Environmental Technology
ISSN: 0959-3330 (Print) 1479-487X (Online) Journal homepage: http://www.tandfonline.com/loi/tent20
Impact of green algae on the measurement of Microcystis aeruginosa populations in lagoontreated wastewater with an algae online analyser Thang Nguyen, Felicity A. Roddick & Linhua Fan To cite this article: Thang Nguyen, Felicity A. Roddick & Linhua Fan (2015) Impact of green algae on the measurement of Microcystis aeruginosa populations in lagoon-treated wastewater with an algae online analyser, Environmental Technology, 36:5, 556-565, DOI: 10.1080/09593330.2014.953212 To link to this article: http://dx.doi.org/10.1080/09593330.2014.953212
Accepted author version posted online: 09 Sep 2014. Published online: 15 Sep 2014. Submit your article to this journal
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Date: 06 November 2015, At: 13:46
Environmental Technology, 2015 Vol. 36, No. 5, 556–565, http://dx.doi.org/10.1080/09593330.2014.953212
Impact of green algae on the measurement of Microcystis aeruginosa populations in lagoon-treated wastewater with an algae online analyser Thang Nguyen ∗ , Felicity A. Roddick and Linhua Fan School of Civil, Environmental and Chemical Engineering, Water: Effective Technologies and Tools (WETT) Centre, RMIT University, Melbourne, Victoria, Australia
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(Received 26 February 2014; final version received 29 July 2014 ) Tests on the algae online analyser (AOA) showed that there was a strong direct linear correlation between cell density and in vivo Chl-a concentration for M. aeruginosa over the range of interest for a biologically treated effluent at a wastewater treatment plant (25,000–65,000 cells mL−1 , equivalent to a biovolume of 2–6 mm3 L−1 ). However, the AOA can provide an overestimate or underestimate of M. aeruginosa populations when green algae are present in the effluent, depending on their species and relative numbers. The results from this study demonstrated that the green algae (e.g., Euglena gracilis, Chlorella sp.) in the field phytoplankton population should be considered during calibration. In summary, the AOA has potential for use as an alert system for the presence of M. aeruginosa, and thus potentially of cyanobacterial blooms, in wastewater stabilization ponds. Keywords: Cyanobacterial bloom; lagoon-treated wastewater; Chlorophyll a; Fluorescence; Microcystis aeruginosa; algal online analyser
1. Introduction Melbourne Water’s Western Treatment Plant (WTP) treats approximately 52% and 72% of Melbourne’s domestic sewage and industrial wastewater, respectively. The WTP process comprises a sequence of biological processes: anaerobic, activated sludge and lagoon treatment. The treated effluent for recycling is then held in the Head of the Road Storage (HORS) pond before being released as Class C (without disinfection) or Class A recycled water (after disinfection with ultraviolet (UV) and chlorine). Typical algal groups found in sewage stabilization ponds include cyanobacteria, diatoms, flagellated algae (e.g., cryptophytes) and green algae. Microcystis aeruginosa is the most common cyanobacterium found in lagoons.[1] In the warmer months, blooms of the potentially toxic M. aeruginosa periodically occur within the lagoons and the HORS storage pond at WTP. As M. aeruginosa produces microcystin, a hepatotoxin and a possible carcinogen,[2,3] water supply outages result. Therefore, by monitoring and implementing measures to prevent or deal with their occurrence, interruption to the supply of recycled water at WTP could be minimized. Traditionally, monitoring cyanobacterial blooms in drinking water sources has been through the collection of water samples which are sent to laboratories for microscopic algal enumeration and chlorophyll measurement using spectrophotometry. This usually takes several days before the results become available. Another
*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
technique, ‘in vivo fluorometry’, has been used for the continuous measurement of chlorophyll a (Chl-a) in natural populations and cultures of seawater/freshwater phytoplankton.[4–9] The technique is based on the direct measurement of the fluorescence of the Chl-a in the algal cells. No special sample handling or processing is required and thus in vivo fluorometry using on-line instrument systems is suitable for profiling and for real-time data collection. In vivo fluorometry has been used for continuous measurement of Chl-a since the early 1960s [10] and has the advantage of non-invasive and non-destructive sampling.[11] Chl-a is the main photosynthetic pigment in phytoplanktonic organisms. Other pigments (so-called accessory pigments) are also present in photosystem II. These differ, depending on the taxonomic group of the organisms. For example, chlorophyll b is the marker pigment of chlorophytes, chlorophyll c and fucoxanthin for diatoms, perinidin for dinoflagellates, and phycobilins for cyanobacteria and cryptomonads.[12] The various accessory pigments are excited at different electromagnetic radiation wavelengths and the interaction of these pigments results in a specific excitation spectrum for the different algal taxonomic classes. The excited pigments then transfer their excitation energy to Chl-a, which then fluoresces. Consequently, algal groups can be differentiated according to their specific fluorescence excitation spectra [13,14] and the fluorescence intensities at the different excitation
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Environmental Technology wavelengths are then associated with the in vivo Chl-a content of the different algal groups.[15,16] The algal online analyser (AOA) measures the fluorescence of Chl-a in algae and can be used to distinguish the different classes in water according to their excitation spectra.[17] The AOA uses five light emitting diodes to emit pulsed light at 450, 525, 570, 590 and 610 nm to excite the pigments and the fluorescence emission of the Chl-a is measured at 680 nm. Consequently, the AOA can measure the Chl-a concentrations for Chlorophyceae, Cyanophyceae, diatoms and Cryptophyceae in water. A UV-excitation source (380 nm) is used for differentiation between algal fluorescence and the fluorescence of yellow substances (Y.S.) (coloured dissolved organic matter) which may also be present in the water. Y.S. are characterized by strong absorption in the UV and blue range and so tails over into the visible region and attenuates photosynthetically active radiation (400–700 nm). Since they may interfere with the measurement of Chl-a due to the overlap of the excitation spectra with that of phytoplankton, they are measured (in terms of relative units, r.u.) and correction for this background absorption is made to the output of the AOA. The main advantages of the AOA are its ease of handling, low maintenance costs, and that it provides real-time monitoring of algae. A study by Izydorczyk et al.[18] showed that the AOA is a useful tool for monitoring potentially toxic M. aeruginosa blooms in a reservoir. However, the effectiveness of the AOA for monitoring M. aeruginosa in wastewater stabilization ponds has not yet been evaluated. The aim of this work was to investigate the
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Table 1. Characteristics of the lagoon-treated wastewater (HORS). Parameter
Range
pH Turbidity (NTU) Dissolved organic carbon (mg L−1 ) Total suspended solids (mg L−1 ) Total dissolved solids (mg L−1 ) Total nitrogen (mg L−1 ) Phosphorus (mg L−1 ) Fe content (mg L−1 )
7–7.5 1–8 9–12 2–11 870–1200 13–30 7–12 0.1–0.3
effectiveness of the AOA for detecting M. aeruginosa in the lagoon-treated effluent with the view to developing an alert level framework for potentially toxic cyanobacterial blooms. The concentration of M. aeruginosa was measured in terms of Chl-a and the result was compared with that obtained from the spectrophotometric method. The effect of green algae on the measurement of in vivo Chl-a concentration for M. aeruginosa was also investigated using E. gracilis and Chlorella sp., at cell density/biovolume ranges typical for WTP. 2. Materials and methods 2.1. Lagoon-treated wastewater Lagoon-treated wastewater was collected from the HORS pond at WTP (Figure 1) during summer (January 2010 and February 2011). The characteristics of the HORS water are shown in Table 1.
Anaerobic Pond (1)
Anoxic Zone (2)
Activated Sludge (3)
Secondary Settling (4) Class C Recycled Water
Head of the Road Storage Pond (HORS) (6)
UV and Chlorination (7)
Lagoon System (5)
Class A Recycled Water
Figure 1. Flow diagram of WTP. Notes: (1) The retention time for each stage from 1 to 4 is 3 days. (2) The retention time in the lagoon system (5) is 15 days. (3) The retention time in the HORS (6) is 5–7 days.
