CN100564512C - A kind of photosynthetic bacterium enriched substratum - Google Patents
A kind of photosynthetic bacterium enriched substratum Download PDFInfo
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- CN100564512C CN100564512C CNB2007100119970A CN200710011997A CN100564512C CN 100564512 C CN100564512 C CN 100564512C CN B2007100119970 A CNB2007100119970 A CN B2007100119970A CN 200710011997 A CN200710011997 A CN 200710011997A CN 100564512 C CN100564512 C CN 100564512C
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Abstract
Description
技术领域： Technical field:
本发明涉及一种富集细菌的培养基，特别涉及一种富集光合细菌的培养基。The invention relates to a culture medium for enriching bacteria, in particular to a culture medium for enriching photosynthetic bacteria.
背景技术： Background technique:
光合细菌是一大类能进行光合作用的原核生物的总称。研究应用的实践表明，光合细菌能利用多种硫化物和有机物作为其光合作用的供氢体和有机碳源，在高浓度的有机废水处理与资源化、水产养殖的水质调控与促进健康生长、在农业中作为高效活性菌肥等方面，发挥着十分有益的和令人瞩目的作用。Photosynthetic bacteria is a general term for a large group of prokaryotic organisms capable of photosynthesis. The practice of research and application shows that photosynthetic bacteria can use a variety of sulfides and organic matter as hydrogen donors and organic carbon sources for photosynthesis, and can be used in the treatment and recycling of high-concentration organic wastewater, water quality regulation and promotion of healthy growth in aquaculture, As a high-efficiency active bacterial fertilizer in agriculture, it plays a very beneficial and remarkable role.
光合细菌是一类不产氧的特殊生理类群的原核微生物的总称，是最早出现的具有原始光合成体系的原核微生物，在自然界的碳素、氮素、硫素转化循环中起重要作用。光合细菌营养丰富，细胞干物质中蛋白质含量高达60％以上，光合细菌还含有多种维生素，尤其是B族维生素极为丰富，VB12、叶酸、泛酸、生物素的含量远高于酵母。此外，光合细菌还含有大量的类胡萝卜素、辅酶Q等生理活性物质。因此，光合细菌具有很高的营养价值，在水产和畜禽养殖上有着很高的应用价值。Photosynthetic bacteria is a general term for a special physiological group of prokaryotic microorganisms that do not produce oxygen. They are the earliest prokaryotic microorganisms with a primitive photosynthetic system and play an important role in the transformation cycle of carbon, nitrogen and sulfur in nature. Photosynthetic bacteria are rich in nutrients, and the protein content in cell dry matter is as high as 60%. Photosynthetic bacteria also contain a variety of vitamins, especially B vitamins. The content of VB12, folic acid, pantothenic acid, and biotin is much higher than that of yeast. In addition, photosynthetic bacteria also contain a large amount of physiologically active substances such as carotenoids and coenzyme Q. Therefore, photosynthetic bacteria have high nutritional value and have high application value in aquaculture and livestock breeding.
光合细菌的推广和应用，关键在于需要合适的培养基质，但目前经典的用于富集光合细菌的常规培养基，主要配方为：氯化铵1g，碳酸氢钠1g，磷酸氢二钾0.2g，乙酸钠1～5g，七水硫酸镁0.2g，氯化钠0.5～2g，生长因子10ml，微量元素溶液10ml，蒸馏水980ml，(见《微生物学实验教程》)，其平均富集的时间为2～8周，周期长，且存在培养基成分种类多，成本较高的缺点。The key to the promotion and application of photosynthetic bacteria is the need for a suitable culture medium, but the current classic conventional medium for enriching photosynthetic bacteria, the main formula is: ammonium chloride 1g, sodium bicarbonate 1g, dipotassium hydrogen phosphate 0.2g , sodium acetate 1-5g, magnesium sulfate heptahydrate 0.2g, sodium chloride 0.5-2g, growth factor 10ml, trace element solution 10ml, distilled water 980ml, (see "Microbiology Experiment Tutorial"), the average enrichment time is 2 to 8 weeks, the cycle is long, and there are many types of medium components and the disadvantages of high cost.
发明内容： Invention content:
为了解决上述问题，本发明提供一种富集光合细菌的培养基，用本发明的培养基培养光合细菌，培养速度快，成本低。In order to solve the above-mentioned problems, the present invention provides a culture medium for enriching photosynthetic bacteria. The culture medium of the invention is used to cultivate photosynthetic bacteria, and the culture speed is fast and the cost is low.
