CN110511882A - A salt-tolerant strain with electricity-generating properties and its application in microbial fuel cells - Google Patents
A salt-tolerant strain with electricity-generating properties and its application in microbial fuel cells Download PDFInfo
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Abstract
Description
技术领域technical field
本发明属于生物技术领域,尤其是一种耐盐且具有产电特性的Shewanella algae菌株及其在微生物燃料电池中的应用。The invention belongs to the field of biotechnology, in particular to a salt-tolerant and electricity-producing Shewanella algae bacterial strain and its application in microbial fuel cells.
背景技术Background technique
微生物燃料电池(Microbial Fuel Cells,MFCs)是利用微生物的催化作用氧化有机物(包括废水中的污染物)直接将化学能转化成电能的新型装置。由于具有废水处理和产生电能的双重作用,它在清洁能源生产、废水资源化处理、生物传感器开发及环境生物修复等领域表现出巨大的应用前景。Microbial Fuel Cells (MFCs) is a new type of device that uses the catalytic action of microorganisms to oxidize organic matter (including pollutants in wastewater) and directly convert chemical energy into electrical energy. Due to its dual functions of wastewater treatment and electricity generation, it shows great application prospects in the fields of clean energy production, wastewater resource treatment, biosensor development, and environmental bioremediation.
产电微生物是微生物燃料电池系统的核心组成,其电化学活性由于菌种产生电子和传递电子的机制不同而表现出明显的差异。目前大部分产电微生物的来源为污水处理厂的活性污泥、沉积物、土壤和生物垃圾等。已发现的产电微生物主要集中在变形菌门(Proteobacteria)和厚壁菌门(Firmicutes)。变形菌门主要包括假单胞菌属(Pseudomonas)、希瓦氏菌属(Shewanella)、克雷伯氏菌属(Klebsiella)、地杆菌属(Geobacter)、苍白杆菌属(Ochrobactrum)、红育菌属(Rhodoferax)、嗜酸菌属(Acidiphilium)、气单胞菌属(Aeromonas)、柠檬酸杆菌属(Citrobacter)、弧形杆菌属(Arcobacter)和脱硫弧菌属(Desulfovibrio)等,厚壁菌门主要包括芽孢杆菌属(Bacillus)和梭菌属(Clostridium)等。尽管,近年来研究者开始从近海的淤泥样品和海域的潮间带表面沉积物中分离和筛选来源于海洋的产电微生物,但这些菌株在盐条件下的产电特性缺乏进一步的分析。Electrogenic microorganisms are the core components of microbial fuel cell systems, and their electrochemical activities show obvious differences due to the different mechanisms of producing and transferring electrons. At present, most of the sources of electricity-producing microorganisms are activated sludge, sediment, soil and biological waste in sewage treatment plants. The discovered electrogenic microorganisms are mainly concentrated in Proteobacteria and Firmicutes. Proteobacteria mainly include Pseudomonas, Shewanella, Klebsiella, Geobacter, Ochrobactrum, Rhodobacter Rhodoferax, Acidiphilium, Aeromonas, Citrobacter, Arcobacter, Desulfovibrio, etc., Firmicutes Phylum mainly includes Bacillus (Bacillus) and Clostridium (Clostridium) and so on. Although, in recent years, researchers have begun to isolate and screen electrogenic microorganisms from the ocean from offshore silt samples and intertidal surface sediments in the sea, but there is a lack of further analysis of the electrogenic characteristics of these strains under saline conditions.
海藻希瓦氏菌最早于1985年由Yuichi Kotati等人从红藻表面分离出来,当时被命名为OK-1。1990年,Simidu分析了OK-1的G+C含量和16S rRNA碱基序列特点后发现OK-1与希瓦氏菌属有较近的亲缘关系,但因OK-1与腐败希瓦氏菌(Shewanellaputrefaciens)的碱基序列间仍存在显著差异,所以将OK-1定义为一种新的希瓦氏菌,即海藻希瓦氏菌(Shewanella alga)。目前,海藻希瓦氏菌大多应用在发酵生产河豚毒素以及抑制对金属材料的腐蚀性能方面。该种内微生物的耐盐性能、产电特性及其在微生物燃料电池方面的应用在国内外尚未发现相关报道。Algae Shewanella was first isolated from the surface of red algae by Yuichi Kotati et al. in 1985, and was named OK-1 at that time. In 1990, Simidu analyzed the G+C content and 16S rRNA base sequence characteristics of OK-1 Later, it was found that OK-1 has a close relationship with Shewanella, but because there is still a significant difference in the base sequence between OK-1 and Shewanella putrefaciens, OK-1 is defined as a A new species of Shewanella, Shewanella alga. At present, Shewanella algae is mostly used in fermentative production of tetrodotoxin and inhibition of corrosion to metal materials. The salt-tolerance performance, electricity production characteristics and application in microbial fuel cells of the microorganisms in this species have not been reported at home and abroad.
通过检索,尚未发现与本发明专利申请相关的专利公开文献。Through searching, no patent publications related to the patent application of the present invention have been found.
发明内容Contents of the invention
本发明目的在于克服现有技术中目前耐盐产电微生物报道较少的不足之处,提供一种耐盐且具有产电特性的Shewanella algae菌株及其在微生物燃料电池中的应用,该菌株是一株耐盐的产电微生物。这是该种内微生物具有产电特性的首次报道,成果丰富了耐盐产电微生物的多样性,为微生物燃料电池在海水资源化处理方面的应用提供新的实验材料。The purpose of the present invention is to overcome the deficiencies in the prior art that current reports of salt-tolerant and electricity-producing microorganisms are few, and provide a kind of Shewanella algae bacterial strain that is salt-tolerant and has electricity-producing characteristics and its application in microbial fuel cells. A salt-tolerant electrogenic microorganism. This is the first report that this species of microorganism has electricity-generating properties. The results enrich the diversity of salt-tolerant electricity-generating microorganisms and provide new experimental materials for the application of microbial fuel cells in seawater resource treatment.