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2.2. Algal cultures Microcystis aeruginosa (CS 566/01-A01, originally isolated from the lagoons at WTP) was obtained from the CSIRO Microalgae Research Centre, Hobart. Euglena gracilis and Chlorella sp. were obtained from Southern Biological, Nunawading, Victoria, Australia. Microcystis aeruginosa and Chlorella sp. were grown in MLA culture medium [19] and E. gracilis was grown in CM5 medium which was supplied by Southern Biological. All algal cultures were grown in a laboratory incubator (Labec Illuminated Incubator Model ICCBOD) at a constant temperature (22°C) under a 16/8 h light/dark cycle with a light intensity of 3400 Lux. The cell density was determined using a hemocytometer and the Chl-a concentration was measured using the AOA and the spectrophotometric method given below. Cell suspensions were counted in the laboratory immediately prior to the tests. Suspensions (1 L) of algae were prepared in HORS water collected on the day of the tests for onsite in vivo measurement of Chl-a at WTP. The equivalent biovolumes for M. aeruginosa and E. gracilis were calculated based on mean cell volume data which have been used by South Australian Water and Melbourne Water for calculation of the biovolume of M. aeruginosa in reservoirs (Burch M. South Australian Water, Australia. Personal communication, 2011.) and E. gracilis in stabilization ponds. (Mulcare M. Melbourne Water, Australia. Personal communication; 2010.) The equivalent biovolume of Chlorella sp. was calculated according to Bellinger and Sigee.[20]
2.3. Fluorescence measurement of Chl-a Fluorescence measurements were conducted using the AOA (Model BG82000F, bbe Moldaenke, Kiel, Germany). The AOA was originally calibrated by the manufacturer. After addition of the cells, the HORS water was mixed thoroughly before measurement of in vivo Chl-a. The parameters measured by the AOA include the total chlorophyll (as μg Chl-a L−1 ), the Chl-a concentrations of bluegreen algae, green algae in μg Chl-a L−1 , and concentration of Y.S. in r.u. The cuvette of the AOA has an automatic internal mechanical cleaning device which operates at hourly intervals.
2.4.
Spectrophotometric determination of Chl-a
Suspensions of the specific algae were prepared using a known cell density of algal culture and Milli-Q water. Milli-Q water was used so that the Chl-a content of the target algae could be determined without interference from other phytoplankton. The Chl-a of the algae was extracted with acetone (90% v/v) overnight. The absorbance of the extract was measured at 750, 663, 645 and 630 nm using a UV/Vis spectrophotometer (Unicam UV2) and
the Chl-a concentration was determined according to ESS Method.[21] The relationship between cell density determined by cell counting using a hemocytometer and the Chl-a concentration measured by the spectrophotometric method for the different species is given by Cell density (cells mL−1 ) = a × [Chl-a](µg L−1 ). With • [Chl-a] = Chl-a concentration measured by spectrophotometric method • a = ratio between cell density (cells mL−1 ) and Chla concentration (μg L−1 ). It was found that: ◦ a = 6283 for M. aeruginosa, up to 20,000,000 cells mL−1 (R2 = 0.988) ◦ a = 3364 for Chlorella sp., up to 20,000,000 cells mL−1 (R2 = 0.987) ◦ a = 138 for E. gracilis, up to 2,000,000 cells mL−1 (R2 = 0.988) 2.5.
Fluorescence intensity of Y.S. in a filtered algal suspension
In order to measure the fluorescence intensity of Y.S. in a specific algal suspension, a suspension of a known cell density (equivalent to the lowest density at WTP) of the culture was prepared in Milli-Q water and was filtered immediately after preparation (1 μm glass fibre, Whatman GF/B) to remove all cells. The fluorescence intensity of the filtered solution containing Y.S. released by the algae was measured at Ex = 450 nm and Em = 680 nm using a L55 Perkin Elmer fluorescence spectrometer.
3. Results and discussion 3.1. Response of the AOA to cell density of M. aeruginosa The in vivo Chl-a concentrations for M. aeruginosa in the range 23,000–94,000 cells mL−1 (equivalent to biovolume of 2.0–8.2 mm3 L−1 , a typical range in HORS water at WTP during summer) measured on 14 January 2010 and 16 March 2011 are shown in Figure 2. A direct correlation between cell density and the in vivo Chl-a concentration was observed (R2 = 0.992). The background in vivo Chl-a concentrations for cyanobacteria in HORS water on 14 January 2010 and 16 March 2011 were 0.99 and 0 μg L−1 , respectively. If the background in vivo Chl-a concentrations are taken into account, the response of the AOA to cell density of M. aeruginosa in both tests was fairly similar (Figure 2). The concentrations of Y.S. in HORS water on 14 January 2010 and 16 March 2011 were 1.50 and 1.12 r.u., respectively, and increased only slightly to average values of 1.53 ± 0.15 r.u. and 1.27 ± 0.01 r.u.,
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Figure 2. Response of the AOA to increasing cell density of M. aeruginosa on 14 January 2010 and 16 March 2011. (Background in vivo Chl-a concentrations for cyanobacteria in HORS water before adding M. aeruginosa cells on 14 January 2010 and 16 March 2011 were 0.99 and 0 μg L−1 , respectively).
respectively, after addition of M. aeruginosa over the range of 23,000 to 94,000 cells mL−1 to the HORS water. It should be noted that when the AOA is used for continuous monitoring of algae in a wastewater lagoon, a biofilm may develop within the cuvette due to the elevated C, N and P content of the treated wastewater (Table 1) and this will most likely affect the performance of the AOA. The implementation of an appropriate cleaning regime would be necessary. 3.2.
Determination of Chl-a from M. aeruginosa, Chlorella sp. and E. gracilis by AOA and spectrophotometry Euglena gracilis (a member of the Euglenophyta) and Chlorella sp. (a member of the Chlorophyta) are common green algae found within the lagoons at WTP. Euglena gracilis cells (approximately 50 μm long and 10 μm wide [22]) are markedly larger than Chlorella sp. cells (which are spherical with a diameter of 2–5 μm [20]), whereas Chlorella sp. cells are approximately similar in shape but smaller than M. aeruginosa (which has a diameter of 2–10 μm [20]). The in vivo Chl-a concentration in suspensions of the individual species in HORS water, at cell density ranges typical for WTP, was determined and the results were compared with those obtained from spectrophotometric analysis for suspensions of the same culture in Milli-Q water at the same cell density range. For M. aeruginosa at low cell densities, the Chl-a values measured by the two methods were fairly similar. However, as the cell density increased, the in vivo Chl-a
concentrations did not increase in the same proportion (Figure 3). This may be due to the different principle employed in the two methods. The spectrophotometric method measures the absorbance of the Chl-a after destruction of the cells in the acetone extraction, whereas the AOA fluorometric method measures the fluorescence of the Chla of the intact cells in suspension. Consequently, some cells are shaded by others (shading or screening effect) during the fluorometric measurement and the shading effect increased with increasing cell density, as observed by Izydorczyk et al.[18] The presence of other green algae already present in the HORS water may also have contributed to the lower in vivo Chl-a concentration for M. aeruginosa (the Chl-a for green algae in HORS water measured by the AOA was 0.5 μg/L). The result was consistent with that obtained by Rolland et al.[23] at a reservoir. They used a bbe Fluoroprobe™ in vivo analyser and obtained a good correlation (R2 = 0.97) for M. aeruginosa when the Chl-a concentration was less than 3.9 μg L−1 (equivalent to < 25,000 cells L−1 using our empirically derived relationship, Section 2.4), and lower Chl-a values at higher cell densities compared with spectrophotometric analysis. Figure 2 also shows that there was little change in Y.S. ( ± 0.01 r.u.) with increasing cell density of M. aeruginosa, the results implying that the effect of Y.S. on the measurement of in vivo Chl-a for M. aeruginosa was negligible. For the suspensions of Chlorella sp. (5000–65,000 cells mL−1 , equivalent to a biovolume of 0.15–1.95 mm3 L−1 and typical range for WTP) in the HORS water a strong linear relationship between the in vivo Chl-a concentration and cell density (R2 = 0.986) was shown; similar to
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Figure 3. Chl-a concentration of M. aeruginosa measured by the AOA and spectrophotometric method (background in vivo Chl-a for blue green algae and total Y.S. concentration for HORS water before adding M. aeruginosa cells were 0 μg L−1 and 1.28 r.u., respectively).
