CN103194395B - Method for culturing microalgae by using biogas residues as raw materials - Google Patents
Method for culturing microalgae by using biogas residues as raw materials Download PDFInfo
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- CN103194395B CN103194395B CN201310139884.4A CN201310139884A CN103194395B CN 103194395 B CN103194395 B CN 103194395B CN 201310139884 A CN201310139884 A CN 201310139884A CN 103194395 B CN103194395 B CN 103194395B
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Abstract
The invention discloses a method for culturing microalgae by using biogas residues as raw materials. The method is characterized by comprising the following steps of: (1) adjusting the pH value of the biogas residues to 10 by using a sodium hydroxide solution with the concentration of 5mol/L, standing the biogas residues at the temperature of 25 DEG C for 12h, placing the biogas residues into a high-pressure sterilizing pot, heating at the temperature of 115 DEG C for 60min, cooling, and then, making a microalga culture medium by using a liquid which is collected through vacuum filtration; (2) microalga culture: selecting chlorella as a microalga strain; and (3) biogas residue nutrient solution culture: adjusting the pH value of the microalga culture medium obtained in the step (1) to 7 by using a hydrochloric acid solution with the concentration of 5mol/L, sterilizing at high pressure for 20min, inoculating the microalga strain in the sterilized microalga culture medium through aseptic operation, and then, placing the microalga strain into a swing bed with the rotating speed of 180rpm to be subjected to shaking culture at the temperature of 28 DEG C and the illumination intensity of 6000Lux for 7 days.
Description
Technical field
The invention belongs to biomass energy source domain, is that a kind of natural pond slag cultivates with micro-algae the technology combining.
Background technology
Along with the development of Biodiesel; the comprehensive exploitation of oil-rich microalgae and utilization have caused increasing concern; microalgae grease can solve current biodiesel raw material source and lack and unsettled problem; the scientific research institution in every country and area has carried out the research work of micro-algae cultivation aspect in succession, make its become gradually many countries increase income energy-conservation, turn bane into boon and the important means of protection of the environment.Equally, country continued to increase the construction support dynamics of big-and-middle-sized biogas engineering in recent years, that a large amount of by product is as the generation of natural pond slag obtaining incident in a large amount of clean gas, how reasonably this part product of disposal and utilization for the recycling of waste with alleviate environmental pollution and have important impact, will play positive promoter action if can realize both combinations to the development of energy field.
The at present common approach that utilizes has exploitation biogas residue organic fertilizer as farm-forestry crop fertilizer, as patent CN101774848B and CN101229982A etc., the method of preparing fertilizer taking natural pond slag as main raw material is disclosed, patent 201010100163.9 has reported that natural pond slag prepares the method for gac, there is not yet natural pond slag and cultivates with micro-algae the document and the patent report that combine.Because natural pond slag in this patent is mainly made up of the algae-residue (in algae-residue, protein content exceedes 50%) of active sludge and abundant degraded, wherein still contain abundant organism as carbohydrate and protein etc., and various nutrient elements is as N, P etc., process by rational technique means, can be used as the effective constituent that micro-algae is cultivated, and can reduce natural pond slag and deal with the secondary pollution problem of bringing improperly.
Summary of the invention
The present invention is intended to by adopting suitable means to process natural pond slag, realizes the combination of cultivating with micro-algae, for micro-algae provides novel nourishing source and widens the approach that utilizes of natural pond slag.
For achieving the above object, the working method that the present invention adopts is as follows:
(1) anaerobically fermenting
In tool plug wide-necked bottle, add anaerobic grain sludge and fermentation substrate algae-residue, stir on bonnet with the bottle stopper of conduit, pass into residual air in nitrogen purging bottle falling, conduit outlet connects the collection of airbag for gas, wide-necked bottle is put into 35 DEG C of water-baths and is started anaerobic digestion, stops fermentation after 15-20 days.
(2) separation of natural pond slag
Anaerobic fermented liquid is removed supernatant liquor after 4000rpm rotating speed is centrifugal, collects lower floor's residuum and is natural pond slag.
(3) processing of natural pond slag
The means that adopt physical chemistry to combine are processed natural pond slag.Be the sodium hydroxide solution adjusting natural pond slag pH to 10 of 5 mol/L by concentration, place 12 h at 25 DEG C, put into high-pressure sterilizing pot and at 115 DEG C, heat 60 min, cooling rear employing vacuum filtration is collected liquid portion as micro-algae culture medium.
(4) micro-algae is cultivated
The freshwater microalgae that the present invention cultivates comprises the autotrophic freshwater microalgae after wild environments such as coming from fresh water or domestication, can be in each large algae kind preservation center or commercially available acquisition, as chlorella, grid algae and spirulina etc.Described micro-algae algae kind be chlorella (
chlorella vulgaris), be once recorded in disclosed U.S. Patent Application Publication specification sheets US 20090288223 November 19 in 2009, also can obtain in Texas ,Usa algae kind preservation center, its deposit number is UTEX 2714.
