Algal growth in photo-bioreactors: Impact of illumination strategy and nutrient availability

Ecological Engineering 77 (2015) 202–215

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Algal growth in photo-bioreactors: Impact of illumination strategy and nutrient availability Amritanshu Shriwastav * , Purnendu Bose Environmental Engineering and Management Program, Department of Civil Engineering, Indian Institute of Technology Kanpur, Kanpur 208 016, India

A R T I C L E I N F O

A B S T R A C T

Article history: Received 5 August 2014 Received in revised form 13 January 2015 Accepted 20 January 2015 Available online xxx

The objectives of this study were to first identify illumination strategies which support long term sustainable algal growth in photo-bioreactors, and second to address contradictions in literature regarding the algal nutrient uptake behavior under a sustainable growth regime. Experiments were conducted at different light intensities with continuous illumination and intermittent illumination of 12h light and dark periods. Sustainable algal growth was characterized with algal specific chlorophyll-a content of more than
20 m g g1. Experiments were also conducted to address contradictions in literature regarding Droop versus Monod formulations for nutrient uptake, and representation of growth limiting factors using Liebig’s law of the minimum versus the multiplicative rule. Intermittent illumination of 12-h light and dark periods at a light intensity of 246 mmol m2 s1 was found to be the most optimal strategy for sustainable algal-growth. It was further concluded that the impact of several growth limiting factors on algal growth could be best described by the Liebig’s law of the minimum with Droop’s formulation rather than multiplicative rule and Monod formulation. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Algal growth Photo-bioreactor Photo inhibition Nutrient-limitation Droop model Liebig’s law of the minimum

1. Introduction Numerous applications of algal photo-bioreactors for wastewater treatment, hazardous contaminant removal, for hydrogen production, algal biomass production for bio-fuel recovery, carbon sequestration, etc., have been reported in the literature (Ai et al., 2008; Di Termini et al., 2011; Griffiths, 2009; Muñoz et al., 2009; Perales-Vela et al., 2006; Tamburic et al., 2011). The advantages of such photo-bioreactors over the traditional open algal ponds are numerous and extensively reviewed elsewhere (Rawat et al., 2013; Singh and Sharma, 2012). There are some instances of algal photo-bioreactors being operated under continuous illumination at low light intensities (Kim et al., 2013; Ryu et al., 2014; Wang et al., 2010). However in most cases, algal photo-bioreactors are operated with alternate light and dark periods, with duration of light and dark periods ranging from milliseconds to hours (Cabanelas et al., 2013; Ji et al., 2014; Wang et al., 2010; Yoshimoto et al., 2005). Irrespective of the illumination strategy adopted, the main focus during the operation of such reactors is on maximizing the algal growth. Light intensity and illumination strategy have been accepted as important factors which govern the algal growth dynamics (Soletto et al., 2008). At

* Corresponding author. Tel.: +91 9839457878; fax: +91 512 259 7395. E-mail address: [email protected] (A. Shriwastav). http://dx.doi.org/10.1016/j.ecoleng.2015.01.034 0925-8574/ ã 2015 Elsevier B.V. All rights reserved.

low light intensities, algal growth is limited by light availability. However, algal growth under high light intensity conditions is also potentially problematic and phenomena such as algae photo inhibition and photo toxicity have been reported (Béchet et al., 2013). Literature reports also suggest that chlorophyll-a content of the algal cell declines at higher light intensities (Bonente et al., 2012). This is because the Photo System II (PS-II) in algal cells is stressed at high light intensities and may under certain circumstances be irreversibly damaged (Mulo et al., 2012). Depletion of algal chlorophyll content beyond a certain threshold may thus jeopardize the long-term viability of algal culture maintained in photo-bioreactors (Shriwastav et al., 2014). The review of literature reveals that most studies investigating the effect of illumination regime on algal growth dynamics focus solely on algal biomass growth. However, there is enough evidence in the literature to indicate that prolonged exposure to light or exposure to light of high intensity may result in irreversible damage to the long-term viability of the algal cell as indicated by depleted algal chlorophyll content. Often the evidence of such damage may not be apparent in short-duration experiments which only monitor algal biomass growth. Hence it is important to identify illumination strategies which maximize the algal growth while maintaining the culture in sustainable state to ensure long term viability of algal systems. Additionally, once the illumination regimes suitable for sustainable algal growth are identified, further studies on the

