CN103966102A - Culture medium for culturing Chlamydomonas reinhardtii by urea plant wastewater and culture method of Chlamydomonas reinhardtii - Google Patents
Culture medium for culturing Chlamydomonas reinhardtii by urea plant wastewater and culture method of Chlamydomonas reinhardtii Download PDFInfo
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- CN103966102A CN103966102A CN201410222838.5A CN201410222838A CN103966102A CN 103966102 A CN103966102 A CN 103966102A CN 201410222838 A CN201410222838 A CN 201410222838A CN 103966102 A CN103966102 A CN 103966102A
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Abstract
The invention discloses a method for culturing Chlamydomonas reinhardtii by urea plant wastewater. According to the method, urea plant wastewater can be treated in low cost, Chlamydomonas reinhardtii with important economic value can also be obtained, the waste is changed into treasures, harm is turned into a good and many things are achieved at one stroke. A traditional method for culturing Chlamydomonas reinhardtii comprises the step of culturing Chlamydomonas reinhardtii in an open concrete runway pool by virtue of a TAP culture medium, and has the disadvantages of low growth speed, low output, great investment, easy pollution and low efficiency. According to the invention, Chlamydomonas reinhardtii is cultured in a household mineral water bucket to achieve the advantages of low cost, small possibility of pollution, convenience in cleaning, reutilization and convenience in operation; since nutritional ingredients, such as urea plant wastewater, yeast extract powder, sodium deoxycholate, sorbitol, mannitol, sodium thioglycolate, soil extract liquid, sheep manure leachate, chicken manure leachate, L-arginine, potassium dihydrogen phosphate, yeast extract, and diatomite powder are added into the culture medium, and an organic fertilizer is used in combination with an inorganic fertilizer, nutrition is more comprehensive and balanced, the growth rate and yield of Chlamydomonas reinhardtii are substantially increased and the yield is increased by 280%.
Description
Technical field
The invention belongs to aquaculture field, relate in particular to one and utilize urea plant waste water to cultivate Chlamydomonas reinhardtii method.
Background technology
At present, water pollution and shortage of water resources problem are day by day serious, become the important factor of restriction economy and social development, and the body eutrophication being caused by nitrogen and phosphorus pollution has more brought serious threat to water surrounding, and the improvement of nitrogenous effluent is caused to people greatly pay close attention to.Fertilizer waste water is ammonia and nitrogen pollution main source, and what wherein in urea production process, produce there is no ripe improvement technology containing the high ammonia waste water of urea.Begin from the 1950's, China has built nearly 200 cover urea plants successively, what wherein 90% device was taked urea waste water is direct discharge, and wherein 40% device waste discharge does not reach national environmental standard, and only 10% device can be accomplished to recycle.Within 2013, China produces urea approximately 6,000 ten thousand t, wastewater discharge approximately 4,000 ten thousand m
3.A large amount of direct discharging of waste water have been polluted environment, have wasted resource.
Urea technique waste liquid mainly refers to the cleaning relief liquor in the time of the process condensate of the evaporation section generation of urea plant and start-stop vehicle overhauling equipment.Process condensate is moisture, ammonia, carbonic acid gas, urea and biuret.The source of water is mainly that the driving steam of liquefied ammonia and carbonic acid gas urea synthesis water generation reaction, steam injector enters water that evaporative condenser system produces and raw materials ammonia and carbon dioxide gas and brings the water of system into.1 ton of urea of general every production will produce 380-530kg process condensate, and its composition is roughly: urea 0.4-2%, and ammonia 3.5-5.5%, carbonic acid gas 2-3%, all the other are water.In a small amount of cleaning relief liquor producing when parking maintenance equipment, containing ammonia, carbonic acid gas, urea and biuret etc., in this part waste liquid, each component concentration and quantity discharged are because of condition of production difference.At present, have in the urea technique technology of China application: Dutch Stamicarbon company carbonic acid gas air lift technology, Italian Snamprogetti company ammonia are put forward the Urea with Total Circulation Water Solution Method technology of technology, the Japanese ACES of Toyo Engineering Corporation technology and China's exploitation.These technology have larger difference to the treatment effect of urea technique waste liquid.Carbonic acid gas air lift technology, ammonia put forward technology and ACES technology is furnished with desorption tower and hydrolysis tower, and treatment effect is better.Water solution total cycling method technology has only designed desorption system and has not considered hydrolysis of urea system, and treatment effect is poor.China's urea industry is passed through the development of decades, the general layout that has formed large, medium and small type urea plant and deposited at present.In 27 cover Large Scale Urea Plant, adopt CO
2air lift law technology have 15 covers, ammonia is carried 10 covers of technology, 2 covers of ACES technology.Large Scale Urea Plant is substantially all supporting desorb and hydrolysis system,, to process the process waste liquor self producing.In 52 medium-sized nitrogenous fertilizer enterprises that are constructed and put into operation in the 60-80 age in 20th century, there are 44 cover urea plants all to adopt water solution total cycle method urea process.After the eighties, the domestic medium-sized urea plant of tens covers on new, all adopts CO substantially
2air lift method and NH
3air lift method urea technique.