本发明解决其技术问题所采用的技术方案是:The technical solution adopted by the present invention to solve its technical problems is:
一种耐盐且具有产电特性的Shewanella algae菌株,名称为E-1,分类名称为:海藻希瓦氏菌Shewanella algae,保藏编号为:CGMCC No.17857,保藏日期:2019年5月27日,北京市朝阳区北辰西路1号院3号,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心。A salt-tolerant and electrogenic Shewanella algae strain, named E-1, taxonomic name: Shewanella algae, preservation number: CGMCC No.17857, preservation date: May 27, 2019 , No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, preservation unit: General Microbiology Center of China Committee for the Collection of Microbial Cultures.
而且,所述菌株的16S rDNA基因具有如序列SEQ ID No.1所示的核苷酸序列,序列长度为1450bp,在Genbank上的登录号为MK787267.1。Moreover, the 16S rDNA gene of the strain has a nucleotide sequence as shown in SEQ ID No. 1, the sequence length is 1450 bp, and the accession number on Genbank is MK787267.1.
而且,所述菌株筛选自南海的海水;通过对南海的海水经过简单的沉淀处理后进行富集、分离、纯化和筛选得到。Moreover, the strain is selected from the seawater of the South China Sea; it is obtained by enriching, separating, purifying and screening the seawater of the South China Sea after simple sedimentation treatment.
而且,所述菌株为兼性厌氧菌、短杆状、长2.5um、宽0.5um、革兰氏阴性;适宜生长的pH范围为5~10、温度范围25~40℃,能够在NaCl质量浓度为0~7%的范围内生长。Moreover, the strain is a facultative anaerobic bacterium, short rod-shaped, 2.5um long, 0.5um wide, and Gram-negative; the pH range suitable for growth is 5-10, and the temperature range is 25-40°C, and it can grow in the presence of NaCl mass. The concentration ranges from 0 to 7%.
而且,所述菌株在盐胁迫下具有生长性能;所述菌株具有产电性能。Moreover, the strain has growth performance under salt stress; the strain has electricity production performance.
而且,所述菌株在含盐的条件下具有产电性能。Moreover, the strain has electrogenic properties under saline conditions.
而且,所述菌株能够利用乳酸、乙酸、琥珀酰胺酸、L-丙氨酸、尿苷,不能利用L-阿拉伯糖、麦芽糖、蔗糖、甘油。Furthermore, the strain can utilize lactic acid, acetic acid, succinamic acid, L-alanine, and uridine, but cannot utilize L-arabinose, maltose, sucrose, and glycerin.
如上所述的耐盐且具有产电特性的Shewanella algae菌株在微生物燃料电池进行产电方面中的应用。Application of the above-mentioned salt-tolerant and electricity-generating Shewanella algae strain in microbial fuel cells for electricity generation.
如上所述的耐盐且具有产电特性的Shewanella algae菌株在制作微生物燃料电池方面中的应用。The application of the above-mentioned salt-tolerant and electricity-generating Shewanella algae strains in the production of microbial fuel cells.
而且,应用方法的步骤为:And, the steps to apply the method are:
⑴菌种活化:从LB斜面培养基上挑取一环Shewanella algae菌株接种到50mL的LB液体培养基中,30-37℃、160-200r/min下振荡培养20-48h,取菌液6000×g离心10min后,收集菌体并用质量浓度为0.9%的生理盐水洗涤3次;⑴ Strain activation: Pick a ring of Shewanella algae strain from LB slant medium and inoculate it into 50mL LB liquid medium, shake and culture at 30-37℃, 160-200r/min for 20-48h, and take the bacterial solution 6000× After centrifugation at g for 10 min, the bacteria were collected and washed 3 times with 0.9% normal saline;
⑵接种液制备:将菌体重悬于含有不同NaCl浓度的阳极液中,以0.8-2.0g/L葡萄糖作为底物,使其初始OD600为0.8-1.5,然后接种到MFCs的反应器中;(2) Preparation of inoculum: resuspend the bacteria in anolyte containing different NaCl concentrations, use 0.8-2.0g/L glucose as the substrate, make the initial OD 600 0.8-1.5, and then inoculate into the reactor of MFCs;
⑶产电性能检测:反应器分别和外电阻以及数据采集装置连接好,在37±1℃恒温条件下运行,运行过程中定期更换接种液;待出现连续两个稳定电压时即认为启动成功;当监测的输出电压低于50mV时,开始更换阳极液,至获得稳定可重复的输出电压。(3) Electricity production performance test: The reactor is connected to the external resistance and the data acquisition device respectively, and operated at a constant temperature of 37±1°C, and the inoculation solution is regularly replaced during the operation; when two consecutive stable voltages appear, the start-up is considered successful; When the monitored output voltage is lower than 50mV, start to replace the anolyte until a stable and repeatable output voltage is obtained.
本发明取得的优点和积极效果为:Advantage and positive effect that the present invention obtains are:
1、本发明菌株适宜生长的温度范围为25~40℃,pH范围为5~10,同时可在NaCl浓度0~7%范围内生长,表现出良好的耐盐能力。将其接种于不添加NaCl和添加NaCl的微生物燃料电池中均能产生电能,说明它是一株耐盐的产电微生物。这是该种内微生物具有产电特性的首次报道,成果丰富了耐盐产电微生物的多样性,为微生物燃料电池在海水资源化处理方面的应用提供新的实验材料。1. The temperature range suitable for the growth of the strain of the present invention is 25-40° C., the pH range is 5-10, and it can grow in the NaCl concentration range of 0-7%, showing good salt tolerance. Inoculation of it in microbial fuel cells without adding NaCl and adding NaCl can generate electricity, indicating that it is a salt-tolerant electricity-producing microorganism. This is the first report that this species of microorganism has electricity-generating properties. The results enrich the diversity of salt-tolerant electricity-generating microorganisms and provide new experimental materials for the application of microbial fuel cells in seawater resource treatment.
