CN104388315A - Scnedesmus quadricauda for efficiently treating typical domestic sewage, and culture method and application thereof - Google Patents

Scnedesmus quadricauda for efficiently treating typical domestic sewage, and culture method and application thereof Download PDF

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CN104388315A
CN104388315A CN201410625552.1A CN201410625552A CN104388315A CN 104388315 A CN104388315 A CN 104388315A CN 201410625552 A CN201410625552 A CN 201410625552A CN 104388315 A CN104388315 A CN 104388315A
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quadricauda
scenedesmus quadricauda
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裴海燕
宋明明
胡文容
韩琳
张硕
韩飞
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Abstract

The invention discloses a Scnedesmus quadricauda for efficiently treating typical domestic sewage, and a culture method and application thereof. Scnedesmus quadricauda SDEC-8 is cultured in a culture medium prepared according to the nitrogen and phosphorus concentrations of the typical domestic sewage, and is collected in the stabilization period. The result proves that the growth rate, oil content and biodiesel yield of the Scnedesmus quadricauda SDEC-8 in the prepared typical domestic sewage are respectively higher than those of the Scnedesmus quadricauda SDEC-8 cultured in a BG11 culture solution. When the total nitrogen and total phosphorus concentrations of the domestic sewage are respectively 40mg.L<-1> and 8mg.L<-1>, the oil content, fatty acid methyl ester content and fatty acid methyl ester yield of the Scnedesmus quadricauda SDEC-8 are maximum, and the biodiesel has the best properties. Besides, after the prepared typical domestic sewage is treated by the Scnedesmus quadricauda SDEC-8, the nitrogen and phosphorus can be efficiently removed, and the effluent water can reach Grade A (TN<15mg.L<-1>, and TP<0.5mg.L<-1>) in Discharge Standard of Pollutants for Municipal Sewage Treatment Plant. By using the typical domestic sewage to culture the Scnedesmus quadricauda SDEC-8, the nitrogen and phosphorus are efficiently removed, abundant microalgae biodiesel is efficiently obtained, and the microalgae culture cost is lowered.

Description

The grid algae of the typical sanitary sewage of a kind of efficient process and cultural method thereof and application
Technical field
The invention belongs to technical field of microalga biology, particularly the grid algae of the typical sanitary sewage of a kind of efficient process and cultural method thereof and application
Background technology
In recent years; because worldwide petrochemical rate of fuel consumption amount is increased sharply; the minimizing of petrochemical industry reserves; cause worldwide energy dilemma; add the environmental problem that fossil fuel burning and exhausting carbonic acid gas brings; therefore, the Renewable green energy finding a kind of cheapness has become Science & Society's problem of countries in the world government and common people's extensive concern.Research finds, it is high that some algae has oleaginousness, is easy to cultivate, yield per unit is large, do not strive with agricultural etc. advantage, be regarded as a new generation, or even uniquely can realize the biodiesel raw material of substitute fossil fuels.The lipid acid of its 16 carbon contained and 18 carbon is the most favourable for the production of biofuel.But production cost height is the principal element of restriction microalgae biodiesel scale production.And the algae kind that screening acquisition is easily cultivated, growth is fast, produce oil is high is the primary step overcoming micro-algae large-scale production bottleneck.As far back as the seventies in last century, the U.S. just starts the aquatic organism species project that cost 2,500 ten thousand dollars is operated by National Renewable Energy laboratory (NREL).NREL is separated to more than 3000 kind of algae from ocean and lake, therefrom filter out growth fast, comprise algae kind kinds more than 300 such as diatom, green alga and blue-green algae containing high, but unresolved Cost Problems at that time.The investigator of other various countries also utilizes various means to screen the excellent algae kind found and can adapt to commercially produce energetically in the world.In recent years, domestic Duo Jia laboratory started respectively the Department of Science and Technology, Natural Science Fund In The Light entrust and large-lot producer subsidy under, carry out the work gathering and screen the strain of high produce oil algae on a large scale, and have made some progress.The micro-algae being selected from physical environment often has certain resistance in temperature, illumination and salinity adaptation, is suitable for mass propgation and produces.Simultaneously for reduce production cost further, utilize waste water and gas to cultivate study hotspot and difficult point that micro-algae starts to become the micro-algae development of resources of current energy source.Algae utilizes waste gas and waste water by photosynthesis, not only reaches the object that waste water and gas reduces discharging, but also can cultivate micro-algae to reduce production cost by utilizing waste water and gas, has the feasibility that commercialization reduces discharging exploitation.
