CN103571876A - Method for producing biogas with efficient utilization of solar energy by utilizing blue-green algae - Google Patents
Method for producing biogas with efficient utilization of solar energy by utilizing blue-green algae Download PDFInfo
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Abstract
Description
技术领域technical field
本发明涉及一种利用蓝藻高效利用太阳能生产生物燃气的方法,属于生物技术领域。The invention relates to a method for efficiently utilizing solar energy to produce biogas by using cyanobacteria, and belongs to the field of biotechnology.
背景技术Background technique
蓝藻(Blue green algae)是一种能进行光合自养作用的原核生物,其光合作用的过程和机理与高等植物类似,可利用光能产生O2并通过固定CO2来合成有机物。蓝藻是一种细胞结构简单,遗传背景清晰的光合生物,随着两株模式蓝藻基因组序列测定完成,人们开始普遍从分子生物学水平研究蓝藻代谢相关的机理。Cyanobacteria (Blue green algae) is a prokaryotic organism capable of photoautotrophy. Its photosynthetic process and mechanism are similar to those of higher plants. It can use light energy to produce O 2 and synthesize organic matter by fixing CO 2 . Cyanobacteria is a photosynthetic organism with simple cell structure and clear genetic background. With the completion of the genome sequence of two model cyanobacteria, people began to study the mechanism of cyanobacteria metabolism from the level of molecular biology.
近年来,随着能源危机和环境污染给人类社会带来的压力愈演愈烈,蓝藻的另一个重要代谢—光合产氢也受到人们的重视。In recent years, as the energy crisis and environmental pollution have intensified the pressure on human society, another important metabolism of cyanobacteria—photosynthetic hydrogen production has also attracted people's attention.
前人的研究表明蓝藻光合产氢是一种经济的,可再生的产氢方式,获得更高的产氢效率是应用蓝藻光合产氢的必经途径。同时,微藻细胞生物质被证明是一种高效,低成本的厌氧发酵产甲烷的底物。因此,将光合产氢后的藻细胞进行进一步的资源再利用无疑是一条创新性的,可有效提高太阳能转化效率和降低蓝藻产氢成本的途径。Previous studies have shown that photosynthetic hydrogen production by cyanobacteria is an economical and renewable way of hydrogen production, and obtaining higher hydrogen production efficiency is the only way to apply photosynthetic hydrogen production by cyanobacteria. At the same time, microalgal cell biomass has been proved to be an efficient and low-cost substrate for anaerobic fermentation of methane. Therefore, the further resource reuse of algal cells after photosynthetic hydrogen production is undoubtedly an innovative approach that can effectively improve the efficiency of solar energy conversion and reduce the cost of hydrogen production by cyanobacteria.
因此,一直以来,蓝藻光合产氢被视为低碳、经济的产氢方式。目前认为,蓝藻的光合作用效率是高等植物的十几倍至上百倍,即便如此,其光合产氢过程中的光能转化效率依然低下,无法满足生产应用的要求。为此,我们首先选择从光合作用机制上对蓝藻的光合产氢的能量传递过程进行调控,通过提高藻细胞对太阳能的转化效率,促进蓝藻光合产氢能力。根据调控的情况,我们在分子生物学水平对藻细胞光合产氢的关键代谢途径进行监测,然后依据监测的结果,我们希望通过基因工程的手段定向的改造蓝藻细胞,从而获得高产氢能力的工程藻种,为蓝藻光合产氢的工程化应用提供基础材料和调控手段。Therefore, photosynthetic hydrogen production by cyanobacteria has always been regarded as a low-carbon and economical hydrogen production method. It is currently believed that the photosynthesis efficiency of cyanobacteria is ten to hundreds of times that of higher plants. Even so, the conversion efficiency of light energy in the process of photosynthetic hydrogen production is still low, which cannot meet the requirements of production and application. To this end, we first choose to regulate the energy transfer process of photosynthetic hydrogen production of cyanobacteria from the perspective of photosynthesis mechanism, and promote the photosynthetic hydrogen production capacity of cyanobacteria by improving the conversion efficiency of algae cells to solar energy. According to the regulation, we monitor the key metabolic pathways of photosynthetic hydrogen production in algae cells at the level of molecular biology, and then based on the monitoring results, we hope to transform cyanobacteria cells in a targeted manner by means of genetic engineering, so as to obtain engineering with high hydrogen production capacity. Algae species provide basic materials and control means for the engineering application of cyanobacteria photosynthetic hydrogen production.