2、本发明来自海洋的耐盐产电菌,该菌株分别接种到不添加NaCl及添加6.6%NaCl的微生物燃料电池中均能表现出产电能力,功率密度分别达到51.69mW·m-2和26.56mW·m-2。其适宜生长的温度范围为25~40℃,pH范围为5~10,表现出多样的底物利用能力,这是首次对S.algae种内微生物产电性能及其在微生物燃料电池中应用的报道,为微生物燃料电池在海水资源化处理方面的应用提供新的实验材料。2. The salt-tolerant electrogenic bacteria from the ocean of the present invention can exhibit electricity production capacity when inoculated into microbial fuel cells without adding NaCl and adding 6.6% NaCl, and the power densities reach 51.69mW·m -2 and 26.56 mW·m -2 . The temperature range suitable for its growth is 25-40°C, the pH range is 5-10, and it shows a variety of substrate utilization capabilities. The report provides new experimental materials for the application of microbial fuel cells in seawater resource treatment.
附图说明Description of drawings
图1为本发明菌株Shewanella algae E-1在无外源添加盐压力的条件下运行MFCs产生的电压变化曲线(a)、极化曲线和功率密度曲线(b);Fig. 1 is the voltage change curve (a), polarization curve and power density curve (b) that bacterial strain Shewanella algae E-1 of the present invention operates MFCs to produce under the condition of no exogenous added salt pressure;
图2为本发明菌株Shewanella algae E-1在不同盐浓度下的生长曲线;Fig. 2 is the growth curve of bacterial strain Shewanella algae E-1 of the present invention under different salt concentrations;
图3为本发明菌株Shewanella algae E-1在外源添加盐压力6.6%的条件下运行MFCs产生的电压变化曲线(a)、极化曲线和功率密度曲线(b);Fig. 3 is the voltage change curve (a), polarization curve and power density curve (b) that bacterial strain Shewanella algae E-1 of the present invention operates MFCs under the condition of exogenous added salt pressure 6.6%;
图4为本发明菌株Shewanella algae E-1的菌落形态图(a,b);革兰氏染色结果(c);菌体扫描电镜图(d);Fig. 4 is the colony morphology diagram (a, b) of bacterial strain Shewanella algae E-1 of the present invention; Gram staining result (c); Bacteria scanning electron microscope diagram (d);
图5为本发明菌株Shewanella algae E-1与GenBank中收录的相近菌株的16SrDNA序列进行同源性比对所构建的系统发育树。Fig. 5 is a phylogenetic tree constructed by homology comparison between the strain Shewanella algae E-1 of the present invention and the 16S rDNA sequences of similar strains recorded in GenBank.
具体实施方式Detailed ways
下面详细叙述本发明的实施例,需要说明的是,本实施例是叙述性的,不是限定性的,不能以此限定本发明的保护范围。The embodiments of the present invention will be described in detail below. It should be noted that the embodiments are illustrative, not restrictive, and cannot limit the protection scope of the present invention.
本发明中所使用的原料,如无特殊说明,均为常规的市售产品;本发明中所使用的方法,如无特殊说明,均为本领域的常规方法。The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.
一种耐盐且具有产电特性的Shewanella algae菌株,名称为E-1,分类名称为:海藻希瓦氏菌Shewanella algae,保藏编号为:CGMCC No.17857,保藏日期:2019年5月27日,北京市朝阳区北辰西路1号院3号,保藏单位:中国微生物菌种保藏管理委员会普通微生物中心。A salt-tolerant and electrogenic Shewanella algae strain, named E-1, taxonomic name: Shewanella algae, preservation number: CGMCC No.17857, preservation date: May 27, 2019 , No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, preservation unit: General Microbiology Center of China Committee for the Collection of Microbial Cultures.
较优地,所述菌株的16S rDNA基因具有如序列SEQ ID No.1所示的核苷酸序列,序列长度为1450bp,在Genbank上的登录号为MK787267.1。Preferably, the 16S rDNA gene of the strain has a nucleotide sequence as shown in SEQ ID No.1, the sequence length is 1450bp, and the accession number on Genbank is MK787267.1.
较优地,所述菌株筛选自南海的海水;通过对南海的海水经过简单的沉淀处理后进行富集、分离、纯化和筛选得到。Preferably, the strain is selected from the seawater of the South China Sea; it is obtained by enriching, separating, purifying and screening the seawater of the South China Sea after simple sedimentation treatment.
较优地,所述菌株为兼性厌氧菌、短杆状、长2.5um、宽0.5um、革兰氏阴性;适宜生长的pH范围为5~10、温度范围25~40℃,能够在NaCl质量浓度为0~7%的范围内生长。该菌株可以作为微生物燃料电池的阳极催化剂,进行催化底物产电,从而实现了本发明的目的。Preferably, the strain is a facultative anaerobic bacteria, short rod-shaped, 2.5um in length, 0.5um in width, and Gram-negative; the pH range suitable for growth is 5-10, and the temperature range is 25-40°C, and can be grown in The NaCl mass concentration grows within the range of 0-7%. The bacterial strain can be used as an anode catalyst of a microbial fuel cell to catalyze substrate electricity generation, thereby realizing the purpose of the present invention.
较优地,所述菌株在盐胁迫下具有良好的生长性能;所述菌株具有良好的产电性能。Preferably, the bacterial strain has good growth performance under salt stress; the bacterial strain has good electricity production performance.
较优地,所述菌株在含盐的条件下具有良好的产电性能。Preferably, the strain has good electricity production performance under saline conditions.
较优地,所述菌株能够利用乳酸、乙酸、琥珀酰胺酸、L-丙氨酸、尿苷,不能利用L-阿拉伯糖、麦芽糖、蔗糖、甘油。Preferably, the strain can utilize lactic acid, acetic acid, succinamic acid, L-alanine, and uridine, but cannot utilize L-arabinose, maltose, sucrose, and glycerin.
如上所述的耐盐且具有产电特性的Shewanella algae菌株可以应用在微生物燃料电池进行产电方面中。The above-mentioned salt-tolerant and electricity-generating Shewanella algae strains can be used in microbial fuel cells for electricity generation.
如上所述的耐盐且具有产电特性的Shewanella algae菌株可以应用在制作微生物燃料电池方面中。The above-mentioned salt-tolerant and electricity-generating Shewanella algae strains can be used in the production of microbial fuel cells.