本发明采用的技术方案是：一种富集光合细菌的培养基，其特征在于含有基础培养基、酵母膏和羟基丁二酸(苹果酸)；在基础培养基中，每1000ml基础培养基加入0.1g～3.0g的酵母膏和0.5g～2.0g的羟基丁二酸。The technical scheme adopted in the present invention is: a culture medium for enriching photosynthetic bacteria, characterized in that it contains basal medium, yeast extract and hydroxysuccinic acid (malic acid); in the basal medium, every 1000ml of basal medium is added 0.1g-3.0g of yeast extract and 0.5g-2.0g of hydroxysuccinic acid.
优选的，在基础培养基中，每1000ml基础培养基加入1.0g的酵母膏和1.5g的羟基丁二酸。Preferably, in the basal medium, 1.0 g of yeast extract and 1.5 g of hydroxysuccinic acid are added per 1000 ml of basal medium.
基础培养基是光合细菌的常用培养基。也就是现有技术中经常用的，并已公开的光合细菌培养基。Basal medium is a common medium for photosynthetic bacteria. That is the commonly used and disclosed photosynthetic bacteria culture medium in the prior art.
调节本发明培养基pH为7～9，121℃灭菌30min。将光合细菌接种于培养基中，振荡混匀，置于光照培养箱中光照培养，光照条件为1500～2000lux，温度为30±1℃。Adjust the pH of the culture medium of the present invention to 7-9, and sterilize at 121° C. for 30 minutes. Inoculate the photosynthetic bacteria into the culture medium, shake and mix well, and place them in a light incubator for light culture. The light conditions are 1500-2000 lux and the temperature is 30±1°C.
酵母膏对光合细菌的生长量的影响随着酵母膏的浓度的增加而增加，经过7天的光照培养后，当酵母膏的用量小于3g/L时(每1000ml基础培养基加入3.0g)，其菌体繁殖速度很快，菌液OD660达1.9，当酵母膏的用量大于3g/L时，其OD660值无明显变化，菌液OD660为2.0～2.1。因此，酵母膏的用量以0.1g/L～3.0g/L的酵母膏为宜。The influence of yeast extract on the growth of photosynthetic bacteria increases with the increase of the concentration of yeast extract. After 7 days of light culture, when the amount of yeast extract is less than 3g/L (add 3.0g per 1000ml basal medium), The bacterial growth rate is very fast, and the OD 660 of the bacterial liquid reaches 1.9. When the dosage of yeast extract is greater than 3g/L, the OD 660 value does not change significantly, and the OD 660 of the bacterial liquid is 2.0-2.1. Therefore, the dosage of yeast extract is preferably 0.1g/L-3.0g/L yeast extract.
羟基丁二酸对光合细菌的生长量的影响，随着羟基丁二酸的浓度的增加而增加，经过7天的光照培养后，当羟基丁二酸的用量小于2.0g/L(每1000ml基础培养基加入2.0g)时，其菌体繁殖速度很快，菌液OD660达1.7，当羟基丁二酸的用量大于2.0g/L时，其OD660值无明显变化，菌液OD660为1.8～1.9。因此，羟基丁二酸的用量以0.5g/L～2.0g/L为宜。The influence of hydroxysuccinic acid on the growth of photosynthetic bacteria increases with the increase of the concentration of hydroxysuccinic acid. After 7 days of light culture, when the amount of hydroxysuccinic acid is less than 2.0g/L (per 1000ml basis When adding 2.0g to the culture medium, its bacterium propagates very fast, and the OD 660 of the bacterium solution reaches 1.7. When the amount of hydroxysuccinic acid is greater than 2.0g/L, its OD 660 value has no obvious change, and the OD 660 of the bacterium solution is 1.8～1.9. Therefore, the dosage of hydroxysuccinic acid is preferably 0.5g/L-2.0g/L.
本发明的有益效果是：The beneficial effects of the present invention are:
1、富集培养速度快。由于在基础培养基中加入了酵母膏、羟基丁二酸等营养成分，为光合细菌创造了一个更好的厌氧环境，使光合细菌生长速度快，培养5天后，菌液颜色逐渐变深，由浅红转变为粉红最后变为深红，培养其OD660为1.7～2.0。1. Fast enrichment and culture. Because the nutrients such as yeast extract and hydroxysuccinic acid were added to the basic medium, a better anaerobic environment was created for photosynthetic bacteria, which made the photosynthetic bacteria grow faster. After 5 days of cultivation, the color of the bacterial solution gradually became darker. From light red to pink and finally to deep red, the OD 660 of the culture is 1.7-2.0.
