Method for detecting optical density of microalgae biomass

Abstract

The invention relates to a microalgae biomass optical density detection method which comprises the following steps: taking a certain volume of algae liquid obtained under different batches or different culture conditions, repeatedly filtering the algae liquid by using a filter screen dried to constant weight, drying the algae body and the filter screen to constant weight, representing the dry weight of microalgae biomass by using a difference value before and after drying, and calculating the optical density of the microalgae biomass according to a calculation result. Taking a certain volume of algae solutions obtained in different batches or under different culture conditions, and fixing the volume to the same dry weight to obtain an algae solution to be detected; carrying out full-wavelength scanning on the algae liquid to be detected to obtain full-spectrum characteristics; performing variable coefficient analysis on the full-spectrum characteristics to obtain a screening scanning wave band; and selecting the wavelength corresponding to the minimum variable coefficient from the screening scanning wavebands as the final detection wavelength. By performing variable coefficient analysis on the absorption spectrum of the microalgae liquid, an optimal wavelength interval for detecting the optical density of the biomass can be selected, the influence of photosynthetic pigment composition of the microalgae on the optical density value is avoided to a great extent, and the real biomass condition is better reflected.

Description

Technical Field

[0001] The invention belongs to the field of microalgae biomass detection, and particularly relates to a microalgae biomass optical density detection method. Background Art

[0002] Microalgae usually refers to tiny algae whose morphology can only be identified under a microscope, such as Chlorella, Spirulina, Nostoc, etc. Since microalgae are small and often evenly distributed in liquid culture media such as water bodies, their biomass detection methods mainly include cell counting based on microscopic observation, dry weight method based on drying to obtain algae dry matter, and optical density (OD) method based on the spectral characteristics of algae liquid. The cell counting method takes a long time to detect, and some microalgae are difficult to accurately count under a microscope. The dry weight method is a more accurate biomass detection method, but it often requires steps such as filtration and drying, which is time-consuming and requires more operations. In addition, the samples are disposable consumables, and multiple tests require a large amount of samples. At present, the optical density method is widely used, using equipment such as a spectrophotometer to measure the optical density of the algae liquid to indirectly reflect the relationship between cell density and biomass. This method has the advantages of real-time, fast, simple operation, and small sample demand.

[0003] The working principle of the microalgae biomass optical density detection method is based on the Lambert-Beer law of the spectrophotometer, that is, the optical density of a substance at a certain wavelength is proportional to the concentration and optical path length of the substance. Microalgae liquid is a suspension composed of culture medium and algae cells suspended therein. In addition to the absorption effect on incident light, it also has reflection, refraction and scattering effects, which may not fully meet the conditions for the Lambert-Beer law to be established. In addition, the absorption of light by algae liquid is not only directly related to biomass or cell density, but also to the size, shape and photosynthetic pigment composition of the algae. Therefore, when the algae morphology or photosynthetic pigment composition changes, the absorption spectrum characteristics will also change accordingly.

[0004] Studies have shown that the spectral characteristics of microalgae vary significantly within and between species, which may be related to the algae particle size and photosynthetic pigment composition. In addition to algae species, growth environmental conditions such as season, light, temperature, nutrition and water body have a significant impact on the morphology and photosynthetic pigment content of microalgae, which will affect the spectral characteristics of the algae solution, and then affect the accuracy of the OD value as an indicator of microalgae biomass. Therefore, when comparing the biomass of microalgae under different culture conditions or batches, the spectral characteristics are often different due to differences in algae particle size and photosynthetic pigment composition, resulting in a decrease in the accuracy of the optical density method, and a significant difference in optical density at the same dry weight. Therefore, it is necessary to develop a universal microalgae biomass detection method. Summary of the invention

[0005] The purpose of the present invention is to solve the problem that the accuracy of the optical density method is reduced due to the different spectral characteristics caused by the differences in algae particle size and pigment composition in the existing microalgae optical density detection method.