较优地,应用方法的步骤为:Preferably, the steps of applying the method are:
⑴菌种活化:从LB斜面培养基上挑取一环Shewanella algae菌株接种到装有50mL的LB液体培养基中,30-37℃、160-200r/min下振荡培养20-48h,取菌液6000×g离心10min后,收集菌体并用质量浓度为0.9%的生理盐水洗涤3次;⑴ Strain activation: Pick a ring of Shewanella algae strain from LB slant culture medium and inoculate it into 50mL LB liquid medium, shake and culture at 30-37℃, 160-200r/min for 20-48h, and take the bacteria solution After centrifugation at 6000×g for 10 min, the cells were collected and washed 3 times with 0.9% normal saline;
⑵接种液制备:将菌体重悬于含有不同NaCl浓度的阳极液中,以0.8-2.0g/L葡萄糖作为底物,使其初始OD600为0.8-1.5,然后接种到MFCs的反应器中;(2) Preparation of inoculum: resuspend the bacteria in anolyte containing different NaCl concentrations, use 0.8-2.0g/L glucose as the substrate, make the initial OD 600 0.8-1.5, and then inoculate into the reactor of MFCs;
⑶产电性能检测:反应器分别和外电阻以及数据采集装置连接好,在37±1℃恒温条件下运行,运行过程中定期更换接种液;待出现连续两个稳定电压时即认为启动成功;当监测的输出电压低于50mV时,开始更换阳极液,至获得稳定可重复的输出电压。(3) Electricity production performance test: The reactor is connected to the external resistance and the data acquisition device respectively, and operated at a constant temperature of 37±1°C, and the inoculation solution is regularly replaced during the operation; when two consecutive stable voltages appear, the start-up is considered successful; When the monitored output voltage is lower than 50mV, start to replace the anolyte until a stable and repeatable output voltage is obtained.
所述海藻希瓦氏菌(Shewanella algae)E-1通过16SrDNA序列比对分析,与已知菌株Shewanella algae Hiro-1具有100%的同源性。该菌株16S rRNA的Genbank登录号为MK787267.1。The algae Shewanella algae (Shewanella algae) E-1 has 100% homology with the known strain Shewanella algae Hiro-1 through 16SrDNA sequence alignment analysis. The Genbank accession number of the strain 16S rRNA is MK787267.1.
海藻希瓦氏菌(Shewanella algae)E-1是一种革兰氏阴性、兼性厌氧的细菌。到目前为止,未有能产电的海藻希瓦氏菌的报道。所述海藻希瓦氏菌(Shewanella algae)E-1能在0~7%NaCl浓度下产电。Shewanella algae E-1 is a Gram-negative, facultative anaerobic bacterium. So far, no electricity-producing algal Shewanella has been reported. The seaweed Shewanella algae (Shewanella algae) E-1 can produce electricity at a concentration of 0-7% NaCl.
更具体地,所述海藻希瓦氏菌(Shewanella algae)E-1在制备微生物燃料电池中的应用,具体包括如下步骤:More specifically, the application of the seaweed Shewanella algae (Shewanella algae) E-1 in the preparation of microbial fuel cells specifically includes the following steps:
1)菌种活化:从LB斜面培养基上挑取一环菌体接种到装有50mL的LB液体培养基的250mL三角瓶中,30-37℃、160-200r/min下振荡培养20-48h,取菌液6000×g离心10min后收集菌体并用0.9%的生理盐水洗涤3次。1) Strain activation: Pick a ring of bacteria from the LB slant medium and inoculate it into a 250mL Erlenmeyer flask filled with 50mL of LB liquid medium, and culture it with shaking at 30-37°C and 160-200r/min for 20-48h , the bacteria liquid was collected by centrifugation at 6000×g for 10 min, and the bacteria were collected and washed three times with 0.9% normal saline.
接种液制备:将菌体重悬于含有不同NaCl浓度的阳极液中(以0.8-2.0g/L葡萄糖作为底物),使其初始OD600为0.8-1.5,然后接种到MFCs的反应器中。Preparation of inoculum: Resuspend the bacteria in anolyte containing different NaCl concentrations (with 0.8-2.0 g/L glucose as the substrate) so that the initial OD 600 is 0.8-1.5, and then inoculate them into the reactor of MFCs.
产电性能检测:反应器分别和外电阻以及数据采集装置连接好,在(37±1)℃恒温条件下运行,运行过程中定期更换接种液。待出现连续两个稳定电压时即认为启动成功。当监测的输出电压低于50mV时,开始更换阳极液。当获得稳定可重复的输出电压后测定功率密度和计算系统内阻等。Electricity production performance test: The reactor is connected to the external resistance and the data acquisition device respectively, and operates at a constant temperature of (37±1)°C, and the inoculation solution is replaced regularly during the operation. When two consecutive stable voltages appear, the startup is considered successful. When the monitored output voltage is lower than 50mV, start to replace the anolyte. When a stable and repeatable output voltage is obtained, the power density is measured and the internal resistance of the system is calculated.