The cultivation of algae kind: algal species cultivation based component is NH
4cl 400 mg/L, KH
2pO
456 mg/L, K
2hPO
4108 mg/L, MgSO
47H
2o 100 mg/L, Glacial acetic acid 1 mg/L, micro-1 ml/L, algae kind is inoculated in sterilized substratum through aseptic technique, is then placed in illumination box and leaves standstill cultivation, shake 3-4 every day, culture temperature is 15-30 DEG C, and intensity of illumination is 2000-4000 Lux, cultivates 7 days.
Natural pond slag nutritive medium is cultivated: by 10 times of distilled water dilutings for gained micro-algae culture medium in (3), then regulate pH to 7 with 5 mol/L hydrochloric acid solns, carry out autoclaving 20min, algae kind is inoculated in sterilized micro-algae culture medium through aseptic technique, then being placed in rotating speed is the shaking table concussion cultivation of 180rpm, culture temperature is 28 DEG C, and intensity of illumination is 6000 Lux, cultivates 7 days.
Beneficial effect of the present invention is as follows: natural pond slag tradition utilizes mode mainly contain compost as the fertilizer of farm-forestry crop or return in anaerobic fermentation system as inoculum etc., but in this patent, alkaline condition causes the solubleness of residual algae-residue protein in the slag of natural pond to increase and preliminary hydrolysis occurs, then the in the situation that of certain temperature, be decomposed into micromolecular amino acid, polypeptide is even further degraded to short chain fatty acid, carbohydrate decomposed is monose, and in this process, mud flora cell wall structure is produced to certain destruction, thereby discharge composition and intracellular organic matter in cell walls, thereby natural pond slag can provide a kind of new nutrition source for micro-algae.At natural pond slag without any processing in the situation that, the nutritive substance that adopts the mode of simple water logging to obtain is barrenr, before and after cultivating the biomass cultivation of finding micro-algae, there is hardly any variation, by process after to these wherein release of contained nutritive substance, biomass reaches 9.0g/L, and fat content reaches 35.8%, has realized natural pond slag and micro-algae and has cultivated and organically combine, and the approach that utilizes of further having widened natural pond slag, has reduced the secondary pollution of natural pond slag.
Embodiment
Embodiment:
Step 1: anaerobically fermenting.In the tool plug wide-necked bottle of 1L, add 300g anaerobic grain sludge (to take from Binzhou Xiang Chi group starch wastewater anaerobic fermentation system, its total solids material content is 9.44%, volatile solid material is 84.58%) and algae-residue 26g(total solids material content 97.8%, volatile solid substances content 92.6%), wherein the concrete acquisition pattern of algae-residue is: on the basis of step 3 algal species cultivation, by cultivate after micro-algae through centrifugal removal supernatant liquor, collect after the wet algae of lower floor through high pressure homogenizer (NS3015H, Niro Soavi company of Italy) carry out cytoclasis, homogenization pressure is 120 Mpa, circular treatment 2 times.Select normal hexane as extraction solvent, broken wall cell is added in single port flask, ratio according to 1 ml/g broken wall cell adds solvent, on 55 DEG C of lower magnetic force heating and stirring devices, leaches 1h, finishes rear it to be carried out to standing separation, collect the organic solvent phase on upper strata, and reclaim frond and under identical condition, carry out secondary lixiviate, finally wet frond steams de-24h in vacuum drying oven (service temperature is 40 DEG C, and vacuum tightness is 2000 pa), remove residual solvent and moisture, finally obtain dry algae-residue.In mud and algae-residue, adding water and regulating solids content is 8%, on the bonnet that stirs, with the bottle stopper of conduit, passes into residual air in nitrogen purging bottle falling, and conduit outlet connects the collection of airbag for gas, and temperature is controlled at 35 ± 1 DEG C, ferments 15 days.
Step 2: natural pond slag processing.After fermentation ends, anaerobic fermented liquid is removed to supernatant liquor after 4000 rpm rotating speeds are centrifugal, residue soup compound is natural pond slag.The treatment process that adopts alkali-Re to combine is processed natural pond slag, with 5 mol/L sodium hydroxide solutions adjusting pH to 10, at 25 DEG C, place 12h, put into high-pressure sterilizing pot and heat 60min at 115 DEG C, cooling rear employing vacuum filtration is collected liquid portion as micro-algae culture medium.
