，利用矢野口鞘氨醇杆菌对硝酸氧化低阶煤的好氧降解产物进行产气，可以极大地提高产气量。It should be noted that, using the degradation method of Sphingobacter yanoguchi determined in the present invention, at least 120 mL of degradation products can be prepared from 1 g of nitric acid oxidation coal. According to the data in Table 4, every 10mL of degradation products can produce 16.1mL of methane, 120mL of degradation products can produce 16.1×12=193.2mL of methane, and the methane production per unit mass of pulverized coal reaches 193.2÷1=193.2mL/g coal ; while the amount of methane produced by unoxidized coal is 2.1÷5=0.42mL/g coal, direct use of nitric acid to oxidize coal cannot produce gas. It can be seen that using Sphingobacter yanoguchi to produce gas from the aerobic degradation products of nitric acid oxidation of low-rank coal can greatly increase the gas production.

实施例8硝酸和矢野口鞘氨醇杆菌联合处理的优势分析Advantage analysis ofembodiment 8 nitric acid and Sphingobacter yanoguchi combined treatment

利用pH计对实施例7中各实验组第36天发酵液和实施例3中获得的降解产物进行pH值测定，结果如表5所示。从表5可以看出，添加未氧化煤和好氧降解产物的实验组发酵液pH值均在产甲烷菌最适宜的pH范围（6.8~7.5）内。硝酸氧化煤中含有大量吸附的硝酸，因而使得发酵液pH值呈现明显的酸性，不适宜产甲烷菌生长，因而没有甲烷产生。The pH value of the fermented liquid of each experimental group in Example 7 on the 36th day and the degradation product obtained in Example 3 was measured with a pH meter, and the results are shown in Table 5. It can be seen from Table 5 that the pH values of the fermentation broth of the experimental group added with unoxidized coal and aerobic degradation products were all within the optimum pH range (6.8-7.5) for methanogens. Nitric acid oxidation coal contains a large amount of adsorbed nitric acid, which makes the pH value of the fermentation broth significantly acidic, which is not suitable for the growth of methanogens, so no methane is produced.

表5 发酵液和降解产物pH值Table 5 pH values of fermentation broth and degradation products

好氧降解产物的pH为8.86，呈现明显的碱性，这说明矢野口鞘氨醇杆菌对硝酸氧化煤降解过程中，中和了煤中的酸性物质，去除了酸性物质对生物产气的不利影响。同时，结合实施例5中有关降解产物组分分析结果来看，利用矢野口鞘氨醇杆菌对硝酸氧化的低阶煤进行降解，可以为产甲烷菌群提供丰富的营养物质。The pH of the aerobic degradation product is 8.86, showing obvious alkalinity, which shows that during the degradation of coal by Sphingobacter yanoguchi on nitric acid oxidation, it neutralizes the acidic substances in coal and removes the adverse effects of acidic substances on biological gas production . At the same time, combined with the analysis results of the degradation product components in Example 5, it can be seen that the degradation of low-rank coal oxidized by nitric acid by Sphingobacter yanoguchi can provide rich nutrients for the methanogenic flora.

Claims (4)

Translated fromChinese

1.一种提高低阶煤生物产气量的方法，其特征在于：包括如下步骤：1. A method for improving low-rank coal biogas production, characterized in that: comprises the steps:第一步，取低阶煤粉碎，用硝酸进行氧化，得到硝酸氧化煤；In the first step, the low-rank coal is crushed and oxidized with nitric acid to obtain nitric acid oxidized coal;第二步，用培养过矢野口鞘氨醇杆菌的培养基的上清液清洗硝酸氧化煤；In the second step, the nitric acid oxidized coal is cleaned with the supernatant of the culture medium of Sphingobacter yanoguchi;第三步，用矢野口鞘氨醇杆菌对清洗后的硝酸氧化煤进行好氧降解，收集降解产物；In the third step, the cleaned nitric acid oxidized coal is aerobically degraded with Sphingobacter yanoguchi, and the degradation products are collected;第四步，用产甲烷菌群对降解产物进行降解，产甲烷。The fourth step is to degrade the degradation products with methanogenic flora to produce methane.2.根据权利要求1所述的一种提高低阶煤生物产气量的方法，其特征在于：第一步中所述低阶煤粉碎至10-20目，所述硝酸的浓度为6mol/L。2. A method for improving the biological gas production of low-rank coal according to claim 1, characterized in that: the low-rank coal is pulverized to 10-20 mesh in the first step, and the concentration of the nitric acid is 6mol/L .3.根据权利要求1所述的一种提高低阶煤生物产气量的方法，其特征在于：第二步中所述矢野口鞘氨醇杆菌的培养基为LB培养基，包括固体培养基和液体培养基，所述固体培养基包括如下组分：蛋白胨10g、酵母粉5g、NaCl 10g、琼脂15g、蒸馏水1000mL；液体培养基包括如下组分：蛋白胨10g、酵母粉5g、NaCl 10g、蒸馏水1000mL。3. a kind of method for improving low-rank coal biogas production according to claim 1, is characterized in that: the substratum of Sphingobacterium yanoguchi described in the second step is LB substratum, comprises solid substratum and liquid substratum Medium, the solid medium includes the following components: peptone 10g, yeast powder 5g, NaCl 10g, agar 15g, distilled water 1000mL; liquid medium includes the following components: peptone 10g, yeast powder 5g, NaCl 10g, distilled water 1000mL.4.根据权利要求1所述的一种提高低阶煤生物产气量的方法，其特征在于：所述上清液取矢野口鞘氨醇杆菌发酵第9天的培养基的上清液。4 . The method for increasing the biological gas production of low-rank coal according to claim 1 , wherein the supernatant is the supernatant of the medium fermented by Sphingobacter yanoguchi on the 9th day.

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