Say technically, urea technique waste liquid can be accomplished to recycle after advanced treatment.Urea in waste liquid, NH
3and CO
2be distillated after concentrated and return to Urea Conversion System as raw material at desorb and hydrolysis system, in remaining waste water, urea and ammonia content are about 0.5-0.6%, can be used as process water, even can be used as oiler feed.Current domestic urea wastewater disposition is very uneven, large-lot producer's processing horizontal is higher, medium and small sized enterprises are lower, the enterprise recycling after advanced treatment is few, the enterprise that only meets qualified discharge is many, also have the middle-size and small-size urea plant of more than 100 covers there is no auxiliary construction hydrolysis system, cannot reach the advanced treatment requirement to urea waste water, even one arranged it.
No matter see technically, be external patented technology or the desorb of domestic independent development and technology for hydrolyzing, can make urea waste water after advanced treatment, reach discharging standards.But urea production enterprise of China is extremely low to the recycling ratio of urea waste water at present, even qualified discharge is all difficult to accomplish, its problem and reason are as follows:
1, enterprise operates and manages not according to design requirements.The pressure rating of the quality of urea technique liquid waste disposal effect and the residence time of waste liquid in hydrolysis tower and heating steam used is closely related.The existing urea plant of China conventionally selects the steam of 3.9MPa or 2.5MPa to make thermal source in the time carrying out desorb and hydrolysis system design, and in urea waste water after treatment, the control indexes of ammonia and urea content is at 0.5-0.6%.And in actual moving process, control index and often do not reach.Trace it to its cause, the fed distance of some enterprise's steam is long, piping insulation weak effect, and the vapor pressure and the temperature that enter hydrolysis system reduce gradually, do not reach design requirements, and some enterprise reduces steam grade standard voluntarily.Because enterprise does not operate and manages according to design requirements, cause the treatment effect of urea technique waste liquid obviously to decline, in urea waste water after treatment, ammonia and urea content are often higher than design controlling index 0.5-0.6%, conventionally at 0.6-1.0%.If such waste water is recycled and also must be for further processing as service water, and enterprise is unwilling to increase processing costs, only has and has arranged it, this is one of basic reason causing body eutrophication.
2, in urea plant design, there is defect.The middle-size and small-size urea plant that adopts water solution total cycle method urea process to build, in the time of design, only have desorption system and there is no supporting hydrolysis system, even if such device has reached very high operational administrative level in process of production, be subject to the restriction of desorption system state of the art, urea waste water after treatment also cannot reach the requirement of reuse, can only serve as discharge of wastewater, environment is caused to larger pollution.
3, urea plant volume increase transformation does not match with the transformation of desorption and hydrolysis system.Be provided with desorb and hydrolysis system or newly increased afterwards the urea plant of hydrolysis system for when design, enterprise in the time increasing production transformation at every turn, often desorb and hydrolysis system are not synchronously transformed, make original desorb and hydrolysis system can not adapt to the demand that the rear process waste liquor treatment capacity of urea plant volume increase also increases thereupon, cause desorb and the hydrolysis degree of depth inadequate, in urea waste water after treatment, ammonia and urea content increase, and cannot recycle.This phenomenon is at each urea production producer ubiquity.
In order to solve the deficiency of current technology, I have invented one and have utilized urea plant waste water to cultivate Chlamydomonas reinhardtii method, can low-cost processes urea plant waste water, can obtain again biological product-Chlamydomonas reinhardtii that Important Economic is worth, turn waste into wealth, turn harm into good, comprehensive utilization benefit is good, can significantly reduce sewage treatment equipment investment and running expense, achieve many things at one stroke, be the optimal selection of urea plant wastewater treatment.