Figure 4. Chl-a concentration of Chlorella sp. measured by the AOA and spectrophotometric method (background in vivo Chl-a for green algae and total Y.S. concentration for HORS water were 0.5 μg L−1 and 1.2 r.u, respectively).
M. aeruginosa, the difference between the Chl-a concentration obtained from both AOA and spectrophotometric methods increased with increasing cell density (Figure 4). The difference could be due to the shading effect as well as interference from chlorophyll b (Chl-b), a common accessory pigment in green algae, and may also be from phaeophytin, the breakdown product of chlorophyll, in HORS water. The presence of these pigments tends to decrease the apparent Chl-a concentration when using the fluorometric method as reported by Gibbs [24] and Pinto et al.[25] The lower in vivo Chl-a concentrations for Chlorella sp. at high cell density were therefore attributed to the shading effect (similar to that obtained for M. aeruginosa) and/or the
presence of Chl-b and phaeophytin (it should be noted that the AOA cannot measure phaeophytin or Chl-b). The change in Y.S. concentration was insignificant ( ± 0.05 r.u.) when the cell density of Chlorella sp. increased from 5000 to 65,000 cells L−1 , indicating that the effect of Y.S. was also compensated for during in vivo Chl-a measurement for Chlorella sp. A strong linear relationship between the in vivo Chla concentration and cell density (R2 = 0.992) was shown for the suspensions of E. gracilis (670–3500 cells mL−1 , equivalent to a biovolume of 2.0–10.5 mm3 L−1 and typical range for WTP) in the HORS water. In contrast to M. aeruginosa and Chlorella sp., the Chl-a concentrations
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Figure 5. Chl-a concentration of E. gracilis measured by the AOA and spectrophotometric method (background in vivo Chl-a of green algae and total Y.S. concentration for HORS water were 0.5 μg L−1 and 1.2 r.u, respectively).
for the E. gracilis were higher for the AOA than for the spectrophotometric method, however as before, the difference between the two methods increased with increasing cell density (Figure 5). The presence of carotenoid pigments, which occur in E. gracilis, tends to increase the level of Chl-a fluorescence [26]; the difference in size (and thus less shading effect due to lower abundance) and cell structure of E. gracilis compared with Chlorella sp. and M.
aeruginosa may also play a role. There was a significant increase in concentration of Y.S. measured by the AOA with increasing cell density of E. gracilis, which appears to have had an impact on the in vivo Chl-a values (Figure 5). Determination of the absorbance of the individual filtered (i.e., cell-free) suspension of E. gracilis, M. aeruginosa and Chlorella sp. in Milli-Q water showed that there was absorbance at 350–370 nm for E. gracilis whereas there
Figure 6. Effect of green algae on the response of the AOA to increasing cell density of M. aeruginosa.
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was no absorbance in this range for Chlorella sp., and very little for M. aeruginosa. Therefore, the absorbance in this region may affect the measurement of the Y.S. and thus interfere with the in vivo Chl-a measurement for E. gracilis. As the AOA was originally calibrated by the manufacturer, it was possible that E. gracilis was not chosen for calibration of the AOA and hence the effect of the Y.S. was not compensated for during in vivo Chl-a measurement for E. gracilis.
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3.3.
Impact of E. gracilis and Chlorella sp. on in vivo Chl-a of M. aeruginosa The impact of the presence of E. gracilis and Chlorella sp. on the measurement of in vivo Chl-a for M. aeruginosa was investigated. Suspensions of M. aeruginosa in the cell density range of 35,000–65,000 cells mL−1 (equivalent to a biovolume of 3.05–5.66 mm3 L−1 ) were prepared using HORS water containing E. gracilis or Chlorella sp. with an in vivo Chl-a concentration of 5.2 μg L−1 (equivalent to approx. 670 E. gracilis cells mL−1 , the lowest cell concentration for E. gracilis at WTP, or equivalent to approx. 23,000 Chlorella sp. cells mL−1 ). The presence of Chlorella sp. decreased the apparent in vivo Chl-a concentration for M. aeruginosa by approximately 12%, whereas the presence of E. gracilis increased the apparent in vivo Chl-a concentration for M. aeruginosa by approximately 20% (Figure 6). This may be due to the shading effect caused by the presence of Chlorella sp. in the suspensions leading to the in vivo Chl-a concentration for M. aeruginosa to decrease, and insufficient compensation for the effect of Y.S. released by E. gracilis causing the Chl-a reading for M. aeruginosa to increase. As shown in Figure 6, there was a large increase in Y.S. concentration when E. gracilis was added (from 1.27 ± 0.01 r.u. to 3.45 ± 0.06 r.u.), but very little change when Chlorella sp. was added (from 1.27 ± 0.01 r.u. to 1.32 ± 0.02 r.u) to the suspensions of M. aeruginosa. The results demonstrated that the AOA can give an overestimate or underestimate of M. aeruginosa populations when green algae are present in the lagoon-treated wastewater, depending on their species and their pigment (such as Chl-b) content, and possibly their size. The results are consistent with the findings of Zamyadi et al.[27] who reported that the presence of other phytoplankton such as Scenedesmus and Anabaena circinalis can affect the Chl-a reading from in vivo fluorescence probes for M. aeruginosa. 3.4.
Response of the AOA to a fixed density of M. aeruginosa at varying cell densities of Chlorella sp. The in vivo Chl-a concentrations of suspensions of M. aeruginosa with 25,000 and 50,000 cells mL−1 (equivalent biovolumes of 2.2 and 4.4 mm3 L−1 , respectively, half and the acceptable limit for recycled water at WTP) in
the HORS water in the presence of Chlorella sp. (5000– 65,000 cells mL−1 ) were measured. At both cell densities of M. aeruginosa a strong linear relationship (R2 = 0.945 and 0.989, respectively) between the in vivo Chl-a and cell density for Chlorella sp. was shown (Figure 7). The in vivo Chl-a concentration of the M. aeruginosa suspension (25,000 cells mL−1 ) was decreased to 3.86 μg L−1 (by 7.2%) from 4.16 μg L−1 on addition of 5000 cells mL−1 Chlorella sp. However, when the cell density of Chlorella sp. increased from 5000 to 65,000 cells mL−1 , the change in in vivo Chl-a concentration for M. aeruginosa was negligible (the average in vivo Chl-a concentration for M. aeruginosa was 3.69 ± 0.09 μg L−1 ; Figure 7(a)). Similarly, there was little change (only ± 0.06 r.u.) in the concentration of Y.S. when the cell density of Chlorella sp. increased from 5000 to 65,000 cells mL−1 . A similar trend was observed for the M. aeruginosa suspension of 50,000 cells mL−1 : the in vivo Chl-a concentration decreased from 8.07 to 7.74 μg L−1 (a decrease of 4.1%) when 5000 cells mL−1 Chlorella sp. was added. There was little change in in vivo Chl-a concentration for M. aeruginosa and Y.S. when the density of Chlorella sp. increased from 5000 to 65,000 cells mL−1 (Figure 7(b)), with values of 7.61 ± 0.23 μg L−1 and 1.29 ± 0.02 r.u., respectively. The results indicated that increasing cell density of Chlorella sp. had little effect on the measurement of in vivo Chl-a concentration for a fixed cell density of M. aeruginosa, and the effect of Y.S. when Chlorella sp. was added was taken into account.