At present, the body eutrophication caused due to nitrogen and phosphorus pollution has become one of global question of common concern.Denitrogenation dephosphorizing has become a ring important in sewage disposal.Traditional physics, chemical denitrification dephosphorization technique are difficult to reach efficient in sewage disposal application, economy, low consumption requirement.Because algae has the advantage that photosynthetic rate is high, growth velocity is fast, environmental compatibility is strong, adsorption is fast, utilize it to dispose of sewage and can realize efficient, nutritive substance such as low cost removal nitrogen, phosphorus etc., traditional wastewater treatment process can be overcome easily cause secondary pollution if be used to algae, the drawbacks such as potential nutritive substance is lost, resource can not utilize completely.Therefore, the research direction that algae purification sewage is just becoming important in sewage disposal is utilized.
Therefore, if the novel method that a kind of be coupled sewage production microalgae biodiesel and micro-algae purify waste water can be sought, can sewage disposal be realized, algae bio mass-energy source can be gathered in the crops again simultaneously, there is very important application prospect.
Summary of the invention
The object of the invention is just to overcome of the prior art above not enough, provides the grid algae of the typical sanitary sewage of a kind of efficient process and cultural method thereof and application.The method both can improve microalgae biodiesel productive rate and quality, reduction microdisk electrode cost, can realize again sewage recycling and utilize.The typical sanitary sewage of preparation is after Scenedesmus quadricauda SDEC-8 process, and nitrogen phosphorus can realize efficient removal, and water outlet can reach " urban wastewater treatment firm pollutant emission standard " (GB 18918-2002) one-level A limit value TN<15mgL -1, TP<0.5mgL -1.Technical scheme is as follows:
The present invention provide firstly the grid algae of the typical sanitary sewage of a kind of efficient process, be located away from the eutrophic lake of Quan Cheng park, Jinan City, called after Scenedesmus quadricauda SDEC-8, be preserved in China typical culture collection center, its deposit number is: CCTCC NO:M 2014448.Preservation date: on September 28th, 2014.Address: Wuhan, China typical culture collection center (Wuhan University); Postcode: 430072; Deposit number: CCTCC NO:M 2014448.Scenedesmus quadricauda SDEC-8 is rich in high-quality grease, and fat content can reach 31.8% of dry cell weight, and in lipid acid, saturated fatty acid can reach 80%, can be used as biodiesel raw material.High hexadecane value (61.38) and low iodine number (29.38gI2/100g) determine with the micro-algae of this strain as biofuel prepared by raw material has good oxidation stability.
In an aspect, the invention provides a kind of Scenedesmus quadricauda SDEC-8 artificial sewage nutrient solution, its composition and consumption are (NH 4) 2cO 368.5 ~ 291.1mgL -1, K 2hPO 422.5 ~ 84.3mgL -1, MgSO 47H 2o 75mgL -1, CaCl 22H 2o 36mgL -1, citric acid 6mgL -1, ferric ammonium citrate 6mgL -1, EDTANa 21mgL -1, Na 2cO 320mgL -1, H 3bO 32.86mgL -1, MnCl 24H 2o 1.86mgL -1, ZnSO 47H 2o 0.22mgL -1, Na 2moO42H 2o 0.39mgL -1, CuSO 45H 2o 0.08mgL -1, Co (NO 3) 26H 2o 0.05mgL -1, initial pH value is between 7 ~ 8, and postvaccinal algae cell density is 0.35 ~ 0.40 × 10 6cells.mL -1.
Preferably, described Media Components and consumption are: (NH 4) 2cO 3137mgL -1, K 2hPO 445mgL -1, MgSO 47H 2o 75mgL -1, CaCl 22H 2o 36mgL -1, citric acid 6mgL -1, ferric ammonium citrate 6mgL -1, EDTANa 21mgL -1, Na 2cO 320mgL -1, H 3bO 32.86mgL -1, MnCl 24H 2o 1.86mgL -1, ZnSO 47H 2o 0.22mgL -1, Na 2moO42H 2o 0.39mgL -1, CuSO 45H 2o 0.08mgL -1, Co (NO 3) 26H 2o 0.05mgL -1, initial pH value=7.0, postvaccinal algae cell density is 0.38 × 10 6cells.mL -1.Be (NH in concentration sanitary sewage in preparation 4) 2cO 3concentration is 137mgL -1, K 2hPO 4concentration is 45mgL -1time, the grease productive rate that Scenedesmus quadricauda SDEC-8 obtains is 53.84mgL -1d -1, fatty acid methyl ester productive rate 17.53mgL -1d -1
In one aspect of the method, the invention provides a kind of application of Scenedesmus quadricauda SDEC-8 artificial sewage nutrient solution, described nutrient solution cultivates Scenedesmus quadricauda SDEC-8, obtains Scenedesmus quadricauda SDEC-8 cell.