[0006] The purpose of the present invention is to adopt the following technical solutions to achieve:

[0007] A method for detecting the optical density of microalgae biomass comprises the following steps:

[0008] Take a certain volume of algae liquid obtained from different batches or under different culture conditions, filter it repeatedly with a filter that has been dried to a constant weight, and then dry the algae together with the filter to a constant weight. The difference before and after drying represents the dry weight of the microalgae biomass. According to the calculation results, take another certain volume of algae liquid obtained from different batches or under different culture conditions, and adjust the volume to the same dry weight to obtain the algae liquid to be tested;

[0009] Performing full-wavelength scanning on the algae liquid to be tested to obtain full-spectrum characteristics;

[0010] Performing coefficient of variation analysis on the full spectrum characteristics to obtain a screening scanning band;

[0011] The wavelength corresponding to the minimum coefficient of variation is selected from the screening scanning band as the final detection wavelength.

[0012] Preferably, the wavelength band of the full wavelength scan is 300-800 nm.

[0013] Preferably, the coefficient of variation analysis specifically includes: calculating the coefficient of variation of the optical density values ​​of microalgae in different batches or under different culture conditions at the same wavelength, and drawing a fitting curve with the obtained coefficient of variation as the ordinate and the corresponding wavelength as the abscissa; the calculation formula of the coefficient of variation is:

[0014]

[0015] Where CV is the coefficient of variation, σ is the standard deviation, and μ is the mean.

[0016] Preferably, the screening scanning band is a region that is not within the photosynthetic pigment absorption peak range and where the coefficient of variation is relatively stable.

[0017] Preferably, the final detection wavelength is a visible light wavelength.

[0018] Preferably, the microalgae include one or more of unicellular algae species, multicellular algae species, a single algae species, a mixture of multiple algae species, algae species from the same batch, and a mixture of multiple batches of algae species.

[0019] Preferably, when the microalgae include non-unicellular algae species, the step of taking a certain volume of microalgae obtained from different batches or under different culture conditions and fixing the volume to the same dry weight to obtain the algae solution to be tested further comprises:

[0020] A certain volume of algae solution obtained from different batches or under different culture conditions is taken, fixed to the same dry weight, and homogenized to obtain the algae solution to be tested.

[0021] Preferably, the homogenization treatment specifically includes breaking the microalgae by ultrasound to achieve morphological homogenization of the microalgae.

[0022] Preferably, the ultrasonic conditions of the ultrasonic wave are determined according to the homogenization degree and cell disruption rate of the algal filaments after treatment, and ultrasonic conditions with high homogenization degree and low cell disruption rate are preferred.

[0023] The specific conditions of ultrasound refer to specific conditions such as ultrasonic power, ultrasonic duration, ultrasonic interval and ultrasonic frequency. Different algae species and different equipment may be suitable for different ultrasonic conditions. The degree of homogenization of the algae filaments needs to be observed and compared under a microscope. The closer their morphology is, the higher the degree of homogenization is. It is generally recommended that the algae filaments be at the single cell level (several microns) for best results.

[0024] Preferably, the fragmentation rate of the algae filaments is not higher than 25%, and the calculation formula of the cell fragmentation rate is:

[0025] S=R t / R max

[0026] Where S is the cell breakage rate, R t is the amount of dissolved matter released under the test conditions, R max The maximum amount of released extracts.

[0027] The cell disruption rate refers to the detection of a cell lysate such as the content of photosynthetic pigments in the supernatant of the algae solution obtained under ultrasonic conditions by centrifugation. The higher the content of the cell lysate, the higher the degree of cell disruption.