The rapid minimizing of the Nonrenewable resources such as worldwide oil, Sweet natural gas, coal, forces the mankind to have to find renewable and clean energy resource.Chlamydomonas reinhardtii (Chamydomonas reinhardtii) forms the pattern species into biological hydrogen production research because sun power being converted into Hydrogen Energy.Chlamydomonas reinhardtii belongs to Chlorophyta, volvocales, chlamydomonas section, it is a kind of unicellular eucaryon Flagellatae, it is the model animals of the multiple vital movement of research (as photosynthesis, flagellum assembling, phototaxis and physiological rhythm etc.), there are many common features with yeast cell, have the title of " photosynthetic yeast ".Chlamydomonas reinhardtii is simple in structure, and plasmalemma is close to cell walls, and top length has 2 flagellums, and side has 1 eyespot, and most cells also have 1 or multiple vacuole, and 1 accounts for the large-scale cup-shaped chloroplast(id) of cell cumulative volume nearly 40%.Its genetic background is clear, and genomics research closely completes, and is current unique biomaterial of setting up nucleus, chloroplast(id) and plastosome three and overlapping genetic transformation system.Unicellular individuality, cell is spherical in shape, subsphaeroidal, ellipse or Long Circle, 1 layer of thinner cell walls being made up of Mierocrystalline cellulose and glycoprotein of tool.Have 1 pair of tinsel type flagellum that top is raw, isometric, active, the motion of flagellum depends on starting of matrix, and cell is moved forwards or backwards.In cell, be full of tenuigenin, have 1 nucleus; 1 chromatoplast, side position, larger, often account for cell very most of, cup-shaped or ampuliform; In chloroplast(id), bury 1 to several outer have starch sheath, interior pyrenoids and 1 eyespot for protein; Eyespot, because there being carotenoid to take on a red color, having photoperceptivity, makes chlamydomonas tool photopositive; Also have 1 to drain refuse in born of the same parents to several contractile vacuole.Distribute all over the world, polar region also has, and is common algae.Mostly move in all kinds of poisons in freshwaters, comprise some contaminated water body; Minority moves in salt solution and seawater, also has some to be born on slowly drained soil, pond or well head wall.Monogony is cell lobe, overflows after forming the protoplasma of 2,4 or 8 each tool 1 cores in parent cell, produces new wall, grows up to new individuality.Under special conditions, these cell envelopes, in 1 common glue quilt, are colloid group, treat environment transition, then grow flagellum, overflow and become new individuality from parent cell.Daughter cell forms chlamydospore in the time that environment is unfavorable.Syngenesis has with joining, different join and ovum is joined point.When environment is applicable to, each cell can become isogamete, carries out mating.Zygote all passes through dormancy, when sprouting, through reduction division, produces 4-8 swimming cell, and each cell can bud into new individuality.It is diploid that chlamydomonas only has zygote, and gamete, without fertilization, also can carry out monogenesis.Optimum temperuture 17-28 DEG C, optimal light intensity 1800-7000Lux, optimum pH 7-8.5.
Chlamydomonas reinhardtii tradition cultural method is to adopt Tris-Acetate-Phosphate (TAP) substratum and use open cement track pond to cultivate, growth is slow, yield poorly, investment is large, easily pollute, benefit is low, I adopt home-use mineral water bottle to cultivate now, cost is low, be difficult for polluting, scrub conveniently, can reuse, easy handling, and I have increased urea plant waste water in substratum, yeast soaks powder, Sodium desoxycholate, sorbyl alcohol, N.F,USP MANNITOL, sodium thioglycolate, soil Extract, sheep excrement leach liquor, chicken manure leach liquor, L-arginine, potassium primary phosphate, medical stone powder, ironic citrate, S3 vitamin solution, casein peptone, glycerine, maltose, lactose, Zulkovsky starch, yeast extract paste, the nutritive ingredients such as diatomite in powder, fertilizer and inorganic fertilizer mix use, nutrition is more comprehensive, balanced, the Chlamydomonas reinhardtii speed of growth and output significantly improve, culture cycle shortens, output improves 280%.
Summary of the invention
In order to overcome the deficiency of current technology, the invention provides a kind of substratum that utilizes urea plant waste water to cultivate Chlamydomonas reinhardtii, fill a prescription as follows:
85 milliliters of urea plant waste water, yeast soaks 10 milligrams, powder, 5 milligrams of Sodium desoxycholates, 12 milligrams of sorbyl alcohols, 8 milligrams, N.F,USP MANNITOL, 4 milligrams of sodium thioglycolates, 1.8 milliliters of soil Extracts, 2.5 milliliters of sheep excrement leach liquors, 1.2 milliliters of chicken manure leach liquors, 3 milligrams of L-arginines, 6 milligrams of potassium primary phosphates, 10 milligrams of medical stone powders, 7 milligrams of ironic citrates, 1.5 milliliters of S3 vitamin solutions, 8 milligrams of casein peptones, 15 milligrams of glycerine, 20 milligrams of maltose, 16 milligrams of lactose, 25 milligrams of Zulkovsky starches, 15 milligrams of yeast extract pastes, 12 milligrams of diatomites in powder, 1000 milliliters of pure fresh water after clorox sterilization,