In an embodiment scheme, the fatty acid methyl ester of described Scenedesmus quadricauda SDEC-8 cell is 9.55mgg -1, saturated fatty acid content is 80%, and cetane value is 61.38, and iodine number is 29.38gI 2/ 100g.Cultivate after 18 days, centrifugation results Scenedesmus quadricauda SDEC-8 biomass, algae powder, after 60 DEG C of cryodryings, carries out detection to fatty acid component wherein and analyzes, and finds to use typical sanitary sewage ((NH 4) 2cO 3137mgL -1, K 2hPO 445mgL -1) the Scenedesmus quadricauda SDEC-8 that cultivates is rich in optimum fatty acid component C18:1, be greater than the C18:1 content of other bibliographical informations, and the low temperature flowability of Scenedesmus quadricauda SDEC-8 biofuel is greatly improved, the common Scenedesmus quadricauda SDEC-8 therefore using this method to turn out is a kind of potential high-quality biodiesel raw material.
Present invention also offers a kind of cultural method cultivating Scenedesmus quadricauda SDEC-8, comprise the steps:
A) be inoculated in BG11 substratum by Scenedesmus quadricauda SDEC-8 and carry out sterile culture, obtained Scenedesmus quadricauda SDEC-8 seed liquor, culture condition is temperature 25 ± 1 DEG C, illumination 2000-3000Lux;
B) according to typical sanitary sewage water quality, preparation artificial sewage, its composition and consumption are (NH 4) 2cO 368.5 ~ 291.1mgL -1, K 2hPO 422.5 ~ 84.3mgL -1, MgSO 47H 2o 75mgL -1, CaCl 22H 2o 36mgL -1, citric acid 6mgL -1, ferric ammonium citrate 6mgL -1, EDTANa 21mgL -1, Na 2cO 320mgL -1, H 3bO 32.86mgL -1, MnCl 24H 2o 1.86mgL -1, ZnSO 47H 2o 0.22mgL -1, Na 2moO42H 2o 0.39mgL -1, CuSO 45H 2o 0.08mgL -1, Co (NO 3) 26H 2o 0.05mgL -1, and by after its pH regulator to 7.0 in 120 DEG C of high-temperature sterilization 30min, obtain typical sanitary sewage nutrient solution;
C) be inoculated in typical sanitary sewage nutrient solution by obtained Scenedesmus quadricauda SDEC-8 seed liquor, postvaccinal algae cell density is about 0.35 ~ 0.40 × 10 6cells.mL -1, constant temperature culture of ventilating under whole system continuous illumination condition, culture cycle is 18 days, and after reaching selected incubation time, centrifugal namely obtaining cultivates rear Scenedesmus quadricauda SDEC-8 cell.
Preferably, step c) middle culture condition: culture temperature is 25 ± 1 DEG C, and illumination is the continuous illumination of 2500Lux, and aeration vapour-liquid ratio is 0.2vvm.Grease productive rate and the biodiesel production rate of Scenedesmus quadricauda SDEC-8 acquisition are the highest, are respectively 53.84mg.L -1d -1, 17.53mg.L -1d -1, after inoculation the 3rd day, nitrogen phosphorus ligands was obvious, and total nitrogen total phosphorus clearance reaches 74.46%, 65.80% respectively, and when being cultured to the 18th day, in sewage, nitrogen phosphorus is almost removed completely.
We are bright provides the application of Scenedesmus quadricauda SDEC-8 in process polluted lake.Scenedesmus quadricauda SDEC-8 carries out the application of the purification of nitrogenous and/or phosphorus-containing wastewater.The production application of Scenedesmus quadricauda SDEC-8, for the production of the application of high-efficiency grease, lipid acid, protein, starch, pigment, polysaccharide and/or nucleic acid.Scenedesmus quadricauda SDEC-8 essentially comprising the lipid acid such as C14:0, C16:0, C18:0 and C18:1, and C16 and C18 system content is up to 96%, the wherein rich content of C16:0 and C18:1, analyzes from above lipid acid composition the requirement meeting preparation biofuel.
Beneficial effect
1. the present invention simulates typical sanitary sewage and cultivates Scenedesmus quadricauda SDEC-8, provide a kind of new way of sewage recycling efficiency utilization, nitrogen phosphorus efficiency obtains microalgae biodiesel while removing in large quantities, greatly reduce the comprehensive cost of Scenedesmus quadricauda SDEC-8 production and wastewater treatment, improve grease productive rate and the yield of biodiesel of Scenedesmus quadricauda SDEC-8, bring larger economic benefit.
2. the present invention simulates typical sanitary sewage and cultivates Scenedesmus quadricauda SDEC-8; not only increase the grease productive rate of Scenedesmus quadricauda SDEC-8; also improve the biodiesel quality of Scenedesmus quadricauda SDEC-8; under good Oxidation Stability; optimize low temperature flowability, the large-scale production for biofuel provides a kind of high-quality biodiesel raw material.
Accompanying drawing explanation
Fig. 1: Scenedesmus quadricauda SDEC-8 phylogenetic relationship tree in position.