[0028] The technical principle of the present invention is as follows: the present invention provides an optical density biomass detection method based on algae homogenization treatment and screening scanning bands. The influence of microalgae morphology on the detection results is reduced by using ultrasound under specific conditions to homogenize cells, and the influence of photosynthetic pigment composition on the optical density value is screened by full spectrum scanning and coefficient of variation analysis of algae liquid, thereby improving the reliability of the detection results.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The method for detecting the optical density of microalgae biomass of the present invention comprises the following steps: taking a certain volume of algae liquid obtained from different batches or under different culture conditions, filtering repeatedly with a filter screen dried to constant weight, drying the algae together with the filter screen to constant weight, and taking the difference before and after drying to represent the dry weight of the microalgae biomass; according to the calculation results, taking a certain volume of algae liquid obtained from different batches or under different culture conditions, and fixing the volume to the same dry weight to obtain the algae liquid to be tested; performing full wavelength scanning on the algae liquid to be tested to obtain full spectrum characteristics; performing coefficient of variation analysis on the full spectrum characteristics to obtain a screening scanning band; and selecting the wavelength corresponding to the minimum coefficient of variation from the screening scanning band as the final detection wavelength. By performing coefficient of variation analysis on the absorption spectra of algae liquids from different batches or under different culture conditions, the preferred wavelength range for biomass optical density detection can be selected, which can avoid the influence of the photosynthetic pigment composition of microalgae on the optical density value to the greatest extent, better reflect the actual biomass situation, and has wide applicability.

[0031] 2. When the microalgae is a non-unicellular algae species, the microalgae biomass optical density detection method of the present invention further includes breaking up the algae liquid by ultrasound to achieve morphological homogenization of the microalgae and avoid the influence of the microscopic morphology of the microalgae on the optical density value, which has high credibility and good universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a technical flow chart of the present invention;

[0033] Figure 2 The full spectrum characteristics of algae liquid with the same dry weight under different culture conditions in Example 1 of the present invention;

[0034] Figure 3 is a curve showing the relationship between the coefficient of variation and wavelength of the algae solution in Example 1 of the present invention;

[0035] Figure 4 Comparison of the optical density of algae liquid measured by the present method and the conventional method in Example 1 of the present invention;

[0036] Figure 5 is the algae solution OD in Example 1 of the present invention 742 Correlation curve with biomass;

[0037] Figure 6 The microscopic morphology of Spirulina in Example 2 of the present invention;

[0038] Figure 7 The microscopic morphology of Spirulina after homogenization treatment in Example 2 of the present invention;

[0039] Figure 8 It is the full spectrum characteristics of the homogenized algae liquid with the same dry weight under different culture conditions in Example 2 of the present invention;

[0040] Fig. 9 is a relationship curve between the coefficient of variation and wavelength of the homogenized algae solution in Example 2 of the present invention;

[0041] Fig.10 Comparison of the optical density of algae liquid measured by the present method and the conventional method in Example 2 of the present invention;

[0042] Fig.11 is the OD of the homogenized algae solution in Example 2 of the present invention 520 Correlation curve with biomass. DETAILED DESCRIPTION

[0043] The specific implementation methods of the present invention are described in detail below with reference to examples, but the implementation examples do not limit the present invention in any form. For those skilled in the art, any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0044] In order to solve the problem that the optical density of microalgae obtained under different culture conditions is significantly different at the same biomass (dry weight), the present invention provides an optical density biomass detection method based on algae homogenization treatment and screening scanning bands. Figure 1 Technical process.

[0045] Example 1 (single-cell algae species)

[0046] In this embodiment, the microalgae is Chlorella vulgaris, which is a unicellular algae species. The conventional detection method is to directly measure the optical density (OD) of the algae solution at 680 nm. 680 ).

[0047] Use the method described in this application to re-determine the appropriate detection wavelength, refer to Figure 1 The technical process, the specific steps are as follows:

[0048] 1) Algae liquid treatment: Take 20mL of algae liquid obtained under different light quality (red and blue light) culture conditions of T1, T2, T3, T4, and T5, and filter it three times through a filter with a pore size of 5μm that has been dried to constant weight, and dry the algae together with the filter to constant weight. The difference before and after drying represents the dry weight of microalgae biomass (g / L). According to the calculated dry weight results, take a certain volume of algae liquid obtained under different culture conditions, adjust the volume to the same dry weight, and obtain the algae liquid to be tested.