Wherein S3 vitamin solution formula: 50 milligrams of thiamine salt hydrochlorates, 10 milligrams of Nicotinicum Acidums, 10 milligrams of calcium pantothenate, to 1.0 grams of ammonia M-nitro benzoic acids, 0.1 gram of vitamin H, 0.5 gram of inositol, folic acid 0.2 microgram, 0.3 gram of pyridine between chest, 1000 milliliters of distilled water;
Wherein S3 vitamin solution formula can also be: 50 milligrams of thiamine salt hydrochlorates, 20 milligrams, fused(calcium magnesium)phosphate, 10 milligrams of calcium superphosphate, 10 milligrams of Nicotinicum Acidums, 10 milligrams of calcium pantothenate, to 1.0 grams of ammonia M-nitro benzoic acids, 0.1 gram of vitamin H, 0.5 gram of inositol, folic acid 0.2 microgram, 0.3 gram of pyridine between chest, 1000 milliliters of distilled water;
One utilizes urea plant waste water to cultivate Chlamydomonas reinhardtii method, and concrete steps are as follows:
Culture vessel is home-use mineral water bottle, 20 liters of volumes, there is a water tap near bottom, water white transparency or band light sky blue (market is on sale), wash, rear with distilled water rinse 3 times, 12 liters of substratum described in packing into funnel, fully shake up 5 minutes, select vigorous, the free of contamination Chlamydomonas reinhardtii of vitality to inoculate, inoculum density 4 × 10
4cell/mL, shakes mineral water bottle 5 minutes gently after inoculation, frustule is uniformly distributed; Bottleneck inserts two clean Glass tubings, and one longer, and 10 centimetres of bottlenecks are exposed on top, and lower end is inserted darker, from the bottom of bottle 5 centimetres, in order to inflation; Another root is shorter, and 16 centimetres, bottleneck is exposed at two ends, in order to exhaust; Two same thicknesses of Glass tubing, 6 millimeters of diameters; Finally use cotton ball soaked in alcohol wiping bottleneck, with the sealing of clean scotch tape and two Glass tubings are fixed; Long Glass tubing upper end materials pipe is connected with aerator pump, is filled with mixed gas, and mixed gas composition and volume ratio are air: ammonia: ozone: nitrogen protoxide=97%: 1%: 1%: 1%; Aeration quantity should not be too large, can not float with frustule; Indoor cultivation, air-conditioning temperature control, 25 ± 2 DEG C of optimum tempss, fluorescent lamp irradiates, intensity of illumination 4000-10000lux, light/dark cycle is 16L/8D, i.e. illumination 16 hours, dark 8 hours; Be cultured to the 6th day, stop inflation, open scotch tape, add 3 liters of fresh cultures with funnel from bottleneck, adhesive tape seals again, and inflation continues cultivation, and to the 9th day, Chlamydomonas reinhardtii density was increased to 600 × 10
4cell/mL, stops cultivating, and emits algae liquid from the water tap of bottom, after culture vessel is washed, add 0.2% peracetic acid disinfectant solution soaking sterilization 2 hours, then use distilled water rinse 2 times, after enter next round and cultivate.
Preferably, after culture vessel is washed, do not adopt " add 0.2% peracetic acid disinfectant solution soaking sterilization 2 hours, then use distilled water rinse 2 times, after enter next round and cultivate, " disinfection way, and adopt ", add disinfection of Chinese drug liquid disinfectant 30 minutes, after enter next round cultivate, described Traditional Chinese medicine disinfectant is made up of the material of following weight ratio: the Chinese medicinal materials that adds following weight part in every 100 part of 85% ethanol: 30～50 parts of Root of Indigowoad, 15～25 parts of radix bupleuri, 15～25 parts of peppermints, 5～15 parts of cape jasmines, 5～15 parts of tarragons, disinfection of Chinese drug liquor manufacture method: by fresh Root of Indigowoad, radix bupleuri, mentha leave, tarragon, mountain Cape jasmine fruit chopping, at 60-70 DEG C, dry, be crushed to respectively 70 orders with Chinese medicine material crushing machine more for subsequent use, take respectively 120 grams of the Root of Indigowoad drying and pulverize with table balance, 60 grams of radix bupleuri, 60 grams of mentha leavves, 30 grams of tarragons, 30 grams of mountain Cape jasmine fruits, be placed in after the abundant mixing of Erlenmeyer flask, add 1000 milliliter of 85% ethanol, soak 48 hours, 200 silk cover filterings, clear liquid is staticly settled to 36 hours, skim liquid level floater, get clear liquid at the middle and upper levels, be compound Chinese medicine disinfection solution, be sealed in black reagent bottle, labelled, be placed in 4 DEG C of refrigerators for subsequent use.”。
Preferably, to the described reactor of selecting, after washing, carry out disinfection, use ultraviolet lamp disinfection, sterilization 10-20 minute.
