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Chiang Mai J. Sci. 2013; 40(X) 1

Chiang Mai J. Sci. 2013; 40(X) : 1-9 http://it.science.cmu.ac.th/ejournal/ Contributed Paper

Chlorophyll is not accurate measurement for algal Rameshprabu Ramaraj **[a], David D-W. Tsai [a] and Paris Honglay Chen *[a] [a] Department of Soil and Water Conservation, National Chung-Hsing University, 402 Taichung, Taiwan. *Author for correspondence; e-mail: [email protected], [email protected]

Received: 1 August 2012 Accepted: 19 March 2013

ABSTRACT are key primary producers and their biomass is widely applied for the production of pharmaceutics, bioactive compounds and . Conventionally, the content of algal is considered an index for algal biomass. However, this study, we estimated algal biomass by direct measurement of total suspended solids (TSS) and correlated it with chlorophyll content. The results showed mean chlorophyll-a equal to 1.05 mg/L; chlorophyll- b 0.51 mg/L and chlorophyll-a+b 1.56 mg/L. Algal biomass as 161 mg/L was measured by dry weight (TSS). In statistical t-tests, F-tests and all the tested growth models, such as linear, quadratic, cubic, power, compound, inverse, logarithmic, exponential, s-curve and logistic models, we did not find any discernible relationship between all chlorophyll indices and TSS biomass. Hence, the conventional method of chlorophyll measurement might not be a good index for biomass estimation.

Keywords: Microalgae; Biomass; Chlorophyll; Total suspended solids; Eco-tech

1. INTRODUCTION Microalgae are highly diverse ideal indicators of the health of aquatic groups of organisms playing vital roles in ecosystems and water quality. Hence, algal ecosystem, not only as the primary producers, biomass measurement is important in many but also as symbionts with other organisms biological and ecological studies and in including bacteria [1-3]. They sequester microalgae industry.

CO2 by and supply food Chlorophyll in , algae, and some and to the consumers of aquatic bacteria is vital to the survival of the , environments, and thus play central roles in animal and other kingdoms in . biogeochemical cycles [4, 5]. Algal biomass Chlorophyll is essential for photosynthesis, serves as a sustainable raw material for since it absorbs energy, which is producing pharmaceuticals, , subsequently converted into chemical energy biofuels and food products [6]. Algae are bound in biomass. Conventionally biologists, ideal organisms for biological monitoring. ecologists and industrialists consider Algal density, abundance, and diversity are chlorophyll as a reliable and standard algal 2 Chiang Mai J. Sci. 2013; 40(X)

biomass measurement [7-9]. Common of Chl amount as an index for algal biomass. chlorophyll (Chl) types in plants and algae Hence, in this study, we focused on Chl-a, -b are a, b, c and d. Chlorophyll-a (Chl-a) is and -a+b, to understand whether the found in all photosynthetic algae [10, 11], chlorophyll amount is a reliable index for algal whereas chlorophyll-b (Chl-b) is confined to biomass. and blue-green algae [12]. Chlorophyll- c is found in traces in green algae and abundant 2. MATERIALS AND METHODS in and brown algae [13, 14]. The methodology adopted in this study Chlorophyll-d is found in marine algae is illustrated in (Figure 1). We performed the [15, 16]. In algal chlorophyll, Chl-a is the gravimetric method of dry weight to measure abundant species, while Chl-b is the minor algal biomass directly, since we assumed that species [17]. algal biomass was in the proportion of total Amount of Chl has been used as a suspended solid (TSS). We also performed measure of algal biomass world-wide the proxy measurement of chlorophyll index [18-21]. However, though there is a algal (Chl-a, Chl-b and Chl-a+b). Subsequently, taxon-specific distribution of Chl species is the values were tested by t-tests and F-tests. evident, there exists a wide species-specific We formed possible growth models such as difference in the cellular concentrations of linear, quadratic, cubic, power, compound, Chl [22, 23]. Such wide variation in Chl inverse, logarithmic, exponential, logistic and concentration questions the reliability of use s-curve. We also did the ANOVA analyses.