具体地,本发明相关的步骤如下:Specifically, the relevant steps of the present invention are as follows:
一、MFCs阳极生物膜上微生物的分离纯化1. Separation and purification of microorganisms on MFCs anode biofilm
取自南海的海水经过简单的沉淀处理后接种于MFCs的阳极室,分别在阳极液含有质量浓度0%和6.6%NaCl的两种条件下连续运行。反应器主体为一个长为2.0cm,横断面直径为3.0cm的有机玻璃柱体,有效容积为14mL,阴阳极的有效面积均为7cm2。阳极和阴极分别置于MFCs主体的两测,并用橡胶圈密封固定。阳极一端用有机玻璃盖盖住,阴极一端的盖子是开口的,且阴极碳布载铂,阴极和阳极都与钛丝连接将电子导出或导入,最后整个装置用螺丝和丝杠拧紧固定。系统于37℃下分别连续运行125h和60h后,负载电阻两端的电压逐渐稳定,阳极上形成肉眼可见的厚的生物膜。将该生物膜上的全部菌体用接种针刮取,并悬浮于质量浓度0.9%的生理盐水中制备成均匀的菌悬液,将其进行梯度稀释后涂布于分离的LB固体培养基上,37℃恒温条件下培养2d,根据菌落形态、颜色、大小、表面和边缘特征,挑取特征差异明显的菌落,分别接种于LB液体培养基中再次富集培养,如此反复传代5~6次,得到纯培养的菌株,并对其进行编号和命名。Seawater from the South China Sea was inoculated into the anode chamber of MFCs after simple precipitation treatment, and operated continuously under two conditions of anolyte containing 0% and 6.6% NaCl in mass concentration. The main body of the reactor is a plexiglass cylinder with a length of 2.0 cm and a cross-sectional diameter of 3.0 cm. The effective volume is 14 mL, and the effective areas of the cathode and anode are both 7 cm 2 . The anode and cathode are respectively placed on the two sides of the MFCs main body, and sealed and fixed with rubber rings. One end of the anode is covered with a plexiglass cover, the cover of the cathode end is open, and the cathode carbon cloth is loaded with platinum. Both the cathode and the anode are connected with titanium wires to export or import electrons. Finally, the whole device is tightened and fixed with screws and leadscrews. After the system was continuously operated at 37°C for 125h and 60h, the voltage across the load resistor gradually stabilized, and a thick biofilm visible to the naked eye formed on the anode. All the bacteria on the biofilm were scraped with an inoculation needle, and suspended in 0.9% saline to prepare a uniform bacterial suspension, which was serially diluted and spread on the isolated LB solid medium , cultured at a constant temperature of 37°C for 2 days, according to the colony shape, color, size, surface and edge characteristics, pick colonies with obvious differences in characteristics, inoculate them in LB liquid medium for enrichment and culture again, and pass 5 to 6 times in this way , to obtain purely cultured strains, and to number and name them.
二、MFCs阳极生物膜上纯种微生物的产电性能分析2. Analysis of the electricity production performance of pure microorganisms on MFCs anode biofilm
以步骤一中分离和筛选得到的各个分离株为对象,将其分别接种到LB液体培养基中,37℃,200r/min下振荡培养24h,取菌液6000×g离心10min后收集菌体并用质量浓度0.9%的生理盐水洗涤3次。Take each isolate isolated and screened in step 1 as the object, inoculate them into LB liquid medium, shake and culture at 37°C and 200r/min for 24h, take the bacterial liquid and centrifuge at 6000×g for 10min, collect the bacterial cells and use Wash with 0.9% saline for 3 times.
将不同分离株的相同质量的菌体分别重悬于阳极液中(以1g/L葡萄糖作为底物),得到接种液(接种了对应分离株菌体的阳极液),使其初始OD600为1.0,然后接种到MFCs反应器中。反应器分别和外电阻(无特殊说明时外电阻均固定在1000Ω)和数据采集装置连接好,在(37±1)℃恒温条件下运行,运行过程中定期更换接种液。待出现连续两个稳定电压时即认为启动成功。启动成功后进入正式运行期,当监测的输出电压低于50mV时,只需要更换阳极液。当输出电压再次达到最高并获得稳定可重复的输出电压后进行电化学参数的测定,之后停止MFCs运行。整个过程中利用PISO-813型数据采集系统对电压进行实时的在线数据监测和记录,分析数据时取30min内的平均值,采样精度为0.001V。根据各个分离株的电压曲线,最终筛选到一株产电性能较好的分离株E-1。The bacteria of the same quality from different isolates were resuspended in the anolyte (using 1 g/L glucose as the substrate) to obtain the inoculum (the anolyte inoculated with the bacteria of the corresponding isolate), so that the initial OD600 was 1.0, and then inoculated into the MFCs reactor. The reactor was connected to the external resistance (the external resistance was fixed at 1000Ω unless otherwise specified) and the data acquisition device, and operated at a constant temperature of (37±1)°C, and the inoculum was replaced regularly during operation. When two consecutive stable voltages appear, the startup is considered successful. After successful startup, it enters the formal operation period. When the monitored output voltage is lower than 50mV, only the anolyte needs to be replaced. When the output voltage reached the highest again and a stable and repeatable output voltage was obtained, the electrochemical parameters were measured, and then the MFCs were stopped. During the whole process, the PISO-813 data acquisition system is used to monitor and record the real-time online data of the voltage. When analyzing the data, the average value within 30 minutes is taken, and the sampling accuracy is 0.001V. According to the voltage curves of each isolate, an isolate E-1 with better electricity production performance was finally screened.
三、菌株E-1在无外源添加盐压力下的产电性能3. Electricity production performance of strain E-1 under no exogenous added salt pressure
由图1可知,在无外源添加盐压力的条件下,分离株E-1接种到MFCs后运行415h后达到稳定,稳定电压约为554mV,功率密度在电流密度为192.14mA/m2时最大,达到51.69mW·m-2。It can be seen from Figure 1 that under the condition of no exogenous added salt pressure, the isolate E-1 reached a steady state after being inoculated into MFCs for 415 hours, and the stable voltage was about 554mV . , reaching 51.69mW·m -2 .
四、菌株E-1在盐压力下的生长性能Four. The growth performance of bacterial strain E-1 under salt pressure
将分离株E-1过夜培养后的菌液分别转接到含有质量浓度0%、2%、4%、6.6%NaCl的50mL的LB液体培养基中,并将其初始OD值调为一致。37℃,200r/min振荡培养,不同时间取样测定OD600并绘制生长曲线。The overnight culture of isolate E-1 was transferred to 50 mL of LB liquid medium containing 0%, 2%, 4%, and 6.6% NaCl, and the initial OD values were adjusted to be consistent. Culture at 37°C with shaking at 200r/min, take samples at different times to measure OD 600 and draw the growth curve.
由图2可知,在NaCl浓度为0%的条件下,菌株的延滞期为0~2.5h,对数期为2.5~16h,培养21h后OD600值达到最大,为1.66。随着NaCl浓度的增加,菌株的延滞期明显延长,达到稳定后的OD600值随之下降。当NaCl浓度为6.6%时,菌株的延滞期为0~6h,对数期为6~23h,培养25h后OD600值达到最大,为1.53。因此,上述数据表明,菌株E-1对盐具有较强的耐受性,在盐压力下生长状态良好。It can be seen from Figure 2 that under the condition of 0% NaCl concentration, the lag phase of the strain is 0-2.5h, the logarithmic phase is 2.5-16h, and the OD 600 value reaches the maximum after 21h of cultivation, which is 1.66. With the increase of NaCl concentration, the lag period of the strain was obviously extended, and the OD 600 value after reaching the stability decreased accordingly. When the NaCl concentration was 6.6%, the lag phase of the strain was 0-6 hours, and the logarithmic phase was 6-23 hours. The OD 600 value reached the maximum after 25 hours of cultivation, which was 1.53. Therefore, the above data show that the strain E-1 has strong tolerance to salt and grows well under salt pressure.