The micromorphology photo of Fig. 2: Scenedesmus quadricauda SDEC-8.
The pcr amplification product electrophoresis photographs of Fig. 3: 18S rRNA.
Fig. 4: Scenedesmus quadricauda SDEC-8 growth curve in three different concentration of nitrogen and phosphorus sewage.
Fig. 5: Scenedesmus quadricauda SDEC-8 denitrification effect in three different concentration of nitrogen and phosphorus sewage.
Fig. 6: Scenedesmus quadricauda SDEC-8 phosphor-removing effect in three different concentration of nitrogen and phosphorus sewage.
Wherein, 1 light micrograph, 6 μm, 2 scanning electron photomicrographs, scale, 3,4 transmission electron micrographs, scale 500nm.c, chloroplast(id); M, plastosome; N, nucleus; Py, pyrenoids; S, starch small grain; V, vacuole; A represents Scenedesmus quadricauda SDEC-8.
Embodiment
Mode by the following examples further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally selects with condition.
Embodiment 1 Scenedesmus quadricauda SDEC-8 isolation identification:
One, sample collecting
In July, 2012 contains the water sample of green alga from the eutrophic lake collection of Quan Cheng park, Jinan City, Shandong Province.
Two, the separation and purification of Scenedesmus quadricauda SDEC-8
Purification procedures: by blood cell plate counting process under the microscope to Botanical gardens water sample counting, liquid-tight for algae degree is diluted to about 3000/ml.On ultra-clean experiment table, BG11 solid culture liquid is down flat plate (placed and spent the night, without varied bacteria growing), algae liquid to be separated use laryngeal atomizer is sprayed on respectively on the flat board marked, then build, be placed in incubator and cultivate more than ten skies, the algal community of isolation mutually just can be there is on substratum, and by microscopy Chun Zao group, then be grafted directly to the transfering loop of sterilizing and nutrient solution be housed and in the 100ml Erlenmeyer flask of sterilizing, with gauze and sterile film sealing, cultivate.BG11; PH controls 7.1.Above-mentioned substratum as made semisolid medium, then adds agar 5 ~ 6 ‰.Examine under a microscope frustule whether to be purified, if having assorted algae by the further separation and purification of 96 orifice plate of the frustule improvement of enlarged culturing: shake well substratum, as frustule becomes polymer to make frustule colony break for unicellular as far as possible.Under the microscope with cell counting count board counting (again counting after need diluting when concentration is large), according to counted algae cell density, nutrient solution is diluted to every 50 μ l containing 0.9 frustule unit.After abundant mixing, draw 50 μ L nutrient solutions successively in 96 well culture plate holes with liquid-transfering gun, be placed with fresh BG11 nutrient solution (300-500 μ l) in each culture hole in advance, add a cover and with sealed membrane, culture plate is sealed, prevent from evaporating.Cultivation plate hole is cultivated in illumination box, after 6 ~ 12h, examines under inverted microscope and mark slender hilum.Slender hilum inner cell to be marked increase to more than 500 or there is color time, can switching enlarged culturing be carried out.Select the hole liquid-transfering gun of single cells grown to be transferred to by its culture in the culturing bottle of the fresh culture that 200mL is housed and carry out enlarged culturing.Obtain the pure algae strain of a strain, called after SDEC-8.
Three, the qualification of Scenedesmus quadricauda SDEC-8
1. the identification of morphology of Scenedesmus quadricauda SDEC-8
The algae of Scenedesmus quadricauda SDEC-8 falls morphological specificity: after cultivating on culture medium flat plate, can be observed green algae and fall, rounded, neat in edge, smooth, glossy.
The morphological features of Scenedesmus quadricauda SDEC-8: observe its form and meet Scenedesmus feature under microscope and scanning electron microscope, there is sheet chloroplast(id), frustule true property coenobium is flat, is made up of 2-4 cell, and colony's cell straight line arranged side by side forms a line; Cell Long Circle, avette, the upper and lower two ends of cell extensively circle, the straight or slightly bending thorn that outside colony, each tool in two ends up and down of cell is outwards oblique, cell walls is more level and smooth.Cell colony length and width depend on growth period, general long 9-26 μm, wide 8-14 μm.Transmission electron microscope observing result shows, and containing an independent chloroplast(id) in each frustule, and occupies half cell, comprises a pyrenoids clearly, by starch small grain bag quilt around pyrenoids in chloroplast(id).Vacuole not of uniform size can be found in cell, the irregular oily mater of Dark grey can be seen in some vacuoles.As Fig. 2;
2. the Molecular Identification of Scenedesmus quadricauda SDEC-8
(1) pcr amplification of 18S rRNA and order-checking:
Laboratory apparatus: compact centrifuge (Eppendorf, rotating speed >12000r/min); Electrophoresis apparatus (Liuyi Instruments Plant, Beijing); PCR thermal cycling amplification instrument (Eppendorf); Gel imaging instrument (Bio-Rad company of the U.S.).