Preferably, to the described reactor of selecting, after washing, carry out disinfection, use high pressure nitrogen to spray sterilization, sterilization 5-10 minute;
Preferably, to the described reactor of selecting, after washing, carry out disinfection, use high-pressure carbon dioxide to spray sterilization, sterilization 5-6 minute;
Preferably, to the described reactor of selecting, point two sterilized phases after washing, first use high pressure nitrogen to spray sterilization, sterilization 2-3 minute; Use high-pressure carbon dioxide to spray sterilization, sterilization 2-3 minute; After sterilization, carry out again algae culture.
Useful result:
In order to solve the deficiency of current technology, having invented one utilizes urea plant waste water to cultivate Chlamydomonas reinhardtii method, can low-cost processes urea plant waste water, can obtain again biological product-Chlamydomonas reinhardtii that Important Economic is worth, turn waste into wealth, turn harm into good, comprehensive utilization benefit is good, can significantly reduce sewage treatment equipment investment and running expense, achieve many things at one stroke, be the optimal selection of urea plant waste water wastewater treatment.Chlamydomonas reinhardtii tradition cultural method is to adopt Tris-Acetate-Phosphate (TAP) substratum and use open cement track pond to cultivate, growth is slow, yield poorly, investment is large, easily pollute, benefit is low, I adopt home-use mineral water bottle to cultivate now, cost is low, be difficult for polluting, scrub conveniently, can reuse, easy handling, and I have increased urea plant waste water in substratum, yeast soaks powder, Sodium desoxycholate, sorbyl alcohol, N.F,USP MANNITOL, sodium thioglycolate, soil Extract, sheep excrement leach liquor, chicken manure leach liquor, L-arginine, potassium primary phosphate, medical stone powder, ironic citrate, S3 vitamin solution, casein peptone, glycerine, maltose, lactose, Zulkovsky starch, yeast extract paste, the nutritive ingredients such as diatomite in powder, fertilizer and inorganic fertilizer mix use, nutrition is more comprehensive, balanced, the Chlamydomonas reinhardtii speed of growth and output significantly improve, culture cycle shortens, output improves 280%.
Brief description of the drawings
Fig. 1 is Chlamydomonas reinhardtii morphological structure figure;
Wherein Reference numeral is: red eyespot 3 chromatoplast 4 pyrenoids 5 nucleus 6 cell walls 7 flagellums of 1 contractile vacuole 2;
Fig. 2 is that one is utilized urea plant waste water cultivation Chlamydomonas reinhardtii method figure;
Wherein Reference numeral is: 8 mineral water bottles, 9 gas-filled valves, 10 vapor pipes, 11 algae liquid, 12 water taps.
Embodiment
Urea plant waste water pretreatment method for wastewater: urea plant waste water needs to carry out pre-treatment before supporting algae, method is: first use 60 object silk cover filtering waste water, remove floating matter, glass bits, hair, silt, leaf, the larger solid substance of plastics bag equal-volume, after sewage is introduced in long 30 meters, wide 20 meters, the cement aeration tank of dark 0.7 meter, it is east-west that pond is, to accept more solar radiation, the depth of water remains on 55 centimetres, (unslaked lime preferably adds the amount of 5 liters of pure water to dissolve in plastic tank by 1 kilogram to unslaked lime in advance to add the amount of 50 grams of unslaked limes evenly to splash by every cubic metre of sewage, dilution, stir, full pool spilling head again), every 60 minutes, (alum preferably dissolves in plastic tank by 100 grams of amounts that add 4 liters of pure water in advance to add the amount full pool spilling head alum of 80 grams by every cubic metre of sewage again, dilute and stir, full pool spilling head again), then stir or air agitation 60 minutes with rake, lime and alum are fully dissolved, mix and with sewage in organism fully there is chemical reaction, then natural sedimentation, fermentation, expose 72 hours, skim the floating matters such as water surface foam, at the bottom of not stirring pond, get sewage at the middle and upper levels, boil 6 minutes, cooling, pack in lighttight black plastic bucket, sealing, 4 DEG C of refrigerators or freezer, preserve, for subsequent use,
1) because of ironic citrate (FeC
6h
5o
75H
2o), compared with indissoluble solution, can add a small amount of tap water low-grade fever on stove and, to 80-90 DEG C, and constantly be stirred to whole thawings;
2) sheep excrement leach liquor preparation method: get 10 kilograms, fresh sheep excrement, add 1 kilogram, wheat bran, 0.5 kilogram of Semen Maydis powder, 1 liter, tap water, fully stir, pack plastics bag fermentation 10 days into, after get 2 kilograms and put into basin, add 6 liters of distilled water, fully stir 15 minutes, leave standstill 100 order silk cover filterings after 7 days, get filtrate heated and boiled 30 minutes, to kill microorganism wherein, cooling, leave standstill 72 hours, get supernatant liquor, be sheep excrement leach liquor, pack grinding port plug reagent bottle into, sealing, is placed in 4 DEG C of refrigerators for subsequent use;
