| Publication number | CN1392251 A |
| Publication type | Application |
| Application number | CN 02134387 |
| Publication date | Jan 22, 2003 |
| Filing date | Jul 18, 2002 |
| Priority date | Jul 18, 2002 |
| Also published as | CN1181194C |
| Publication number | 02134387.X, CN 02134387, CN 1392251 A, CN 1392251A, CN-A-1392251, CN02134387, CN02134387.X, CN1392251 A, CN1392251A |
| Inventors | 刘昕, 王江海, 袁建平, 黄晓霓 |
| Applicant | 中山大学 |
| Export Citation | BiBTeX, EndNote, RefMan |
| Referenced by (2), Classifications (2), Legal Events (4) | |
| External Links: SIPO, Espacenet | |
技术领域 FIELD
本发明涉及一种可调控的全天候封闭式大规模运行系统光生物反应器培养和促进雨生红球藻生长增殖及提高种群密度和调控细胞内次生代谢产物虾青素合成和积累的方法。 The present invention relates to a control system to run large-scale weather enclosed photobioreactor foster and promote pluvialis algae proliferation and increase population density and secondary metabolites in the regulation of cell astaxanthin synthesis and accumulation methods.
背景技术 BACKGROUND
雨生红球藻(Haematococcus pluvialis)是一种单细胞绿藻，细胞呈椭圆形或卵形，大小从5微米左右到20-30微米不等，有时甚至更大些；有两条等长鞭毛，鞭毛的摆动推动细胞自由游动；细胞壁和原生质间具空隙，其间充满胶状物质，细胞壁和原生质体间有细胞质连丝相连，营养繁殖为细胞分裂，形成2、4、8、16甚至更多个子细胞，无性生殖在环境不良时形成胶群体或厚壁孢子。 Pluvialis algae (Haematococcus pluvialis) is a single-cell green algae, elliptical or oval cell size from about 5 microns to 20-30 micron range, sometimes even bigger; there are two equal-length flagella flagellum swing to promote free-swimming cells; a gap between the wall and protoplasm, which is fraught with a gelatinous substance, between the cytoplasm of the cell wall and protoplast have even wire connected vegetative propagation of cell division, the formation of 2,4,8,16 even more multiple sub-cells, the formation of asexual spores thick glue groups or bad environment. 处于生长增殖阶段的雨生红球藻呈绿色；当环境条件的改变对其生长不利时，如在缺乏氮、磷和双价离子镁或在强光照射条件下，雨生红球藻细胞的生长将趋于缓慢并使运动细胞失去鞭毛，形成胶群体或厚壁孢子，同时，促进细胞内次生代谢产物合成，主要是虾青素的大量积累，使雨生红球藻细胞的颜色迅速形成砖红色。 In the proliferation phase pluvialis algae green; when changes in environmental conditions detrimental to their growth when, as in the absence of nitrogen, phosphorus and magnesium bivalent ions or in bright light conditions, pluvialis algal cells will tend to slow the growth and movement of cells lose their flagella, the formation of groups or thick-walled spores gum at the same time, promote the intracellular synthesis of secondary metabolites, mainly massive accumulation of astaxanthin, so pluvialis algal cells rapidly colors the formation of red brick. 影响雨生红球藻的生理、生物量、产量和细胞内次生代谢产物成分和生成量，主要涉及藻的品种和良好的培养条件(如环境的净化程度、温度、光照、溶解氧、营养液组成、流体切变力等)。 Pluvialis algae affect physiology, biomass, yield and generate the amount of intracellular components and secondary metabolites, mainly related to algal species and good culture conditions (such as the degree of purification of the environment, temperature, light, dissolved oxygen, nutrients fluid composition, fluid shear force, etc.). 通过物理或化学以至基因工程及遗传工程手段改造现有藻种获得优良工程菌株，提高藻种的光饱和值、抗光抑制，以及抗逆、抗污染能力，可以获得耐高温、细胞增殖快和虾青素积累能力强的藻种。 By physical or chemical transformation as well as genetic engineering and genetic engineering to get good engineering existing algae strains, increase light saturation value of algae, anti-light suppression, as well as resilience, anti-pollution, you can get high temperature, rapid cell proliferation and astaxanthin accumulation ability of the algae. 有研究表明，经过优化的光生物反应器及良好的培养条件，可促使雨生红球藻中虾青素的含量高达细胞干重的4-5％，而采用细菌或酵母菌等培养生成虾青素的含量最高不足细胞干重的0.8％。 Studies have shown that after a photobioreactor and good optimization of culture conditions, can contribute to the content of the rain pluvialis astaxanthin up to 4-5% of the dry weight of the cells, and the use of bacteria or yeast, such as generating shrimp culture astaxanthin content of the highest cell dry weight of less than 0.8%.
目前，阻碍雨生红球藻大规模生产的主要因素是生物污染和雨生红球藻培养过程种群密度低使单位面积产量低所导致的高成本。 The main factors that currently hinder pluvialis algae bio mass production of pollution and population density pluvialis algal culture process, low cost and low yield per unit area so caused. 小规模由管道式光生物反应器或小型固定化系统组成的生物反应器或气升式生物反应器进行培养的雨生红球藻，存在的缺陷是管道壁易被藻细胞粘连遮蔽导致培养期间透光性下降及清洗困难，虽然已采用不同类型的桨叶轮搅拌混合，以尽量降低藻细胞之间的遮蔽作用及提高光照的利用，但只适应实验室的小规模试验。 Bioreactor or airlift bioreactor small scale by pipeline or small photobioreactor system consisting of immobilized cultured rain pluvialis, defects during the pipe wall is easily obscured cause algal cell adhesion culture translucent drop and difficult to clean, although the use of different types of mixing paddle wheel to minimize the shielding effect between the algal cells and improve the utilization of light, but only accommodate small scale laboratory tests.
发明内容 SUMMARY
本发明的目的是提供一种可调控的全天候封闭式大规模运行系统光生物反应器培养和促进雨生红球藻细胞生长增殖及提高种群密度和调控细胞内次生代谢产物虾青素合成和积累的方法。 The purpose of the present invention is to provide an all-weather control large-scale operation of the system enclosed photobioreactor foster and promote pluvialis algal cell proliferation and increase population density and secondary metabolites in the regulation of cell synthesis and astaxanthin The method of accumulation.
本发明的方法包括建立可调控的全天候封闭式大规模运行系统光生物反应器，在主体反应器底部及四周和封盖采用透光材料及外置可调控的光照设施，调速桨叶轮搅拌器及卷水轮，无菌空气鼓气设施及O2气体系统，咸淡水营养液调配和温度调控系统，清洗和消毒杀菌系统，通过环境条件的改变进行生理诱导调控雨生红球藻生长增殖及提高种群密度和调控雨生红球藻细胞内次生代谢产物虾青素的合成和积累、分离收获藻细胞及低温干燥等工序。 The method of the present invention may include the establishment of a large-scale operation of control-weather enclosed photobioreactor system in the body and around the bottom of the reactor and capping using translucent materials and external adjustable lighting facilities, paddle wheel speed blender and volume water wheel, sterile air and blow facilities and O2 gas system, brackish water nutrient solution deployment and temperature control systems, cleaning and disinfection system for the regulation of physiological induction pluvialis algae by changing environmental conditions and to improve the growth and proliferation population density and regulation of endogenous rain pluvialis cells secondary metabolites astaxanthin synthesis and accumulation of algal cells harvested isolated and low-temperature drying processes.
