CN113930343B - Application of mixed wastewater in chlorella culture and culture method - Google Patents
Application of mixed wastewater in chlorella culture and culture method Download PDFInfo
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- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
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- C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
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Abstract
The invention discloses application of mixed wastewater in chlorella cultivation, wherein the mixed wastewater is soybean product wastewater and dairy product wastewater mixed according to a volume ratio of 1:1-1:9. The invention is based on a mixed wastewater coupled microalgae culture system, explores the capability and influence mechanism of microalgae for purifying wastewater in a single wastewater culture mode and a mixed mode, researches the yield of high-added-value products such as pigment, grease and the like produced by the microalgae, and provides basis for exploring an economically feasible microalgae purification path and obtaining microalgae biomass with higher application value.
Description
Technical Field
The invention belongs to the technical field of agriculture, and particularly relates to an application of mixed wastewater in chlorella culture and a culture method.
Background
Microalgae are recognized as a sustainable and promising biological raw material and can be used for producing various added-value products, including nutritional health products, medicines, cosmetics, biofuels, animal and aquaculture feeds, natural dyes and the like. However, the high production cost of microalgae biomass is a barrier to the widespread use of microalgae biotechnology. The waste water contains a large amount of nutrient elements such as carbon source, nitrogen source, phosphate and various trace minerals, is a free, low-cost and sustainable alternative culture solution, and can be used for culturing various algae.
In recent years, wastewater including municipal wastewater, domestic wastewater, pig farm wastewater, industrial wastewater, and anaerobic digestion wastewater has been applied to cultivation of various algae. The industrial wastewater contains heavy metals or toxic organic compounds, the obtained algae biomass has low added product value, and the accumulated algae biomass can only be used for producing biofuel and other microalgae products with low added value, so that the application range of algae is greatly limited. Compared with the waste water, the food processing waste water is more suitable for microalgae cultivation, because toxic compounds and harmful substances for inhibiting the growth of microalgae are less, the quality of biomass obtained by cultivating the microalgae is relatively better, and the waste water can be further used for producing products with high added value. The treatment of dairy waste water in dairy farms is also one of the environmental problems facing our country. It is counted that about 0.2-10L of waste water is produced per 1L of milk processed. Although physical and chemical techniques can be used for treatment, the cost is high. The biological effect of removing the nutrient substances in the wastewater is good, and the cost is greatly reduced. In recent years, algae cultivation systems have been used to treat soy product wastewater. However, since the waste water of bean products contains excessive organic matters, the waste water cannot be directly utilized by microalgae. In order to provide a suitable growth environment for microalgae, it is often necessary to dilute the wastewater. However, this wastes water resources.
At present, related researches on microalgae cultivation by mixed wastewater are less, so that the invention considers the characteristic differences of organic matter concentration, nutrient salt concentration, pH and the like in different wastewater, selects two typical wastewater of bean product wastewater and dairy product wastewater in a dairy farm to be mixed in different proportions, and then carries out chlorella cultivation, thus not only balancing the defect that single wastewater is unfavorable for microalgae growth, but also carrying out wastewater purification treatment, improving the effect of removing nutrient substances in the wastewater and carrying out biomass production of microalgae expansion propagation cultivation, thereby expanding the application range of microalgae and saving a large amount of clean water.
By searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides an application of mixed wastewater in chlorella culture and a culture method.
The technical scheme adopted for solving the technical problems is as follows:
the application of the mixed wastewater in the aspect of chlorella cultivation is that the mixed wastewater is bean product wastewater and dairy product wastewater which are mixed according to the volume ratio of 1:1-1:9.
Further, the volume ratio of the bean product wastewater to the dairy product wastewater is 1:1, 1:5, 1:9.
Further, the water quality conditions of the bean product wastewater, the dairy product wastewater and the mixed wastewater are as follows:
further, the Chlorella is Chlorellasorokiniana UTEX, chlorella sp.LAMB 166 and LAMB 38, chlorella vulgaris FACHB-1227.
A method for culturing chlorella by using mixed wastewater comprises the following steps:
respectively taking mixed wastewater of bean product wastewater and dairy product wastewater mixed according to the volume ratio of 1:1-1:9, and adjusting the pH value to 7 by using 1M HCl or 1M NaOH after autoclaving to obtain treated mixed wastewater;
culturing, inoculating 4 kinds of Chlorella UTEX1602, L166, L38 and/or FACHB1227 when the initial light absorption value of the culture solution is at 680nm, i.e. the optical density at 680nm, is 0.2, culturing under the conditions of 30+ -1deg.C, 6500Lux illumination and 24hr illumination, and shaking for 3 times per day.