3) clorox sterilization fresh water method: get fresh water, absorbent cotton filters, and by adding the amount of chlorinated lime 50-100 gram to add chlorinated lime in 1 cubic metre of fresh water, makes available chlorine content in fresh water reach 10-20mg/L, inflate after 10-12 hour, use in Sulfothiorine and chlorine residue.Sulfothiorine amount is determined by the number of chlorine residue, until splash into the nondiscoloration of starch potassium iodide liquid;
4) peracetic acid disinfectant liquid and preparation method thereof and application notice: the former liquid hold-up 35% of commercially available Peracetic Acid (by weight), colourless liquid, has intense stimulus smell.When use, apply clean tap water and be diluted to 0.2% solution, soak water tower inwall 2 hours.These product have intense stimulus effect to eyes, skin, mucous membrane and the upper respiratory tract, headache, nauseating and inflammable, and tool explosivity, severe corrosive, strong and stimulating, can cause human body and burn, and is heated to 100 DEG C of i.e. fierce decomposition, meets fiery or be heated, be subject to shake all can detonate.Contact with reductive agent, promotor, organism, combustiblematerials etc. vigorous reaction can occur; Corrodibility is strong, not directly catches tactile, when operation, should be with rubber gloves, mouth mask and glasses, and metal is corrosive, and is not useable for the sterilization of metallic weapon; The long-term placement of stoste can be decomposed, and notes the quality guaranteed period, should store in cool dark place in plastic tank and preserve, away from inflammable substance.
5) Chlamydomonas reinhardtii of inoculation use is purified (this area ordinary method, as long as can realize purification, for example Kai Shi extraction method) in advance, and algae kind is in eugonic exponential phase of growth when guaranteeing to inoculate;
6) various nutritive salt will add by the order providing in formula, to avoid chemical reaction;
7) various inorganics nutritive salt and VITAMIN need be made in advance mother liquor and be placed in 4 DEG C of refrigerator cold-storages and save backup, and do not exceed 45 days storage period;
8) all nutritive salt will be inflated and mix 10 minutes after adding, and inoculates after nutritive salt is mixed;
9) chicken manure leach liquor preparation method: dried poultrymanure, green grass, soya-bean cake, rice bran, grain husk shell (shells of five cereals) and vegetable mould were by 60: 10: 12: the part by weight of 15: 2: 1 mixes, stir, pack airtight fermentation in cylinder into, after 30 days, pour out, at clean water mire, stand lamellar, dry 48 hours, after get 2 kilograms, be placed in plastic bucket, add 10 kilograms, tap water, stir, with 80 order silk cover filterings, clear liquid is boiled 5 minutes, cooling, leave standstill 24 hours, get supernatant liquor, pack reagent bottle into, labelled, be placed in 4 DEG C of refrigerators, for subsequent use,
10) soil Extract preparation method: loose to getting in the woods, be rich in 1 kilogram of organic upper layer of soil, add 5 liters, tap water, boil 20 minutes, mud is poured in beaker, left standstill 24 hours, rear absorption supernatant liquor, pour in Erlenmeyer flask, add cotton plug, boil 15 minutes, cooling, staticly settle 12 hours, get supernatant liquor and obtain soil Extract, pack grinding port plug reagent bottle into, sealing, labelled, be placed in 4 DEG C of refrigerators for subsequent use;
11) medical stone powder is the powder product of medical stone,, is pulverized through screening by medical stone raw ore, is processed into floury, and granular size can be passed through 200 mesh sieve thin,tough silk;
12) pharmaceutical chemicals purity used all needs chemical pure rank;
13) in order to overcome the deficiency of current technology, the invention provides a kind of substratum that utilizes urea plant waste water to cultivate Chlamydomonas reinhardtii, fill a prescription as follows:
14) 85 milliliters of urea plant waste water, yeast soaks 10 milligrams, powder, 5 milligrams of Sodium desoxycholates, 12 milligrams of sorbyl alcohols, 8 milligrams, N.F,USP MANNITOL, 4 milligrams of sodium thioglycolates, 1.8 milliliters of soil Extracts, 2.5 milliliters of sheep excrement leach liquors, 1.2 milliliters of chicken manure leach liquors, 3 milligrams of L-arginines, 6 milligrams of potassium primary phosphates, 10 milligrams of medical stone powders, 7 milligrams of ironic citrates, 1.5 milliliters of S3 vitamin solutions, 8 milligrams of casein peptones, 15 milligrams of glycerine, 20 milligrams of maltose, 16 milligrams of lactose, 25 milligrams of Zulkovsky starches, 15 milligrams of yeast extract pastes, 12 milligrams of diatomites in powder, 1000 milliliters of pure fresh water after clorox sterilization,