Experimental technique: according to RNA isolation kit, centrifugal and extract the STb gene of algae kind from proper amount of fresh algae liquid, 18S rRNA amplification is carried out to DNA sample.Amplimer is as follows.
Forward primer is: 18S-F (CCTGGTTGATCCTGCCAG);
Reverse primer is: 18S-R (TTGATCCTTCTGCAGGTTCA).
PCR reaction is carried out in 50 μ L systems.Consisting of of reaction system: template DNA 1 μ L; 1.0U Taq PCR mixture; Forward primer 2 μ L; Reverse primer 0.5 μ L; Distilled water 20 μ L.
Pcr amplification condition: 94 DEG C of sex change 4min; 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1.5min.Circulate 30 times; 72 DEG C extend 10min, 4 DEG C of placements.The 18S rRNA amplified production of sepharose with 1% to bacterial strain does electrophoresis detection, cuts adhesive tape after checking, reclaims test kit (Shanghai Sheng Gong biotechnology company limited) purified pcr product with DNA gel.Pcr amplification product after recovery entrusts Shanghai Sheng Gong biotechnology company limited to check order.
18S rRNA sequential analysis and Phylogenetic Analysis:
The pcr amplification product electrophoresis photographs of 18S rRNA as shown in Figure 3.By contrasting with both sides Marker, the fragment length of known target amplification product is about 1.8kb.
The 18S rRNA length obtaining Scenedesmus quadricauda SDEC-8 after order-checking is the sequence of 1054bp, be submitted to Genbank and (accession number is KF999643) is compared in other algae strains, find that the evolutionary distance of Scenedesmus quadricauda SDEC-8 and Scenedesmus sp. is the most close, determine that it belongs to Scenedesmus, identify in conjunction with microscopic morphology, preliminary identification its be Scenedesmus quadricauda, called after Scenedesmus quadricauda SDEC-8.
Based on the result of identification of morphology and Molecular Identification, SDEC-8 is accredited as Scenedesmus quadricauda (Scenedesmus quadricauda SDEC-8), be preserved in China typical culture collection center, its deposit number on September 28th, 2014: CCTCC NO:M 2014448.Address: Wuhan, China typical culture collection center (Wuhan University); Postcode: 430072
The BG11 culture medium culturing of embodiment 2 Scenedesmus quadricauda SDEC-8
In order to the growth making Scenedesmus quadricauda SDEC-8 faster and better, BG-11 medium base is cultivated.
(1) use substratum: BG11 medium component is as shown in table 1, and before using, pH controls 7.1,120 DEG C, sterilizing 30 minutes.
Table 1BG11 medium component
Mother liquor is 1L distilled water, regulates PH to sterilizings after 7.1, for subsequent use with 1M NaOH or HCl.
(2) culture condition: the ratio of the algae seedling solution of preservation 1:3 is by volume added nutrient solution, be positioned in illumination box and cultivate 7 days, temperature is 25 ± 1 DEG C, full exposure, and intensity of illumination 2000-3000Lux can obtain algae strain.
The optimization of embodiment 3 Scenedesmus quadricauda SDEC-8 process typical sanitary sewage growth conditions
In order to the growth making Scenedesmus quadricauda SDEC-8 faster and better, after the condition of embodiment 2 and culture medium culturing Scenedesmus quadricauda SDEC-8, continue to cultivate on typical sanitary sewage basis, prepare high ((NH 4) 2cO 3291.1mgL -1, K 2hPO 484.3mgL -1), in ((NH 4) 2cO 3137mgL -1, K 2hPO 445mgL -1), low ((NH 4) 2cO 368.5mgL -1, K 2hPO 422.5mgL -1) artificial sewage of three kinds of different concentration of nitrogen and phosphorus, the quality of three kinds of culture condition is assessed.
(1) sanitary sewage preparation: according to typical sanitary sewage water quality, prepare high ((NH 4) 2cO 3291.1mgL -1, K 2hPO 484.3mgL -1), in ((NH 4) 2cO 3137mgL -1, K 2hPO 445mgL -1), low ((NH 4) 2cO 368.5mgL -1, K 2hPO 422.5mgL -1) artificial sewage of three kinds of different concentration of nitrogen and phosphorus, other compositions and the following MgSO of content 47H 2o 75mgL -1, CaCl 22H 2o 36mgL -1, citric acid 6mgL -1, ferric ammonium citrate 6mgL -1, EDTANa 21mgL -1, Na 2cO 320mgL -1, H 3bO 32.86mgL -1, MnCl 24H 2o 1.86mgL -1, ZnSO 47H 2o 0.22mgL -1, Na 2moO42H 2o 0.39mgL -1, CuSO 45H 2o 0.08mgL -1, Co (NO 3) 26H 2o 0.05mgL -1, regulate its pH to neutral, then carry out high-temperature sterilization half an hour; The liquid seeds liquid obtained by above-mentioned example is inoculated in the sanitary sewage of preparation;
(2) culture condition: 25 ± 1 DEG C, illumination is the continuous illumination of 2500Lux, aeration vapour-liquid ratio 0.2vvm, and culture cycle is 18 days, harvested by centrifugation frustule after 18 days, obtains microalgae biomass.