Further, the volume ratio of the bean product wastewater to the dairy product wastewater is 1:1, 1:5, 1:9.
Further, the water quality conditions of the bean product wastewater, the dairy product wastewater and the mixed wastewater are as follows:
further, the Chlorella is Chlorellasorokiniana UTEX, chlorella sp.LAMB 166 and LAMB 38, chlorella vulgaris FACHB-1227.
Further, when the mixing ratio was 1:1, the biomass dry weights of the Chlorella Chlorellasorokiniana UTEX, chlorella sp.LAMB166 and LAMB 38, chlorella vulgaris FACHB-1227 were 4.27g/L, 4.09g/L, 2.63g/L and 2.2g/L, respectively; the removal rate of TN by 4 kinds of chlorella is 69.03% -80.30%; chlorella UTEX1602, L166, L38 and FACHB1227 removed 231.56mg/L, 218.46mg/L, 261.07mg/L and 235.85mg/L, respectively, of TN in the solution; the removal rate of TP by 4 kinds of chlorella is 92.41% -95.48%, wherein the purifying effect of UTEX1602 is the best; the removal rate of the 4 kinds of chlorella to COD reaches 55.58% -63.06%, the removal effect of the UTEX1602 and the L38 in the 4 kinds of chlorella to COD is best, and 6555.50mg/L and 6522.50mg/L of chlorella are purified respectively; the high value added products produced by UTEX1602 are at most: chlorophyll a reaches 70.83mg/L, carbohydrate reaches 271.57mg/L, polysaccharide reaches 117mg/L, protein exceeds other ratios by 6-7.5 times, and oil enrichment capability is better than that of other algae.
Further, the UTEX1602 protein has the highest yield and rich protein content, and can be developed for animal feed production; the UTEX1602 has the strongest ability to enrich for lipids; the UTEX1602 has the highest carbohydrate production and is capable of developing and utilizing fats and carbohydrates as an important energy source in algal biomass to produce additional products.
The invention has the advantages and positive effects that:
1. the invention is based on a mixed wastewater coupled microalgae culture system, explores the capability and influence mechanism of microalgae for purifying wastewater in a single wastewater culture mode and a mixed mode, researches the yield of high-added-value products such as pigment, grease and the like produced by the microalgae, and provides basis for exploring an economically feasible microalgae purification path and obtaining microalgae biomass with higher application value.
2. The invention discovers that in a single wastewater culture system, the growth of chlorella in bean product wastewater is limited, nutrient salts and organic carbon in solution can not be utilized, TN and NH in water 4 + The N, TP and COD concentrations were not significantly changed. The chlorella has no obvious purification effect on the water quality of the bean product wastewater. In dairy raw water in a dairy farm, TN, TP and COD concentrations decrease to different degrees along with the increase of chlorella in 0-2 days, but the descending speed is slowed down after 2 days. The removal rates of TN, TP and COD in the dairy waste water by the chlorella are respectively 37.15%, 61.09% and 16.16%, and the purification effect of the chlorella on the dairy waste water is poor.
3. The invention discovers that in the mixed wastewater coupled microalgae culture system, the increase of the ratio of bean product wastewater can promote the growth of chlorella. The algae growth condition and the wastewater nutrient removal effect shown by the 1:1 mixed wastewater are most remarkable. Wherein, the biomass dry weight accumulated by the chlorella is highest, TN, TP and COD in the mixed wastewater all show good removal effect, the highest removal rate TN is 80.3%, TP is 92.41% -95.48%, and the highest removal amount of COD is 6555.5mg/L.
4. The invention finds out an optimal cultivated chlorella strain, the chlorella UTEX1602 not only generates the highest biomass dry weight, but also has good water purification capability, has higher yields of chlorophyll a, grease, carbohydrate, protein, polysaccharide and the like, has the leaf green a of 70.83mg/L, the grease yield of up to 534.15mg/L, the carbohydrate yield of up to 271.57mg/L, the protein yield of up to 2.03g/L, and the polysaccharide of up to 117.00mg/L, and can be developed and utilized as important additional products for energy production.