发明内容Contents of the invention
本发明提供了一种利用蓝藻高效利用太阳能生产生物燃气的方法,解决蓝藻对太阳能转化效率低的问题。The invention provides a method for efficiently utilizing solar energy to produce biogas by using blue algae, and solves the problem of low conversion efficiency of blue algae to solar energy.
为了解决上述问题,本发明的技术方案如下:(1)藻种的培养;(2)光合产氢的诱导;(3)收集氢气。In order to solve the above problems, the technical scheme of the present invention is as follows: (1) cultivation of algal species; (2) induction of photosynthetic hydrogen production; (3) collection of hydrogen.
上述光合产氢的诱导剂为一种光合抑制剂二氯苯基二甲脲(DCMU)。The above-mentioned inducer of photosynthetic hydrogen production is a photosynthetic inhibitor dichlorophenyldimethylurea (DCMU).
上述诱导剂的添加量为1μM。The above-mentioned inducer was added in an amount of 1 μM.
本发明的步骤如下:(1)藻种的培养;(2)光合产氢的诱导;(3)收集氢气;(4)厌氧发酵产沼气。The steps of the invention are as follows: (1) cultivation of algal species; (2) induction of photosynthetic hydrogen production; (3) collection of hydrogen; (4) anaerobic fermentation to produce biogas.
上述蓝藻为鱼腥藻7120,在中科院水生生物研究所的编号为FACHB-418。The above-mentioned cyanobacteria is Anabaena 7120, and the serial number in the Institute of Hydrobiology, Chinese Academy of Sciences is FACHB-418.
上述步骤(4)是利用产氢结束的藻细胞进行厌氧发酵降解产生沼气的。The above step (4) uses the algae cells that have finished hydrogen production to perform anaerobic fermentation and degradation to generate biogas.
一种蓝藻高产氢气的方法的步骤如下:The steps of a method for cyanobacteria to produce hydrogen are as follows:
1)鱼腥藻7120的培养,恒温并持续光照,液体通气培养,藻种保存在固体琼脂BGII(+N)培养基平板上;1) Cultivation of Anabaena 7120, constant temperature and continuous light, liquid aeration culture, algae species are stored on solid agar BGII (+N) medium plate;
2)用培养基培养使得藻细胞生长到一定浓度,分装到玻璃样品管中,然后保持厌氧环境进行诱导,密闭试管;2) Cultivate the algae cells to a certain concentration by culturing them in the culture medium, divide them into glass sample tubes, then maintain an anaerobic environment for induction, and seal the test tubes;
3)收集完成光合产氢过程的藻细胞,再取藻泥和种泥,加入一定量的蒸馏水和PBS缓冲液,密闭后置于37℃水浴中进行持续发酵降解产沼气过程。3) Collect the algae cells that have completed the photosynthetic hydrogen production process, then take the algae mud and seed mud, add a certain amount of distilled water and PBS buffer, seal it and place it in a 37°C water bath for continuous fermentation and degradation to produce biogas.
为了实现上述目的,本发明要用以下技术措施:藻种的保存和培养、藻细胞光合产氢诱导、种泥发酵菌群活性的诱导、蓝藻生物质厌氧降解产沼气、氢气的检测和定量、沼气主要成分的检测和定量。主要流程如图1所示,由三部分构成:藻细胞的培养,光合产氢的诱导,以及厌氧发酵产沼气。In order to achieve the above object, the present invention will use the following technical measures: preservation and cultivation of algae species, induction of photosynthetic hydrogen production in algal cells, induction of activity of seed sludge fermentation flora, anaerobic degradation of cyanobacteria biomass to produce biogas, detection and quantification of hydrogen , Detection and quantification of the main components of biogas. The main process is shown in Figure 1, which consists of three parts: the cultivation of algae cells, the induction of photosynthetic hydrogen production, and the anaerobic fermentation to produce biogas.
