JP2014108101A - Method for culturing algae using peritoneal dialysis wastewater as culture medium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for utilizing peritoneal dialysis wastewater and promoting algae biomass production efficiency by increasing the growth rate of algae.SOLUTION: Provided is a method for culturing algae using peritoneal dialysis wastewater(PDW) as the culture medium with addition of vitamin Band PIV metals. Also provided is a biomass production method where algae is grown in a culture medium that is PDW to which vitamin Band PIV metals are added. Also provided a method for purifying PDW using algae characterized in that the phosphate concentration of the PDW is decreased by growing algae in the PDW to which vitamin Band PIV metals are added.

Description

  The present invention provides a method for culturing algae, particularly Euglena algae using dialysis wastewater as a medium, a method for producing biomass using algae using dialysis wastewater as a medium, and a method for purifying dialysis wastewater using algae.

  In recent years, biomass obtained from products produced by biological cells, particularly biofuels, has attracted attention due to problems such as global warming or depletion of reserve resources. Among these biomasses, oils or polysaccharides such as hydrocarbons and triacylglycerol produced by microalgae do not compete with food and can be cultivated in large quantities. The acquisition of biofuels and other useful components is promising. However, in order to industrially cultivate a large amount of algae, it is necessary to solve economic problems such as operating costs and securing of a large amount of water resources and nutrient salts used in the culture medium.

  On the other hand, the amount of discharged wastewater has been increasing year by year due to population growth and industrial development, and now that water shortages have become serious, various efforts have been made to reuse these wastewater as resources. Since algae can absorb nutrients contained in wastewater and grow, algae biomass production that achieves low cost, energy saving, and reduction of carbon dioxide emissions can be achieved by using wastewater for algae culture. It seems possible.

One of the drains is dialysis drainage generated by peritoneal dialysis. Peritoneal dialysis is a convenient and less constrained treatment method that allows patients with chronic renal failure to be treated with their own hands by exchanging dialysate at home or at work. The amount of dialysis drainage discharged during peritoneal dialysis is large, and peritoneal dialysate exchange is usually performed 4 to 6 times a day, and the amount of drainage per time is as large as 1,500 to 2,000 ml. Disposal was a serious problem for patients. In particular, when APD therapy using an automatic peritoneal dialysis device (APD) is selected, the amount of drainage that a patient must process at one time is 8,000 to 10,000 ml for an adult.
Dialysis wastewater contains abundant elements necessary for algae culture such as glucose, nitrogen, phosphorus, etc., but these are released into sewage after dialysis treatment through pH adjustment and organic substance decomposition treatment, and their effective use has been studied little. Not. In addition, the number of patients receiving dialysis treatment in 2012 is over 300,000 in Japan, and the dialysis drainage discharged is huge.

Bioresour Technol. 2011 Jan; 102 (1): 17-25. Epub 2010 Jul 1

  There has been no research on the application of dialysis wastewater to algae culture. If the dialysis wastewater can be effectively used for algae culture without being discarded, the wastewater problem and the efficiency of algal biomass production can be improved. Therefore, in this study, for the purpose of evaluating the effectiveness of algae culture using dialysis drainage, optimization of dialysis drainage medium conditions by growth test, water quality analysis of the culture medium after culturing, and quantification of oil content in algae after recovery Went.

Specifically, in this study, algae was cultured using peritoneal dialysis wastewater (PWD). Since the growth of peritoneal dialysis effluent diluted with distilled water was not so high, AF-6 synthetic medium was used as the diluent, and media with different concentrations of peritoneal dialysis effluent were filtered and autoclaved. A test was conducted. As a result, the medium subjected to autoclave sterilization grew better than the one subjected to filter sterilization, and a wastewater concentration of about 25% was optimal. From these results, it was found that the components required for algae culture contained in AF-6 medium are insufficient in peritoneal dialysis drainage. Next, after removing each component from AF-6 medium as a dilute solution with a drainage concentration of 25%, no growth was seen with vitamin B ₁₂ and PIV metal (trace metal) removed. This result, missing the peritoneal dialysis effluent, the addition of vitamin B ₁₂ and trace metal components, it is suggested that similar growth to that added AF-6 synthetic medium is obtained, actually culture Confirmed by experiment. In addition, as a result of water quality analysis, it was revealed that phosphorus and nitrogen contained in the medium in which vitamin B ₁₂ and PIV metal were added to the dialysis effluent were almost removed in two weeks after the culture.