Drawings
FIG. 1 is a graph showing the growth of chlorella at different mixing ratios in the present invention;
FIG. 2 is a graph showing the effect of removing total nitrogen from 4 kinds of chlorella in the mixed wastewater;
FIG. 3 is a graph showing the effect of removing ammonia nitrogen from 4 kinds of chlorella in the mixed wastewater;
FIG. 4 is a graph showing the effect of 4 kinds of chlorella in mixed wastewater on total phosphorus removal in the invention;
FIG. 5 is a graph showing the COD removal effect of 4 kinds of chlorella in the mixed wastewater;
FIG. 6 is a graph showing pigment yields of 4 kinds of Chlorella at different mixing ratios in the present invention;
FIG. 7 is a graph showing the oil yields of 4 kinds of Chlorella at different mixing ratios in the present invention;
FIG. 8 is a graph showing carbohydrate yields of 4 Chlorella species at different mixing ratios in the present invention;
FIG. 9 is a graph showing protein yields of 4 kinds of Chlorella at different mixing ratios in the present invention;
FIG. 10 is a graph showing polysaccharide yields from 4 Chlorella species at different mixing ratios in accordance with the present invention;
Detailed Description
The following describes the embodiments of the present invention in detail, but the present embodiments are illustrative and not limitative, and are not intended to limit the scope of the present invention.
The raw materials used in the invention are conventional commercial products unless specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The application of the mixed wastewater in the aspect of chlorella cultivation is that the mixed wastewater is bean product wastewater and dairy product wastewater which are mixed according to the volume ratio of 1:1-1:9.
Preferably, the volume ratio of the bean product wastewater to the dairy product wastewater is 1:1, 1:5, 1:9.
Preferably, the water quality of the bean product wastewater, the dairy product wastewater and the mixed wastewater is as follows:
preferably, the Chlorella is Chlorellasorokiniana UTEX, chlorella sp.LAMB 166 and LAMB 38, chlorella vulgaris FACHB-1227. The relevant species may be selected from species described in the following documents:
(1)Cs A,Zl A,Cw A,et al.Different interaction performance between microplastics and microalgae:The bio-elimination potential ofChlorella sp.L38 and Phaeodactylum tricornutum MASCC-0025-ScienceDirect[J].Science ofThe Total Environment,723.
(2)X Zheng,H Niu,Yu J,et al.Responses ofAlpha-linolenic acid strain(C-12)from Chlorella sp.L166 to Low Temperature Plasma Treatment[J].Bioresource Technology,2021(1):125291.
a method for culturing chlorella by using mixed wastewater comprises the following steps:
respectively taking mixed wastewater of bean product wastewater and dairy product wastewater mixed according to the volume ratio of 1:1-1:9, and adjusting the pH value to 7 by using 1M HCl or 1M NaOH after autoclaving to obtain treated mixed wastewater;
culturing, inoculating 4 kinds of Chlorella UTEX1602, L166, L38 and/or FACHB1227 when the initial light absorption value of the culture solution is at 680nm, i.e. the optical density at 680nm, is 0.2, culturing under the conditions of 30+ -1deg.C, 6500Lux illumination and 24hr illumination, and shaking for 3 times per day.
Preferably, the volume ratio of the bean product wastewater to the dairy product wastewater is 1:1, 1:5, 1:9.
Preferably, the water quality of the bean product wastewater, the dairy product wastewater and the mixed wastewater is as follows:
preferably, the Chlorella is Chlorellasorokiniana UTEX, chlorella sp.LAMB 166 and LAMB 38, chlorella vulgaris FACHB-1227.
Preferably, when the mixing ratio is 1:1, the biomass dry weights of the Chlorella are Chlorellasorokiniana UTEX, chlorella sp.LAMB166 and LAMB 38, chlorella vulgaris FACHB-1227 are 4.27g/L, 4.09g/L, 2.63g/L and 2.2g/L, respectively; the removal rate of TN by 4 kinds of chlorella is 69.03% -80.30%; chlorella UTEX1602, L166, L38 and FACHB1227 removed 231.56mg/L, 218.46mg/L, 261.07mg/L and 235.85mg/L, respectively, of TN in the solution; the removal rate of TP by 4 kinds of chlorella is 92.41% -95.48%, wherein the purifying effect of UTEX1602 is the best; the removal rate of the 4 kinds of chlorella to COD reaches 55.58% -63.06%, the removal effect of the UTEX1602 and the L38 in the 4 kinds of chlorella to COD is best, and 6555.50mg/L and 6522.50mg/L of chlorella are purified respectively; the high value added products produced by UTEX1602 are at most: chlorophyll a reaches 70.83mg/L, carbohydrate reaches 271.57mg/L, polysaccharide reaches 117mg/L, protein exceeds other ratios by 6-7.5 times, and oil enrichment capability is better than that of other algae.