3.5.
Response of the AOA to a fixed cell density of M. aeruginosa at varying cell densities of E. gracilis Suspensions of M. aeruginosa (25,000 and 50,000 cells mL−1 ) were prepared using HORS water containing E. gracilis cells of various cell densities (670–3500 cells mL−1 , equivalent to a biovolume of 2.0–10.5 mm3 L−1 , the typical range at WTP). For both suspensions of M. aeruginosa a strong linear relationship (R2 = 0.970 and 0.983 for 25,000 and 50,000 cells mL−1 , respectively) between in vivo Chl-a concentration and cell density for E. gracilis was shown (Figure 8(a) and (b)). The in vivo Chl-a concentration of the M. aeruginosa suspension (25,000 cells mL−1 ) was increased from 4.16 to 5.73 μg L−1 (by 27%) on addition of 670 cells mL−1 E. gracilis. Although the cell density of M. aeruginosa in all samples was constant, the in vivo Chl-a concentration for M. aeruginosa increased from 5.73 to 9.47 μg L−1 (Figure 8(a)) when the cell density of E. gracilis increased from 670 to 3500 cells mL−1 . Thus when the cell density of E. gracilis increased five times, the apparent cell density for M. aeruginosa measured by the AOA was almost doubled.
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(a)
(b)
Figure 7. Relationship between the in vivo Chl-a and cell density for a fixed cell density of M. aeruginosa in suspensions of Chlorella sp. (a) 25,000 M. aeruginosa cells mL−1 and (b) 50,000 M. aeruginosa cells mL−1 (in vivo Chl-a concentration for suspensions of M. aeruginosa at 25,000 cells mL−1 and 50,000 cells mL−1 when no Chlorella sp. was present were 4.16 and 8.07 μg L−1 , respectively).
A similar trend was found for the M. aeruginosa suspension of 50,000 cells mL−1 , with an increase in apparent in vivo Chl-a concentration from 8.07 to 9.97 μg L−1 (more than 19%) on addition of 670 cells mL−1 E. gracilis, and from 9.97 to 14.23 μg L−1 when 3500 cells mL−1 of E. gracilis were added, an increase of approx. 30% (Figure 8(b)). The Y.S. measured by the AOA increased from 1.45 and 1.48 r.u. for M. aeruginosa at 25,000 and 50,000 cells mL1 , respectively, to 3.06 and 3.08 r.u., respectively, on addition of E. gracilis at 670 cells mL−1 and then increased with increasing cell density of E. gracilis. The results demonstrated that increasing cell density of E. gracilis had an effect on the measurement of in vivo
Chl-a concentration for a fixed cell density of M. aeruginosa, and the effect of Y.S. released by E. gracilis was not compensated by the AOA.
3.6. Reproducibility of the AOA results The relative standard deviation (%STDEV) for three Chla measurements for each M. aeruginosa suspension in the cell density range of 23,000–94,000 cells mL−1 is shown in Table 2. The %STDEV for Chl-a measured by the AOA for M. aeruginosa suspensions of 25,000 and 50,000 cells mL−1 can be estimated as 10% and 12%, respectively, which was acceptable.
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Figure 8. Relationship between in vivo Chlorophyll-a concentration and cell density for a fixed density of M. aeruginosa in suspensions of E. gracilis (a) 25,000 M. aeruginosa cells mL−1 and (b) 50,000 M. aeruginosa cells mL−1 (in vivo Chl-a concentration for suspensions of M. aeruginosa at 25,000 cells mL−1 and 50,000 cells mL−1 without E. gracilis were 4.16 and 8.07 μg L−1 , respectively). Table 2. Mean, STD and % STDEV of in vivo Chl-a concentration for M. aeruginosa in HORS water in the cell density range of 23,000–94,000 cells mL −1 (after chemical cleaning). Chl-a concentration measured by the AOA (μg L−1 ) Density (cells mL−1 ) 23,000 45,000 48,000 68,000 80,000 94,000
Equivalent biovolume (mm3 L−1 )
Mean
STD
%STDEV
2.0 3.9 4.2 5.9 7.0 8.2
3.24 6.97 7.19 9.63 10.98 12.25
0.32 0.80 0.83 1.10 1.30 1.27
< 10 (9.9) 11 12 11 12 > 10 (10.4)
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Environmental Technology 4. Conclusions This study demonstrated that the AOA has potential to be used as an alert system for cyanobacterial blooms in a wastewater stabilization pond. However, the effect of the presence of some green algae on the apparent cell density of M. aeruginosa needs to be taken into account. Furthermore, the accuracy of the AOA in terms of in vivo Chl-a concentration should be cross-checked periodically with a laboratory-based method such as spectrophotometry, and calibration for Y.S. over an appropriate range of green algae in the pond needs to be maintained. Although the AOA readings may be affected by these factors, the AOA provides real-time monitoring of algal populations and an indication of change in cyanobacterial densities, e.g., imminent blooms. Ease of handling and relatively low maintenance costs are further advantages compared with manual cell counts which are time-consuming, and the accuracy of which is dependent on the sampling time, sample management and operator error. Acknowledgements This research was funded by the Smart Water Fund Victoria, Australia (Project Number 62M-2026)
References [1] Palmer CM. Algae and water pollution: the identification, significant and control of algae in water supplied and in polluted water, Tunbridge Wells: Caste House Publications Ltd; 1980. [2] Carmichael WW. Cyanobacteria secondary metabolites – the Cyanotoxins. J Appl Bacteriol. 1992;73:445–459. [3] Fujiki H, Sueoka E, Suganuma M. Carcinogenesis of microcystins. In: Watanabe MF, Harada K, Camichael WW, Fujiki H, editors. Toxic microcystis. Boca Raton, FL: CRC Press; 1996. p. 296. [4] Yentsch CS, Menzel DW. A method for the determination of phytoplankton chlorophyll and pheophytin by fluorescence. Deep Sea Res Oceanogr Abstracts. 1963;10:221–231. [5] Holm-Hansen O, Lorenzen CJ, Holmes RW, Strickland JDH. Fluorometric determination of chlorophyll. Journal du Conseil Permanent International pour l’Exploration de la Mer. 1965;30:3–15. [6] Strickland JDH. Continuous measurement of in vivo chlorophyll: a precaution note. Deep Sea Res. 1968;15:225–227. [7] Loftus ME, Seliger HH. Some limitations of the in vivo fluorescence techniques. Chesapeake Science. 1975;16:79–92. [8] Gregor J, Maršálek B. Freshwater phytoplankton quantification by chlorophyll a: a comparative study of in vitro, in vivo and in situ methods. Water Res. 2004;38:517–522. [9] Matorin DN, Antal TK, Ostrowska M, Rubin AB, Ficek D, Majchrowski R. Chlorophyll fluorometry as a method for studying light absorption by photosynthetic pigments in marine algae. Oceanologia. 2004;46:519–531. [10] Lorenzen CJ. A method for the continuous measurement of in vivo chlorophyll concentration. Deep Sea Res. 1966;13:223–227.