五、菌株E-1在盐压力下的产电性能5. Electricity production performance of strain E-1 under salt pressure
由图3可知,将分离株E-1接种到外源添加盐质量浓度6.6%的MFCs运行98h后达到稳定,稳定电压约为384mV,其功率密度在电流密度79.54mA/m2时最大,达到26.56mW·m-2。与无压力条件相比,NaCl添加后的系统达到稳定的时间缩短了76.4%。说明该菌株是一株耐盐的产电微生物,在海水资源化处理方面表现出巨大的应用潜力。It can be seen from Figure 3 that the isolate E-1 was inoculated into MFCs with an exogenous added salt concentration of 6.6% and then stabilized after 98 hours of operation. The stable voltage was about 384mV, and its power density was the largest when the current density was 79.54mA /m2, reaching 26.56mW·m -2 . The time to stabilize the system after NaCl addition was shortened by 76.4% compared with the unstressed condition. It shows that the strain is a salt-tolerant electrogenic microorganism, which shows great application potential in seawater resource treatment.
六、菌株E-1的生物学鉴定6. Biological Identification of Strain E-1
1.菌株的形态特征1. Morphological characteristics of the strain
(1)菌落形态特征:菌株E-1在LB固体培养基上,37℃恒温培养24-48h后,菌落呈圆形,为浅棕黄色,边缘整齐,表面透明光滑,呈油脂状,直径2mm,分泌某种褐色物质使培养基呈现浅棕黄色(见图4(a,b))(1) Morphological characteristics of the colony: After the strain E-1 was cultivated on LB solid medium at a constant temperature of 37°C for 24-48 hours, the colony was round, light brown, with neat edges, transparent and smooth surface, oily, and 2mm in diameter. , to secrete a certain brown substance to make the medium appear light brown (see Figure 4 (a, b))
(2)菌体形态特征:革兰氏染色的结果表明菌株E-1为革兰氏阴性菌。利用扫描电子显微镜观察菌株,菌体呈短杆状,长度约2.5μm,宽度约0.5μm(见图4(c,d))。(2) Morphological characteristics of bacteria: the results of Gram staining showed that strain E-1 was a Gram-negative bacterium. Observing the strain with a scanning electron microscope, the bacteria were short rod-shaped with a length of about 2.5 μm and a width of about 0.5 μm (see Figure 4(c, d)).
2.Biolog分析2. Biolog analysis
按照革兰氏阴性菌Biolog鉴定参数选择合适的培养条件。参考文献进行Biolog分析。具体方法为:将单菌落接种到BUA+B培养平板上,37℃培养24h,用无菌牙签挑取少量新鲜菌落制成菌悬液,和标准菌悬液进行对照,误差范围小于±2,用八道移液枪进行BiologGN II板接种,每孔接种菌悬液150μL,加盖30℃培养24h,打开微孔板盖放入结果自动读数仪,显色结果与Biolog数据库进行比对分析。Biolog鉴定结果有三个重要的参数需要考虑:可能性Probability(PROB),相似性Similarity(SIM)和位距Distance(DIST)。当DIST<5.0,SIM>0.75为良好的匹配;SIM值越接近于1,检定结果的可靠性越高。The appropriate culture conditions were selected according to the Gram-negative bacteria Biolog identification parameters. References were subjected to Biolog analysis. The specific method is: inoculate a single colony on the BUA+B culture plate, incubate at 37°C for 24 hours, pick a small amount of fresh colonies with a sterile toothpick to make a bacterial suspension, and compare it with the standard bacterial suspension, the error range is less than ±2, Use an eight-channel pipette gun to inoculate the BiologGN II plate, inoculate 150 μL of the bacterial suspension in each well, cover and incubate at 30°C for 24 hours, open the microplate cover and put it into the automatic result reader, and compare the color results with the Biolog database. There are three important parameters to consider in the Biolog identification results: Probability (PROB), Similarity (SIM) and Distance (DIST). When DIST<5.0, SIM>0.75 is a good match; the closer the SIM value is to 1, the higher the reliability of the test results.
由表1知,分离株E-1的SIM值=0.94>0.75;DIS值=0.89<5.0;PROB为100%。系统得到的三个重要参数比较理想,与数据库匹配良好,SIM值越接近1.00,说明鉴定结果的可靠性更高。在物种栏显示出一个最佳匹配名称:Shewanella algae。菌种鉴定出阳性反应33种,阴性反应50种,边界反应12种。阳性反应表示目标菌株与数据库的匹配程度>80%,阴性反应表示样品菌与数据库匹配程度<20%,边界反应多于15个为鉴定结果不好,实验中为12。各种数据指标都表明,鉴定结果准确,和数据库有很好的匹配,初步鉴定菌株E-1为海藻希瓦氏菌。Known from Table 1, the SIM value of isolate E-1=0.94>0.75; DIS value=0.89<5.0; PROB is 100%. The three important parameters obtained by the system are ideal and match well with the database. The closer the SIM value is to 1.00, the higher the reliability of the identification results. The species column shows a best matching name: Shewanella algae. There were 33 positive reactions, 50 negative reactions and 12 borderline reactions identified. A positive reaction indicates that the matching degree between the target strain and the database is >80%, a negative reaction indicates that the matching degree between the sample bacteria and the database is <20%, and more than 15 borderline reactions indicate a poor identification result, which was 12 in the experiment. Various data indicators show that the identification result is accurate and has a good match with the database, and the strain E-1 is preliminarily identified as Shewanella algae.