[0049] 2) Full spectrum scanning: The algae solution of the Chlorella vulgaris is transferred to a cuvette, and clear water is used as a blank control. The algae solution is subjected to full spectrum scanning using a spectrophotometer, and the scanning band is 300-800 nm to obtain full spectrum characteristics, such as Figure 2 shown.

[0050] 3) Spectral feature analysis:

[0051] 301. Calculate the coefficient of variation: According to the algae liquid absorption spectrum obtained in step 1), the coefficient of variation of the optical density of the microalgae at the same wavelength is analyzed to evaluate the degree of deviation between the data. The calculation formula is:

[0052]

[0053] Where CV is the coefficient of variation, σ is the standard deviation, and μ is the mean.

[0054] 302. Draw a fitting curve: Use the coefficient of variation obtained in step 301 as the ordinate and the corresponding wavelength as the abscissa to draw a relationship curve, such as Figure 3 shown.

[0055] 303. Screening scanning band: by Figure 3 It can be seen that the coefficient of variation in the 700-760 nm band is low and the amplitude of variation is relatively stable, indicating that the absorption spectrum in this band is less affected by photosynthetic pigments.

[0056] 4) Determine the detection wavelength: Analyze the screening scanning band obtained in step 3), and preferentially select 742 nm with the smallest coefficient of variation in the visible light range as the final detection wavelength.

[0057] Verification and result analysis:

[0058] Comparative analysis: The optical density values ​​of algae liquid with the same dry weight under different culture conditions were measured at 680nm and 742nm respectively. The results are as follows: Figure 4 As shown, OD 680 The range and variance of the algae solution were 0.072 and 0.0008 respectively; 742 The range and variance are 0.035 and 0.0002, respectively, which is higher than OD 680 The decreases were 51.15% and 75.90%, respectively. It can be seen that at the detection wavelength obtained by this method, the difference in absorbance of algae liquid under different culture conditions at the same dry weight was significantly reduced.

[0059] Draw a regression equation: The algae solution under a certain culture condition was fixed to different dry weights, transferred to a cuvette, and water was used as a control. The optical density of the homogenized algae solution at 742 nm was measured using a spectrophotometer, and the OD was plotted. 742 The relationship curve between (x) and dry weight (y) is as follows Figure 5 As shown, the regression equation is: y = 0.5573x + 0.0021, and the linear correlation coefficient R 2 =0.9993, algae solution OD 742There is a good linear relationship with biomass. This shows that the optical density value of microalgae measured by this method has a high correlation with biomass, avoiding the influence of photosynthetic pigment composition of microalgae on the optical density value and better reflecting the actual biomass situation.

[0060] Example 2 (Multicellular algae species)

[0061] In this embodiment, the microalgae is Spirulina, which is a multicellular algae species, and its microscopic morphology is as follows: Figure 6 As shown, it can be seen that there are differences in the length of the algal filaments. The conventional detection method is to directly measure the optical density (OD) of the algal liquid at 560nm. 560 ).

[0062] Use the method described in this application to perform homogenization and re-determine the appropriate detection wavelength, refer to Figure 1 The technical process, the specific steps are as follows:

[0063] 1) Algae liquid treatment:

[0064] 101. Obtain algae liquid of the same dry weight: Take 20 mL of algae liquid obtained under different light quality (red and blue light) culture conditions of T1, T2, T3, T4, and T5, and filter it three times through a filter with a pore size of 5 μm that has been dried to constant weight, and dry the algae together with the filter to constant weight. The difference before and after drying represents the dry weight of microalgae biomass (g / L). According to the calculated dry weight results, take a certain volume of algae liquid obtained under different culture conditions, adjust the volume to the same dry weight, and obtain algae liquid under the same dry weight treatment.