From the above results, it was clarified that in the culture of algae using peritoneal dialysis drainage, the addition of vitamin B ₁₂ and PIV metal is the optimum medium in terms of cell growth and nutrient removal. This study suggested the possibility of algae culture using dialysis wastewater.

Accordingly, the present application provides the following inventions.
(1) A method for culturing algae using dialysis wastewater added with vitamin B ₁₂ and PIV metal as a medium.
(2) The culture method according to (1), wherein the dialysis waste water is heat-treated.
(3) The culture method according to (1) or (2), wherein the dialysis waste water is diluted to 5 to 50%.
(4) The culture method according to (3), wherein the dialysis waste water is diluted to 10 to 40%.
(5) The culture method according to (4), wherein the dialysis waste water is diluted to 20 to 30%.
(6) The culture method according to any one of (1) to (5), wherein the algae is Euglena algae.
(7) A method for producing biomass from algae using dialysis wastewater to which vitamin B ₁₂ and PIV metal are added as a medium.
(8) The biomass production method according to (7), wherein the dialysis waste water is heat-treated.
(9) The method for producing biomass according to (7) or (8), wherein the dialysis waste water is diluted to 5 to 50%.
(10) The method for producing biomass according to (9), wherein the dialysis waste water is diluted to 10 to 40%.
(11) The biomass production method according to (10), wherein the dialysis waste water is diluted to 20 to 30%.
(12) The biomass production method according to any one of (7) to (11), wherein the algae is Euglena algae.
(13) A method for purifying dialysis wastewater using algae, characterized in that the phosphate concentration of dialysis wastewater is reduced by adding vitamin B ₁₂ and PIV metal to the dialysis wastewater to grow algae, Dialysis wastewater purification method.
(14) The dialysis drainage purification method according to (13), wherein the dialysis drainage is heat-treated.
(15) The dialysis drainage purification method according to (13) or (14), wherein the dialysis drainage is diluted to 5 to 50%.
(16) The dialysis drainage purification method according to (15), wherein the dialysis drainage is diluted to 10 to 40%.
(17) The dialysis drainage purification method according to (16), wherein the dialysis drainage is diluted to 20 to 30%.
(18) The method for purifying dialysis waste water according to any one of (13) to (17), wherein the algae is Euglena algae.

  According to the present invention, the dialysis waste water can be effectively used, and the algal biomass production efficiency can be improved by increasing the growth rate of the algae. In addition, efficient purification of dialysis wastewater is also achieved.

The growth rate of algae when the concentration of dialysis wastewater is changed to the algal culture medium AF-6 and the dialysis wastewater is autoclaved or filtered is shown. The growth rate of algae when various components are removed from the algae culture medium AF-6 and dialysis drainage is added and cultured is shown. The growth rate of algae when vitamin B12 and PIV metal are added to dialysis wastewater is shown. It shows that the phosphate concentration of dialysis wastewater decreases by adding vitamin B ₁₂ and PIV metal to dialysis wastewater to grow algae.

In the present invention, dialysis drainage refers to drainage generated by peritoneal dialysis. Examples of components of peritoneal dialysis drainage include glucose, urea, creatinine, uric acid, sodium ion, potassium ion, calcium ion, magnesium ion, phosphorus, ammonium ion, and 3-indoxyl sulfate as shown in the following table. However, it is not limited to them.