本发明在调控蓝藻光合产氢的基础上,创造性地将蓝藻的自身光合产氢过程与藻细胞生物质厌氧发酵产甲烷过程进行结合,在国际上首次尝试开发蓝藻光合产氢和厌氧降解产甲烷偶联工艺。这对于提高太阳能转化效率,开发低成本的蓝藻光合产氢新工艺具有重大意义。本发明与现有工艺技术相比具有以下优点和效果:On the basis of regulating the photosynthetic hydrogen production of cyanobacteria, the present invention creatively combines the self-photosynthetic hydrogen production process of cyanobacteria with the anaerobic fermentation methane production process of algal cell biomass, and is the first attempt in the world to develop photosynthetic hydrogen production and anaerobic degradation of cyanobacteria Methanogenic coupling process. This is of great significance for improving the efficiency of solar energy conversion and developing a new low-cost process for photosynthetic hydrogen production by cyanobacteria. Compared with the existing technology, the present invention has the following advantages and effects:
1、见效快,效果显著,效率高。一般在几小时之内就能起到促进蓝藻光合产氢的效果,并且能够持续长时间产氢,十分有效地提高了氢气的产量;1. Quick effect, remarkable effect and high efficiency. Generally, within a few hours, it can promote the photosynthetic hydrogen production of cyanobacteria, and can continue to produce hydrogen for a long time, which effectively increases the hydrogen production;
2、沼气生产过程能耗低。使用废水处理厂污泥作为种泥,使用光合自养的微藻生物质作为底物;2. Low energy consumption in the biogas production process. Using wastewater treatment plant sludge as seed sludge and photoautotrophic microalgal biomass as substrate;
3、步骤简易、过程清洁环保。3. The steps are simple and the process is clean and environmentally friendly.
说明书附图Instructions attached
图1本发明的流程图。Figure 1 is a flow chart of the present invention.
图2蓝藻一级种子液培养器皿示意图。Fig. 2 Schematic diagram of a cyanobacteria primary seed liquid culture vessel.
图3蓝藻二级种子液培养器皿简图。Fig. 3 Schematic diagram of a vessel for cultivating secondary seed liquid of cyanobacteria.
图4氢气产量定量分析标准曲线图。Figure 4. Standard curve diagram for quantitative analysis of hydrogen production.
图5产氢蓝藻-鱼腥藻40倍光学显微镜照片Fig. 5 40 times optical microscope photo of hydrogen-producing cyanobacteria Anabaena
具体实施方式Detailed ways
下面结合具体实施例,进一步阐述本发明。应当理解,这些实施例仅用于说明本发明而不用于限制本发明要求保护的范围。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.
实施例1 藻种的保存与培养:Preservation and cultivation of embodiment 1 algal species:
A).藻种的来源:工艺所选蓝藻藻种为鱼腥藻7120,购自中科院水生生物研究所,编号为FACHB-418。A). The source of the algae species: the cyanobacterium species selected in the process is Anabaena 7120, which was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences, and the number is FACHB-418.
B).鱼腥藻7120藻种的保存:藻种保存在琼脂浓度为1.5%的固体BGII(+N)培养基平板上。保存条件为恒温30℃,持续30μmol photon m–2s–1(Model MQ-100)光照强度以及空气环境中,如图5所示。B). Preservation of Anabaena 7120 algae species: the algae species were preserved on solid BGII(+N) medium plates with an agar concentration of 1.5%. The storage conditions were a constant temperature of 30°C, continuous light intensity of 30 μmol photon m –2 s –1 (Model MQ-100) and air environment, as shown in Figure 5.