本发明的方法具体步骤如下：一、建立全天候封闭式大规模运行系统光生物反应器：改造并优化通常工业培养微藻使用的浅水道光生物反应器成为封闭式光生物反应器，或设计光利用率高的全天候封闭式大规模运行系统光生物反应器，其中包括支撑式主体底座，设置封闭式可调控排放系统的主体反应器(水槽)，设置可调控的光照设施，设置调速桨叶轮搅拌器及卷水轮，设置有进排气口的无菌空气设施及O2气体系统，设置有回流装置的咸淡水营养液调配和温度调控系统，清洗和消毒杀菌系统及分离收获藻细胞和低温干燥设施。 The method of the present invention the following steps: First, the establishment of large-scale operation of the system clock enclosed photobioreactor: transform and optimize the shallow dynasty bioreactor cultured microalgae industry generally used to become a closed photobioreactor, or design light use Closed-weather running high scale photobioreactor system, including base-supporting body, set up a closed drainage system can regulate the body of the reactor (sink), setting adjustable lighting facilities, setting speed stirring paddle wheel Volume control and water wheels provided with sterile air intake and exhaust port facilities and O2 gas system, provided with a reflux device of brackish water nutrient solution deployment and temperature control systems, cleaning and disinfection systems and separate algal cells harvested and low-temperature drying facilities. 主体反应器呈长方形，长宽为200-1500cm×100-300cm，通常为220-500cm×110-150cm；深度为450-700cm，通常为550-650cm。 Main reactor rectangular, length and width of 200-1500cm × 100-300cm, usually 220-500cm × 110-150cm; depth of 450-700cm, usually 550-650cm. 亦可将全天候封闭式大规模运行系统光生物反应器包括咸淡水营养液调配、控光、控温、清洗、消毒、杀菌、搅拌、鼓气、分离收获藻细胞及干燥等工序由计算机控制实现长期半连续生产。 The weather can also be a large-scale operation of the system enclosed photobioreactor include brackish water nutrient solution deployment, controlled light, temperature, cleaning, disinfection, sterilization, mixing, drum gas, separation of algal cells harvested and drying processes implemented by a computer-controlled Long-term semi-continuous production.
二、在主体反应器底部及四周和封盖采用透光材料及外置可调控光照设施：透光材料可选用高抗张力及抗压力的无色透明钢化玻璃或特种玻璃，厚度为0.8-3cm，通常为1.5-2cm；封盖部分透光材料可选用市售透明塑料薄膜或复合材料透明薄膜，厚度为0.01-0.6mm，通常为0.03-0.3mm；透明薄膜用作主体反应器封盖并用胶带纸封贴或胶粘剂封贴；封盖部分透光材料也可选用有机玻璃等透光材料；在主体反应器四周及上下安装多盏可调亮度的照明用的日光灯、白炽灯、太阳灯或节能射灯，每盏灯功率为40-2000w，通常为40-300w；可单独使用或组合使用；雨生红球藻生长增殖及提高种群密度调控阶段光照强度为800-1800勒克斯，通常为1000-1500勒克斯；调控细胞内次生代谢产物虾青素合成和积累阶段光照强度为1900-3200勒克斯，通常为2000-2500勒克斯；光照强度和光照周期可根据自然日光光照强度及通过灯的数量和功率及光照时间进行调控。 Second, in the main reactor at the bottom and the sides and cover the use of translucent material and external adjustable lighting facilities: a translucent material could be high tensile strength and resistance to pressure of colorless transparent glass or special glass, thickness 0.8-3cm , typically 1.5-2cm; closure part translucent material transparent plastic film can be used a commercially available transparent film or composite material, having a thickness of 0.01-0.6mm, usually 0.03-0.3mm; transparent film used as the cover with the main body of the reactor paper tape or adhesive seal affixed seal affixed; cover partially transmissive material can also be used, such as translucent plexiglass material; installation of multi-lamp dimmable fluorescent lighting used in the main reactor around and up and down, incandescent lamp or the sun energy-saving lighting, each lamp power 40-2000w, usually 40-300w; can be used alone or in combination; pluvialis algae proliferation and increase the population density of the regulation stage 800-1800 lux light intensity, typically 1000 -1500 lux; in the regulation of cell secondary metabolites astaxanthin synthesis and accumulation phase for 1900-3200 lux light intensity, typically 2000-2500 lux; photoperiod and light intensity according to the amount of natural daylight illumination and by light and Power and light time regulation.
三、调速桨叶轮搅拌器及卷水轮：调速浆叶轮搅拌器可使主体反应器咸淡水营养液处于轻微流动状态并保持咸淡水营养液组成及溶解氧浓度和PH值的可调控性，使藻细胞的生长处于悬浮状态；雨生红球藻在合适的流体切变力作用下，如咸淡水营养液以0.01-0.08m/s流速循环，可有效减少藻细胞之间的遮蔽作用及提高光照的利用。 Third, the speed and volume water wheel paddle wheel blender: Speed pulp agitator impeller allows the main reactor in slightly brackish water nutrient solution flowing state and maintain brackish liquid nutrient composition and dissolved oxygen concentration and PH Regulatability that the growth of algal cells in suspension; pluvialis algae shear forces acting under a suitable fluid, such as brackish water nutrient solution to 0.01-0.08m / s velocity loop, which can effectively reduce the shadowing effect between algal cells and increase the use of lighting. 采用工业上使用的可调速桨叶轮搅拌器，转速为10-400r/m，通常为20-60r/m；卷水轮为工业上使用的卷水轮，转速为10-300r/m，通常为20-80r/m；卷水轮装置于主体反应器的一测，调控咸淡水营养液循环流动的流速。 Use can be used industrially speed paddle wheel agitator speed was 10-400r / m, usually 20-60r / m; volume waterwheel volume on the turbine for industrial use, speed of 10-300r / m, usually To 20-80r / m; roll waterwheel device in the body of a test reactor, regulation of salt and fresh water circulating nutrient solution flow rate.
四、设置有进排气口的无菌空气设施及O2气体系统：采用工业上使用的空压机并经常规的滤油滤水滤菌处理成为无菌空气；O2气体采用工业氧气瓶，并采用过滤法除菌。 Four provided with sterile air intake and exhaust port facilities and O2 gas system: The air compressor used in the industry and by the conventional oil filter filtration treatment become sterile air filter bacteria; O2 gas using oxygen tanks, and sterilized by filtration method. 通过调节阀及分布于主体反应器底部管道式微孔导流孔鼓入无菌空气及氧气。 By adjusting the valve and distributed in the body of the reactor bottom of the pipe microporous diversion hole blown into the sterile air and oxygen. 雨生红球藻生长增殖阶段无菌空气鼓入量为0.02-2m3/m，通常为0.03-0.2m3/m；调控雨生红球藻细胞内次生代谢产物虾青素合成和积累阶段无菌空气鼓气量为0.01-0.08m3/m，通常为0.02-0.05m3/m；O2气体按咸淡水营养液所需溶解氧的浓度进行调节。 Pluvialis algae proliferation phase sterile air blown into the amount 0.02-2m3 / m, usually 0.03-0.2m3 / m; internal regulation pluvialis algal secondary metabolites astaxanthin synthesis and accumulation phase without Bacteria air drum volume of 0.01-0.08m3 / m, usually 0.02-0.05m3 / m; O2 gas concentration of dissolved oxygen required by brackish water nutrient solution can be adjusted.