  In the present invention, the dialysis waste water may be used without being diluted, but is preferably used after being diluted. For dilution of the dialysis waste water, water such as distilled water, ion exchange water, sterilized water, or various cell culture media suitable for cell culture can be selected. For example, the algae culture medium includes a freshwater algae culture medium (for example, AF6 culture medium, C culture medium, URO culture medium, VT culture medium, etc.), or a marine algae culture medium (ESM culture medium, f / 2 culture medium, IMR culture medium, MNK culture medium, etc.) ) In the present invention, the dialysis drainage is 5 to 50 v / v%, preferably 10 to 40 v / v%, more preferably 20 to 30 v / v%, most preferably 25 v / v% in water or a suitable cell culture medium. Dilute as follows.

  The dialysis waste water is preferably sterile filtered or heat sterilized, for example, heated and autoclaved by an autoclave and then used for culturing algae. Although not particularly bound by theory, autoclave sterilization is preferred because the urea contained in the dialysis effluent is thermally decomposed, resulting in an increase in the amount of ammonia nitrogen and promotion of algal growth. It is done.

Culture of algae in the present invention uses an added dialysis drainage of vitamin B ₁₂ and PIV metals (trace metals) as medium. Preferably, the concentration of vitamin B ₁₂ dialysis drainage 100ml per 0.01～10Myug, will preferably be 0.01～1Myug. The PIV metal preferably comprises the composition of Table 2 below.

  Preferably, each component of the PIV metal is within the range of 1% to 500% of the values shown in the table above, preferably within the range of 1% to 500%, more preferably within the range of 1% to 100%. Set as follows.

  As used herein, algal cells include prokaryotes such as eubacteria or archaea and all cells of eukaryotes such as algae, protozoa, plants, fungi or animals. The cells are preferably algae, plants, fungi cells, and in particular algal cells. As used herein, `` algae '' includes cyanobacteria, prokaryotic green algae, red algae, gray algae, crypto algae, dinoflagellates, golden algae, diatoms, brown algae, rabin linchula, yellow green algae, There are haptoalgae, rafidoalgae, true-eyed point algae, chloralachnion algae, euglena algae, plastinolgae, green algae, axle algae, etc., preferably called microalgae, cyanobacteria, diatoms, rabinchuras, true eyes There are point algae, crypt algae, dinoflagellates, golden algae, hapto algae, rafido algae, euglena algae, plasino algae and green algae. The algae suitable for the present invention are preferably Euglena, Botryococcus, Spirulina, Microkistis aeruginosa, Spirulina, Aurantiochytrium and Schizochytrium. Particularly preferred are Euglena algae, more preferred is Euglena gracilis, and most preferred is Euglena gracilis strain NIES-48.

Purification of culture or dialysis drainage algae in the present invention, were seeded in addition dialysis drainage of vitamin B ₁₂ and PIV metals refers to the strain in the present invention is carried out by culturing in accordance with a conventional method. The medium can further contain a carbon source or nitrogen source, vitamins, protease peptone, yeast extract and the like as appropriate. After preparation, the dialysis wastewater can be appropriately adjusted in pH by adding an appropriate acid or base if necessary. The preferred pH of the medium will depend on the type of algae being cultured. The culture conditions also depend on the type of algae to be cultured, and the temperature is 5 to 40 ° C., preferably 10 to 35 ° C., more preferably 10 to 30 ° C., usually 1 to 10 days, preferably 3 to 7 days. It can be performed by aeration or anaerobic stirring culture, shaking culture or stationary culture.

The algal culture used in the present invention may be cultured in a culture apparatus having a suitable cell culture means. “Cell culture means” means means having all functions for culturing cells, for example, a culture tank, which comprises a stirrer, a vibration device, a temperature control device, a pH control device, a turbidity device. One or more devices selected from a degree measuring device, a light control device, a specific gas concentration measuring device such as CO ₂ , and a pressure measuring device may be included. The culture tank may be the same tank as the concentration / separation tank or may be a tank different from the concentration / separation tank. When the tank is separate from the concentration / separation tank, it may be connected by an appropriate means, for example, a flow path.