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[11] Richardson TL, Lawrenz E, Pinckney JL, Guajardo RC, Walker EA, Paerl HW, MacIntyre HL. Spectral fluorometric characterization of phytoplankton community composition using the Algae Online Analyser. Water Res. 2010;44:2461–2472. [12] Gregor J, Maršálek B. A simple in vivo fluorescence method for the selective detection and quantification of freshwater cyanobacteria and eukaryotic algae. Acta Hydrochimica et Hydrobiologica. 2005;33:142–148. [13] Yentsch CS, Phinney DA. Spectral fluorescence: a taxonomic tool for studying the structure of phytoplankton populations. J Plankt Res. 1985;7:617–632. [14] Hilton J, Rigg E, Jaworski G. Algal identification using in vivo fluorescence spectra. J Plankt Res. 1989;11:65–74. [15] Beutler M, Wiltshire KH, Meyer B, Moldaenke C, Lüring C, Meyerhöfer M, Hansen U-P, Dau H. A fluorometric method for the differentiation of algal populations in vivo and in situ. Photosynth Res. 2002a;72:39–53. [16] Beutler M, Wiltshire KH, Lüring C, Moldaenke C. Fluorometric depth-profiling of chlorophyll corrected for yellow substances. Actes de Colloques. 2002b;34:231–238. [17] Bbe Moldaenke 2010. Available from: http://www.bbemoldaenke.de/chlorophyll/algaeonlineanalyser (accessed 18 January 2010). [18] Izydorczyk K, Carpentier C, Mrówczy´nski J, Wagenvoort A, Jurczak T, Tarczy´nska M. Establishment of an alert level framework for cyanobacteria in drinking water sources by using the algae online analyser for monitoring cyanobacterial chlorophyll a. Water Res. 2009;43:989–996. [19] Bolch CJS, Blackburn SI. Isolation and purification of Australian isolates of the toxic cyanobacterium Microcystis aeruginosa Kütz. J Appl Phycol. 1996;8:5–13. [20] Bellinger EG, Sigee DC. Freshwater algae: identification and use as bioindicators. New York: Wiley; 2010. p. 74–232. [21] “ESS Method 150.1: Chlorophyll – Spectrophotometric” 1991. Madison: Environmental Sciences Section, Inorganic Chemistry Unit, Wisconsin State Lab of Hygiene, Available from: http://www.epa.gov/glnpo/lmmb/methods/methd 150.pdf (accessed 29 September 2010). [22] Buetow ED, editor. The biology of Euglena. New York: Academic press; 1982. Vol. III, p. 29–51. [23] Rolland A, Rimet F, Jacquet S. A 2-year survey of phytoplankton in the Marne reservoir (France): a case study to validate the use of an in situ spectrofluorometer by comparison with algal taxonomy and chlorophyll a measurements. Knowl Manage Aquat Ecosyst. 2010;398:2–17. [24] Gibbs CF. Chlorophyll b interference in the fluorometric determination of chlorophyll a and ‘phaeo-pigments’. Austral J Mar Freshw Res. 1979;30:597–606. [25] Pinto AMF, Von Sperling E, Moreira RM. Chlorophylla determination via continuous measurement of plankton fluorescence: methodology development. Water Res. 2001;35:3977–3981. [26] Gruszecki WI, Matula M, My´sliwa-Kurdziel B, Kernen P, Krupa Z, Strzalka K. Effect of xanthophyll pigments on fluorescence of chlorophyll a in LHC II embedded to liposomes. J Photochem Photobiol B. 1997;37:84–90. [27] Zamyadi A, McQuaid N, Dorner S, Bird DF, Burch M, Barker P, Hobson P, Prévost M. Cyanobacterial detection using in vivo fluorescence probes: managing interferences for improved decision-making. J Amer Water Works Assoc. 2012;104:466–478.
ISSN: 0959-3330 (Print) 1479-487X (Online) Journal homepage: http://www.tandfonline.com/loi/tent20
Impact of green algae on the measurement of Microcystis aeruginosa populations in lagoontreated wastewater with an algae online analyser Thang Nguyen, Felicity A. Roddick & Linhua Fan To cite this article: Thang Nguyen, Felicity A. Roddick & Linhua Fan (2015) Impact of green algae on the measurement of Microcystis aeruginosa populations in lagoon-treated wastewater with an algae online analyser, Environmental Technology, 36:5, 556-565, DOI: 10.1080/09593330.2014.953212 To link to this article: http://dx.doi.org/10.1080/09593330.2014.953212
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Date: 06 November 2015, At: 13:46
Environmental Technology, 2015 Vol. 36, No. 5, 556–565, http://dx.doi.org/10.1080/09593330.2014.953212
Impact of green algae on the measurement of Microcystis aeruginosa populations in lagoon-treated wastewater with an algae online analyser Thang Nguyen ∗ , Felicity A. Roddick and Linhua Fan School of Civil, Environmental and Chemical Engineering, Water: Effective Technologies and Tools (WETT) Centre, RMIT University, Melbourne, Victoria, Australia
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(Received 26 February 2014; final version received 29 July 2014 ) Tests on the algae online analyser (AOA) showed that there was a strong direct linear correlation between cell density and in vivo Chl-a concentration for M. aeruginosa over the range of interest for a biologically treated effluent at a wastewater treatment plant (25,000–65,000 cells mL−1 , equivalent to a biovolume of 2–6 mm3 L−1 ). However, the AOA can provide an overestimate or underestimate of M. aeruginosa populations when green algae are present in the effluent, depending on their species and relative numbers. The results from this study demonstrated that the green algae (e.g., Euglena gracilis, Chlorella sp.) in the field phytoplankton population should be considered during calibration. In summary, the AOA has potential for use as an alert system for the presence of M. aeruginosa, and thus potentially of cyanobacterial blooms, in wastewater stabilization ponds. Keywords: Cyanobacterial bloom; lagoon-treated wastewater; Chlorophyll a; Fluorescence; Microcystis aeruginosa; algal online analyser
1. Introduction Melbourne Water’s Western Treatment Plant (WTP) treats approximately 52% and 72% of Melbourne’s domestic sewage and industrial wastewater, respectively. The WTP process comprises a sequence of biological processes: anaerobic, activated sludge and lagoon treatment. The treated effluent for recycling is then held in the Head of the Road Storage (HORS) pond before being released as Class C (without disinfection) or Class A recycled water (after disinfection with ultraviolet (UV) and chlorine). Typical algal groups found in sewage stabilization ponds include cyanobacteria, diatoms, flagellated algae (e.g., cryptophytes) and green algae. Microcystis aeruginosa is the most common cyanobacterium found in lagoons.[1] In the warmer months, blooms of the potentially toxic M. aeruginosa periodically occur within the lagoons and the HORS storage pond at WTP. As M. aeruginosa produces microcystin, a hepatotoxin and a possible carcinogen,[2,3] water supply outages result. Therefore, by monitoring and implementing measures to prevent or deal with their occurrence, interruption to the supply of recycled water at WTP could be minimized. Traditionally, monitoring cyanobacterial blooms in drinking water sources has been through the collection of water samples which are sent to laboratories for microscopic algal enumeration and chlorophyll measurement using spectrophotometry. This usually takes several days before the results become available. Another
*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
technique, ‘in vivo fluorometry’, has been used for the continuous measurement of chlorophyll a (Chl-a) in natural populations and cultures of seawater/freshwater phytoplankton.[4–9] The technique is based on the direct measurement of the fluorescence of the Chl-a in the algal cells. No special sample handling or processing is required and thus in vivo fluorometry using on-line instrument systems is suitable for profiling and for real-time data collection. In vivo fluorometry has been used for continuous measurement of Chl-a since the early 1960s [10] and has the advantage of non-invasive and non-destructive sampling.[11] Chl-a is the main photosynthetic pigment in phytoplanktonic organisms. Other pigments (so-called accessory pigments) are also present in photosystem II. These differ, depending on the taxonomic group of the organisms. For example, chlorophyll b is the marker pigment of chlorophytes, chlorophyll c and fucoxanthin for diatoms, perinidin for dinoflagellates, and phycobilins for cyanobacteria and cryptomonads.[12] The various accessory pigments are excited at different electromagnetic radiation wavelengths and the interaction of these pigments results in a specific excitation spectrum for the different algal taxonomic classes. The excited pigments then transfer their excitation energy to Chl-a, which then fluoresces. Consequently, algal groups can be differentiated according to their specific fluorescence excitation spectra [13,14] and the fluorescence intensities at the different excitation
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Environmental Technology wavelengths are then associated with the in vivo Chl-a content of the different algal groups.[15,16] The algal online analyser (AOA) measures the fluorescence of Chl-a in algae and can be used to distinguish the different classes in water according to their excitation spectra.[17] The AOA uses five light emitting diodes to emit pulsed light at 450, 525, 570, 590 and 610 nm to excite the pigments and the fluorescence emission of the Chl-a is measured at 680 nm. Consequently, the AOA can measure the Chl-a concentrations for Chlorophyceae, Cyanophyceae, diatoms and Cryptophyceae in water. A UV-excitation source (380 nm) is used for differentiation between algal fluorescence and the fluorescence of yellow substances (Y.S.) (coloured dissolved organic matter) which may also be present in the water. Y.S. are characterized by strong absorption in the UV and blue range and so tails over into the visible region and attenuates photosynthetically active radiation (400–700 nm). Since they may interfere with the measurement of Chl-a due to the overlap of the excitation spectra with that of phytoplankton, they are measured (in terms of relative units, r.u.) and correction for this background absorption is made to the output of the AOA. The main advantages of the AOA are its ease of handling, low maintenance costs, and that it provides real-time monitoring of algae. A study by Izydorczyk et al.[18] showed that the AOA is a useful tool for monitoring potentially toxic M. aeruginosa blooms in a reservoir. However, the effectiveness of the AOA for monitoring M. aeruginosa in wastewater stabilization ponds has not yet been evaluated. The aim of this work was to investigate the
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Table 1. Characteristics of the lagoon-treated wastewater (HORS). Parameter
Range
pH Turbidity (NTU) Dissolved organic carbon (mg L−1 ) Total suspended solids (mg L−1 ) Total dissolved solids (mg L−1 ) Total nitrogen (mg L−1 ) Phosphorus (mg L−1 ) Fe content (mg L−1 )
7–7.5 1–8 9–12 2–11 870–1200 13–30 7–12 0.1–0.3
effectiveness of the AOA for detecting M. aeruginosa in the lagoon-treated effluent with the view to developing an alert level framework for potentially toxic cyanobacterial blooms. The concentration of M. aeruginosa was measured in terms of Chl-a and the result was compared with that obtained from the spectrophotometric method. The effect of green algae on the measurement of in vivo Chl-a concentration for M. aeruginosa was also investigated using E. gracilis and Chlorella sp., at cell density/biovolume ranges typical for WTP. 2. Materials and methods 2.1. Lagoon-treated wastewater Lagoon-treated wastewater was collected from the HORS pond at WTP (Figure 1) during summer (January 2010 and February 2011). The characteristics of the HORS water are shown in Table 1.