五、设置有回流装置的咸淡水营养液调配和温度调控系统：咸淡水营养液调配及温度调控系统由配置冷水循环制冷装置、加热装置、营养液循环泵、回流管道、测温控温设施及带搅拌器的工业上使用的夹层贮液罐组成。 Five, there is provided a device for reflux brackish nutrient solution deployment and temperature control systems: brackish water nutrient solution deployment and configuration of the cold water temperature control system by circulating cooling units, heating units, nutrient solution circulating pump, return line temperature measurement and control facilities and Sandwich storage tank used in the industry with a stirrer composed. 每组贮液罐为2至3只，贮液罐容量为100-10000L，通常为1000-6000L；搅拌器转速为20-300r/m，通常为60-80r/m。 Each group of storage tank 2-3, the reservoir tank capacity 100-10000L, usually 1000-6000L; stirrer speed of 20-300r / m, usually 60-80r / m.
处于细胞生长增殖阶段的雨生红球藻咸淡水营养液组成：咸淡水配比为清水30-70％，通常为45-65％；海水25-75％，通常为35-55％；亦可采用人工调配海水；营养液配比为硝酸钾0.1-0.42g/L，通常为0.2-0.3g/L；醋酸钠0.3-0.65g/L，通常为0.45-0.55g/L；亦可以加入尿素、硫酸镁、磷酸二氢钾等营养盐，可以是一种或一种以上，加入量为0.01-1g/L；咸淡水营养液溶解氧浓度为40-55％饱和度；雨生红球藻生长增殖阶段最适pH值为中性；藻细胞生长增殖阶段最适温度为14-27℃，通常为20-24℃；藻细胞迅速生长增殖处于对数生长期的培养周期为48-96h，通常为60-72h。 In cell growth and proliferation stages pluvialis algae nutrient solution composed of brackish water: brackish water ratio of 30-70%, typically 45-65%; 25-75% seawater, typically 35-55%; also artificial seawater deployment; nutrient solution ratio of potassium nitrate 0.1-0.42g / L, typically 0.2-0.3g / L; sodium acetate 0.3-0.65g / L, usually from 0.45-0.55g / L; also may be added urea , magnesium, potassium dihydrogen phosphate and other nutrients, which can be one or more, add the amount of 0.01-1g / L; brackish nutrient solution concentration of 40-55% oxygen saturation; pluvialis algae proliferation stage optimum pH neutral; algal cell proliferation phase the optimum temperature is 14-27 ℃, usually 20-24 ℃; the rapid proliferation of algal cells in the logarithmic growth phase of the training cycle for 48-96h, usually 60-72h.
通过环境条件的改变进行生理诱导调控促进雨生红球藻细胞内次生代谢产物虾青素合成和积累阶段最适培养温度为21-32℃，通常为26-29℃，最适pH值为微碱性；培养周期为50-80h，通常为60-72h。 Physiological regulation induced by changes in environmental conditions promote within pluvialis algal secondary metabolites astaxanthin synthesis and accumulation stage optimum cultivation temperature was 21-32 ℃, usually 26-29 ℃, the optimum pH slightly alkaline; training cycle for 50-80h, usually 60-72h.
六、清洗和消毒杀菌系统：投入生产之前应将光生物反应器彻底清洗消毒，每次清洗时均应清除封盖部分的透明薄膜并更换新的透明薄膜，咸淡水营养液调配过程应保持洁净无污染，咸淡水中清水及海水可采用工业上使用的臭氧发生器进行杀菌；营养液可采用加热法杀菌，加热温度为70-80℃，时间15-25分钟，或95-100℃，时间5-15分钟；亦可采用化学杀菌法进行杀菌。 Six, cleaning and disinfection system: light should be put into production before the bioreactor thoroughly cleaned and disinfected every time cleaning should remove the transparent film cover parts and replace with new transparent film, brackish nutrient solution deployment process should be kept clean pollution, brackish water and sea water can be used on industrial ozone generator for sterilization; nutrient solution can be heated sterilization method, the heating temperature is 70-80 ℃, time is 15-25 minutes, or 95-100 ℃, time 5-15 minutes; we can use chemical sterilization method for sterilization. 循环使用的咸淡水营养液可采用加热法杀菌后再投入使用。 Brackish water circulating nutrient solution can be used in heating sterilization and then put into use.
七、收获雨生红球藻细胞及低温干燥：离心是目前应用最广的收获藻细胞的方法，通常采用工业上连续式浆渣分离装置，转速为3000-30000r/m，通常为8000-15000r/m。 Seven, harvesting pluvialis algae cells and low-temperature drying: Centrifugal is currently the most widely used method of algal cells harvested, usually on an industrial continuous slurry residue separation devices, speed is 3000-30000r / m, usually 8000-15000r / m. 亦可采用其他分离设备或过滤法收获藻细胞。 We can use other separation equipment or filtration harvested algal cells. 将分离的雨生红球藻细胞采用工业上使用的低温干燥设施，如真空冷冻干燥、真空干燥、低温热风干燥、低温喷雾干燥等设备进行干燥，干燥温度为38-85℃，通常为43-48℃。 The isolated pluvialis algal cells using the industry's use of low-temperature drying facilities, such as vacuum freeze-drying, vacuum drying, low temperature hot air drying, spray drying and other equipment for low temperature drying, drying temperature is 38-85 ℃, usually 43- 48 ℃. 要求干燥后产物含水份低于3％，通常为1％以下。 After the dried product moisture content require less than 3%, usually 1% or less.
封闭式大规模运行系统中光生物反应器的设计对于提供可调控的环境条件促进雨生红球藻迅速生长增殖和提高种群密度及促进细胞内次生代谢产物虾青素的合成和积累至关重要。 Optical design of a closed bioreactor system for large-scale operation to provide adjustable environmental conditions promote rapid pluvialis algae proliferation and increasing population density and promote intracellular secondary metabolites astaxanthin synthesis and accumulation to close important. 培养雨生红球藻由于非生物因素的变量太多，如生物污染、光照强度、溶解氧、温度和营养液组成等多种因子的影响和应变环境促进细胞内目标产物的合成和积累而变得非常复杂。 Pluvialis algae culture because too many variables abiotic factors, such as environmental impact and strain various factors of biological contamination, light intensity, dissolved oxygen, temperature and composition of the nutrient solution to promote the synthesis and accumulation of intracellular target product becomes very complex. 光照是雨生红球藻生长的重要因素。 Light is an important factor pluvialis algae. 最适于雨生红球藻生长增殖的光照度为中等强度光照1000-1600勒克斯；加大光照强度在1900-3200勒克斯，可促进雨生红球藻细胞内次生代谢生物虾青素合成和积累；大于3500勒克斯的光照强度将抑制雨生红球藻的生长，分裂的细胞也由营养生长转为休眠状态。 The most suitable pluvialis algae growth and proliferation of moderate intensity illumination lux illumination 1000-1600; 1900-3200 lux light intensity increased, can promote within pluvialis algal secondary metabolites biological astaxanthin synthesis and accumulation ; greater than 3500 lux light intensity will inhibit pluvialis algae growth, division of cells into a dormant state by vegetative growth. 昼夜连续光照有利于雨生红球藻的生长增殖并明显提高细胞体积产率。 Continuous day and night illumination is conducive pluvialis algae proliferation and cell volume significantly increased yield. 培养过程光照不足及藻细胞相互之间的遮蔽作用，将导致藻细胞生长受到抑制而增加了其他生物污染的机会。 Insufficient light shielding effect of the training process and algal cells between them, will lead to algal cell growth inhibition and increases the chance of the other biological contamination. 在室外人工培养微藻采用的桨叶轮搅拌的浅水道光生物反应器，构造上只接受正面光的光照，对光线的充分利用及光照的调控受到局限，特别是昼夜和阴晴天与正常日光光照强度差别大的影响成为大规模培养雨生红球藻及调控细胞内次生代谢产物虾青素的合成和积累的关键性技术难题。 In the outdoor artificial cultivation of microalgae used in stir paddle wheel shallow dynasty bioreactor only accept positive light on the structure of light, to make full use of light regulation and lighting has been limited, especially in day and night and confront with normal daylight illumination Large differences affect large-scale cultivation has become pluvialis algae and secondary metabolites in the regulation of cell astaxanthin synthesis and accumulation of key technical problems.