Preferably, the UTEX1602 protein has the highest yield and rich protein content, and can be developed for animal feed production; the UTEX1602 has the strongest ability to enrich for lipids; the UTEX1602 has the highest carbohydrate production and is capable of developing and utilizing fats and carbohydrates as an important energy source in algal biomass to produce additional products.
Specifically, the relevant preparation and detection examples are as follows:
1 materials and methods
1.1 microalgae and wastewater samples
Test 4 kinds of chlorella: chlorellasorokiniana UTEX1602 the Chlorella vulgaris (Chlorella sp.LAMB 166 and LAMB 38) were purchased from the algae seed center of the Ostin division of the university of Texas from the applied microalgae biology laboratory of the university of ocean (Qingdao China); chlorella vulgaris FACHB1227 was purchased from China institute of aquatic organisms. The stored Chlorella strain cells were activated in liquid BG-11 medium and cultured at 25℃for 15 days under illumination of 6000Lux white fluorescent lamp.
Test wastewater: the bean product waste water (raw water) was taken from a Tianjin bean product processing plant, the dairy product waste water (raw water) was taken from a Tianjin bean product processing plant, and the water quality of the bean product waste water and dairy product waste water and the water quality of the mixed waste water in this study are shown in Table 1. Mixing bean product wastewater and dairy product wastewater in volume ratios of 1:1, 1:5 and 1:9, and adjusting the pH value to 7 by using 1M HCl or 1M NaOH after autoclaving to form mixed wastewater with different proportions.
TABLE 1 two wastewater raw water and mixed wastewater quality conditions
1.2 method
Placing 200mL of single waste water (100% bean product waste water and 100% dairy product waste water) or mixed waste water with different mixing ratios (1:1, 1:5 and 1:9) into a 250mL conical flask for culture, and obtaining an OD (optical density) when the initial light absorption value (680 nm wavelength) of each culture solution is equal to the OD 680 4 kinds of chlorella UTEX1602, L166, L38 and FACHB1227 were inoculated and added respectively to 0.2 (i.e., biomass dry weight of 0.04, 0.06, 0.02 and 0.05g/L, respectively), and incubated at 30+ -1deg.C under light intensity 6500Lux for 24hr, and the Erlenmeyer flask was manually shaken 3 times per day. Each treatment was repeated 3 times.
The culture of single waste water chlorella is totally 5 days, and the microalgae growth condition and water quality change are measured by sampling every day during the period. The mixed wastewater chlorella culture is carried out for 10 days, sampling is carried out every 2 days (days 0, 2, 4, 6, 8 and 10) during the period, the growth condition and water quality change of the microalgae are measured, and the contents of pigment, grease, carbohydrate, polysaccharide and protein are measured at the end of the culture.
1.3 determination of wastewater quality and high added value products
The pH value is measured by a pH meter and the total nitrogen(TN) was measured by persulfate oxidation, total Phosphorus (TP) was measured by digestion-molybdenum-antimony resistance, NH4 + N is determined by the Navier reagent method and the Chemical Oxygen Demand (COD) is determined according to the potassium dichromate method (Hach spectrophotometer).
Chlorophyll a, chlorophyll b and carotenoids in pigments were determined according to methods reported in the literature (J.Xiong, M.B.Kurade, J.R.Kim, H.Roh, B.Jeon, et al ciprofloxacin toxicity and its co-metabolic removal by afreshwater microalga Chlamydomonas Mexicana j. Hazard. Mater, 323 (2017), pp. 212-219). Polysaccharide and carbohydrate in microalgae were measured using the modified phenol-sulfuric acid method (P.Rao, T.N.Pattabiraman.Reevaluation of the phenol-sulfuric acid reaction for the estimation ofhexoses and assisted biochem, 181 (1989), pp.18-22); lipid concentrations were determined using nile red staining (l.zhao.isolation and Optimization Involved an Oleaginous Microalgae Doctoral disruption.tianjin University (2014)). After the microalgae after the culture period is finished are sufficiently dried and ground, all samples are analyzed by an elemental analyzer. The nitrogen content obtained by the measurement of the elemental analyzer was multiplied by 6.25 to estimate the protein content in the algal powder (ParkW K, moon M, kwak M S, et al, use oforange peel extract for mixotrophic cultivation ofChlorella vulgaris: increased production ofbiomass and FAMEs [ J ]. Bioresource Technology,2014,171 (171C): 343-349.).