  In general, a cell culture process includes a logarithmic growth phase in which the number of cells grows logarithmically with respect to time and a stationary phase after the logarithmic growth phase. For example, in the case of algae, it can be divided into a growth phase in which logarithmic growth is caused by nutrients in the photosynthesis and growth medium and a biomass production phase in which biomass is produced. Preferably, the medium is exchanged between the growth phase and the biomass production phase.

The present invention further provides a method for producing biomass using algae using dialysis wastewater as a culture medium. The biomass production method of the present invention is characterized by culturing algae using a dialysis wastewater to which vitamin B ₁₂ and PIV metal are added as a medium, and causing the algae to produce biomass.

  As used herein, “biomass” refers to any biomass produced by cells, and specifically, for example, biomass and raw materials required for fuel, food, medical supplies, and other industrial or daily necessities. Say that. The biomass includes hydrocarbons, saccharides, proteins, amino acids, and the like that are the basis of resins, fuels, or surfactant sugars. For example, as biomass produced by algae, there are oils and polysaccharides such as hydrocarbons produced using organic carbon sources. Specific examples of the oil include hydrocarbons such as alkanes, epoxyalkanes, alkadienes, alkatrienes, triterpenoids, and tetreterpenoids, and triacylglycerols in which three fatty acid molecules of one glycerin are bonded. When the algae is Euglena gracilis NIES-48, there are lipids such as triacylglycerol and wax esters produced under anaerobic conditions. Examples of polysaccharides include polysaccharides containing fucose, and carotenoids as natural pigments.

  Biomass produced by the algae of the present invention can be extracted and analyzed by methods known to those skilled in the art. For example, the wet alga bodies cultured and grown as described above and collected from the obtained culture solution by centrifugation or filtration are dried by freeze drying or drying by heating. Alternatively, the cultured medium may be used as it is in the biomass extraction step.

  Biomass, which is a lipid, can be extracted from the obtained cell dry product or a medium containing cultured cells using an organic solvent. The extraction may be performed twice or more using different organic solvents. The organic solvent will depend on the type of biomass. For example, a mixed solvent of a polar solvent such as chloroform / methanol mixed solvent (for example, 1: 1, 1: 2) or ethanol / diethyl ether mixed solvent and a weakly polar solvent. A liquid can be used. After the extraction, for example, extraction is performed using n-hexane from a sample concentrated and dried under a nitrogen stream. The resulting extract is purified by methods known to those skilled in the art. For example, it can be purified by adsorbing polar lipids using silica gel or acidic clay. The purified biomass is analyzed by NMR, IR, gas chromatography, GC / MS and the like.

  The algae can produce at least 10%, at least 30%, preferably at least 50%, more preferably at least 70%, and even more preferably at least 80% of the biomass per dry weight.

The present invention further provides a method for purifying dialysis wastewater using algae. This method is characterized by decreasing the phosphate concentration of the dialysis effluent by growing by adding vitamin B ₁₂ and PIV metal dialysis drainage algae as described above. Phosphoric acid can optionally be removed by 85-95% or more in 2 weeks of culture.

  Hereinafter, the present invention will be specifically described by way of examples. However, the technical scope of the present invention is not limited by these examples.

Algae used in the experiment and dialysis drainage In this experiment, Euglena gracilis NIES-48 strain (strain Z) was used as a target for microalgae. Dialysis drainage was peritoneal dialysis wastewater (PDW). The component composition is listed in Table 1.