通过改造并优化浅水道光生物反应器系统成为封闭式光生物反应器，在主体反应器的底部及四周和封盖采用透光材料提高光生物反应器内整体光照强度，改善整体的培养条件为可调控的高净化度的环境条件，利用日光光照并外置可调控的人工光源照射使藻细胞昼夜能均匀地接受到中等以上强度光照，对光能利用率较高，既可促进雨生红球藻细胞生长增殖，又能调控雨生红球藻细胞内次生代谢产物虾青素的合成和积累，雨生红球藻细胞体积产量高达4-6g/L以上，其中虾青素含量可高达细胞干重的4-5％以上，同时又能克服开放式大规模运行系统光生物反应器运转期间昼夜和阴晴天的影响及其他杂藻和生物沾污以及大气污染。 The light through the transformation and optimization shallow bioreactor system becomes closed photo bioreactor, and around the body at the bottom of the reactor using light-transmitting material and the closure within the photobioreactor improve overall light intensity, improve the overall culture conditions to be regulation of the environmental conditions of high degree of purification, illumination and external use of daylight and artificial light irradiation can be regulated so that the circadian algal cells uniformly received above average light intensity of high energy efficiency, can promote pluvialis algal cell growth and proliferation, but also in the regulation of cell pluvialis algal secondary metabolites astaxanthin synthesis and accumulation pluvialis algal cell volume output of up to 4-6g / L or more, which can be as high astaxanthin content cell dry weight of more than 4-5%, while the open during a large-scale operation to overcome the photobioreactor system operation and confront affect circadian day and other miscellaneous algae and bio-contamination and air pollution. 由于主体反应器封盖部分透光材料更换方便，使主体反应器整体清洗更加便利快捷，有利于实现由计算机控制长期半连续化生产。 Because the main part of the reactor lid translucent material easy to replace, so the main reactor overall cleaning more convenient and efficient, is conducive to long-term by a computer-controlled semi-continuous production. 因此，建立可调控的全天候封闭式大规模运行系统光生物反应器比浅水道光生物反应器培养和促进雨生红球藻细胞生长增殖及提高种群密度和调控细胞内次生代谢产物虾青素合成和积累更具优越性。 Therefore, the establishment of large-scale adjustable-weather running a closed photobioreactor system than the shallow dynasty bioreactor culture and promote pluvialis algal cell proliferation and increase population density and secondary metabolites in the regulation of cell astaxanthin synthesis and accumulate more advantages.
具体实施方式 DETAILED DESCRIPTION
下面结合实施例对本发明作进一步详细说明。 Below in connection with embodiments of the present invention will be further described in detail.
实施例一：建立全天候封闭式大规模运行系统光生物反应器，主体反应器(水槽)长宽深为500×150×65cm，底部及四周采用2cm厚的无色透明钢化玻璃，封盖部分采用厚度为0.2mm的透明薄膜并用胶带纸封贴，主体反应器四周及上下安装可调亮度的功率为100W太阳灯20盏，40W节能射灯20盏；反应器内调速浆叶轮搅拌器转速为30r/m；卷水轮转速为50r/m，咸淡水营养液以0.04m/s流速循环；咸淡水营养液调配及温度调控系统配备3只容量4000L的贮液罐，搅拌器转速为80r/m。 Embodiment 1: establish a large-scale operation of the system clock enclosed photobioreactor, the main reactor (sink) length and width of deep 500 × 150 × 65cm, 2cm thick at the bottom and around the use of colorless transparent glass, cover part of the adoption 0.2mm thick transparent film and paper seal affixed with adhesive tape, around and up and down the main reactor installed power of 100W adjustable brightness sunlamps 20, 40W energy saving spotlights 20; governor slurry reactor stirrer speed of the impeller 30r / m; volume waterwheel speed is 50r / m, brackish water nutrient solution to 0.04m / s velocity loop; brackish nutrient solution deployment and temperature control system is equipped with three storage tank capacity 4000L, the stirrer speed is 80r / m. 藻细胞生长增殖及提高种群密度调控阶段的培养条件为，咸淡水营养液组成：清水65％，海水35％，硝酸钾0.21g/L，醋酸钠0.52g/L，硫酸镁0.04g/L，磷酸二氢钾0.04g/L，尿素0.9g/L；清水及海水可经臭氧发生器杀菌；营养液采用经100℃加热杀菌5分钟；藻细胞生长增殖温度为20℃；光照强度为1300-1700勒克斯；无菌空气鼓入量为0.3m3/m；咸淡水营养液溶解氧浓度为43％饱和度；最适藻细胞生长增殖pH值为6.8，培养周期为60h，藻细胞生长增殖处于对数生长期，藻细胞呈绿色。 Algal cell proliferation and increase the population density of the culture conditions for the regulation stage, brackish nutrient solution composition: 65% water, 35% sea water, potassium nitrate 0.21g / L, sodium acetate, 0.52g / L, magnesium 0.04g / L, potassium dihydrogen phosphate 0.04g / L, urea 0.9g / L; water and seawater may be an ozone generator sterilization; nutrient solution using heat sterilization at 100 ℃ for 5 minutes; algal cell proliferation temperature is 20 ℃; light intensity for 1300- 1700 lux; sterile air blown into the amount of 0.3m3 / m; brackish nutrient solution dissolved oxygen concentration of 43% saturation; optimal algal cell growth and proliferation of pH 6.8, the culture period 60h, cell growth and proliferation of algae in the right logarithmic growth, green algal cells. 通过环境条件的改变进行生理诱导调控促进雨生红球藻细胞内次生代谢产物虾青素合成和积累，此阶段培养条件：培养温度为28℃；光照强度为2300-2800勒克斯；无菌空气鼓入量为0.1m3/m；最适pH值为8.2；培养周期为72h，藻细胞呈砖红色。 Through changes in environmental conditions induced physiological regulation of promoting within pluvialis algal secondary metabolites astaxanthin synthesis and accumulation of culture conditions at this stage: the culture temperature was 28 ℃; 2300-2800 lux light intensity; sterile air Drums intake of 0.1m3 / m; optimum pH 8.2; training cycle for 72h, algal cells was brick red. 经连续式浆渣分离装置收获雨生红球藻细胞，转速为10000r/m，然后再经45℃真空干燥处理，使藻细胞水份含量低于1％。 After continuous slurry residue separation device harvest pluvialis algal cells, speed is 10000r / m, then by 45 ℃ vacuum drying, so that the moisture content of the algal cells less than 1%. 经分析，藻细胞虾青素含量高达细胞干重的3.9％。 After analysis, the algal cells astaxanthin content of up to 3.9% of cell dry weight.