Step 3: the cultivation of algae kind: the algae kind of employing is
chlorella vulgaris(purchased from Texas ,Usa algae kind preservation center, UTEX 2714).Algal species cultivation based component is NH
4cl 400 mg/L, KH
2pO
456 mg/L, K
2hPO
4108 mg/L, MgSO
47H
2o 100 mg/L, Glacial acetic acid 1 mg/L, micro-1 ml/L, in this substratum, total fat content can reach the more than 40% of dry cell weight, is oil-rich microalgae.Concrete operations are: substratum is joined respectively in Erlenmeyer flask (tool breathable sealing film), after 121 DEG C of sterilizings, access algae kind through aseptic technique, then be placed in illumination box and leave standstill cultivation, shake every day 3 times, culture temperature is 28 DEG C, intensity of illumination is 4000 Lux(fluorescent lamps), cultivate 7 days.
10 times of distilled water dilutings for gained micro-algae culture medium in step 2 then, with 5 mol/L hydrochloric acid solns adjusting pH to 7, are respectively charged in Erlenmeyer flask after 115 DEG C of sterilizing 20 min, by algae kind according to 10% inoculum size (algae kind OD
680be 1.0) be inoculated in sterilized micro-algae culture medium through aseptic technique, being then placed in rotating speed is the shaking table concussion cultivation of 180rpm, culture temperature is 28 DEG C, intensity of illumination is 6000 Lux(fluorescent lamps), cultivate 7 days.Sampling and measuring biomass is 9.0 g/L, and fat content is 35.8%.
Claims (1)
1. one kind is carried out the method for micro-algae cultivation taking natural pond slag as raw material, it is characterized in that it comprises the steps: that (1) regulates natural pond slag pH to 10 with the sodium hydroxide solution that concentration is 5 mol/L, at 25 DEG C, place 12 h, put into high-pressure sterilizing pot and at 115 DEG C, heat 60 min, cooling rear employing vacuum filtration is collected liquid portion as micro-algae culture medium;
Described natural pond slag is mainly made up of the algae-residue of active sludge and abundant degraded, in algae-residue, protein content exceedes 50%, the concrete acquisition pattern of described algae-residue is: on the basis of algal species cultivation, by cultivate after micro-algae through centrifugal removal supernatant liquor, after collecting the wet algae of lower floor, carry out cytoclasis through high pressure homogenizer, homogenization pressure is 120 MPa, circular treatment 2 times; Select normal hexane as extraction solvent, broken wall cell is added in single port flask, add solvent according to the ratio of 1 mL/g broken wall cell, on 55 DEG C of lower magnetic force heating and stirring devices, leach 1h, after end, it is carried out to standing separation, collect the organic solvent phase on upper strata, and reclaim frond and under identical condition, carry out secondary lixiviate, finally wet frond is at vacuum drying oven, service temperature is 40 DEG C, vacuum tightness is in 2000 Pa, to steam de-24h, removes residual solvent and moisture, finally obtains dry algae-residue; In mud and algae-residue, adding water and regulating solids content is 8%, on the bonnet that stirs, with the bottle stopper of conduit, passes into residual air in nitrogen purging bottle falling, and conduit outlet connects the collection of airbag for gas, and temperature is controlled at 35 ± 1 DEG C, ferments 15 days; After fermentation ends, anaerobic fermented liquid is removed to supernatant liquor after 4000 rpm rotating speeds are centrifugal, residue soup compound is natural pond slag;
(2) micro-algae is cultivated
Described micro-algae algae kind be chlorella (
chlorella vulgaris), being preserved in Texas ,Usa algae kind preservation center, deposit number is UTEX 2714;
The cultivation of algae kind: algal species cultivation based component is NH
4cl 400 mg/L, KH
2pO
456 mg/L, K
2hPO
4108 mg/L, MgSO
47H
2o 100 mg/L, Glacial acetic acid 1 mg/L, micro-1 ml/L, algae kind is inoculated in sterilized substratum through aseptic technique, is then placed in illumination box and leaves standstill cultivation, shake 3-4 every day, culture temperature is 15-30 DEG C, and intensity of illumination is 2000-4000 Lux, cultivates 7 days;
(3) slag nutritive medium in natural pond is cultivated: gained micro-algae culture medium in (1) is regulated to pH to 7 with 5 mol/L hydrochloric acid solns, carry out autoclaving 20min, algae kind is inoculated in sterilized micro-algae culture medium through aseptic technique, then being placed in rotating speed is the shaking table concussion cultivation of 180rpm, culture temperature is 28 DEG C, intensity of illumination is 6000 Lux, cultivates 7 days.
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| CN101074144A (en) * | 2006-05-17 | 2007-11-21 | 郭文瑞 | Ecological treatment and recovering utilization for farm |
| CN101285075A (en) * | 2008-05-27 | 2008-10-15 | 南京工业大学 | Coupling process of marsh gas fermentation and culture of autotrophic freshwater microalgae |
| CN101921811A (en) * | 2010-07-20 | 2010-12-22 | 山东省科学院能源研究所 | Method for culturing microalgae |
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