1.4 data analysis
Statistical analysis was performed using one-way analysis of variance (ANOVA). Results are expressed as mean ± standard error of mean based on parallel experiments and are considered to be within 95% confidence intervals.
2 results and discussion
2.1 Effect of Mixed wastewater of different proportions on Chlorella growth and Water purification
2.1.1 Effect of Mixed wastewater on Chlorella biomass
As shown in figure 1, when the mixing ratio of the bean product wastewater to the dairy product wastewater is 1:5 and 1:9, the chlorella cultured for 0-2 days has obvious growth arrest phenomenon. This is related to the increase in the proportion of dairy waste water which makes the mixed waste water system alkaline (pH 9.60 and 10.25 respectively), thus affecting the growth of chlorella to some extent. When the mixing ratio was 1:1 (i.e., the ratio of bean product wastewater was increased), biomass dry weights of chlorella UTEX1602, L166, L38 and FACHB1227 cultivated to day 10 all reached the highest, 4.27g/L, 4.09g/L, 2.63g/L and 2.2g/L, respectively, indicating that the mixed cultivation of the two wastewater 1:1 was most suitable for the growth of chlorella.
In the experiment, the ratio (1:1) of bean product wastewater in the mixed wastewater system is increased, and the growth of 4 kinds of chlorella is promoted. This is related to the fact that the waste water of the bean products is rich in various organic or inorganic components, such as monosaccharides, oligosaccharides, vitamins, organic acids, phosphates, sulphates, metal ions and other nutrients. These substances provide sufficient nutrition for the growth of algae, in particular mono-and oligosaccharides as carbon sources for chlorella, which can be effectively utilized by them. In this case, the nutrient system of chlorella in the culture solution containing the bean product wastewater is a mode of autotrophy, that is, a metabolic system having autotrophy and heterotrophy. The growth rate of microalgae in the concurrently culture mode is about the sum of the growth rates of autotrophic growth and heterotrophic growth, and is the culture mode with the fastest growth of algae biomass. Therefore, in the mixed food waste water culture system of the experiment, the proportion of bean product waste water is increased, the organic carbon source of the mixing system is increased, the time for mixedly culturing the chlorella is relatively prolonged, the growth of 4 kinds of chlorella is promoted, and the method is the best reason for the growth condition of four strains of chlorella under the mixing proportion of 1:1.
2.1.2 removal effect of Nitrogen in Mixed wastewater
Nitrogen is an essential element for chlorella life metabolism, and chlorella can utilize ammonia nitrogen, nitrate nitrogen or nitrogen-containing compounds to promote the formation of proteins and amino acids in cells of the chlorella. TN and NH in 4 Chlorella UTEX1602, L166, L38 and FACHB1227 in systems of different mixing ratios 4 + The removal effect of N is shown in fig. 2 and 3. Indicating that the 4 kinds of chlorella all lead to TN value and NH in the mixed wastewater 4 + -a decrease in N concentration. Wherein, in the wastewater with the mixing ratio of 1:1, the growth condition of the chlorella is excellent and no hysteresis condition exists in the culture for 10 days, the TN concentration is continuously reduced, and the TN removal rate of 4 chlorella is realized69.03% -80.30%; chlorella UTEX1602, L166, L38 and FACHB1227 removed 231.56mg/L, 218.46mg/L, 261.07mg/L and 235.85mg/L, respectively, of TN in the solution. When the mixing ratio is 1:5, the removal rate of TN is 63.95-76.00 percent. When the mixing ratio is 1:9, the removal rate of TN is 53.30% -69.73%. Comparing FIGS. 1 and 2, it can be seen that the rate of decrease in TN concentration is proportional to the rate of increase in Chlorella.
In different mixing systems, due to different contents of bean product wastewater, different NH are formed at different mixing ratios 4 + -an initial concentration of N. NH in wastewater with a mixing ratio of 1:1 4 + The removal rate of N is 58.27% -65.97%. At a mixing ratio of 1:5, NH 4 + The removal rate of N is 74.38% -85.50%. At a mixing ratio of 1:9, NH 4 + The removal rate of N is highest, reaching 79.17% -86.99%, possibly related to the pH in solution. The nitrogen source in the wastewater has various forms and NH 4 + N is the nitrogen source most readily available to algal cells, NH 4 + The removal of N results in ammonia volatilization mainly by direct assimilation of microalgae or by a pH rise of the solution.