实施例二：建立全天候封闭式大规模运行系统光生物反应器，主体反应器(水槽)长宽深为220×110×55cm，底部及四周采用1.5cm厚的无色透明特种玻璃，封盖部分采用厚度为0.1mm的透明薄膜并用胶带纸封贴，主体反应器四周及上下安装可调亮度的功率为40W日光灯20盏；反应器内调速浆叶轮搅拌器转速为20r/m；卷水轮转速为30r/m，咸淡水营养液以0.02m/s的流速循环；咸淡水营养液调配及温度调控系统配备2只容量1200L的贮液罐，搅拌器转速为60r/m。 EXAMPLE II: establish a large-scale operation of the system clock enclosed photobioreactor, the main reactor (sink) length and width of deep 220 × 110 × 55cm, 1.5cm thick at the bottom and around the use of special glass colorless transparent, cover part transparent film with a thickness of 0.1mm and seal affixed with adhesive tape, around and up and down the main reactor installed power adjustable brightness 40W fluorescent lamp 20; slurry reactor speed impeller stirrer speed is 20r / m; volume waterwheel speed is 30r / m, brackish water nutrient solution to 0.02m / s velocity loop; brackish nutrient solution deployment and temperature control system is equipped with two liquid storage tank capacity 1200L, stirrer speed is 60r / m. 藻细胞生长增殖及提高种群密度调控阶段的培养条件为，咸淡水营养液组成：清水40％，海水60％，硝酸钾0.2g/L，醋酸钠0.6g/L，磷酸二氢钾0.4g/L，硫酸镁0.2g/L，尿素1g/L；清水及海水可经臭氧发生器杀菌；营养液采用经95℃加热杀菌10分钟；藻细胞生长增殖温度为23℃；光照强度为1100-1500勒克斯；无菌空气鼓入量为0.3m3/m；咸淡水营养液溶解氧浓度为53％饱和度；最适藻细胞生长增殖pH值为中性，培养周期为48h，藻细胞呈绿色。 Algal cell proliferation and increase the population density of the culture conditions for the regulation stage, brackish nutrient solution composition: 40% water, 60% of the water, potassium nitrate 0.2g / L, sodium acetate 0.6g / L, potassium dihydrogen phosphate 0.4g / L, magnesium sulfate 0.2g / L, urea, 1g / L; water and sterilized seawater may be an ozone generator; nutrient solution adopted by 95 ℃ heat sterilization 10 minutes; algal cell proliferation temperature was 23 ℃; light intensity of 1100-1500 Lux; sterile air blown into the amount of 0.3m3 / m; brackish nutrient solution dissolved oxygen concentration of 53% saturation; optimal cell growth and proliferation of algae neutral pH training cycle for 48h, green algal cells. 通过环境条件的改变进行生理诱导调控促进雨生红球藻细胞内次生代谢产物虾青素合成和积累，此阶段培养条件：培养温度为28.5℃；光照强度为2400-2700勒克斯；无菌空气鼓入量为0.1m3/m；最适pH值为8；培养周期为72h，藻细胞呈砖红色。 Through changes in environmental conditions induced physiological regulation of promoting within pluvialis algal secondary metabolites astaxanthin synthesis and accumulation of culture conditions at this stage: the culture temperature is 28.5 ℃; 2400-2700 lux light intensity; sterile air Drums intake of 0.1m3 / m; optimum pH 8; training cycle for 72h, algal cells was brick red. 经过滤装置收获雨生红球藻细胞，然后再经45℃真空干燥处理，使藻细胞水份含量低于1％。 Harvested by filtration device pluvialis algal cells, and then by 45 ℃ vacuum drying, so that the moisture content of the algal cells less than 1%. 经分析，藻细胞虾青素含量高达细胞干重的4.1％。 After analysis, the algal cells astaxanthin content of up to 4.1% dry cell weight.
实施例三：建立全天候封闭式大规模运行系统光生物反应器，主体反应器(水槽)长宽深为200×100×65cm，底部及四周采用1cm厚的无色透明钢化玻璃，封盖部分采用厚度为0.2mm的透明薄膜并用胶粘剂封贴，主体反应器四周及上下安装可调亮度的功率为40W日光灯7盏，100W太阳灯5盏；反应器内调速浆叶轮搅拌器转速为10r/m；卷水轮转速为10r/m，咸淡水营养液以0.03m/s流速循环；咸淡水营养液调配及温度调控系统配备2只容量800L的贮液罐，搅拌器转速为20r/m。 EXAMPLE III: establish a large-scale operation of the system clock enclosed photobioreactor, the main reactor (sink) length and width of deep 200 × 100 × 65cm, bottom and around the use of 1cm thick colorless transparent glass, cover part of the adoption 0.2mm thick transparent film and sealed with adhesive stickers, around and up and down the main reactor installed power adjustable brightness 40W fluorescent 7, 100W sun lamp 5; slurry reactor speed impeller stirrer speed is 10r / m ; volume water wheel speed is 10r / m, brackish water nutrient solution to 0.03m / s velocity loop; brackish nutrient solution deployment and temperature control system is equipped with two storage tank capacity of 800L, the stirrer speed is 20r / m. 藻细胞生长增殖及提高种群密度调控阶段的培养条件为，咸淡水营养液组成：清水68％，海水32％，硝酸钾0.4g/L，醋酸钠0.4g/L，尿素0.3g/L，硫酸镁0.04g/L；清水及海水可经臭氧发生器杀菌；营养液采用经98℃加热杀菌8分钟；藻细胞生长增殖温度为24℃；光照强度为800-1600勒克斯；无菌空气鼓入量为0.05m3/m；咸淡水营养液溶解氧浓度为51％饱和度；最适藻细胞生长增殖的pH值为6.6，培养周期为60h，藻细胞生长增殖处于对数生长期，藻细胞呈绿色。 Algal cell proliferation and increase the population density of the culture conditions for the regulation stage, brackish nutrient solution composition: 68% water, 32% of the water, potassium nitrate 0.4g / L, sodium acetate 0.4g / L, urea 0.3g / L, sulfuric acid Magnesium 0.04g / L; water and seawater may be an ozone generator sterilization; nutrient solution adopted by 98 ℃ heat sterilization 8 minutes; algal cell proliferation temperature is 24 ℃; 800-1600 lux light intensity; sterile air blown into the volume of 0.05m3 / m; brackish nutrient solution dissolved oxygen concentration of 51% saturation; optimal algal cell growth and proliferation of a pH 6.6, the culture period 60h, algal cell growth and proliferation in the logarithmic growth phase, cells were green algae . 通过环境条件的改变进行生理诱导调控促进雨生红球藻细胞内次生代谢产物虾青素合成和积累，此阶段培养条件：培养温度为28℃；光照强度为2400-2900勒克斯；无菌空气鼓入量为0.08m3/m；最适pH值为8.3；培养周期为68h，藻细胞呈砖红色。 Through changes in environmental conditions induced physiological regulation of promoting within pluvialis algal secondary metabolites astaxanthin synthesis and accumulation of culture conditions at this stage: the culture temperature was 28 ℃; 2400-2900 lux light intensity; sterile air Drums intake of 0.08m3 / m; optimum pH 8.3; training cycle for 68h, algal cells was brick red. 经连续式浆渣分离装置收获雨生红球藻细胞，转速为12000r/m，然后再经42℃真空干燥处理，使藻细胞水份含量低于1.5％。 After continuous slurry residue separation device harvest pluvialis algal cells, speed is 12000r / m, then by 42 ℃ vacuum drying, so that the moisture content of the algal cells less than 1.5%. 经分析，藻细胞虾青素含量高达细胞干重的4.3％。 After analysis, the algal cells astaxanthin content of up to 4.3% of cell dry weight.