Anaerobic Pond (1)
Anoxic Zone (2)
Activated Sludge (3)
Secondary Settling (4) Class C Recycled Water
Head of the Road Storage Pond (HORS) (6)
UV and Chlorination (7)
Lagoon System (5)
Class A Recycled Water
Figure 1. Flow diagram of WTP. Notes: (1) The retention time for each stage from 1 to 4 is 3 days. (2) The retention time in the lagoon system (5) is 15 days. (3) The retention time in the HORS (6) is 5–7 days.
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2.2. Algal cultures Microcystis aeruginosa (CS 566/01-A01, originally isolated from the lagoons at WTP) was obtained from the CSIRO Microalgae Research Centre, Hobart. Euglena gracilis and Chlorella sp. were obtained from Southern Biological, Nunawading, Victoria, Australia. Microcystis aeruginosa and Chlorella sp. were grown in MLA culture medium [19] and E. gracilis was grown in CM5 medium which was supplied by Southern Biological. All algal cultures were grown in a laboratory incubator (Labec Illuminated Incubator Model ICCBOD) at a constant temperature (22°C) under a 16/8 h light/dark cycle with a light intensity of 3400 Lux. The cell density was determined using a hemocytometer and the Chl-a concentration was measured using the AOA and the spectrophotometric method given below. Cell suspensions were counted in the laboratory immediately prior to the tests. Suspensions (1 L) of algae were prepared in HORS water collected on the day of the tests for onsite in vivo measurement of Chl-a at WTP. The equivalent biovolumes for M. aeruginosa and E. gracilis were calculated based on mean cell volume data which have been used by South Australian Water and Melbourne Water for calculation of the biovolume of M. aeruginosa in reservoirs (Burch M. South Australian Water, Australia. Personal communication, 2011.) and E. gracilis in stabilization ponds. (Mulcare M. Melbourne Water, Australia. Personal communication; 2010.) The equivalent biovolume of Chlorella sp. was calculated according to Bellinger and Sigee.[20]
2.3. Fluorescence measurement of Chl-a Fluorescence measurements were conducted using the AOA (Model BG82000F, bbe Moldaenke, Kiel, Germany). The AOA was originally calibrated by the manufacturer. After addition of the cells, the HORS water was mixed thoroughly before measurement of in vivo Chl-a. The parameters measured by the AOA include the total chlorophyll (as μg Chl-a L−1 ), the Chl-a concentrations of bluegreen algae, green algae in μg Chl-a L−1 , and concentration of Y.S. in r.u. The cuvette of the AOA has an automatic internal mechanical cleaning device which operates at hourly intervals.
2.4.
Spectrophotometric determination of Chl-a
Suspensions of the specific algae were prepared using a known cell density of algal culture and Milli-Q water. Milli-Q water was used so that the Chl-a content of the target algae could be determined without interference from other phytoplankton. The Chl-a of the algae was extracted with acetone (90% v/v) overnight. The absorbance of the extract was measured at 750, 663, 645 and 630 nm using a UV/Vis spectrophotometer (Unicam UV2) and
the Chl-a concentration was determined according to ESS Method.[21] The relationship between cell density determined by cell counting using a hemocytometer and the Chl-a concentration measured by the spectrophotometric method for the different species is given by Cell density (cells mL−1 ) = a × [Chl-a](µg L−1 ). With • [Chl-a] = Chl-a concentration measured by spectrophotometric method • a = ratio between cell density (cells mL−1 ) and Chla concentration (μg L−1 ). It was found that: ◦ a = 6283 for M. aeruginosa, up to 20,000,000 cells mL−1 (R2 = 0.988) ◦ a = 3364 for Chlorella sp., up to 20,000,000 cells mL−1 (R2 = 0.987) ◦ a = 138 for E. gracilis, up to 2,000,000 cells mL−1 (R2 = 0.988) 2.5.
Fluorescence intensity of Y.S. in a filtered algal suspension
In order to measure the fluorescence intensity of Y.S. in a specific algal suspension, a suspension of a known cell density (equivalent to the lowest density at WTP) of the culture was prepared in Milli-Q water and was filtered immediately after preparation (1 μm glass fibre, Whatman GF/B) to remove all cells. The fluorescence intensity of the filtered solution containing Y.S. released by the algae was measured at Ex = 450 nm and Em = 680 nm using a L55 Perkin Elmer fluorescence spectrometer.
3. Results and discussion 3.1. Response of the AOA to cell density of M. aeruginosa The in vivo Chl-a concentrations for M. aeruginosa in the range 23,000–94,000 cells mL−1 (equivalent to biovolume of 2.0–8.2 mm3 L−1 , a typical range in HORS water at WTP during summer) measured on 14 January 2010 and 16 March 2011 are shown in Figure 2. A direct correlation between cell density and the in vivo Chl-a concentration was observed (R2 = 0.992). The background in vivo Chl-a concentrations for cyanobacteria in HORS water on 14 January 2010 and 16 March 2011 were 0.99 and 0 μg L−1 , respectively. If the background in vivo Chl-a concentrations are taken into account, the response of the AOA to cell density of M. aeruginosa in both tests was fairly similar (Figure 2). The concentrations of Y.S. in HORS water on 14 January 2010 and 16 March 2011 were 1.50 and 1.12 r.u., respectively, and increased only slightly to average values of 1.53 ± 0.15 r.u. and 1.27 ± 0.01 r.u.,
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Figure 2. Response of the AOA to increasing cell density of M. aeruginosa on 14 January 2010 and 16 March 2011. (Background in vivo Chl-a concentrations for cyanobacteria in HORS water before adding M. aeruginosa cells on 14 January 2010 and 16 March 2011 were 0.99 and 0 μg L−1 , respectively).
respectively, after addition of M. aeruginosa over the range of 23,000 to 94,000 cells mL−1 to the HORS water. It should be noted that when the AOA is used for continuous monitoring of algae in a wastewater lagoon, a biofilm may develop within the cuvette due to the elevated C, N and P content of the treated wastewater (Table 1) and this will most likely affect the performance of the AOA. The implementation of an appropriate cleaning regime would be necessary. 3.2.