(3) mensuration of biomass: sample 10ml every day and monitor algae cell density, determine the growth curve of algae, after frustule growth phase enters stationary phase, algae liquid is gathered in the crops.Determine to gather in the crops volume, with 50ml centrifuge tube splendid attire algae liquid, put into large capacity refrigerated centrifuge and carry out centrifugal, remove supernatant liquor and retain bed mud Scenedesmus quadricauda, obtain algae powder at 60 DEG C of oven drying at low temperatures.Biomass calculates as equation:
In formula: m-algae dry weight, g;
V-algae liquid amasss, L.
(4) fat content measures:
Take 0.1g dry algae powder in 50ml centrifuge tube, add 10ml chloroform/methanol (2:1) solution, with the broken 10min of Ultrasonic Cell Disruptor, then 4000r/min centrifugation 10min, retain organic phase, organic phase transferred in separating funnel, whole leaching process repeats twice.According to the organic phase volume be separated, add the sodium chloride solution (1:5) of 0.9%, fully shake up 1min, and stratification 15min, reclaim lower floor's grease extract solution and measure its volume, get 5ml and reclaim solution in 10ml glass test tube, dry up with nitrogen, Glass tubing is positioned in 80 DEG C of baking ovens and is dried to weight (about 30 minutes).Equation for Calculating fat content:
LW = ( m 2 - m 0 ) &times; V 5 &times; m 1 - - - ( 2 )
In formula: LW-based on the fat content under dry weight, g/g;
M 1-algae dried bean noodles weight, g;
M 0-10ml Glass tubing dry weight, g;
M 2-with the 10ml Glass tubing dry weight of grease, g;
V-grease volume, ml.
(5) Fatty Acid Methyl Esters analysis:
Take algae powder (200mg) Duplicate Samples of equal in quality, put into centrifuge tube, add the solid acid of algae opaque amount 10%; Add methyl alcohol 2mL water-bath 40 minutes under 60 degree, then each sample adds the 4%KOH-methanol solution with methanol phase same volume 2ml, and water-bath 60 minutes; Mixed solution is poured into from centrifuge tube in the Glass tubing (measuring the 10mL point end tool cover glass pipe of chlorophyll) with a scale of having dried, add 1mL normal hexane, ultrasonication 10 minutes; After layering, get supernatant liquor 200 μ L in the tool plug glass tubule of having dried, add mark liquid (2mg/mL) in 25 μ L methyl margarates in each sample, sealing, get 1 μ L and carry out gas chromatography combined with mass spectrometry (GC-MS) mensuration; Remaining fatty acid methyl ester, shifts out with liquid-transfering gun, and metered volume.Gas chromatographicanalyzer device: Trace GC-DSQII (thermoelectricity) gas chromatographicanalyzer; Chromatographic column: VF-23ms quartz capillary column, its specification is 30m × 0.25mm × 0.25 μm; Splitting ratio: 50:1; Injector temperature: 210 DEG C; Helium flow velocity: constant current, 1mL/min; Heating schedule:: 150 DEG C keep 1min, are raised to 165 DEG C with the speed of 1 DEG C of per minute; Ion source temperature: 250 DEG C; Sample size: l μ L; Data are by NIST mass spectrum database analysis.Adopt internal standard quanitation.
Embodiment 4 Scenedesmus quadricauda SDEC-8 biofuel properties evaluations
One, the cultivation of Scenedesmus quadricauda SDEC-8
The condition optimized according to embodiment 2,3 and culture medium culturing Scenedesmus quadricauda SDEC-8, culture temperature 25 ± 1 DEG C, illumination
For the continuous illumination of 2500Lux, cultivate 18 days under aeration vapour-liquid ratio 0.2vvm condition.
Two, the determination of grid algae grease productive rate and fatty acid methyl ester productive rate
Average degree of unsaturation according to following formulae discovery algae fatty acid methyl ester admixture:
ADU＝∑M╳Y i
Wherein, ADU refers to the average degree of unsaturation of algae oil;
Y irefer to the massfraction of each fatty acid component;
M is the number of carbon-carbon double bond in each fatty acid component.