C).培养基的配置:(1)所用培养基:BGII(-N)及BGII(+N)培养基,其成分如下:C). The configuration of the medium: (1) The medium used: BGII(-N) and BGII(+N) medium, the composition of which is as follows:
[A]BGII(-N):[A]BGII(-N):
StockⅠ(1000mL):NaCl—20.6g;K2HPO4·3H2O—0.8g;Na2CO3—0.4g;StockⅠ(1000mL): NaCl—20.6g; K 2 HPO 4 ·3H 2 O—0.8g; Na 2 CO 3 —0.4g;
StockⅡ(100mL):柠檬酸—0.3g;柠檬酸铁铵—0.3g;EDTA-二钠—0.005gStockⅡ(100mL): citric acid—0.3g; ferric ammonium citrate—0.3g; EDTA-disodium—0.005g
StockⅢ(100mL):CaCl2—1.36g;StockⅢ(100mL): CaCl 2 —1.36g;
StockⅣ(100mL):MgSO4·7H2O—3.75gStockⅣ(100mL): MgSO 4 7H 2 O—3.75g
StockⅤ(1000mL):H3BO3—2.86g;MnCl2.4H2O—1.81g;Stock Ⅴ (1000mL): H 3 BO 3 —2.86g; MnCl 2 .4H 2 O—1.81g;
ZnSO4.7H2O—3.75g;Na2MoO4.2H2O—0.391g;ZnSO 4 .7H 2 O—3.75g; Na 2 MoO 4 .2H2O—0.391g;
CuSO4.H2O—0.0079g;Co(NO3)2.6H2O—0.0494g。CuSO 4 .H 2 O—0.0079g; Co(NO 3 ) 2 .6H 2 O—0.0494g.
[B]BGII(+N):[B]BGII(+N):
StockⅠ(1000mL):NaCl—30.0g;K2HPO4·3H2O—0.8g;Na2CO3—0.4g;StockⅠ(1000mL): NaCl—30.0g; K 2 HPO 4 ·3H2O—0.8g; Na 2 CO 3 —0.4g;
StockⅡ(100mL):柠檬酸—0.3g;柠檬酸铁铵—0.3g;EDTA-二钠—0.005gStockⅡ(100mL): citric acid—0.3g; ferric ammonium citrate—0.3g; EDTA-disodium—0.005g
StockⅢ(100mL):CaCl2—1.36g;StockⅢ(100mL): CaCl 2 —1.36g;
StockⅣ(100mL):MgSO4·7H2O—3.75gStockⅣ(100mL): MgSO 4 7H 2 O—3.75g
StockⅤ(1000mL):H3BO3—2.86g;MnCl2.4H2O—1.81g;Stock Ⅴ (1000mL): H 3 BO 3 —2.86g; MnCl 2 .4H 2 O—1.81g;
ZnSO4.7H2O—3.75g;Na2MoO4.2H2O—0.391g;ZnSO 4 .7H 2 O—3.75g; Na 2 MoO 4 .2H2O—0.391g;
CuSO4.H2O—0.0079g;Co(NO3)2.6H2O—0.0494g。CuSO 4 .H 2 O—0.0079g; Co(NO 3 ) 2 .6H 2 O—0.0494g.
具体配置方法如下:在约800mL双蒸水中一次加入50mL StockⅠ、2mL StockⅡ、2mLStockⅢ、2mL StockⅣ、1mL StockⅤ以及0.61g Tris碱,定容至1000mL,调pH到8.0,然后高温高压灭菌,冷却后备用。The specific configuration method is as follows: Add 50mL Stock Ⅰ, 2mL Stock Ⅱ, 2mL Stock Ⅲ, 2mL Stock Ⅳ, 1mL Stock Ⅴ and 0.61g Tris base to about 800mL double-distilled water at one time, set the volume to 1000mL, adjust the pH to 8.0, then sterilize under high temperature and high pressure, and cool for backup use.