Wherein S3 vitamin solution formula: 50 milligrams of thiamine salt hydrochlorates, 10 milligrams of Nicotinicum Acidums, 10 milligrams of calcium pantothenate, to 1.0 grams of ammonia M-nitro benzoic acids, 0.1 gram of vitamin H, 0.5 gram of inositol, folic acid 0.2 microgram, 0.3 gram of pyridine between chest, 1000 milliliters of distilled water;
Wherein S3 vitamin solution formula can also be: 50 milligrams of thiamine salt hydrochlorates, 20 milligrams, fused(calcium magnesium)phosphate, 10 milligrams of calcium superphosphate, 10 milligrams of Nicotinicum Acidums, 10 milligrams of calcium pantothenate, to 1.0 grams of ammonia M-nitro benzoic acids, 0.1 gram of vitamin H, 0.5 gram of inositol, folic acid 0.2 microgram, 0.3 gram of pyridine between chest, 1000 milliliters of distilled water;
15) one utilizes urea plant waste water to cultivate Chlamydomonas reinhardtii method, and concrete steps are as follows:
Foster container is home-use mineral water bottle, 20 liters of volumes, there is a water tap near bottom, water white transparency or band light sky blue (market is on sale), wash, rear with distilled water rinse 3 times, 12 liters of substratum described in packing into funnel, fully shake up 5 minutes, select vigorous, the free of contamination Chlamydomonas reinhardtii of vitality to inoculate, inoculum density 4 × 10
4cell/mL, shakes mineral water bottle 5 minutes gently after inoculation, frustule is uniformly distributed; Bottleneck inserts two clean Glass tubings, and one longer, and 10 centimetres of bottlenecks are exposed on top, and lower end is inserted darker, from the bottom of bottle 5 centimetres, in order to inflation; Another root is shorter, and 16 centimetres, bottleneck is exposed at two ends, in order to exhaust; Two same thicknesses of Glass tubing, 6 millimeters of diameters; Finally use cotton ball soaked in alcohol wiping bottleneck, with the sealing of clean scotch tape and two Glass tubings are fixed; Long Glass tubing upper end materials pipe is connected with aerator pump, is filled with mixed gas, and mixed gas composition and volume ratio are air: ammonia: ozone: nitrogen protoxide=97%: 1%: 1%: 1%; Aeration quantity should not be too large, can not float with frustule; Indoor cultivation, air-conditioning temperature control, 25 ± 2 DEG C of optimum tempss, fluorescent lamp irradiates, intensity of illumination 4000-10000lux, light/dark cycle is 16L/8D, i.e. illumination 16 hours, dark 8 hours; Be cultured to the 6th day, stop inflation, open scotch tape, add 3 liters of fresh cultures with funnel from bottleneck, adhesive tape seals again, and inflation continues cultivation, and to the 9th day, Chlamydomonas reinhardtii density was increased to 600 × 10
4cell/mL, stops cultivating, and emits algae liquid from the water tap of bottom, after culture vessel is washed, add 0.2% peracetic acid disinfectant solution soaking sterilization 2 hours, then use distilled water rinse 2 times, after enter next round and cultivate;
Preferably, after culture vessel is washed, do not adopt " add 0.2% peracetic acid disinfectant solution soaking sterilization 2 hours, then use distilled water rinse 2 times, after enter next round and cultivate, " disinfection way, and adopt ", add disinfection of Chinese drug liquid disinfectant 30 minutes, after enter next round cultivate, described Traditional Chinese medicine disinfectant is made up of the material of following weight ratio: the Chinese medicinal materials that adds following weight part in every 100 part of 85% ethanol: 30～50 parts of Root of Indigowoad, 15～25 parts of radix bupleuri, 15～25 parts of peppermints, 5～15 parts of cape jasmines, 5～15 parts of tarragons, disinfection of Chinese drug liquor manufacture method: by fresh Root of Indigowoad, radix bupleuri, mentha leave, tarragon, mountain Cape jasmine fruit chopping, at 60-70 DEG C, dry, be crushed to respectively 70 orders with Chinese medicine material crushing machine more for subsequent use, take respectively 120 grams of the Root of Indigowoad drying and pulverize with table balance, 60 grams of radix bupleuri, 60 grams of mentha leavves, 30 grams of tarragons, 30 grams of mountain Cape jasmine fruits, be placed in after the abundant mixing of Erlenmeyer flask, add 1000 milliliter of 85% ethanol, soak 48 hours, 200 silk cover filterings, clear liquid is staticly settled to 36 hours, skim liquid level floater, get clear liquid at the middle and upper levels, be compound Chinese medicine disinfection solution, be sealed in black reagent bottle, labelled, be placed in 4 DEG C of refrigerators for subsequent use.”