And calculating biofuel character as viscosity, density, cloud point, cetane value, iodine number and calorific value by biofuel degree of unsaturation, their relation is respectively by following formulate.
y＝－0.6316x+5.2065
y＝0.0055x+0.8726
y＝－13.356x+19.994
y＝－6.6684x+62.876
y＝74.373x+12.71
y＝1.7601x+38.534
With reference to " water and waste water determination method " (the 4th edition) that China Environmental Science Press publishes, according to TN: peroxide potassium sulfate-ultraviolet spectrophotometry; TP, ammonium molybdate-ultraviolet spectrophotometry.Every day, the total phosphorus and total nitrogen content to the algae liquid cultivated in each concentration waste water measured, until algae liquid results.Total nitrogen total phosphorus results change is as Fig. 5,6.
Result is as shown in table 2, is (NH in preparation in concentration sanitary sewage 4) 2cO 3concentration is 137mgL -1, K 2hPO 4concentration is 45mgL -1time, the grease productive rate that Scenedesmus quadricauda SDEC-8 obtains is 53.84mgL -1d -1, fatty acid methyl ester productive rate 17.53mgL -1d -1, in contrasting with BG11 and other concentration sewage, grease productive rate is compared with fatty acid methyl ester productive rate, and middle concentration sanitary sewage is the accumulation that substratum greatly facilitates Scenedesmus quadricauda SDEC-8 biofuel.Simultaneously optimum biodiesel fuel component C18:1 in preparation under concentration sewage up to 31.80%, be greater than the C18:1 content of other bibliographical informations.As shown in table 3, compare with BG11 and the contrast of other concentration sewage, in utilization, concentration sewage is cultivated the Scenedesmus quadricauda SDEC-8 obtained and is improve biodiesel quality, keeping under high hexadecane value (59.54) the i.e. condition of good oxidation stability, reduce cloud point temperature, namely optimize low temperature flowability.In Scenedesmus quadricauda SDEC-8 is inoculated in concentration sewage the 3rd day, nitrogen phosphorus ligands was obvious, and total nitrogen total phosphorus clearance reaches 74.46%, 65.80% respectively, and when being cultured to the 18th day, in sewage, nitrogen phosphorus is almost removed completely.Water outlet can reach " urban wastewater treatment firm pollutant emission standard " (GB 18918-2002) one-level A limit value TN<15mgL -1, TP<0.5mgL -1.
Table 2 Scenedesmus quadricauda SDEC-8 grease productive rate under difficult environmental conditions and fatty acid methyl ester productive rate
The biofuel character of table 3 Scenedesmus quadricauda SDEC-8 and the contrast of biofuel, ASTM biofuel and EN Biodiesel Standards
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.

Claims (10)

1. one kind efficiently processes the grid algae of typical sanitary sewage, it is characterized in that, be located away from eutrophic lake, called after Scenedesmus quadricauda SDEC-8, be preserved in China typical culture collection center, its deposit number: CCTCCNO:M 2014448.
2. a Scenedesmus quadricauda SDEC-8 artificial sewage nutrient solution as claimed in claim 1, is characterized in that: composition and consumption are (NH 4) 2cO 368.5 ~ 291.1mgL -1, K 2hPO 422.5 ~ 84.3mgL -1, MgSO 47H 2o 75mgL -1, CaCl 22H 2o 36mgL -1, citric acid 6mgL -1, ferric ammonium citrate 6mgL -1, EDTANa 21mgL -1, Na 2cO 320mgL -1, H 3bO 32.86mgL -1, MnCl 24H 2o 1.86mgL -1, ZnSO 47H 2o 0.22mgL -1, Na 2moO42H 2o 0.39mgL -1, CuSO 45H 2o 0.08mgL -1, Co (NO 3) 26H 2o 0.05mgL -1, initial pH value is between 7 ~ 8, and postvaccinal algae cell density is 0.35 ~ 0.40 × 10 6cells.mL -1.
3. a Scenedesmus quadricauda SDEC-8 artificial sewage nutrient solution as claimed in claim 2, is characterized in that: Media Components and consumption are: (NH 4) 2cO 3137mgL -1, K 2hPO 445mgL -1, MgSO 47H 2o 75mgL -1, CaCl 22H 2o 36mgL -1, citric acid 6mgL -1, ferric ammonium citrate 6mgL -1, EDTANa 21mgL -1, Na 2cO 320mgL -1, H 3bO 32.86mgL -1, MnCl 24H 2o 1.86mgL -1, ZnSO 47H 2o 0.22mgL -1, Na 2moO42H 2o 0.39mgL -1, CuSO 45H 2o 0.08mgL -1, Co (NO 3) 26H 2o 0.05mgL -1, initial pH value=7.0, postvaccinal algae cell density is 0.38 × 10 6cells.mL -1.
4. an application for Scenedesmus quadricauda SDEC-8 artificial sewage nutrient solution as claimed in claim 3, is characterized in that: described nutrient solution cultivates Scenedesmus quadricauda SDEC-8, obtains Scenedesmus quadricauda SDEC-8 cell.