2、光合细菌生长期延长，最大生长量提高。随着培养时间的增加，菌液OD660逐渐增加，培养4天时，其OD660为1.2～1.5，比使用基础培养基培养光合细菌时的OD660有所提高，培养8～12天时，菌液生长量达最高峰，其OD660为2.0～3.0。光合细菌的生长期延长了3～7天，对其繁殖更有促进作用。2. The growth period of photosynthetic bacteria is extended and the maximum growth rate is increased. With the increase of culture time, the OD 660 of the bacterial solution gradually increased. When cultured for 4 days, its OD 660 was 1.2-1.5, which was higher than the OD 660 when using basic medium to cultivate photosynthetic bacteria. When cultured for 8-12 days, the bacterial liquid The growth amount reaches the highest peak, and its OD 660 is 2.0-3.0. The growth period of photosynthetic bacteria is extended by 3 to 7 days, which promotes their reproduction more.
3、使用本发明的培养基培养光合细菌，pH值上升较常规基础培养基缓慢。光合细菌生长过程中，随着光合细菌的生长pH值不断升高，当基础培养基中pH值上升到9时，细菌生长受抑制。而本发明的培养基相对于基础培养基pH值上升缓慢，当pH值上升到9时，光合细菌的OD660值开始下降。主要是由于培养基的pH值影响了培养基中营养物质的离子化程度，从而影响微生物对营养物质的吸收，而且还会影响代谢反应中各种酶的活性。3. Using the culture medium of the present invention to cultivate photosynthetic bacteria, the pH value rises more slowly than conventional basic culture medium. During the growth of photosynthetic bacteria, with the growth of photosynthetic bacteria, the pH value continued to increase. When the pH value in the basal medium rose to 9, the growth of bacteria was inhibited. However, the pH value of the culture medium of the present invention rises slowly relative to the base culture medium, and when the pH value rises to 9, the OD 660 value of the photosynthetic bacteria begins to decline. The main reason is that the pH value of the medium affects the ionization degree of nutrients in the medium, which affects the absorption of nutrients by microorganisms, and also affects the activities of various enzymes in metabolic reactions.
4、成本低。在常规培养基中，由于加入了生长因子和微量元素溶液，使用成本太高，而本发明用酵母膏和羟基丁二酸替代了生长因子和微量元素溶液，使成本降低。4. Low cost. In the conventional culture medium, the use cost is too high due to the addition of growth factors and trace element solutions, but the present invention uses yeast extract and hydroxysuccinic acid to replace the growth factor and trace element solutions to reduce the cost.
5、用本发明培养基培养的光合细菌，用于净化和改善养殖水体。按光合细菌的30kg/hm2用量一次性洒入鱼塘，试验池水质比对照组有明显改善，其中溶解氧增加68％，COD下降21％，氨氮下降58.7％，硝酸氮下降29.4％，硫化物下降77.4％。5. The photosynthetic bacteria cultivated with the culture medium of the present invention are used to purify and improve aquaculture water. According to the amount of photosynthetic bacteria 30kg/hm 2 sprinkled into the fish pond at one time, the water quality of the test pond was significantly improved compared with the control group, in which the dissolved oxygen increased by 68%, the COD decreased by 21%, the ammonia nitrogen decreased by 58.7%, the nitrate nitrogen decreased by 29.4%. The goods dropped by 77.4%.
附图说明： Description of drawings:
图1是本发明与常规培养基，在培养基富集培养过程中，光密度OD660的变化图；Fig. 1 is the present invention and conventional culture medium, in the medium enrichment culture process, the change figure of optical density OD 660 ;
图2是本发明与常规培养基，在培养基富集培养过程中，pH变化图。Fig. 2 is a diagram of pH changes during the medium enrichment culture process of the present invention and conventional medium.