D).鱼腥藻7120培养:在如图2所示的通气试管中装入一定体积的BGII(-N)培养基,接入一定量的藻种,将新鲜藻液置于30℃温度和30μmol photon m–2s–1(Model MQ-100)光照强度下,并通以CO2:Air=2:98(v:v)混合气体进行培养。D). Cultivation of Anabaena 7120: Put a certain volume of BGII(-N) medium into the ventilated test tube shown in Figure 2, insert a certain amount of algae species, and place the fresh algae liquid at a temperature of 30°C and Under the light intensity of 30μmol photon m –2 s –1 (Model MQ-100), the culture was carried out with the mixed gas of CO 2 :Air=2:98(v:v).
实施例2 鱼腥藻7120产氢的诱导Example 2 Induction of hydrogen production by
A).诱导:取上述培养条件下生长至对数期的藻液,转接入含有一定体积BGII(-N)培养基的通气试剂瓶中,如图3所示,将叶绿素浓度定到0.15μg/mL,然后将藻液置于30℃温度和30μmol photon m–2s–1(Model MQ-100)光照强度下,并通以CO2:Air=2:98(v:v)混合气体进行培养;A). Induction: Take the algae liquid grown to the logarithmic phase under the above-mentioned culture conditions, transfer it into an aeration reagent bottle containing a certain volume of BGII(-N) medium, as shown in Figure 3, set the chlorophyll concentration to 0.15 μg/mL, and then put the algae solution under the temperature of 30℃ and the light intensity of 30μmol photon m –2 s –1 (Model MQ-100), and pass through the mixed gas of CO 2 :Air=2:98(v:v) to cultivate;
B).分装:72h的连续诱导培养之后,将藻液分装于60mL规格的玻璃样品管中,每管按40mL藻液的量进行分装;B). Dispensing: After 72 hours of continuous induction culture, the algae solution is divided into 60mL glass sample tubes, and each tube is divided into 40mL algae solution;
D).厌氧诱导:用纯度为(99.99%)的纯氩气将管内剩余空间的空气全部排尽,以确保管内气体环境为厌氧状态。将处理好的样品管置于30℃温度和100μmol photon m–2s–1(ModelMQ-100)光照强度下诱导藻细胞光合产氢。D). Anaerobic induction: Use pure (99.99%) pure argon to exhaust all the air in the remaining space in the tube to ensure that the gas environment in the tube is in an anaerobic state. The treated sample tubes were placed at a temperature of 30°C and a light intensity of 100 μmol photon m –2 s –1 (ModelMQ-100) to induce photosynthetic hydrogen production in algal cells.
实施例3 产氢速率的测定:Embodiment 3 The mensuration of hydrogen production rate:
A).氢气含量的检测:以实例3中所描述的处理条件持续特定时间后,用微量进样器从样品管上部空间取500μL的气体样品,使用山东鲁南厂生产的SP6890型高压气相色谱仪进行检测和分析;A). Detection of hydrogen content: after the processing conditions described in Example 3 continued for a specific period of time, a 500 μL gas sample was taken from the upper space of the sample tube with a micro-sampler, and the SP6890 high-pressure gas chromatograph produced by Shandong Lunan Factory was used. instrument for detection and analysis;
B).产氢速率的定量:使用外标法绘制氢气含量与峰面积之间的标准曲线,如图4所示。使用该标准曲线将所测得样品峰面积数值换算为氢气的物质的量,然后在样品叶绿素浓度以及产氢积累时间的基础上最终换算出鱼腥藻7120的产氢速率,氢气产率达到约17mL/mg。B). Quantification of hydrogen production rate: use the external standard method to draw a standard curve between hydrogen content and peak area, as shown in Figure 4. Use this standard curve to convert the measured sample peak area value into the amount of hydrogen gas, and then finally convert the hydrogen production rate of
实施例4 蓝藻生物质厌氧降解产沼气Example 4 Anaerobic degradation of cyanobacterial biomass to produce biogas
将结束光合产氢过程的藻细胞进行收集,取鲜重15g左右的藻泥(包含约0.3g干重的蓝藻生物质),加入10g左右的种泥,加入一定量的蒸馏水和PBS缓冲液。密闭后置于37℃水浴中进行持续发酵降解产沼气过程。Collect the algae cells that have completed the photosynthetic hydrogen production process, take about 15g of algae mud (including about 0.3g of dry weight of cyanobacterial biomass), add about 10g of seed mud, and add a certain amount of distilled water and PBS buffer. After being airtight, place it in a 37°C water bath for continuous fermentation and degradation to produce biogas.