2.1.3 removal effect of phosphorus in Mixed wastewater
Phosphorus element is an essential element for maintaining normal life activities and metabolism of microalgae, and plays an important role in the synthesis of nucleic acid and protein and in the energy transfer process. FIG. 4 shows that the TP concentration in each mixed culture liquid tended to decrease. When the mixing ratio is 1:1, the chlorella has the best effect of removing TP, the removal rate is 92.41% -95.48%, and the UTEX1602 has the best purifying effect. At a ratio of 1:5, the removal rate is 74.38% -85.50%. The removal rate is 65.67-83.58% when the mixing ratio is 1:9. The waste water with different mixing ratios has the highest removal rate with the mixing ratio of 1:1. This may be related to the relatively low concentrations of TP in the water (16.74 mg/L and 8.71mg/L, respectively) in the soy product wastewater diluted in the 1:5 and 1:9 wastewater systems, due to the small initial concentrations, the total phosphorus content in the water after the end of the incubation period is 0.6-1.86mg/L and 0.25-1.43mg/L. In a word, 4 kinds of chlorella have better TP removal effect on three kinds of mixed wastewater with different proportions.
2.1.4 removal effect of COD in Mixed wastewater
As shown in FIG. 5, the COD concentration in the mixed wastewater in different proportions is continuously reduced along with the extension of the culture time, wherein the initial COD concentration in the mixed solution in the mixing proportion of 1:1 is 10344mg/L, 3821.5-4595mg/L remains at the end of the culture (10 days of the culture), and the removal rate reaches 55.58% -63.06%. UTEX1602 and L38 showed the best removal effect among the 4 kinds of Chlorella, and were purified by 6555.50mg/L and 6522.50mg/L, respectively. The initial COD concentration of the mixed ratio 1:5 is 3895.00mg/L, and the residual concentration at the end of the culture period is 1555.80-1637.00mg/L. The initial COD concentration of the mixed ratio of 1:9 is 2471.50mg/L, and the residual concentration at the end of the culture is 985.60-1444.20mg/L.
Under the condition of 3 mixing ratios, the COD removal rate of each chlorella strain is about 60%, but the COD removal amount of the chlorella under the condition of 1:1 is 6555.50mg/L due to different initial concentrations, and the purifying effect is best. Of the 4 chlorella species, UTEX1602 has the strongest removal of COD. The chlorella in the mixed solution with the ratio of 1:1 has good COD removal effect, and is related to the increase of the waste water content of bean products in the mixed proportion, the availability of organic carbon for the chlorella is more, and the growth of the chlorella is good. The better the growth of chlorella, the larger the biomass, the more vigorous the metabolism, and the more COD is degraded.
2.2.2 changes in the content of Chlorella high added value products in wastewater with different mixing ratios
2.2.1 pigment yield
The most abundant pigments in chlorella are chlorophyll a, chlorophyll b and carotenoids, which have various therapeutic properties such as antioxidant activity, regulation of cholesterol in blood, enhancement of immune system, etc.
FIG. 6 shows the contents of chlorophyll a, chlorophyll b and carotenoids produced by 4 kinds of Chlorella at different mixing ratios after the end of cultivation. The highest concentration of chlorophyll a is 1:1 mixed solution, and the concentrations in UTEX1602, L166, L38 and FACHB1227 culture solutions are 70.83mg/L, 57.01mg/L, 62.28mg/L and 51.51mg/L respectively, which are far greater than 1:5 mixed solution (14.00-23.73 mg/L) and 1:9 mixed solution (12.57-14.49 mg/L). Wang et al therefore also used chlorophyll a concentration in the aqueous environment as an indicator reflecting the growth of chlorella. Chlorophyll b is also one of photosynthetic natural pigments, light energy is absorbed and transmitted in a water body, the ratio of chlorophyll a to chlorophyll b can be used for measuring the growth condition of algae, and the algae with high ratio grow well, which is the best consistent with the growth condition of 4 kinds of chlorella in a high mixing ratio (1:1) in the experiment.
In the wastewater with 3 mixing ratios, the carotenoid content of the 4 kinds of chlorella culture solutions is low, and Kurade et al consider that the carotenoid concentration is a sensitive biomarker of water environment pollutants. The carotenoid content is high, the number of sensitive biological species or quantity in the water body is high, and the pollutant is high. In the experiment, the chlorella rapidly degrades pollutants such as organic matters, which probably is the reason for the lower concentration of carotenoid in the water body. In addition, changes in nutrient (nitrogen and phosphorus) and environmental (salinity and stress) factors are another cause of lower carotenoid concentrations in water bodies.