16) preferably, to the described reactor of selecting, after washing, carry out disinfection, use ultraviolet lamp disinfection, sterilization 10-20 minute.
Preferably, to the described reactor of selecting, after washing, carry out disinfection, use high pressure nitrogen to spray sterilization, sterilization 5-10 minute;
Preferably, to the described reactor of selecting, after washing, carry out disinfection, use high-pressure carbon dioxide to spray sterilization, sterilization 5-6 minute;
Preferably, to the described reactor of selecting, point two sterilized phases after washing, first use high pressure nitrogen to spray sterilization, sterilization 2-3 minute; Use high-pressure carbon dioxide to spray sterilization, sterilization 2-3 minute; After sterilization, carry out again algae culture.
Finally it should be noted that: above embodiment only, in order to technical scheme of the present invention to be described, is not intended to limit; Although the present invention is had been described in detail with reference to previous embodiment, those of ordinary skill in the art is to be understood that: its technical scheme that still can record previous embodiment is modified, or part technical characterictic is wherein equal to replacement; And these amendments or replacement do not make the essence of appropriate technical solution depart from the spirit and scope of embodiment of the present invention technical scheme.
Claims (2)
1. utilize urea plant waste water to cultivate a substratum for Chlamydomonas reinhardtii, it is characterized in that formula is as follows:
85 milliliters of urea plant waste water, yeast soaks 10 milligrams, powder, 5 milligrams of Sodium desoxycholates, 12 milligrams of sorbyl alcohols, 8 milligrams, N.F,USP MANNITOL, 4 milligrams of sodium thioglycolates, 1.8 milliliters of soil Extracts, 2.5 milliliters of sheep excrement leach liquors, 1.2 milliliters of chicken manure leach liquors, 3 milligrams of L-arginines, 6 milligrams of potassium primary phosphates, 10 milligrams of medical stone powders, 7 milligrams of ironic citrates, 1.5 milliliters of S3 vitamin solutions, 8 milligrams of casein peptones, 15 milligrams of glycerine, 20 milligrams of maltose, 16 milligrams of lactose, 25 milligrams of Zulkovsky starches, 15 milligrams of yeast extract pastes, 12 milligrams of diatomites in powder, 1000 milliliters of pure fresh water after clorox sterilization,
Wherein S3 vitamin solution formula: 50 milligrams of thiamine salt hydrochlorates, 10 milligrams of Nicotinicum Acidums, 10 milligrams of calcium pantothenate, to 1.0 grams of ammonia M-nitro benzoic acids, 0.1 gram of vitamin H, 0.5 gram of inositol, folic acid 0.2 microgram, 0.3 gram of pyridine between chest, 1000 milliliters of distilled water.
2. utilize urea plant waste water to cultivate a Chlamydomonas reinhardtii method, concrete steps are as follows:
Culture vessel is home-use mineral water bottle, 20 liters of volumes, there is a water tap near bottom, water white transparency or band light sky blue, wash, rear with distilled water rinse 3 times, pack 12 liters of substratum claimed in claim 1 into funnel, fully shake up 5 minutes, select vigorous, the free of contamination Chlamydomonas reinhardtii of vitality to inoculate, inoculum density 4 × 10
4cell/mL, after inoculation, shake gently mineral water bottle 5 minutes, frustule is uniformly distributed, bottleneck inserts two clean Glass tubings, one longer, 10 centimetres of bottlenecks are exposed on top, lower end is inserted darker, from at the bottom of bottle 5 centimetres, in order to inflation, another root is shorter, 16 centimetres, bottleneck is exposed at two ends, in order to exhaust, two same thicknesses of Glass tubing, 6 millimeters of diameters, finally use cotton ball soaked in alcohol wiping bottleneck, with the sealing of clean scotch tape and two Glass tubings are fixed, long Glass tubing upper end materials pipe is connected with aerator pump, be filled with mixed gas, mixed gas composition and volume ratio are air: ammonia: ozone: nitrogen protoxide=97%: 1%: 1%: 1%, aeration quantity should not be too large, can not float indoor cultivation, air-conditioning temperature control with frustule, 25 ± 2 DEG C of optimum tempss, fluorescent lamp irradiates, intensity of illumination 4000-10000Lux, light/dark cycle is 16L/8D, i.e. illumination 16 hours, dark 8 hours, be cultured to the 6th day, stop inflation, open scotch tape, add 3 liters of fresh cultures from bottleneck with funnel, adhesive tape seals again, inflation, continue to cultivate, to the 9th day, Chlamydomonas reinhardtii density was increased to 600 × 10
4cell/mL, stops cultivating, and emits algae liquid from the water tap of bottom, after culture vessel is washed, add 0.2% peracetic acid disinfectant solution soaking sterilization 2 hours, then use distilled water rinse 2 times, after enter next round and cultivate.
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