实施例5 沼气组分的分析The analysis of embodiment 5 biogas components
沼气主要成分的检测和定量基本过称为:使用瑞典自动检测系统来监测发酵过程的产气体积,并用高效气相色谱对其他成分进行测定,以含有已知量的混合标准气体来对所测定的气体样品进行定性和定量的分析。The detection and quantification of the main components of biogas is basically called: use the Swedish automatic detection system to monitor the gas production volume of the fermentation process, and use high-performance gas chromatography to measure other components, and use a mixed standard gas containing a known amount to compare the measured Gas samples are analyzed qualitatively and quantitatively.
实施例6Example 6
一种可提高蓝藻太阳能转化效率的方法的步骤如下:A kind of method step that can improve cyanobacteria solar energy conversion efficiency is as follows:
1)对鱼腥藻7120的培养条件是:以BGII(-N)作为培养基进行液体培养,藻液置于30℃的恒温和30μmol photon m–2s–1的持续24小时的光照强度下,并通以CO2:Air=2:98(v:v)混合气体;菌种鱼腥藻7120采用持续继代的方保存在琼脂浓度为1.5%的固体BGII(+N)培养基平板上,保存条件为恒温30℃,持续30μmol photon m–2s–1光照强度以及空气环境中;1) The culture conditions for
2)将藻液分装到厌氧诱导试管中,加入光合放氧抑制剂,然后持续向藻液中通以高纯氩气,达到厌氧环境,密闭试管,将处理好的样品管置于30℃温度和100μmol photon m–2s–1(Model MQ-100)光照强度下诱导藻细胞光合产氢过程;2) Divide the algae liquid into anaerobic induction test tubes, add photosynthetic oxygen evolution inhibitors, and then continuously pass high-purity argon gas into the algae liquid to achieve anaerobic environment, seal the test tubes, and place the treated sample tubes in Under the temperature of 30℃ and the light intensity of 100μmol photon m –2 s –1 (Model MQ-100), the photosynthetic hydrogen production process of algae cells was induced;
3)将结束光合产氢过程的藻细胞进行收集,取鲜重15g左右的藻泥(包含约0.3g干重的蓝藻生物质),加入10g左右的种泥,加入一定量的蒸馏水和PBS缓冲液。密闭后置于37℃水浴中进行持续发酵降解产沼气过程。3) Collect the algae cells that have completed the photosynthetic hydrogen production process, take about 15g of algae mud (including about 0.3g of dry weight of cyanobacterial biomass), add about 10g of seed mud, and add a certain amount of distilled water and PBS buffer liquid. After being airtight, place it in a 37°C water bath for continuous fermentation and degradation to produce biogas.
本发明通过对藻细胞光合作用放氧活性的调控,光合产氢能力被提高200倍左右,而结束产氢过程的蓝藻生物质在通过活性污泥的厌氧降解过程进一步转化为富含甲烷和氢气的清洁能源-沼气。光能转化效率是微藻生物质能源能否实现应用的根本决定因素。通过计算,整套工艺的光能转化效率超过3%,达到国际先进水平。In the present invention, by regulating the photosynthetic oxygen release activity of algal cells, the photosynthetic hydrogen production capacity is increased by about 200 times, and the cyanobacterial biomass that has completed the hydrogen production process is further converted into methane-rich and Hydrogen clean energy - biogas. Light energy conversion efficiency is the fundamental determinant of the application of microalgae biomass energy. Through calculation, the light energy conversion efficiency of the entire process exceeds 3%, reaching the international advanced level.
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