Figure. 1 A flowchart of methodology Chiang Mai J. Sci. 2013; 40(X) 3

2.1. Algal cultivation and biomass units were shown in (Figure 2). The daily measurements feed of natural freshwater was collected The samples were collected from from the Green River in the vicinity of the triplicated reactors (P1, P2 and P3) in the National Chung Hsing University, Taichung, Sustainable Resources and Sustainable Taiwan. The collected water was filtered Engineering research laboratory, Department through by 0.45 m filter paper and used as of Soil and Water Conservation, National the medium. Chung-Hsing University, Taichung, Taiwan. The algal biomass was measured by The algae were grown as a mixed culture. TSS with Whatman GF/C filter paper [24]. The dominant algal species in the mixed A total suspended cell (TSC) is the popular culture were of the genera Anabaena, Chlorella, measurement in , but TSS is Oedogonium and Oscillatoria. The photo- another way to measure algae biomass by bioreactors were illuminated through weight directly and the measurement is uorescent lamps and the cultures were grown convenient and efficient for bio-engineering in autotrophic conditions with 10 days determination. The amounts of Chl-a, -b with detention time, for 20 months period, in -(a+b) were determined following the batch-fed 4 liters continuously-stirred tank method described by Becker [25]. reactor (CSTR) at room temperature; the

Figure 2. Photo-bioreactor 4 Chiang Mai J. Sci. 2013; 40(X)

3. RESULTS AND DISCUSSION straightforward, the amount of Chl-a is 3.1. Algal biomass indices considered as an index of algal biomass Since the feed (the natural river water) and widely used as a proxy measurement. was filtrated through 0.45  filter paper, there Results for Chl-b (a secondary pigment) and were no algae seeding in the feed. In our Chl-(a+b) were shown in Figure 4B and study, done in triplicate, the mean biomass Figure 4C, respectively. The mean amount of production was 0.16 g/L (Figure 3), while Chl-b was 0.51 mg/L and the mean amount the mean amount of Chl-a average was 1.05 of Chl-(a+b) was 1.56 mg/L. Many earlier mg/L (Figure 4A). Since the determination studies used the Chl-(a+b) to measure algal of the Chl-a is relatively simple and growth, as the biomass index [25–29].

Figure 3. Total suspended solids of feed and reactors

Figure 4. of feed and reactors Chiang Mai J. Sci. 2013; 40(X) 5

3.2. Statistical analysis of biomass indices As a catalyst, Chl is required to carry on the For each paired index comparison of the photosynthesis just by its existence, not by its TSS and Chl-indices, t-tests were performed concentration. as the initial trials in this study. The Chl-a/b/ In photosynthesis, Chl-a serves a dual a+b are the popular indices of algal growth role in oxygenic photosynthesis: light and biomass. However, they are relatively harvesting and converting energy of absorbed less direct, when compared to the indices of photons to chemical energy. While different TSS. Hence, in this study, we compared the chlorophylls are participants in photosynthesis Chl-indices with the direct indices of TSS. in different photosynthetic organisms, Chl-a The t-test results were shown in (Table 1); is present in all photosynthetic organisms all tests were rejected to indicate that each [34]. Chl-b may be necessary for effective index of Chl was different from biomass utilization of light energy, and it may stabilize statistically. This situation can be attributed to the photosynthetic device in certain species the lack of discernible relationship between [35]. However, more rapid oxygen evolution chlorophylls and biomass. (on chlorophyll basis) in the cells with high Amount of algal Chl and its efficiency in Chl-a/Chl-b ratio is suggestive of relatively an aquatic ecosystem varies depending on minor role for Chl-b in photosynthesis [26]. the algal species, taxonomic composition The t-test results revealed the lack of and physicochemical and biological factors. relationship between the amount of Chl and Chl content and could be biomass. However, there was still a certain strongly influenced by physiological shifts chance to make Chl as a good candidate of in intracellular pigmentation in response the index for biomass by distribution to changing growth conditions (light, similarity mathematically. Therefore, we tried temperature, pH and temperature). Hence, to investigate further with F-test, which was though Chl content can reflect the (relative) the best likelihood test of distributions to rate of photosynthesis, it may not account find any potential resemblance. However, the for the total accumulated biomass. results demonstrated that the distributions Moreover, Chl is not a reactant in the were completely different, as shown in photosynthesis, but a biocatalyst [30-32], (Table 2). which may not be reliable index of algal Both t-tests of mean values and F-tests biomass. Though Chl is a vital component of of distribution proved that each index of photosynthesis process, its amount is not Chl and biomass was completely different crucial to the reaction rate [33]. A small quantity and strongly suggest that Chl index may not of Chl is sufficient to maintain photosynthesis. be a reliable biomass index. 6 Chiang Mai J. Sci. 2013; 40(X)