5. apply as claimed in claim 4, it is characterized in that, the fatty acid methyl ester of described Scenedesmus quadricauda SDEC-8 cell is 9.55mgg -1, saturated fatty acid content is 80%, and cetane value is 61.38, and iodine number is 29.38gI 2/ 100g.
6. cultivate a cultural method of Scenedesmus quadricauda SDEC-8 according to claim 1, it is characterized in that, comprise the steps:
A) be inoculated in BG11 substratum by Scenedesmus quadricauda SDEC-8 and carry out sterile culture, obtained Scenedesmus quadricauda SDEC-8 seed liquor, culture condition is temperature 25 ± 1 DEG C, illumination 2000-3000Lux;
B) according to typical sanitary sewage water quality, preparation artificial sewage, its composition and consumption are (NH 4) 2cO 368.5 ~ 291.1mgL -1, K 2hPO 422.5 ~ 84.3mgL -1, MgSO 47H 2o 75mgL -1, CaCl 22H 2o 36mgL -1, citric acid 6mgL -1, ferric ammonium citrate 6mgL -1, EDTANa 21mgL -1, Na 2cO 320mgL -1, H 3bO 32.86mgL -1, MnCl 24H 2o 1.86mgL -1, ZnSO 47H 2o 0.22mgL -1, Na 2moO42H 2o 0.39mgL -1, CuSO 45H 2o 0.08mgL -1, Co (NO 3) 26H 2o 0.05mgL -1, and by after its pH regulator to 7.0 in 120 DEG C of high-temperature sterilization 30min, obtain typical sanitary sewage nutrient solution;
C) be inoculated in typical sanitary sewage nutrient solution by obtained Scenedesmus quadricauda SDEC-8 seed liquor, postvaccinal algae cell density is about 0.35 ~ 0.40 × 10 6cells.mL -1, constant temperature culture of ventilating under whole system continuous illumination condition, culture cycle is 18 days, and after reaching selected incubation time, centrifugal namely obtaining cultivates rear Scenedesmus quadricauda SDEC-8 cell.
7. method as claimed in claim 6, is characterized in that, step c) middle culture condition: culture temperature is 25 ± 1 DEG C, and illumination is the continuous illumination of 2500Lux, and aeration vapour-liquid ratio is 0.2vvm.
8. the application of Scenedesmus quadricauda SDEC-8 according to claim 1 in process eutrophic lake.
9. Scenedesmus quadricauda SDEC-8 according to claim 1 carries out the application of the purification of nitrogenous and/or phosphorus-containing wastewater.
10. Scenedesmus quadricauda SDEC-8 according to claim 1 is for the production of the application of high-efficiency grease, lipid acid, protein, starch, pigment, polysaccharide and/or nucleic acid.
CN201410625552.1A 2014-11-07 2014-11-07 Scnedesmus quadricauda for efficiently treating typical domestic sewage, and culture method and application thereof Pending CN104388315A (en)

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CN106467895B (en) * 2015-08-17 2019-08-06 中国石油化工股份有限公司 A kind of selenium-rich scenedesmus and its culture application
CN105483015A (en) * 2016-01-19 2016-04-13 山东大学 Method for cultivating chroococcus by domestic-wastewater-diluted kitchen waste anaerobic digestion solution
CN106281974A (en) * 2016-10-28 2017-01-04 天津大学 A kind of oil-producing microalgae culture apparatus and cultural method thereof
CN106803599B (en) * 2016-12-30 2019-07-05 山东大学 Microalgae-microorganism battery system method is prepared by raw material of sanitary sewage
CN106803599A (en) * 2016-12-30 2017-06-06 山东大学 The method for preparing microalgae microorganism battery system as raw material with sanitary sewage
CN107140741A (en) * 2017-05-18 2017-09-08 西北农林科技大学 A kind of method that utilization scenedesmus obliquus removes the salt in bitter and oil-producing
CN107345208A (en) * 2017-06-19 2017-11-14 南京师范大学 A kind of low-temperature epitaxy green alga and its application for removing nitrogen and phosphorus in sewage
CN107345208B (en) * 2017-06-19 2020-05-05 南京师范大学 Low-temperature growth green algae and application thereof in removing nitrogen and phosphorus in sewage
CN110668571A (en) * 2019-08-16 2020-01-10 天津大学 Method for constructing phycomycete co-culture system by utilizing artificial municipal sewage to accumulate biomass and measuring content of indoleacetic acid in system
CN113214996A (en) * 2021-05-18 2021-08-06 武汉理工大学 Scenedesmus as well as culture method and application thereof
CN117965311A (en) * 2024-04-01 2024-05-03 山东悦翔生物有限公司 Immobilization method of microalgae
CN117965311B (en) * 2024-04-01 2024-06-25 山东悦翔生物有限公司 Immobilization method of microalgae

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