实施例四：建立全天候封闭式大规模运行系统光生物反应器，主体反应器(水槽)长宽深为500×150×65cm，底部及四周采用2cm厚的无色透明钢化玻璃，封盖部分采用厚度为0.18mm的复合材料透明薄膜并用胶带纸封贴，主体反应器四周及上下安装可调亮度的功率为100W太阳灯27盏；反应器内调速浆叶轮搅拌器转速为30r/m；卷水轮转速为50r/m，咸淡水营养液以0.04m/s流速循环；咸淡水营养液调配及温度调控系统配备3只容量4000L的贮液罐，搅拌器转速为80r/m。 EXAMPLE IV: establish a large-scale operation of the system clock enclosed photobioreactor, the main reactor (sink) length and width of deep 500 × 150 × 65cm, 2cm thick at the bottom and around the use of colorless transparent glass, cover part of the adoption The thickness of the composite material and a transparent film 0.18mm seal affixed with adhesive tape, around and up and down the main reactor installed power of 100W adjustable brightness sunlamps 27; slurry reactor speed impeller stirrer speed is 30r / m; Volume the turbine speed is 50r / m, brackish water nutrient solution to 0.04m / s velocity loop; brackish nutrient solution deployment and temperature control system is equipped with three storage tank capacity 4000L, the stirrer speed is 80r / m. 藻细胞生长增殖及提高种群密度调控阶段的培养条件为，咸淡水营养液组成：清水65％，海水35％，硝酸钾0.21g/L，醋酸钠0.5g/L，尿素0.4g/L，磷酸二氢钾0.01g/L；清水及海水可经臭氧发生器杀菌；营养液采用经70℃加热杀菌25分钟；藻细胞生长增殖温度为17℃；光照强度为1600-1750勒克斯；无菌空气鼓入量为0.3m3/m；咸淡水营养液溶解氧浓度为55％饱和度；最适藻细胞生长增殖的pH值为6.9，培养周期为96h，藻细胞生长增殖处于对数生长期，藻细胞呈绿色。 Algal cell proliferation and increase the population density of the culture conditions for the regulation stage, brackish nutrient solution composition: 65% water, 35% sea water, potassium nitrate 0.21g / L, sodium acetate 0.5g / L, urea 0.4g / L, phosphoric acid Potassium dihydrogen 0.01g / L; water and seawater may be an ozone generator sterilization; nutrient solution adopted by 70 ℃ heat sterilization 25 minutes; algal cell proliferation temperature is 17 ℃; 1600-1750 lux light intensity; sterile air drums the amount of 0.3m3 / m; brackish nutrient solution of dissolved oxygen concentration of 55% saturation; optimal algal cell growth and proliferation of a pH 6.9, the culture period 96h, algal cell growth and proliferation in the logarithmic growth phase, algal cells green. 通过环境条件的改变进行生理诱导调控促进雨生红球藻细胞内次生代谢产物虾青素合成和积累，此阶段培养条件：培养温度为28℃；光照强度为2300-2500勒克斯；无菌空气鼓入量为0.1m3/m；最适pH值为8；培养周期为72h，藻细胞呈砖红色。 Through changes in environmental conditions induced physiological regulation of promoting within pluvialis algal secondary metabolites astaxanthin synthesis and accumulation of culture conditions at this stage: the culture temperature was 28 ℃; 2300-2500 lux light intensity; sterile air Drums intake of 0.1m3 / m; optimum pH 8; training cycle for 72h, algal cells was brick red. 经连续式浆渣分离装置收获雨生红球藻细胞，转速为8000r/m，然后再经80℃低温喷雾干燥处理，使藻细胞水份含量低于1％。 After continuous slurry residue separation device harvest pluvialis algal cells, speed is 8000r / m, then by 80 ℃ low temperature spray drying, moisture content of algal cells to make less than 1%. 以上光生物反应器配置的营养液调配、控光、控温、清洗、消毒、杀菌、搅拌、鼓气、分离、干燥等装置由计算机控制实现半连续生产。 Above photo bioreactor configuration nutrient solution deployment, light control, temperature control, cleaning, disinfection, sterilization, stirring, bubbling, separation, drying and the like means controlled by the computer to realize semi-continuous production. 经分析，藻细胞虾青素含量高达细胞干重的4.2％。 After analysis, the algal cells astaxanthin content of up to 4.2% dry cell weight.
实施例五：建立全天候封闭式大规模运行系统光生物反应器，主体反应器(水槽)长宽深为1500×300×70cm，底部及四周采用3cm厚的无色透明钢化玻璃，封盖部分采用厚度为0.6mm的透明薄膜并用胶粘剂封贴，主体反应器四周及上下安装可调亮度的功率为300W白炽灯20盏，30W节能射灯50盏；反应器内调速浆叶轮搅拌器转速为80r/m；卷水轮转速为100r/m，咸淡水营养液以0.06m/s流速循环；咸淡水营养液调配及温度调控系统配备3只容量10000L贮液罐，搅拌器转速为60r/m。 EXAMPLE V: establish a large-scale operation of the system clock enclosed photobioreactor, the main reactor (sink) length and width of deep 1500 × 300 × 70cm, 3cm thick at the bottom and around the use of colorless transparent glass, cover part of the adoption transparent film thickness of 0.6mm and sealed with adhesive stickers, around and up and down the main reactor installation adjustable brightness power of 300W incandescent lamp 20, 30W energy saving spotlights 50; slurry reactor speed impeller stirrer speed of 80r / m; volume waterwheel speed of 100r / m, brackish water nutrient solution to 0.06m / s velocity loop; brackish nutrient solution deployment and temperature control system is equipped with three capacity fluid storage tank 10000L, stirrer speed is 60r / m. 藻细胞生长增殖及提高种群密度调控阶段的培养条件为，咸淡水营养液组成：清水70％，海水30％，硝酸钾0.33g/L，醋酸钠0.55g/L，硫酸镁0.03g/L，磷酸二氢钾0.08g/L，尿素0.8g/L；清水及海水可经臭氧发生器杀菌；营养液采用经95℃加热杀菌10分钟；藻细胞生长增殖温度为20℃；光照强度为1100-1600勒克斯；无菌空气鼓入量为1m3/m；咸淡水营养液溶解氧浓度为54％饱和度；最适藻细胞生长增殖的pH值为6.9，培养周期为60h，藻细胞生长增殖处于对数生长期，藻细胞呈绿色。 Algal cell proliferation and increase the population density of the culture conditions for the regulation stage, brackish nutrient solution composition: 70% water, 30% sea water, potassium nitrate 0.33g / L, sodium acetate, 0.55g / L, magnesium 0.03g / L, potassium dihydrogen phosphate 0.08g / L, urea 0.8g / L; water and seawater may be an ozone generator sterilization; nutrient solution adopted by 95 ℃ heat sterilization 10 minutes; algal cell proliferation temperature is 20 ℃; light intensity for 1100- 1600 lux; sterile air blown into the amount of 1m3 / m; brackish nutrient solution dissolved oxygen concentration of 54% saturation; optimal algal cell growth and proliferation of a pH 6.9, the culture period 60h, cell growth and proliferation of algae in the right logarithmic growth, green algal cells. 通过环境条件的改变进行生理诱导调控促进雨生红球藻细胞内次生代谢产物虾青素合成和积累，此阶段培养条件：培养温度为29℃；光照强度为2300-2500勒克斯；无菌空气鼓入量为0.16m3/m；最适pH值为8.1；培养周期为72h，藻细胞呈砖红色。 Through changes in environmental conditions induced physiological regulation of promoting within pluvialis algal secondary metabolites astaxanthin synthesis and accumulation of culture conditions at this stage: the culture temperature is 29 ℃; 2300-2500 lux light intensity; sterile air Drums intake of 0.16m3 / m; optimum pH 8.1; training cycle for 72h, algal cells was brick red. 经连续式浆渣分离装置收获雨生红球藻细胞，转速为20000r/m，然后再经43℃真空干燥处理，使藻细胞水份含量低于1％。 After continuous slurry residue separation device harvest pluvialis algal cells, speed is 20000r / m, then by 43 ℃ vacuum drying, so that the moisture content of the algal cells less than 1%. 经分析，藻细胞虾青素含量高达细胞干重的4.4％。 After analysis, the algal cells astaxanthin content of up to 4.4% of cell dry weight.