2.2.2 grease and carbohydrate production
Because of the exhaustion of fossil resources, the continuous exploitation of petroleum reserves to produce traditional fossil fuels is not a long term, it is important to find new energy sources to reduce the social dependence on existing fossil fuels. Microalgae biomass has received great attention as a promising renewable biofuel feedstock, with its ability to be derived into biodiesel. As shown in fig. 7, the yield of important additional product oil in algal biomass was measured, which is a promising renewable biofuel feedstock.
In the 4 chlorella species of the experiment, UTEX1602 has stronger lipid enrichment capability, and the oil yields in the mixed wastewater of 3 proportions are 337.45mg/L, 309.94mg/L and 534.15mg/L respectively. In the 1:1 mixed wastewater, the accumulation of chlorella biomass is the greatest, but the oil yield is lower than the relative oil yields of chlorella UTEX1602, L166 and L38 in the mixed liquid of which the ratio is 1:9. The oil and fat yield of chlorella in wastewater is related to the nitrogen and phosphorus content of the wastewater. Research reports indicate that nitrogen or phosphorus deficiency and other environmental stresses can lead to lipid accumulation in algal cells. That is, low nitrogen and low phosphorus in the culture solution limit synthesis of amino acids and proteins in algal cells, and carbon in photosynthesis is changed from a protein synthesis pathway to a synthesis pathway such as lipid. Therefore, this explains why chlorella grown in a 1:9 mixture of low nitrogen and low phosphorus produces more grease.
In addition, although accumulation of biomass in algal cells can also increase the oil yield. For example, the biomass accumulation amount of chlorella in the 1:1 mixed solution is highest, but the concentration of the nitrogen source is high (2.06-3.67 times of the concentration of other mixed nitrogen sources), so that more amino acids and proteins are synthesized by the algae cells, the carbon source for synthesizing fat is reduced, and then the fat content in the algae cells is lower (the mixed solution lower than 1:9).
Carbohydrates are biochemical components rich in microalgae biomass and can also be used for energy production, for example, ethanol production or biogas production. FIG. 8 shows that the Chlorella carbohydrate yield in the 1:1 mixture was significantly higher than in the 1:5 and 1:9 mixtures, with the highest yielding strain being Chlorella UTEX1602, possibly associated with its ability to produce higher biomass yields. The research on the synthesis of the chlorella carbohydrate and the grease in the wastewater has important significance for the development and the reuse of biological energy.
2.2.3 protein production
The nitrogen source is one of the important factors directly affecting the growth of microalgae. The nitrogen is used by algae cells to synthesize amino acids and proteins and promote the growth of microalgae. In the mixed wastewater culture system, TN concentration in the solution increases as the proportion of the bean product wastewater increases. When the mixed solution with different proportions is cultured until 10 days, the protein yield (shown in figure 9) of 4 kinds of chlorella (UTEX 1602, L166, L38 and FACHB 1227) is proportional to the TN content (shown in figure 2), the total nitrogen concentration in the mixed solution is high, and the protein yield is also high.
Wherein the protein yield of the chlorella in the 1:1 mixed solution is far higher than that of the mixed solution of 1:5 and 1:9. The highest protein yield (2.03 g/L) of Chlorella UTEX1602 in the 1:1 mixture was 6 times the highest protein yield (0.34 g/L) in the 1:5 mixture and 7.5 times the highest protein yield (0.27 g/L) in the 1:9 mixture.
Protein synthesis is critical to microalgae growth. While insufficient nutrition inhibits protein synthesis and microalgae growth. In this study, the initial nitrogen content in the low-concentration soy product wastewater broth (mixing ratios 1:5 and 1:9) did not meet the need for chlorella protein synthesis, resulting in reduced protein content in chlorella cells by day 10 of culture, and reduced final protein yield. The biomass of chlorella grown in the 1:1 mixture has a high protein content, and thus can be considered as an animal feed or the like.
2.2.4 polysaccharide production
Polysaccharide is an important bioactive substance, is used for growth and reproduction of algae, and has special physiological effects. The polysaccharide has wide application in the aspects of biological pharmacy, functional food, environmental protection and the like. Microalgae are considered to be one of the potential sources of polysaccharides. Polysaccharide yields of 4 chlorella UTEX1602, L166, L38 and FACHB1227 cultured in the mixture of different ratios are shown in FIG. 10.
Polysaccharide yields of the 4 chlorella UTEX1602, L166, L38 and FACHB1227 were significantly higher than the 1:5 and 1:9 mixtures, which may be associated with the highest biomass accumulation of chlorella in the 1:1 mixtures.