表1 Biolog微生物鉴定系统鉴定结果Table 1 Identification results of Biolog microbial identification system
3.菌株的16S rDNA测序分析3. 16S rDNA sequencing analysis of strains
采用细菌基因组DNA提取试剂盒,抽提菌株E-1的基因组DNA作为模版,细菌16SrDNA的通用引物(27F,1492R)作为引物,扩增菌株E-1的16S rDNA片段。该菌株的16S rDNA基因的序列长度为1450bp,将获得的16S rDNA基因序列输入GenBank,通过Blast程序对数据库中的所有序列进行比较分析。利用MEGA7.0软件,采用基于Kimura 2-parameter模型的邻接法(Neighbor-joining method)构建系统发育树,进化树分枝稳定性用Bootstrap分析,重复1000次。结果发现,该菌株的16S rDNA基因序列与希瓦氏菌属(Shewanella)的菌株Shewanella algae Hiro-1具有较高的相似性,同源性达到100%。由图5可知,分离株E-1与Shewanella algae Hiro-1处在系统发育树的同一分支,亲缘关系最为接近。The bacterial genomic DNA extraction kit was used to extract the genomic DNA of strain E-1 as a template, and the universal primers (27F, 1492R) of bacterial 16S rDNA were used as primers to amplify the 16S rDNA fragment of strain E-1. The sequence length of the 16S rDNA gene of this strain is 1450bp. The obtained 16S rDNA gene sequence was input into GenBank, and all the sequences in the database were compared and analyzed by the Blast program. Using MEGA7.0 software, the phylogenetic tree was constructed using the Neighbor-joining method based on the Kimura 2-parameter model, and the branch stability of the phylogenetic tree was analyzed with Bootstrap, repeated 1000 times. It was found that the 16S rDNA gene sequence of the strain has a high similarity with the strain Shewanella algae Hiro-1 of the genus Shewanella, and the homology reaches 100%. It can be seen from Figure 5 that the isolate E-1 and Shewanella algae Hiro-1 are in the same branch of the phylogenetic tree, and the genetic relationship is the closest.
经过菌株的形态观察、biolog分以及16S rDNA序列比对,菌株E-1鉴定为Shewanella algae。After strain observation, biolog score and 16S rDNA sequence alignment, strain E-1 was identified as Shewanella algae.
七、菌株E-1的碳源利用情况和生长条件特性7. Carbon source utilization and growth condition characteristics of strain E-1
根据代谢微平板对照相应碳源,将菌株E-1分别涂布到含有不同碳源的基本无机盐固体培养基上,通过观察菌株是否生长分析其对碳源的利用程度。由表2可知,菌株能够利用乳酸、乙酸、琥珀酰胺酸、L-丙氨酸、尿苷等碳源,但不能利用L-阿拉伯糖、麦芽糖、蔗糖和甘油等碳源(表3)。此外,该菌株在不同温度和pH条件下的生长情况表明,适宜生长的温度范围为25~40℃,pH范围为5~10。According to the corresponding carbon source of the metabolic microplate, the strain E-1 was spread on the basic inorganic salt solid medium containing different carbon sources, and the utilization degree of the carbon source was analyzed by observing whether the strain grew. It can be seen from Table 2 that the strains can utilize carbon sources such as lactic acid, acetic acid, succinamic acid, L-alanine, and uridine, but cannot utilize carbon sources such as L-arabinose, maltose, sucrose, and glycerol (Table 3). In addition, the growth of the strain under different temperature and pH conditions showed that the suitable temperature range for growth was 25-40° C., and the pH range was 5-10.
表2菌株E-1能够利用的碳源Table 2 The carbon source that strain E-1 can utilize
表3菌株E-1不能利用的碳源Table 3 The carbon source that strain E-1 cannot utilize
所述菌株的16S rDNA基因具有如序列SEQ ID No.1所示的核苷酸序列,序列长度为1450bp,在Genbank上的登录号为MK787267.1。The 16S rDNA gene of the strain has the nucleotide sequence shown in SEQ ID No.1, the sequence length is 1450bp, and the accession number on Genbank is MK787267.1.
SEQ ID No.1:SEQ ID No. 1:
CGCTTGCGCAGCTACACATGCAGTCGAGCGGTAACATTTCAAAAGCTTGCTTTTGAAGATGACGAGCGGCGGACGGGTGAGTAATGCCTGGGAATTTGCCCATTTGTGGGGGATAACAGTTGGAAACGACTGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGGACCTTCGGGCCTTGCGCTGATGGATAAGCCCAGGTGGGATTAGCTAGTAGGTGAGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAgGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGKTGTAAAGCACTTTCAGCGAGGAGGAAAGGGTGTAAGTTAATACCTTACATCTGTGACGtTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACCGCATTTCGAACTGGCAAACTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTCGGAGTTTGGTGTCTTGAACACTGGGCTCTCAAGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAAcCCTTACCTACTCTTGACATCCASAGAACTTKSCtAGAGATGSATYGGTGCCTTCGGGAACTSTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTACTTGCCAGCGGGTAATGcCCGGGAACTTTAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCGAAGCCGCGAGGTGGAGCTAATCCCATAAAGCCGGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTACCACGGTTGGTCTGCAT。CGCTTGCGCAGCTACACATGCAGTCGAGCGGTAACATTTCAAAAGCTTGCTTTTGAAGATGACGAGCGGCGGACGGGTGAGTAATGCCTGGGAATTTGCCCATTTGTGGGGGATAACAGTTGGAAACGACTGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGGACCTTCGGGCCTTGCGCTGATGGATAAGCCCAGGTGGGATTAGCTAGTAGGTGAGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAgGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGKTGTAAAGCACTTTCAGCGAGGAGGAAAGGGTGTAAGTTAATACCTTACATCTGTGACGtTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACCGCATTTCGAACTGGCAAACTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTCGGAGTTTGGTGTCTTGAACACTGGGCTCTCAAGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAAcCCTTACCTACTCTTGACATCCASAGAACTTKSCtAGAGATGSATYGGTGCCTTCGG GAACTSTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTACTTGCCAGCGGGTAATGcCCGGGAACTTTAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCGAAGCCGCGAGGTGGAGCTAATCCCATAAAGCCGGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTACCACGGTTGGTCTGCAT。
尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例和附图所公开的内容。Although the embodiments of the present invention are disclosed for the purpose of illustration, those skilled in the art will understand that various alternatives, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims, therefore However, the scope of the present invention is not limited to the contents disclosed in the embodiments and drawings.