Experiment 1 Growth test of Euglena gracilis using dialysis drainage medium In this experiment, the concentration of PDW was set to 5, 10, 25% (v / v) and AF-6 synthetic medium (NaNO ₃ 0.14 g / L , NH ₄ NO ₃ 0.022mg / L, MgSO ₄・ 7H ₂ O 0.03mg / L, CaCl ₂・ 2H ₂ O 0.01mg / L, Fe-citrate 2mg / L, Citric acid 2mg / L, KH ₂ PO ₄ 0.01 mg / L, K ₂ HPO ₄ 5 mg / L, Biotin 2 μg / L Thiamine HCl 0.01 mg / L, Vitamin B ₁₂ 1 μg / L, PIV metals (FeCl ₃ · 6H ₂ O 0.98 mg / L, MnCl ₂ · 4H ₂ O _{0.18mg / L, ZnSO 4 · 7H} 2 O 0.11mg / L, CoCl 2 · 6H 2 O 0.02mg / L, Na 2 MoO 4 · 2H 2 O 0.0125mg / L, Na 2 EDTA · 2H 2 O 0.05g / L), diluted with MES 0.04 g / L, pH 6.6) were used. 150 mL of medium was placed in a 300 mL Erlenmeyer flask, and each medium was divided into two sterilization methods, filtration sterilization and autoclave sterilization, to prepare a total of six types of medium. As a control, AF-6 synthetic medium was used.

HUT medium is used for pre-culture, KH ₂ PO ₄ 0.02 g / L, MgSO ₄ 7H ₂ O 0.025 g / L, Sodium acetate 0.4 g / L, Potassium citrate 0.04 mg / L, Polypeptone 0.6 g / L, Yeast Extract 0.4 g / L, Vitamin B ₁₂ 0.5 μg / L, Thiamine HCl 0.4 mg / L, (pH 6.4) were prepared by autoclaving. 150 mL of the prepared medium was placed in a 300 mL Erlenmeyer flask, set to a culture temperature of 25 ° C., a light irradiation intensity of 120 μmol m ⁻² s ⁻¹ (24 hours continuous irradiation), and cultured with shaking at 130 rpm. After culturing for 72 hours, centrifugation was performed at 1500 rpm for 15 minutes. Thereafter, the cells were washed with sterilized water, and E. gracilis was inoculated into the above seven types of media including controls. The culture conditions were the same as in the pre-culture, 25 ° C, light irradiation intensity 120 μmol m ^-2 s ^-1 (24 hours continuous irradiation), shaking culture at 130 rpm, and observed changes in growth

  The time course of proliferation was measured by collecting cells every 24 hours during the culture period and counting the number of cells. For the cell count, a hook slow Zental hemocytometer (Sunlead Glass Co., Ltd.) was used.

  The above results are shown in FIG. A growth test was carried out using AF-6 synthetic medium as the diluent and medium with different concentrations of peritoneal dialysis drainage sterilized by filtration and autoclaving. The medium subjected to autoclave sterilization was subjected to filtration sterilization. The optimal growth rate was about 25%. The growth was promoted by using dialysis effluent sterilized by autoclave rather than filtration sterilization, probably because ammonia nitrogen increased due to thermal decomposition of urea in the dialysis effluent.

  The peritoneal dialysis drainage was diluted with distilled water, and the growth was not observed so much (results are not shown), so the components required for algae culture contained in the AF-6 medium are the peritoneal dialysis drainage. Then it turned out that it was insufficient.

Experiment 2 Identification of supplementary components for determining medium conditions In this experiment, the PDW of all mediums was set to 25% (v / v), and dilution was performed using a modified medium excluding the following components from AF-6. Using. As a control, a medium in which PDW was diluted to the same concentration with an AF-6 synthetic medium was used. 3 types of modified media excluding each component from AF-6 (AF-6 to 1) citric acid, Fe-citrate, 2) citric acid, Fe-citrate, PIV metal, 3) vitamin B ₁₂ 70 ml of the above four types of culture medium diluted with the control medium and the control diluted (n = 3) were subjected to autoclave sterilization. Thereafter, E. gracilis was inoculated 72 hours after the start of preculture (preculture conditions were the same as in Experiment 1). The main culture conditions were culture temperature of 25 ° C., light irradiation intensity of 120 μmol m ⁻² s ⁻¹ (24 hours continuous irradiation), and air culture through an air filter (ADVANTEC). The time course of proliferation was measured using a hemocytometer as in Experiment 1.