实施例六：建立全天候封闭式大规模运行系统光生物反应器，主体反应器(水槽)长宽深为1000×200×68cm，底部及四周采用2.5cm厚的无色透明钢化玻璃，封盖部分采用厚度为0.65mm的透明薄膜并用胶带纸封贴，主体反应器四周及上下安装可调亮度的功率为150W太阳灯18盏，40W日光灯12盏；反应器内调速浆叶轮搅拌器转速为30r/m；卷水轮转速为60r/m，咸淡水营养液以0.04m/s流速循环；咸淡水营养液调配及温度调控系统配备3只容量7000L的贮液罐，搅拌器转速为100r/m。 EXAMPLE VI: establish a large-scale operation of the system clock enclosed photobioreactor, the main reactor (sink) length and width of deep 1000 × 200 × 68cm, 2.5cm thick at the bottom and around the use of colorless transparent glass, cover part transparent film with a thickness of 0.65mm and seal affixed with adhesive tape, around and up and down the main reactor installed power of 150W adjustable brightness sunlamps 18, 40W fluorescent lamp 12; slurry reactor speed impeller stirrer speed is 30r / m; volume waterwheel speed is 60r / m, brackish water nutrient solution to 0.04m / s velocity loop; brackish nutrient solution deployment and temperature control system is equipped with three storage tank capacity 7000L, the stirrer speed of 100r / m . 藻细胞生长增殖及提高种群密度调控阶段的培养条件为，咸淡水营养液组成：清水56％，海水44％，硝酸钾0.38g/L，硫酸镁0.6g/L，醋酸钠0.38g/L，尿素0.9g/L；清水及海水可经臭氧发生器杀菌；营养液采用经98℃加热杀菌6分钟；藻细胞生长增殖温度为21℃；光照强度为1200-1600勒克斯；无菌空气鼓入量为0.9m3/m；咸淡水营养液溶解氧浓度为49％饱和度；最适藻细胞生长增殖的pH值为7.2，培养周期为54h，藻细胞生长增殖处于对数生长期，藻细胞呈绿色。 Algal cell proliferation and increase the population density of the culture conditions for the regulation stage, brackish nutrient solution composition: 56% water, 44% of the water, potassium nitrate 0.38g / L, magnesium 0.6g / L, sodium acetate, 0.38g / L, urea 0.9g / L; water and seawater may be an ozone generator sterilization; nutrient solution adopted by 98 ℃ heat sterilization 6 minutes; algal cell proliferation temperature is 21 ℃; 1200-1600 lux light intensity; sterile air blown into the volume of 0.9m3 / m; brackish nutrient solution dissolved oxygen concentration of 49% saturation; optimal algal cell growth and proliferation of pH value of 7.2, the culture period 54h, algal cell growth and proliferation in the logarithmic growth phase, cells were green algae . 通过环境条件的改变进行生理诱导调控促进雨生红球藻细胞内次生代谢产物虾青素合成和积累，此阶段培养条件：培养温度为28℃；光照强度为2400-2500勒克斯；无菌空气鼓入量为0.13m3/m；最适pH值为8.2；培养周期为72h，藻细胞呈砖红色。 Through changes in environmental conditions induced physiological regulation of promoting within pluvialis algal secondary metabolites astaxanthin synthesis and accumulation of culture conditions at this stage: the culture temperature was 28 ℃; 2400-2500 lux light intensity; sterile air Drums intake of 0.13m3 / m; optimum pH 8.2; training cycle for 72h, algal cells was brick red. 经连续式浆渣分离装置收获雨生红球藻细胞，转速为11000r/m，然后再经45℃真空干燥处理，使藻细胞水份含量低于1％。 After continuous slurry residue separation device harvest pluvialis algal cells, speed is 11000r / m, then by 45 ℃ vacuum drying, so that the moisture content of the algal cells less than 1%. 经分析，藻细胞虾青素含量高达细胞干重的4.5％。 After analysis, the algal cells astaxanthin content of up to 4.5% of cell dry weight.