在图1和图2中，a代表本发明的培养基，b代表常规培养基In Fig. 1 and Fig. 2, a represents the culture medium of the present invention, and b represents conventional culture medium
具体实施方式： Detailed ways:
实施例1Example 1
(一)富集光合细菌培养基的配制(1) Preparation of Enriched Photosynthetic Bacteria Culture Medium
基础培养基：氯化铵1.0g，碳酸氢钠1.0g，磷酸氢二钾0.2g，乙酸钠3.0g，七水硫酸镁0.2g，氯化钠1.0g，用蒸馏水配成1000ml。Basal medium: 1.0g ammonium chloride, 1.0g sodium bicarbonate, 0.2g dipotassium hydrogen phosphate, 3.0g sodium acetate, 0.2g magnesium sulfate heptahydrate, 1.0g sodium chloride, made into 1000ml with distilled water.
在上述基础培养基中，加入0.1g酵母膏和0.5g羟基丁二酸。In the above basal medium, add 0.1 g of yeast extract and 0.5 g of hydroxysuccinic acid.
(二)光合细菌的培养(2) The cultivation of photosynthetic bacteria
将上述培养基的pH调至7，121℃灭菌30min。将光合细菌(市购)按照接种浓度为4％接种于培养基中，振荡混匀，置于光照培养箱中光照培养，光照条件为1500lux，温度30℃。Adjust the pH of the above medium to 7, and sterilize at 121°C for 30 minutes. Photosynthetic bacteria (commercially purchased) were inoculated in the culture medium at an inoculation concentration of 4%, oscillated and mixed, and placed in a light incubator for light culture under the light conditions of 1500 lux and temperature of 30°C.
富集培养结果：光照培养5天后，菌液OD660为1.3，光照培养8天后，达到最大生长量，菌液OD660为1.9，使光合细菌的对数生长期延长3天。Enrichment culture results: After 5 days of light culture, the OD 660 of the bacteria solution was 1.3, and after 8 days of light culture, the maximum growth amount was reached, and the OD 660 of the bacteria solution was 1.9, prolonging the logarithmic growth phase of photosynthetic bacteria for 3 days.
实施例2Example 2
(一)富集光合细菌培养基的配制(1) Preparation of Enriched Photosynthetic Bacteria Culture Medium
基础培养基：氯化铵1.0g，碳酸氢钠1.0g，磷酸氢二钾0.2g，乙酸钠3.0g，七水硫酸镁0.2g，氯化钠1.0g，用蒸馏水配成1000ml。Basal medium: 1.0g ammonium chloride, 1.0g sodium bicarbonate, 0.2g dipotassium hydrogen phosphate, 3.0g sodium acetate, 0.2g magnesium sulfate heptahydrate, 1.0g sodium chloride, made into 1000ml with distilled water.
在上述基础培养基中，加入3.0g酵母膏和2.0g羟基丁二酸。In the above basal medium, add 3.0 g of yeast extract and 2.0 g of hydroxysuccinic acid.
(二)光合细菌的培养(2) The cultivation of photosynthetic bacteria
将上述培养基的pH调至7，121℃灭菌30min。将光合细菌(市购)按照接种浓度为4％接种于培养基中，振荡混匀，置于光照培养箱中光照培养，光照条件为1500lux，温度30℃。Adjust the pH of the above medium to 7, and sterilize at 121°C for 30 minutes. Photosynthetic bacteria (commercially purchased) were inoculated in the culture medium at an inoculation concentration of 4%, oscillated and mixed, and placed in a light incubator for light culture under the light conditions of 1500 lux and temperature of 30°C.
富集培养结果：光照培养5天后，菌液OD660为1.4，光照培养10天后，达到最大生长量，菌液OD660为2.2，使光合细菌的对数生长期延长5天。Enrichment culture results: After 5 days of light culture, the OD 660 of the bacteria solution was 1.4, and after 10 days of light culture, the maximum growth amount was reached, and the OD 660 of the bacteria solution was 2.2, prolonging the logarithmic growth phase of photosynthetic bacteria for 5 days.
实施例3Example 3
(一)富集光合细菌培养基的配制(1) Preparation of Enriched Photosynthetic Bacteria Culture Medium
基础培养基：氯化铵1.0g，碳酸氢钠1.0g，磷酸氢二钾0.2g，乙酸钠3.0g，七水硫酸镁0.2g，氯化钠1.0g，用蒸馏水配成1000ml。Basal medium: 1.0g ammonium chloride, 1.0g sodium bicarbonate, 0.2g dipotassium hydrogen phosphate, 3.0g sodium acetate, 0.2g magnesium sulfate heptahydrate, 1.0g sodium chloride, made into 1000ml with distilled water.