Conclusion 3
(1) In the mixed wastewater coupled microalgae culture system, the increase of the ratio of bean product wastewater can promote the growth of chlorella. The biomass dry weight of chlorella accumulation in the 1:1 mixed wastewater is highest, wherein the biomass dry weight of chlorella UTEX1602 is highest. Compared with other mixing ratios, the 4 kinds of chlorella in the 1:1 mixed wastewater have the best TN and TP removal effects, the highest COD purification capacity is strong, the highest removal rate TN is 80.3%, TP is 92.41-95.48%, and the highest COD removal amount is 6555.5mg/L. Of these, UTEX1602 has the strongest purification capacity for TN, TP, COD.
(2) In the 1:1 mixed wastewater, the content of chlorophyll a produced by 4 kinds of chlorella is highest, wherein the content of phyllanthus green a produced by UTEX1602 reaches 70.83mg/L, and chlorophyll is the necessary pigment for obtaining light and protecting microalgae cells from radiation.
(3) Of the 4 kinds of chlorella, UTEX1602 has the strongest lipid enrichment capacity, and the oil and fat yields in the 3 kinds of mixed solutions are 337.45mg/L, 309.94mg/L and 534.15mg/L respectively. In the 1:1 mixed wastewater, the yield of the chlorella carbohydrate is obviously higher than that of the wastewater with the ratio of 1:5 to 1:9, wherein the algae strain with the highest yield is UTEX1602 (271.57 mg/L). It was found that accumulation of biomass in algal cells can increase the oil yield; the carbohydrate is a biochemical component rich in microalgae biomass, and can be used for energy production, such as ethanol production or biogas production. Grease and carbohydrate can be exploited as important energy sources in algal biomass to produce additional products.
(4) In the 1:1 mixed wastewater, the UTEX1602 protein yield is 6-7.5 times of that of other proportion wastewater, and the wastewater has rich protein content and can be developed for animal feed production.
(5) In the mixed wastewater with the ratio of 1:1, 4 kinds of chlorella have the highest biomass dry weight and highest polysaccharide yield, and can be used for the growth and propagation of algae.
Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.
Claims (2)
1. A method for culturing chlorella by using mixed wastewater is characterized in that: the method comprises the following steps:
respectively taking mixed wastewater of bean product wastewater and dairy product wastewater mixed according to a volume ratio of 1:1, and after autoclaving, regulating the pH value to 7 by using 1MHCl or 1M NaOH to obtain treated mixed wastewater;
culturing, inoculating 4 kinds of Chlorella (Chlorellasorokiniana UTEX, chlorella sp.LAMB 166, chlorella sp.LAMB 38 or Chlorella vulgaris FACHB-1227) when the initial light absorption value of the culture solution is at 680nm (optical density OD 680=0.2), culturing at 30+ -1deg.C under 6500Lux illumination time of 24hr, and shaking for 3 times per day;
the water quality condition of the treated mixed wastewater is as follows:
when the mixing ratio is 1:1 and cultured until day 10, the biomass dry weights produced by the Chlorella Chlorellasorokiniana UTEX1602, chlorella sp.LAMB 166 and Chlorella sp.LAMB 38, chlorella vulgaris FACHB-1227 are 4.27g/L, 4.09g/L, 2.63g/L and 2.2g/L respectively; the removal rate of the TN by the 4 kinds of chlorella is 69.03% -80.30%; the chlorella UTEX1602, LAMB 166, L38 and FACHB1227 removed 231.56mg/L, 218.46mg/L, 261.07mg/L and 235.85mg/L, respectively, of the TN in solution; the removal rate of the TP by the 4 kinds of chlorella is 92.41-95.48%, wherein the UTEX1602 has the best purifying effect; the removal rate of the 4 kinds of chlorella to the COD reaches 55.58% -63.06%, and the UTEX1602 and the LAMB 38 in the 4 kinds of chlorella respectively purify 6555.50mg/L and 6522.50mg/L of the COD; chlorophyll a produced by UTEX1602 reaches 70.83mg/L, carbohydrate reaches 271.57mg/L, and polysaccharide reaches 117mg/L.
2. The method for culturing chlorella using mixed wastewater according to claim 1, wherein: the UTEX1602 protein yield was highest among the 4 chlorella species; the UTEX1602 enriched in lipids was the strongest among the 4 chlorella species; the carbohydrate yield of the UTEX1602 was highest among the 4 chlorella species.
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