Table 1. t-test between TSS and chlorophyll. Biomass / chlorophyll t value p-value test result TSS / Chl-a 33.05056 1.49E-18 reject TSS / Chl-b 33.16413 1.39E-18 reject TSS / Chl-a+b 32.94011 1.58E-18 reject

Table 2. F-test for difference and regression between TSS and chlorophyll. F-test for difference Biomass/ value p-value test result TSS/Chl-a 10443.10433 3.13E-34 reject TSS/Chl-b 14277.56104 1.61E-35 reject TSS/Chl-a+b 4825.55339 4.79E-31 reject F-test for regression dependentvariable independent variable R2 F p-value test result Chl-a 0.036 0.670 0.423 no regression TSS Chl-b 0.002 0.038 0.848 no regression Chl-a+b 0.010 0.190 0.668 no regression

3.3. Linear regression test on chlorophyll and -(a+b) and the directly measured biomass and biomass TSS unfortunately. If Chl-a, -b and -(a+b) have any potential Though Chl method is relatively relationship with biomass, they can be used convenient [36], the wide algal species-specific as biomass indices with the calibration curves. [22, 23,37], age-dependent [38] variations in Accordingly, we checked the most popular Chl content undermine its reliability to linear relationship first. The results of linear consider as index for biomass. Depending regression tests were shown in (Table 2). upon the algal species, the cellular amount There was no relationship between Chl-a, -b chlorophyll may range from 0.1% to 9.7% or -(a+b) and TSS. Consequently there was [36]. When compared to Chl-a, the cellular no linear regression between each Chl index amount of Chl-b is too scarce [39] to be and biomass. considered as a reliable index for biomass. In addition, several external factors, such as 3.4 Further relationships analysis the presence of interfering compounds [37], Further to establish any possible remote nutrition and operation conditions [40], etc relationship between chlorophyll indices and may influence the reliability of Chl amount biomass, we chose several alternative models as an index for biomass. Currently, there is no such as quadratic, cubic, power, compound, single analytical method available to resolve inverse, logarithmic, exponential, s-curve, these problems [41]. Therefore the Chl-a growth and logistic. The results were shown method may not provide correct estimation in (Table 3). All the model tests expressed of algal biomass all the time [42]. no relationship whatsoever between Chl-a,-b Chiang Mai J. Sci. 2013; 40(X) 7

Table 3. Linear regression test. Model F p-value test result linear 2.493 0.132 no regression quadratic 1.991 0.167 no regression cubic 2.188 0.143 no regression power 2.049 0.169 no regression compound 2.356 0.142 no regression inverse 1.917 0.183 no regression log 2.185 0.157 no regression exp 2.356 0.142 no regression s-curve 1.782 0.199 no regression growth 2.356 0.142 no regression logistic 2.356 0.142 no regression

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