Determination of Chl-a from M. aeruginosa, Chlorella sp. and E. gracilis by AOA and spectrophotometry Euglena gracilis (a member of the Euglenophyta) and Chlorella sp. (a member of the Chlorophyta) are common green algae found within the lagoons at WTP. Euglena gracilis cells (approximately 50 μm long and 10 μm wide [22]) are markedly larger than Chlorella sp. cells (which are spherical with a diameter of 2–5 μm [20]), whereas Chlorella sp. cells are approximately similar in shape but smaller than M. aeruginosa (which has a diameter of 2–10 μm [20]). The in vivo Chl-a concentration in suspensions of the individual species in HORS water, at cell density ranges typical for WTP, was determined and the results were compared with those obtained from spectrophotometric analysis for suspensions of the same culture in Milli-Q water at the same cell density range. For M. aeruginosa at low cell densities, the Chl-a values measured by the two methods were fairly similar. However, as the cell density increased, the in vivo Chl-a
concentrations did not increase in the same proportion (Figure 3). This may be due to the different principle employed in the two methods. The spectrophotometric method measures the absorbance of the Chl-a after destruction of the cells in the acetone extraction, whereas the AOA fluorometric method measures the fluorescence of the Chla of the intact cells in suspension. Consequently, some cells are shaded by others (shading or screening effect) during the fluorometric measurement and the shading effect increased with increasing cell density, as observed by Izydorczyk et al.[18] The presence of other green algae already present in the HORS water may also have contributed to the lower in vivo Chl-a concentration for M. aeruginosa (the Chl-a for green algae in HORS water measured by the AOA was 0.5 μg/L). The result was consistent with that obtained by Rolland et al.[23] at a reservoir. They used a bbe Fluoroprobe™ in vivo analyser and obtained a good correlation (R2 = 0.97) for M. aeruginosa when the Chl-a concentration was less than 3.9 μg L−1 (equivalent to < 25,000 cells L−1 using our empirically derived relationship, Section 2.4), and lower Chl-a values at higher cell densities compared with spectrophotometric analysis. Figure 2 also shows that there was little change in Y.S. ( ± 0.01 r.u.) with increasing cell density of M. aeruginosa, the results implying that the effect of Y.S. on the measurement of in vivo Chl-a for M. aeruginosa was negligible. For the suspensions of Chlorella sp. (5000–65,000 cells mL−1 , equivalent to a biovolume of 0.15–1.95 mm3 L−1 and typical range for WTP) in the HORS water a strong linear relationship between the in vivo Chl-a concentration and cell density (R2 = 0.986) was shown; similar to
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Figure 3. Chl-a concentration of M. aeruginosa measured by the AOA and spectrophotometric method (background in vivo Chl-a for blue green algae and total Y.S. concentration for HORS water before adding M. aeruginosa cells were 0 μg L−1 and 1.28 r.u., respectively).
Figure 4. Chl-a concentration of Chlorella sp. measured by the AOA and spectrophotometric method (background in vivo Chl-a for green algae and total Y.S. concentration for HORS water were 0.5 μg L−1 and 1.2 r.u, respectively).
M. aeruginosa, the difference between the Chl-a concentration obtained from both AOA and spectrophotometric methods increased with increasing cell density (Figure 4). The difference could be due to the shading effect as well as interference from chlorophyll b (Chl-b), a common accessory pigment in green algae, and may also be from phaeophytin, the breakdown product of chlorophyll, in HORS water. The presence of these pigments tends to decrease the apparent Chl-a concentration when using the fluorometric method as reported by Gibbs [24] and Pinto et al.[25] The lower in vivo Chl-a concentrations for Chlorella sp. at high cell density were therefore attributed to the shading effect (similar to that obtained for M. aeruginosa) and/or the
presence of Chl-b and phaeophytin (it should be noted that the AOA cannot measure phaeophytin or Chl-b). The change in Y.S. concentration was insignificant ( ± 0.05 r.u.) when the cell density of Chlorella sp. increased from 5000 to 65,000 cells L−1 , indicating that the effect of Y.S. was also compensated for during in vivo Chl-a measurement for Chlorella sp. A strong linear relationship between the in vivo Chla concentration and cell density (R2 = 0.992) was shown for the suspensions of E. gracilis (670–3500 cells mL−1 , equivalent to a biovolume of 2.0–10.5 mm3 L−1 and typical range for WTP) in the HORS water. In contrast to M. aeruginosa and Chlorella sp., the Chl-a concentrations
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Figure 5. Chl-a concentration of E. gracilis measured by the AOA and spectrophotometric method (background in vivo Chl-a of green algae and total Y.S. concentration for HORS water were 0.5 μg L−1 and 1.2 r.u, respectively).
for the E. gracilis were higher for the AOA than for the spectrophotometric method, however as before, the difference between the two methods increased with increasing cell density (Figure 5). The presence of carotenoid pigments, which occur in E. gracilis, tends to increase the level of Chl-a fluorescence [26]; the difference in size (and thus less shading effect due to lower abundance) and cell structure of E. gracilis compared with Chlorella sp. and M.
aeruginosa may also play a role. There was a significant increase in concentration of Y.S. measured by the AOA with increasing cell density of E. gracilis, which appears to have had an impact on the in vivo Chl-a values (Figure 5). Determination of the absorbance of the individual filtered (i.e., cell-free) suspension of E. gracilis, M. aeruginosa and Chlorella sp. in Milli-Q water showed that there was absorbance at 350–370 nm for E. gracilis whereas there
Figure 6. Effect of green algae on the response of the AOA to increasing cell density of M. aeruginosa.
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was no absorbance in this range for Chlorella sp., and very little for M. aeruginosa. Therefore, the absorbance in this region may affect the measurement of the Y.S. and thus interfere with the in vivo Chl-a measurement for E. gracilis. As the AOA was originally calibrated by the manufacturer, it was possible that E. gracilis was not chosen for calibration of the AOA and hence the effect of the Y.S. was not compensated for during in vivo Chl-a measurement for E. gracilis.
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3.3.
Impact of E. gracilis and Chlorella sp. on in vivo Chl-a of M. aeruginosa The impact of the presence of E. gracilis and Chlorella sp. on the measurement of in vivo Chl-a for M. aeruginosa was investigated. Suspensions of M. aeruginosa in the cell density range of 35,000–65,000 cells mL−1 (equivalent to a biovolume of 3.05–5.66 mm3 L−1 ) were prepared using HORS water containing E. gracilis or Chlorella sp. with an in vivo Chl-a concentration of 5.2 μg L−1 (equivalent to approx. 670 E. gracilis cells mL−1 , the lowest cell concentration for E. gracilis at WTP, or equivalent to approx. 23,000 Chlorella sp. cells mL−1 ). The presence of Chlorella sp. decreased the apparent in vivo Chl-a concentration for M. aeruginosa by approximately 12%, whereas the presence of E. gracilis increased the apparent in vivo Chl-a concentration for M. aeruginosa by approximately 20% (Figure 6). This may be due to the shading effect caused by the presence of Chlorella sp. in the suspensions leading to the in vivo Chl-a concentration for M. aeruginosa to decrease, and insufficient compensation for the effect of Y.S. released by E. gracilis causing the Chl-a reading for M. aeruginosa to increase. As shown in Figure 6, there was a large increase in Y.S. concentration when E. gracilis was added (from 1.27 ± 0.01 r.u. to 3.45 ± 0.06 r.u.), but very little change when Chlorella sp. was added (from 1.27 ± 0.01 r.u. to 1.32 ± 0.02 r.u) to the suspensions of M. aeruginosa. The results demonstrated that the AOA can give an overestimate or underestimate of M. aeruginosa populations when green algae are present in the lagoon-treated wastewater, depending on their species and their pigment (such as Chl-b) content, and possibly their size. The results are consistent with the findings of Zamyadi et al.[27] who reported that the presence of other phytoplankton such as Scenedesmus and Anabaena circinalis can affect the Chl-a reading from in vivo fluorescence probes for M. aeruginosa. 3.4.