The above results are shown in FIGS. This result, missing the peritoneal dialysis effluent, the addition of vitamin B ₁₂ and trace metal component (PIV metal), it is suggested that similar growth to that added AF-6 synthetic medium is obtained Actually, it was confirmed by a culture experiment.

Experiment 3 Additional culture and detailed analysis based on the results of Experiment 2 In this experiment, vitamin B ₁₂ 1 μg / L, PIV metals (FeCl ₃ · 6H ₂ O 0.98 mg / L, MnCl ₂ · 4H ₂ O 0.18 mg / L, ZnSO ₄ · 7H ₂ O 0.11mg / L, CoCl ₂ · 6H ₂ O 0.02mg / L, Na ₂ MoO ₄ · 2H ₂ O 0.0125mg / L, Na ₂ EDTA · 2H ₂ O 0.05g / L) A total of 4 mediums diluted with PDW to 25% with diluent, medium diluted with PD-6 to 25% or 50% (v / v) using AF-6, and medium with PDW diluted with distilled water as a control Different types of media were prepared. 150 mL of each medium was dispensed into a 300 mL Erlenmeyer flask and autoclaved. The main culture conditions were a culture temperature of 25 ° C., a light irradiation intensity of 120 μmol m ⁻² s ⁻¹ (24 hours continuous irradiation), and shaking culture at 130 rpm (the preculture conditions for E. gracilis were the same as in Experiment 1).

After 5 mL of the culture solution was collected over time and filtered (GF / C filter: Whatman), the nitrogen concentration and phosphorus concentration in the filtrate were measured by an industrial drainage test method to determine the removal ability by E. gracilis . The culture solution at the end of the culture was filtered using a GF / C filter, dried at 60 ° C. for 24 hours, and then the dry weight was measured.

The above results are shown in FIG. As a result of water quality analysis, it is clear that phosphorus and nitrogen contained in the medium with vitamin B ₁₂ and PIV metal added to the dialysis drainage are almost removed in two weeks after culturing. I understood.

Claims (18)

Using the added dialysis drainage of vitamin B ₁₂ and PIV metals as media, algal culture methods.   The culture method according to claim 1, wherein the dialysis waste water is heat-treated.   The culture method according to claim 1 or 2, wherein the dialysis waste water is diluted to 5 to 50%.   The culture method according to claim 3, wherein the dialysis waste water is diluted to 10 to 40%.   The culture method according to claim 4, wherein the dialysis waste water is diluted to 20 to 30%.   The culture method according to any one of claims 1 to 5, wherein the algae is Euglena algae. Biomass production method using algae using dialysis wastewater containing vitamin B ₁₂ and PIV metal as a medium.   The biomass production method according to claim 7, wherein the dialysis waste water is heat-treated.   The biomass production method according to claim 7 or 8, wherein the dialysis waste water is diluted to 5 to 50%.   The biomass production method according to claim 9, wherein the dialysis waste water is diluted to 10 to 40%.   The method for producing biomass according to claim 10, wherein the dialysis waste water is diluted to 20 to 30%.   The biomass production method according to any one of claims 7 to 11, wherein the algae is Euglena algae. A method for purifying dialysis wastewater using algae, characterized in that the phosphate concentration of dialysis wastewater is reduced by adding vitamin B ₁₂ and PIV metal to the dialysis wastewater to grow algae. Purification method.   The dialysis drainage purification method according to claim 13, wherein the dialysis drainage is heat-treated.   The dialysis drainage purification method according to claim 13 or 14, wherein the dialysis drainage is diluted to 5 to 50%.   The dialysis drainage purification method according to claim 15, wherein the dialysis drainage is diluted to 10 to 40%.   The dialysis drainage purification method according to claim 16, wherein the dialysis drainage is diluted to 20 to 30%.   The method for purifying dialysis drainage according to any one of claims 13 to 17, wherein the algae is Euglena algae.