[0065] 102. Determine the ultrasonic conditions:

[0066] Take 5 ml of algae liquid in a 10 ml centrifuge tube, set the ultrasonic frequency to 20-25 kHz, immerse the front end of the Φ3-type horn 2 cm into the algae liquid, and use the ultrasonic method of 3s / 3s (on / off) intervals, use ultrasonic conditions of different powers and different times for homogenization and microscopic observation, and select the ultrasonic conditions under which the algae filaments are processed into a uniform morphology of several micrometers. The algae liquid obtained under the above ultrasonic conditions was repeatedly filtered three times with a filter with a pore size of 5μm into 5 ml of phosphate buffer, centrifuged at 12000r / min for 5min, and the release of phycocyanin in the supernatant was measured to characterize the degree of cell disruption. The calculation formula is shown below.

[0067]

[0068] Where P is the dry weight of microalgae biomass, R is the release of phycocyanin, and R max is the maximum release amount of phycocyanin (with the increase of ultrasonic power, the release amount of phycocyanin tends to remain unchanged when the cells are almost completely broken. At this time, the measured release amount of phycocyanin is R max), S is the cell disruption rate. Select ultrasonic conditions with good homogenization of algae filaments (the algae filaments are all at the level of several microns) and low cell disruption rate (≤25%). In this example, the selected ultrasonic power is 400W and the time is 1 minute.

[0069] 103. Homogenization treatment: Take 5 ml of the algae solution under the same dry weight treatment in a 10 ml centrifuge tube, set the ultrasonic frequency to 20-25 kHz, the transmission power to 400 W, immerse the front end 2 cm of the Φ3-type horn into the algae solution, and ultrasonicate for 1 min at an interval of 3s / 3s (on / off) to obtain the algae solution to be tested. The algae solution to be tested is an algae solution with a uniform morphology of several micrometers in length of algae filaments, a cell disruption rate of 22.5%, and its microscopic morphology observed under an optical microscope is as follows: Figure 7 shown.

[0070] 2) Full spectrum scanning:

[0071] The algae solution to be tested in step 1) is transferred to a cuvette, and clean water is used as a blank control. The full spectrum is scanned by a spectrophotometer, and the scanning band is 300-800nm ​​to obtain the full spectrum characteristics, such as Figure 8 shown.

[0072] 3) Spectral feature analysis:

[0073] 301. Calculate the coefficient of variation: According to the full spectrum characteristics obtained in step 2), the coefficient of variation of the optical density of microalgae at the same wavelength is analyzed to evaluate the degree of deviation between the data. The calculation formula is:

[0074]

[0075] Where CV is the coefficient of variation, σ is the standard deviation, and μ is the mean.

[0076] 302. Draw a fitting curve: Use the coefficient of variation obtained in step 301 as the ordinate and the corresponding wavelength as the abscissa to draw a relationship curve, such as Fig. 9 shown.

[0077] 303. Screening scanning band: from Fig. 9 It can be seen that the coefficient of variation in the 322-366, 504-566, and 700-712 nm bands is low and the range of variation is relatively stable, indicating that the absorption spectrum in this band is less affected by photosynthetic pigments.

[0078] 4) Determine the detection wavelength: Analyze the scanning band selected in step 3). Considering that the wavelength of 322-366 nm does not belong to the visible light range, and the wavelength range of 700-712 nm is too narrow and the error space is small, 504-566 nm can be selected as the wavelength for Spirulina optical density detection, and 520 nm with the smallest coefficient of variation is selected as the final detection wavelength of optical density.

[0079] Verification and result analysis:

[0080] Comparative analysis: The optical density (OD) of the homogenized algae solution was measured at 520 nm. 520 ), compared with the conventional detection method that directly measures the optical density (OD) at 560 nm without homogenization. 560 ) are compared, and the results are as follows Fig.10 As shown, the OD of algae solution measured by conventional method 560 The range and variance of the algae solution were 0.112 and 0.0021 respectively; the OD of the algae solution measured by the method described in this application 520 The range and variance were 0.035 and 0.0002, respectively, which were reduced by 68.66% and 89.50% compared with the conventional method. It can be seen that the absorbance difference of algae solution under different culture conditions at the same dry weight was significantly reduced after homogenization and screening of the detection wavelength by this method.