实施例七：建立全天候封闭式大规模运行系统光生物反应器，主体反应器(水槽)长宽深为800×150×65cm，底部及四周采用2cm厚的无色透明钢化玻璃，封盖部分采用厚度为0.45mm的透明薄膜并用胶带纸封贴，主体反应器四周及上下安装可调亮度的功率为300W太阳灯8盏，40W日光灯13盏；反应器内调速浆叶轮搅拌器转速为60r/m；卷水轮转速为30r/m，咸淡水营养液以0.06m/s流速循环；咸淡水营养液调配及温度调控系统配备3只容量6000L的贮液罐，搅拌器转速为200r/m。 EXAMPLE VII: establish a large-scale operation of the system clock enclosed photobioreactor, the main reactor (sink) length and width of deep 800 × 150 × 65cm, 2cm thick at the bottom and around the use of colorless transparent glass, cover part of the adoption 0.45mm thick transparent film and paper seal affixed with adhesive tape, around and up and down the main reactor installed power of 300W adjustable brightness sunlamps 8, 40W fluorescent lamp 13; slurry reactor speed impeller stirrer speed is 60r / m; volume waterwheel speed is 30r / m, brackish water nutrient solution to 0.06m / s velocity loop; brackish nutrient solution deployment and temperature control system is equipped with three storage tank capacity 6000L, the stirrer speed of 200r / m. 藻细胞生长增殖及提高种群密度调控阶段的培养条件为，咸淡水营养液组成：清水70％，海水30％，硝酸钾0.33g/L，醋酸钠0.45g/L，尿素0.7g/L；清水及海水可经臭氧发生器杀菌；营养液采用经96℃加热杀菌10分钟；藻细胞生长增殖温度为23℃；光照强度为1300-1600勒克斯；无菌空气鼓入量为0.7m3/m；咸淡水营养液溶解氧浓度为52％饱和度；最适藻细胞生长增殖的pH值为7.1，诱导调控促进细胞内次生代谢产物虾青素合成和积累的培养周期为80h，藻细胞生长增殖处于对数生长期，藻细胞呈绿色。 Algal cell proliferation and increase the population density of the culture conditions for the regulation stage, brackish nutrient solution composition: 70% water, 30% sea water, potassium nitrate 0.33g / L, sodium acetate, 0.45g / L, urea 0.7g / L; Shimizu and seawater may be an ozone generator sterilization; nutrient solution adopted by 96 ℃ heat sterilization 10 minutes; algal cell proliferation temperature is 23 ℃; 1300-1600 lux light intensity; sterile air blown into the amount of 0.7m3 / m; salty freshwater nutrient solution dissolved oxygen concentration of 52% saturation; optimal algal cell growth and proliferation of pH of 7.1, induced cell cycle regulation to foster the development of secondary metabolites astaxanthin synthesis and accumulation of 80h, algal cell growth and proliferation in logarithmic growth phase, green algal cells. 通过环境条件的改变进行生理诱导调控促进雨生红球藻细胞内次生代谢产物虾青素合成和积累，此阶段培养条件：培养温度为28.5℃；光照强度为2400-2600勒克斯；无菌空气鼓入量为0.18m3/m；最适pH值为8.4；培养周期为72h，藻细胞呈砖红色。 Through changes in environmental conditions induced physiological regulation of promoting within pluvialis algal secondary metabolites astaxanthin synthesis and accumulation of culture conditions at this stage: the culture temperature is 28.5 ℃; 2400-2600 lux light intensity; sterile air Drums intake of 0.18m3 / m; optimum pH 8.4; training cycle for 72h, algal cells was brick red. 经连续式浆渣分离装置收获雨生红球藻细胞，转速为15000r/m，然后再经40℃真空冷冻干燥处理，使藻细胞水份含量低于1％。 After continuous slurry residue separation device harvest pluvialis algal cells, speed is 15000r / m, then by 40 ℃ vacuum freeze-drying treatment, the algal cells moisture content of less than 1%. 经分析，藻细胞虾青素含量高达细胞干重的4.5％。 After analysis, the algal cells astaxanthin content of up to 4.5% of cell dry weight.
实施例八：建立全天候封闭式大规模运行系统光生物反应器，主体反应器(水槽)长宽深为900×160×65cm，底部及四周采用2.5cm厚的无色透明钢化玻璃，封盖部分采用厚度为0.5mm的透明薄膜并用胶带纸封贴，主体反应器四周及上下安装可调亮度的功率为200W太阳灯10盏，30W节能灯20盏；反应器内调速浆叶轮搅拌器转速为50r/m；卷水轮转速为30r/m，咸淡水营养液以0.05m/s流速循环；咸淡水营养液调配及温度调控系统配备3只容量8000L的贮液罐，搅拌器转速为200r/m。 EXAMPLE 8: Develop a massive run-weather enclosed photobioreactor system, the main reactor (sink) length and width of deep 900 × 160 × 65cm, 2.5cm thick at the bottom and around the use of colorless transparent glass, cover part transparent film with a thickness of 0.5mm and seal affixed with adhesive tape, around and up and down the main reactor installed power of 200W adjustable brightness sunlamps 10, 30W energy-saving lamps 20; governor slurry reactor stirrer speed of the impeller 50r / m; volume waterwheel speed is 30r / m, brackish water nutrient solution to 0.05m / s velocity loop; brackish nutrient solution deployment and temperature control system is equipped with three capacity 8000L liquid storage tank, agitator speed of 200r / m. 藻细胞生长增殖及提高种群密度调控阶段的培养条件为，咸淡水营养液组成：清水68％，海水32％，硝酸钾0.27g/L，硫酸镁0.01g/L，醋酸钠0.33g/L，尿素0.85g/L；清水及海水可经臭氧发生器杀菌；营养液采用经96℃加热杀菌4分钟；藻细胞生长增殖温度为23℃；光照强度为1400-1600勒克斯；无菌空气鼓入量为0.8m3/m；咸淡水营养液溶解氧浓度为54％饱和度；最适藻细胞生长增殖的pH值为6.8，培养周期为60h，藻细胞生长增殖处于对数生长期，藻细胞呈绿色。 Algal cell proliferation and increase the population density of the culture conditions for the regulation stage, brackish nutrient solution composition: 68% water, 32% of the water, potassium nitrate 0.27g / L, magnesium 0.01g / L, sodium acetate, 0.33g / L, Urea 0.85g / L; water and seawater may be an ozone generator sterilization; nutrient solution adopted by 96 ℃ heat sterilization four minutes; algal cell proliferation temperature is 23 ℃; 1400-1600 lux light intensity; sterile air blown into the volume of 0.8m3 / m; brackish nutrient solution dissolved oxygen concentration of 54% saturation; optimal algal cell growth and proliferation of pH value of 6.8, the culture period 60h, algal cell growth and proliferation in the logarithmic growth phase, cells were green algae . 通过环境条件的改变进行生理诱导调控促进雨生红球藻细胞内次生代谢产物虾青素合成和积累，此阶段培养条件：培养温度为28.5℃；光照强度为2500-2600勒克斯；无菌空气鼓入量为0.1m3/m；最适pH值为8；培养周期为65h。 Through changes in environmental conditions induced physiological regulation of promoting within pluvialis algal secondary metabolites astaxanthin synthesis and accumulation of culture conditions at this stage: the culture temperature is 28.5 ℃; 2500-2600 lux light intensity; sterile air Drums intake of 0.1m3 / m; optimum pH 8; training cycle for 65h. 经连续式浆渣分离装置收获雨生红球藻细胞，转速为13000r/m，然后再经55℃热风干燥处理，使藻细胞水份含量低于1.2％。 After continuous slurry residue separation device harvest pluvialis algal cells, speed is 13000r / m, then by 55 ℃ hot air drying, so the algal cells is less than 1.2% moisture content. 经分析，藻细胞虾青素含量高达细胞干重的4.2％。 After analysis, the algal cells astaxanthin content of up to 4.2% dry cell weight.
| Citing Patent | Filing date | Publication date | Applicant | Title |
|---|---|---|---|---|
| CN104232720A * | Aug 26, 2014 | Dec 24, 2014 | 山东金晶生物技术有限公司 | Method for producing astaxanthin by inducing Haematococcus pluvialis |
| US8834855 | Jan 23, 2006 | Sep 16, 2014 | Promar As | Sunscreen compositions comprising carotenoids |
| Date | Code | Event | Description |
|---|---|---|---|
| Jan 22, 2003 | C06 | Publication | |
| Apr 16, 2003 | C10 | Request of examination as to substance | |
| Dec 22, 2004 | C14 | Granted | |
| Sep 16, 2009 | C19 | Lapse of patent right due to non-payment of the annual fee |