Response of the AOA to a fixed density of M. aeruginosa at varying cell densities of Chlorella sp. The in vivo Chl-a concentrations of suspensions of M. aeruginosa with 25,000 and 50,000 cells mL−1 (equivalent biovolumes of 2.2 and 4.4 mm3 L−1 , respectively, half and the acceptable limit for recycled water at WTP) in
the HORS water in the presence of Chlorella sp. (5000– 65,000 cells mL−1 ) were measured. At both cell densities of M. aeruginosa a strong linear relationship (R2 = 0.945 and 0.989, respectively) between the in vivo Chl-a and cell density for Chlorella sp. was shown (Figure 7). The in vivo Chl-a concentration of the M. aeruginosa suspension (25,000 cells mL−1 ) was decreased to 3.86 μg L−1 (by 7.2%) from 4.16 μg L−1 on addition of 5000 cells mL−1 Chlorella sp. However, when the cell density of Chlorella sp. increased from 5000 to 65,000 cells mL−1 , the change in in vivo Chl-a concentration for M. aeruginosa was negligible (the average in vivo Chl-a concentration for M. aeruginosa was 3.69 ± 0.09 μg L−1 ; Figure 7(a)). Similarly, there was little change (only ± 0.06 r.u.) in the concentration of Y.S. when the cell density of Chlorella sp. increased from 5000 to 65,000 cells mL−1 . A similar trend was observed for the M. aeruginosa suspension of 50,000 cells mL−1 : the in vivo Chl-a concentration decreased from 8.07 to 7.74 μg L−1 (a decrease of 4.1%) when 5000 cells mL−1 Chlorella sp. was added. There was little change in in vivo Chl-a concentration for M. aeruginosa and Y.S. when the density of Chlorella sp. increased from 5000 to 65,000 cells mL−1 (Figure 7(b)), with values of 7.61 ± 0.23 μg L−1 and 1.29 ± 0.02 r.u., respectively. The results indicated that increasing cell density of Chlorella sp. had little effect on the measurement of in vivo Chl-a concentration for a fixed cell density of M. aeruginosa, and the effect of Y.S. when Chlorella sp. was added was taken into account.
3.5.
Response of the AOA to a fixed cell density of M. aeruginosa at varying cell densities of E. gracilis Suspensions of M. aeruginosa (25,000 and 50,000 cells mL−1 ) were prepared using HORS water containing E. gracilis cells of various cell densities (670–3500 cells mL−1 , equivalent to a biovolume of 2.0–10.5 mm3 L−1 , the typical range at WTP). For both suspensions of M. aeruginosa a strong linear relationship (R2 = 0.970 and 0.983 for 25,000 and 50,000 cells mL−1 , respectively) between in vivo Chl-a concentration and cell density for E. gracilis was shown (Figure 8(a) and (b)). The in vivo Chl-a concentration of the M. aeruginosa suspension (25,000 cells mL−1 ) was increased from 4.16 to 5.73 μg L−1 (by 27%) on addition of 670 cells mL−1 E. gracilis. Although the cell density of M. aeruginosa in all samples was constant, the in vivo Chl-a concentration for M. aeruginosa increased from 5.73 to 9.47 μg L−1 (Figure 8(a)) when the cell density of E. gracilis increased from 670 to 3500 cells mL−1 . Thus when the cell density of E. gracilis increased five times, the apparent cell density for M. aeruginosa measured by the AOA was almost doubled.
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(a)
(b)
Figure 7. Relationship between the in vivo Chl-a and cell density for a fixed cell density of M. aeruginosa in suspensions of Chlorella sp. (a) 25,000 M. aeruginosa cells mL−1 and (b) 50,000 M. aeruginosa cells mL−1 (in vivo Chl-a concentration for suspensions of M. aeruginosa at 25,000 cells mL−1 and 50,000 cells mL−1 when no Chlorella sp. was present were 4.16 and 8.07 μg L−1 , respectively).
A similar trend was found for the M. aeruginosa suspension of 50,000 cells mL−1 , with an increase in apparent in vivo Chl-a concentration from 8.07 to 9.97 μg L−1 (more than 19%) on addition of 670 cells mL−1 E. gracilis, and from 9.97 to 14.23 μg L−1 when 3500 cells mL−1 of E. gracilis were added, an increase of approx. 30% (Figure 8(b)). The Y.S. measured by the AOA increased from 1.45 and 1.48 r.u. for M. aeruginosa at 25,000 and 50,000 cells mL1 , respectively, to 3.06 and 3.08 r.u., respectively, on addition of E. gracilis at 670 cells mL−1 and then increased with increasing cell density of E. gracilis. The results demonstrated that increasing cell density of E. gracilis had an effect on the measurement of in vivo
Chl-a concentration for a fixed cell density of M. aeruginosa, and the effect of Y.S. released by E. gracilis was not compensated by the AOA.
3.6. Reproducibility of the AOA results The relative standard deviation (%STDEV) for three Chla measurements for each M. aeruginosa suspension in the cell density range of 23,000–94,000 cells mL−1 is shown in Table 2. The %STDEV for Chl-a measured by the AOA for M. aeruginosa suspensions of 25,000 and 50,000 cells mL−1 can be estimated as 10% and 12%, respectively, which was acceptable.
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Figure 8. Relationship between in vivo Chlorophyll-a concentration and cell density for a fixed density of M. aeruginosa in suspensions of E. gracilis (a) 25,000 M. aeruginosa cells mL−1 and (b) 50,000 M. aeruginosa cells mL−1 (in vivo Chl-a concentration for suspensions of M. aeruginosa at 25,000 cells mL−1 and 50,000 cells mL−1 without E. gracilis were 4.16 and 8.07 μg L−1 , respectively). Table 2. Mean, STD and % STDEV of in vivo Chl-a concentration for M. aeruginosa in HORS water in the cell density range of 23,000–94,000 cells mL −1 (after chemical cleaning). Chl-a concentration measured by the AOA (μg L−1 ) Density (cells mL−1 ) 23,000 45,000 48,000 68,000 80,000 94,000
Equivalent biovolume (mm3 L−1 )
Mean
STD
%STDEV
2.0 3.9 4.2 5.9 7.0 8.2
3.24 6.97 7.19 9.63 10.98 12.25
0.32 0.80 0.83 1.10 1.30 1.27
< 10 (9.9) 11 12 11 12 > 10 (10.4)
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Environmental Technology 4. Conclusions This study demonstrated that the AOA has potential to be used as an alert system for cyanobacterial blooms in a wastewater stabilization pond. However, the effect of the presence of some green algae on the apparent cell density of M. aeruginosa needs to be taken into account. Furthermore, the accuracy of the AOA in terms of in vivo Chl-a concentration should be cross-checked periodically with a laboratory-based method such as spectrophotometry, and calibration for Y.S. over an appropriate range of green algae in the pond needs to be maintained. Although the AOA readings may be affected by these factors, the AOA provides real-time monitoring of algal populations and an indication of change in cyanobacterial densities, e.g., imminent blooms. Ease of handling and relatively low maintenance costs are further advantages compared with manual cell counts which are time-consuming, and the accuracy of which is dependent on the sampling time, sample management and operator error. Acknowledgements This research was funded by the Smart Water Fund Victoria, Australia (Project Number 62M-2026)
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