序列表sequence listing
<110> 天津科技大学<110> Tianjin University of Science and Technology
<120> 一种耐盐且具有产电特性的菌株及其在微生物燃料电池中的应用<120> A salt-tolerant and electrogenic strain and its application in microbial fuel cells
<160> 1<160> 1
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 1450<211> 1450
<212> DNA/RNA<212> DNA/RNA
<213> 菌株的16S rDNA基因(Unknown)16S rDNA gene of <213> strain (Unknown)
<400> 1<400> 1
cgcttgcgca gctacacatg cagtcgagcg gtaacatttc aaaagcttgc ttttgaagat 60cgcttgcgca gctacacatg cagtcgagcg gtaacatttc aaaagcttgc ttttgaagat 60
gacgagcggc ggacgggtga gtaatgcctg ggaatttgcc catttgtggg ggataacagt 120gacgagcggc ggacgggtga gtaatgcctg ggaatttgcc catttgtggg ggataacagt 120
tggaaacgac tgctaatacc gcatacgccc tacgggggaa agcaggggac cttcgggcct 180tggaaacgac tgctaatacc gcatacgccc tacgggggaa agcaggggac cttcggggcct 180
tgcgctgatg gataagccca ggtgggatta gctagtaggt gaggtaaagg ctcacctagg 240tgcgctgatg gataagccca ggtgggatta gctagtaggt gaggtaaagg ctcacctagg 240
cgacgatccc tagctggtct gagaggatga tcagccacac tgggactgag acacggccca 300cgacgatccc tagctggtct gagaggatga tcagccacac tgggactgag acacggccca 300
gactcctacg ggaggcagca gtggggaata ttgcacaatg ggggaaaccc tgatgcaggc 360gactcctacg ggaggcagca gtggggaata ttgcacaatg ggggaaaccc tgatgcaggc 360
catgccgcgt gtgtgaagaa ggccttcggg ktgtaaagca ctttcagcga ggaggaaagg 420catgccgcgt gtgtgaagaa ggccttcggg ktgtaaagca ctttcagcga ggaggaaagg 420
gtgtaagtta ataccttaca tctgtgacgt tactcgcaga agaagcaccg gctaactccg 480gtgtaagtta ataccttaca tctgtgacgt tactcgcaga agaagcaccg gctaactccg 480
tgccagcagc cgcggtaata cggagggtgc gagcgttaat cggaattact gggcgtaaag 540tgccagcagc cgcggtaata cggagggtgc gagcgttaat cggaattact gggcgtaaag 540
cgtgcgcagg cggtttgtta agcgagatgt gaaagccccg ggctcaacct gggaaccgca 600cgtgcgcagg cggtttgtta agcgagatgt gaaagccccg ggctcaacct gggaaccgca 600
tttcgaactg gcaaactaga gtcttgtaga ggggggtaga attccaggtg tagcggtgaa 660tttcgaactg gcaaactaga gtcttgtaga ggggggtaga attccaggtg tagcggtgaa 660
atgcgtagag atctggagga ataccggtgg cgaaggcggc cccctggaca aagactgacg 720atgcgtagag atctggagga ataccggtgg cgaaggcggc cccctggaca aagactgacg 720
ctcaggcacg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa 780ctcaggcacg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa 780
acgatgtcta ctcggagttt ggtgtcttga acactgggct ctcaagctaa cgcattaagt 840acgatgtcta ctcggagttt ggtgtcttga acactgggct ctcaagctaa cgcattaagt 840
agaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca 900agaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca 900
caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa gaacccttac ctactcttga 960caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa gaacccttac ctactcttga 960
catccasaga acttksctag agatgsatyg gtgccttcgg gaactstgag acaggtgctg 1020catccasaga acttksctag agatgsatyg gtgccttcgg gaactstgag acaggtgctg 1020
catggctgtc gtcagctcgt gttgtgaaat gttgggttaa gtcccgcaac gagcgcaacc 1080catggctgtc gtcagctcgt gttgtgaaat gttgggttaa gtcccgcaac gagcgcaacc 1080
cctatcctta cttgccagcg ggtaatgccc gggaacttta gggagactgc cggtgataaa 1140cctatcctta cttgccagcg ggtaatgccc gggaacttta gggagactgc cggtgataaa 1140
ccggaggaag gtggggacga cgtcaagtca tcatggccct tacgagtagg gctacacacg 1200ccggaggaag gtggggacga cgtcaagtca tcatggccct tacgagtagg gctacacacg 1200
tgctacaatg gtcggtacag agggttgcga agccgcgagg tggagctaat cccataaagc 1260tgctacaatg gtcggtacag agggttgcga agccgcgagg tggagctaat cccataaagc 1260
cggtcgtagt ccggattgga gtctgcaact cgactccatg aagtcggaat cgctagtaat 1320cggtcgtagt ccggattgga gtctgcaact cgactccatg aagtcggaat cgctagtaat 1320
cgtggatcag aatgccacgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380cgtggatcag aatgccacgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380
catgggagtg ggctgcacca gaagtagata gcttaacctt cgggagggcg ttaccacggt 1440catgggagtg ggctgcacca gaagtagata gcttaacctt cggggagggcg ttaccacggt 1440
tggtctgcat 1450tggtctgcat 1450
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CN114686404A (en) * | 2022-04-29 | 2022-07-01 | 暨南大学 | Bacterial strain with iron reduction capacity and electricity generation capacity and application thereof |
CN114703104A (en) * | 2022-04-29 | 2022-07-05 | 暨南大学 | A bacterial strain with iron reducing ability and electrochemical activity and its application |
CN114703104B (en) * | 2022-04-29 | 2023-07-18 | 暨南大学 | A bacterial strain with iron reducing ability and electrochemical activity and its application |
CN114686404B (en) * | 2022-04-29 | 2023-07-18 | 暨南大学 | A bacterial strain with iron reduction ability and electricity production ability and its application |
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