在上述基础培养基中，加入1.0g酵母膏和1.5g羟基丁二酸。In the above basal medium, add 1.0 g of yeast extract and 1.5 g of hydroxysuccinic acid.
(二)光合细菌的培养(2) The cultivation of photosynthetic bacteria
将上述培养基的pH调至7，121℃灭菌30min。将光合细菌(市购)按照接种浓度为4％接种于培养基中，振荡混匀，置于光照培养箱中光照培养，光照条件为1500lux，温度30℃。Adjust the pH of the above medium to 7, and sterilize at 121°C for 30 minutes. Photosynthetic bacteria (commercially purchased) were inoculated in the culture medium at an inoculation concentration of 4%, oscillated and mixed, and placed in a light incubator for light culture under the light conditions of 1500 lux and temperature of 30°C.
富集培养结果：光照培养5天后，菌液OD660为1.8，光照培养12天后，达到最大生长量，菌液OD660为2.5，使光合细菌的对数生长期延长7天，最大生长量提高。Enrichment culture results: After 5 days of light culture, the OD 660 of the bacteria solution was 1.8, and after 12 days of light culture, the maximum growth was reached, and the OD 660 of the bacteria solution was 2.5, which prolongs the logarithmic growth phase of photosynthetic bacteria by 7 days and increases the maximum growth. .
用此培养基培养的光合细菌应用于养殖水的净化处理，养殖水COD、氨氮的去除率分别达到了61％、70.48％。The photosynthetic bacteria cultivated with this medium were applied to the purification treatment of aquaculture water, and the removal rates of COD and ammonia nitrogen in the aquaculture water reached 61% and 70.48%, respectively.
对比例comparative example
分别取实施例3本发明的培养基和背景技术中介绍的常规培养基，分别调节培养基的pH为7，121℃灭菌30min。将光合细菌(市购)按照接种浓度为4％接种于培养基中，振荡混匀，置于光照培养箱中光照培养，光照条件为1500lux，温度30℃。光照培养12天，测定培养基富集培养过程中光密度OD660变化(见图1)及pH变化(见图2)。Take the culture medium of the present invention in Example 3 and the conventional culture medium introduced in the background art respectively, adjust the pH of the culture medium to 7, and sterilize at 121° C. for 30 minutes. Photosynthetic bacteria (commercially purchased) were inoculated in the culture medium at an inoculation concentration of 4%, oscillated and mixed, and placed in a light incubator for light culture under the light conditions of 1500 lux and temperature of 30°C. The culture was cultured under light for 12 days, and the change of optical density OD 660 (see Figure 1) and pH change (see Figure 2) were measured during the medium enrichment culture process.
从图1可知，使用常规培养基培养光合细菌，3天后菌液开始转红，第5天时菌液OD660为1.5，5天后其OD660逐渐降低；使用本发明培养基培养光合细菌，随着培养时间的增加，菌液OD660逐渐增加，培养12天，其OD660为2.4，本发明的培养基使光合细菌的对数生长期延长7天，并且对其繁殖更有促进作用。As can be seen from Fig. 1, use conventional substratum to cultivate photosynthetic bacteria, bacterium liquid begins to turn red after 3 days, and in the 5th day, bacterium liquid OD 660 is 1.5 , and its OD after 5 days gradually reduces; Use substratum of the present invention to cultivate photosynthetic bacteria, along with With the increase of the culture time, the OD660 of the bacterial solution increases gradually, and after 12 days of cultivation, its OD660 is 2.4. The culture medium of the present invention prolongs the logarithmic growth phase of photosynthetic bacteria for 7 days, and has a more promoting effect on its reproduction.
由图2可知，随着光合细菌的生长pH值不断升高，当常规培养基中pH值上升到9时，细菌生长受抑制。使用本发明的培养基培养光合细菌相对于常规培养基pH值上升缓慢，使光合细菌的对数生长期延长。It can be seen from Figure 2 that as the pH value of photosynthetic bacteria increases continuously, when the pH value in the conventional medium rises to 9, the growth of bacteria is inhibited. Using the culture medium of the present invention to cultivate photosynthetic bacteria has a slower rise in pH value than conventional culture medium, prolonging the logarithmic growth phase of photosynthetic bacteria.
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