[0081] Draw a regression equation: The algae solution under a certain culture condition was fixed to different dry weights, and after homogenization, it was transferred to a cuvette. Clean water was used as a control. The optical density of the homogenized algae solution at 520nm was measured by a spectrophotometer, and the OD was plotted. 520 The relationship curve between (x) and dry weight (y) is as follows Fig.11 As shown, the regression equation is: y = 0.6932x-0.0092, and the linear correlation coefficient R 2 =0.9996, homogenized algae solution OD 520 There is a good linear relationship with biomass. This shows that the optical density value of microalgae measured by this method has a high correlation with biomass, avoiding the influence of microscopic morphological differences and photosynthetic pigment composition of microalgae on the optical density value, and can better reflect the actual biomass situation.

[0082] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are included in the scope of the claims of the present invention to be approved.

Claims

1. A method for detecting optical density of microalgae biomass, characterized in that: The steps include: Take a certain volume of algae liquid obtained from different batches or under different culture conditions, filter it repeatedly with a filter that has been dried to a constant weight, and then dry the algae together with the filter to a constant weight. The difference before and after drying represents the dry weight of the microalgae biomass. According to the calculation results, take another certain volume of algae liquid obtained from different batches or under different culture conditions, and adjust the volume to the same dry weight to obtain the algae liquid to be tested; Performing full-wavelength scanning on the algae liquid to be tested to obtain full-spectrum characteristics; Performing coefficient of variation analysis on the full spectrum characteristics to obtain a screening scanning band; The wavelength corresponding to the minimum coefficient of variation is selected from the screening scanning band as the final detection wavelength.

2. The method for detecting optical density of microalgae biomass according to claim 1, characterized in that: The wavelength range of the full wavelength scan is 300-800 nm.

3. The method for detecting optical density of microalgae biomass according to claim 1, characterized in that: The coefficient of variation analysis specifically includes: calculating the coefficient of variation of the optical density values ​​of algae liquid obtained from different batches or under different culture conditions at the same wavelength, and drawing a fitting curve with the obtained coefficient of variation as the ordinate and the corresponding wavelength as the abscissa; The calculation formula of the coefficient of variation is: Where CV is the coefficient of variation, σ is the standard deviation, and μ is the mean.

4. The method for detecting optical density of microalgae biomass according to claim 1, characterized in that: The screening scanning band is a region that is not within the photosynthetic pigment absorption peak range and has a relatively stable coefficient of variation.

5. The method for detecting optical density of microalgae biomass according to claim 1, characterized in that: The final detection wavelength is a visible light wavelength.

6. The method for detecting optical density of microalgae biomass according to claim 1, characterized in that: The microalgae include one or more of unicellular algae species, multicellular algae species, a single algae species, a mixture of multiple algae species, algae species from the same batch, and a mixture of multiple batches of algae species.

7. The method for detecting optical density of microalgae biomass according to claim 6, characterized in that: When the microalgae include non-unicellular algae species, the step of taking another certain volume of algae solution obtained from different batches or under different culture conditions and fixing the volume to the same dry weight to obtain the algae solution to be tested further comprises: A certain volume of algae solution obtained from different batches or under different culture conditions is taken, fixed to the same dry weight, and homogenized to obtain the algae solution to be tested.

8. The method for detecting optical density of microalgae biomass according to claim 7, characterized in that: The homogenization treatment specifically includes breaking up the algae liquid by ultrasonic waves to achieve morphological homogenization of the microalgae.

9. The method for detecting optical density of microalgae biomass according to claim 8, characterized in that: The ultrasonic conditions of the ultrasonic wave are determined according to the homogenization degree and cell disruption rate of the algal filaments after treatment, and ultrasonic conditions with high homogenization degree and low cell disruption rate are preferred.

10. The method for detecting optical density of microalgae biomass according to claim 9, characterized in that: The fragmentation rate of the algae filaments is not higher than 25%, and the calculation formula of the cell fragmentation rate is: S=R t / R max Where S is the cell breakage rate, R t is the amount of dissolved matter released under the test conditions, R max The maximum release amount of dissolved substances.

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