Algal Organic Ma tt er Fluorescence Analysis of Chlorella sp. for Biomass Estimation

: Algal Organic Matter (AOM) is derived from the dissolved organic matter composition of the algal species observed. In this study, excitation-emission fluorescence spectroscopy has been used to determine Chlorella sp.’s AOM and pigment characteristics in varying algal biomass concentrations. The AOM and pigment characteristics were observed at 400–600 nm and 600–800 nm fluorescence emission, respectively, with an excitation spectrum of 300-450 nm. F680/450 is computed based on the ratio between the chlorophyll-a at 680 nm and dissolved organic matter contribution at 450 nm. F450/680 positively correlated with algal biomass (r = 0.96) at 405 nm excitation wavelength of Chlorella sp. This study is a good reference in algal biomass estimation and production in natural waters.


Introduction
Algae are mostly abundant in rivers and reservoirs connected to drinking water facilities and factories [1,2].With rapid urbanization and industrialization, effluents can severely affect the environment.This may result in an increase in algal organic matter (AOM) in surface waters.Organic substances produced by algae lead to water discoloration, odor and toxicity problems, and algal blooms [3][4][5].
Different techniques have been discussed to characterize organic matter in aquatic systems, which can be used to understand the composition of AOM in natural waters.These include specific ultraviolet absorption (SUVA), excitation-emission matrix (EEM) fluorescence spectroscopy, and lidar systems [6][7][8][9].These techniques are promising tools in understanding the behaviors and composition of AOM and chlorophyll-a pigment in natural waters.
The behavior and composition of Chlorella sp. has been studied using spectroscopic techniques.Our interest aims to study excitation-emission (Ex/Em) pairs for algal organic matter and pigment measurement in natural waters.This EEM fluorescence analysis may improve our existing portable fluorescence lidar systems used in algal biomass estimation.Fluorescence ratio was used to understand the contributions of chlorophyll-a and AOM in the different growth phases of microalgae.The in-situ and real-time monitoring technique is currently being studied as it provides new information with faster interpretation.

Algal Preparation
Chlorella sp.inoculum was provided by the Microalgae Systematics and Applied Phycology Research Unit of De La Salle University using a BG-11 culture medium.The initial optical density is 0.6, with a pH between 8 and 9.The light/dark duration was controlled at 12 h/12 h with a light intensity of 75 µmol/m 2 /s.
The extracted AOM was obtained with the following processes.The algal supernatant was processed through centrifugation at 8000 r•min −1 for 5 min.It was then filtered with deionized water using 0.47 µm glass filter fiber (Whatman, USA).

UV-Vis Measurement by Absorbance Spectroscopy
Aromaticity, size, and aromatic substances of algae can be interpreted by measuring SUVA254.The protein-like structures can also be characterized by measuring SUVA280.The method performed was based on the characterization of water quality in rivers and estuaries [10,11].

Fluorescence Measurement by Excitation-Emission Spectroscopy
The fluorescence measurements expressed in normalized units are EEM of AOM and pigment at varying biomass concentrations.The excitation-emission pair is obtained by changing the excitation wavelength at 5 nm intervals.A 3D EEM of varying algal biomass and pigments of Chlorella sp. was analyzed.A detailed discussion on the fluorescence setup and data analysis is provided in our previous paper on algal growth and real-time monitoring in natural waters [12][13][14].

Application of EEM to Fluorescence Lidar Measurements
The fluorescence EEM provides preliminary guidelines for constructing a fluorescence lidar by identifying excitation-emission combinations.This study explores the possibility of fluorescence intensity profiles using 380 nm and 405 nm excitation wavelengths.The region of interest for AOM is 400-600 nm and 600-800 nm for pigment measurements.Developing a fluorescence lidar system for biomass estimation is recommended since it is an in-situ and non-invasive technique.

Comparison of Fluorescence EEM
In recent studies, Biomass measurements use indirect or direct techniques.The results from the introduced fluorescence ratio are correlated (Pearson's r) with optical density measurements, biomass estimation, and estimated chlorophyll-a concentrations.

Results and Discussion
The growth of Chlorella sp. in controlled culture media was analyzed for its AOM and pigments for biomass estimation.The fluorescence EEM is crucial in developing the new fluorescence lidar system for biomass estimation.

Spectral Characterization of Chlorella sp.
The variance in SUVA254 and SUVA280 in varying biomass are shown in Figure 1a.The SUVA254 measured in AOM ranges from 0.3 to 1.4 L mg −1 •m −1 .This means that the aromaticity of AOM is low, indicating a smaller number of protein-like substances [15].The same trend was observed between SUVA254 and SUVA280, which shows a positive correlation between aromaticity and protein-like structures (r = 0.89, p < 0.05).These changes in organic matter from the logarithmic phase to the decline phase are helpful in algal growth monitoring.The fluorescence intensity results from 3D EEMs at varying algal biomass are presented in Figure 1b.The fluorescence EEMs based on Ex/Em peaks are valuable for distinguishing different types of organic matter components and types of natural waters.All algal biomass showed similar fluorescence EEM trends for AOM and pigments.The total organic matter contribution is measured at 450 nm fluorescence emission [16].The fluorescence emission is commonly used for surface water and terrestrial systems [17].The studied excitation/emission pair for algal organic matter are 385 nm/405 nm and 405 nm/450 nm.Higher fluorescence intensity profiles were measured using 405 nm/450 nm but showed no significant difference at 385 nm/450 nm (p < 0.05).
The same trend was reflected in the 405 nm/680 nm and 385nm/680 nm pairs for chlorophyll-a measurement (p < 0.05).The mean values were compared using Pearson's r correlation (p < 0.05) as shown in Figure 2. The Ex/Em 385 nm/450 nm pair showed a higher correlation as compared to Ex/Em 405 nm/450 nm with the algal biomass concentration.This is also identical to using 680 nm fluorescence emission.On the other hand, the fluorescence ratio showed opposite results.F450/680 at 405 nm excitation wavelength (r = 0.96) positively correlates to F450/680 at 385 nm excitation wavelength (r = 0.94) with algal biomass.This F-ratio is recommended to understand the dry-weight algal biomass in natural waters.

Analysis of Fluorescence EEM to the Development of the Lidar System
Developing a portable fluorescence lidar system is vital for real-time monitoring and estimating algal biomass in natural waters.With the guidance of excitation-emission pairs used in this study, improving the existing fluorescence lidar system with a new excitation wavelength at 405 nm is recommended.Simultaneous measurements at 450 and 680 nm are also suggested to understand the behavior of AOM and chlorophyll concentration of microalgae, respectively (Figure 3).F450/680 is used to calculate dry weight measurements of algal biomass of Chlorella sp. for 385 nm and 405 nm excitation wavelengths.Similar trends were observed, but a higher positive correlation was computed at 405 nm excitation wavelength.The developed fluorescence lidar system discussed in our previous study showed its unique features and robustness [13,14].It uses a pulsed LED circuit at 385 nm excitation wavelength, which serves as a transmitting system.With the new understanding of Ex/Em pairs shown, we suggest the development of a portable pulsed laser diode (LD) at 405 nm excitation wavelength.This study paves the discovery of a new technique for developing a fluorescence lidar system using excitation-emission pairs [8].

Figure 1 .
Figure 1.(a) Whisker and box plot of AOM in different growth phases.It represents the absorbance at SUVA254 and SUVA280 (n = 6).(b) Fluorescence intensity from 3D EEM at varying algal biomass concentration.

Figure 2 .
Figure 2. Correlation heat map of Ex/Em pairs, F ratio, and algal biomass of Chlorella sp.