A. Shriwastav, P. Bose / Ecological Engineering 77 (2015) 202–215

sustainability of algal cultures and thus identify illumination regimes suitable for sustained algal growth in photo-bioreactors. - To investigate the nutrient uptake and algal growth dynamics in photo-bioreactors under illumination regimes suitable for sustained algal growth and thus gain insight into the limiting factors impacting algal growth. This study links algal chlorophyll content with light induced stresses and thus suggests that algal chlorophyll content can be used as proxy for culture viability in algal systems where autotrophic growth is predominant. In addition, a systematic analysis was carried out to address contradictions in literature regarding the mechanism for algal nutrient uptake. Achieving a comprehensive understanding of all factors associated with algal growth in photo-bioreactors will lead to the development of a realistic model for describing algal growth dynamics. 2. Materials and methods 2.1. Light chamber Standard BOD bottles of 300 mL capacity were used as photobioreactors. These reactors were placed inside a wooden light chamber (dimensions: 0.85 m  0.6 m  0.45 m). Mixing of the photo-bioreactor contents is important for efficient light utilization by algae cells during photosynthesis (Mitsuhashi et al., 1995), however high levels of mixing result in cell damage (Gudin and Chaumont,1991). Hence, for mixing, the base of the light chamber on which the reactors were placed was mounted on bearings. This base was attached to a crankshaft operating at a crank speed of 30 rpm. The mixing thus imparted was sufficient to keep the photobioreactor contents completely mixed, without creating violently agitated conditions. Compact fluorescent lamps (CFL) were fitted inside the light chamber to provide the required illumination. Two types of lamps were used, 14 W CFL lamps (make: Philips, India), 100 W CFL lamps (make: Ajanta, India). The lamps could be turned on and off as required using a timer fitted to the electrical circuit. 2.2. Characterization of the light sources To determine the light emission spectra from a typical light source of each type, i.e., 14 W and 100 W CFL lamps, the optical

2.3. Measurement of light intensity inside a photo-bioreactor The average value of the rate of photon incidence inside the reactors was determined using potassium ferrioxalate actinometry (Hatchard and Parker, 1956; Parker, 1953). Actinometry experiments were conducted in a room illuminated with red light. Seven reactors were each filled with 270 mL of 0.006 M potassium ferrioxalate solution in 0.1 N H2SO4. One bottle was immediately kept in dark and used as reference. Remaining six bottles were kept in the light chamber under mixed conditions and exposed to light of a particular intensity for different exposure times. After exposure for the required duration, 10 mL aliquots were removed from all bottles (in duplicate) and mixed with 5 mL of acidic acetate buffer and 2 mL of 0.1% w/w 1,10-phenanthroline solution and kept in complete darkness. Fe+2-1,10-phenanthroline complex formation was complete in 1 h, after which the absorbance of the solutions was measured at 510 nm using a UV–visible spectrophotometer (Helios Epsilon, Thermo Scientific, USA) and a 1 cm path length quartz cuvette. The number rate of incident photons (in quanta s1) in the 253–430 nm range was calculated using the equation below (Rabek, 1982). na ¼

D  V 1  V 3  103  NL t  f  L  e  V2

(1)

where, na = number of photons absorbed by the actinometer per second, quanta s1.

25x106

Counts per second

- To use algal chlorophyll content as an indicator for the long-term

probe extension of a spectro-fluorometer (Fluorolog-3, JOBIN VYON HORIBA, USA) was placed inside a typical reactor. The reactor was then placed in the vicinity of the light source in a dark room. Typical spectra obtained from the CFL lamps are presented in Fig. 1a. Based on the emission spectra as shown in Fig. 1a, cumulative fractional photon distribution curve, i.e., plot of the fraction of photons below a particular wavelength versus the wavelength, was generated for both sources as shown in Fig. 1b.

a

20x106

100W CFL Light Source 14W CFL Light Source

15x106 10x106 5x106 0 250 300 350 400 450 500 550 600 650 700 750 800 850

Wavelength, nm 1.2

Cumulative Photon Fraction

algal growth dynamics and nutrients uptake under these conditions are necessary for the comprehensive understanding of all factors required to achieve a sustained and high rate algal growth in photo-bioreactors. Uptake of nutrients (N and P) and consequent algal growth has been studied by several researchers (Termini et al., 2011; Wang and Lan, 2011). The fact that algae often take up nutrients in excess of their immediate growth requirements and store excess nutrients internally has also been known for some time (Droop, 1974; Nambiar, 1979; Rhee, 1973). Despite such insights, many experimental and modeling studies still utilize the Monod formulations, which do not account for internal storage of nutrients (He et al., 2012; James and Boriah, 2010; Malve et al., 2007). Further, the effect of multiple growth limitation factors on algal growth is still debatable, as studies supporting the multiplicative effect of several growth limiting factors (James and Boriah, 2010; Malve et al., 2007) as well as those advocating Liebig’s law of the minimum (Chapra et al., 2007; Klausmeier et al., 2008) are both widely used. Such contradictions need to be addressed in a systematic manner in order to gain a comprehensive understanding of sustained and high rate algal growth. Specifically the objectives of the present study were the following:

203

b

1.0 0.8 0.6 0.4 0.2

100 W CFL Light Source 14 W CFL Light Source

0.0 250 300 350 400 450 500 550 600 650 700 750 800 850

Wavelength, nm Fig. 1. Characterization of light sources: (a) typical emission spectra; (b) cumulative fractional photon distribution diagram.

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D = optical density at 510 nm. V1 = volume of potassium ferrioxalate solution irradiated, 270 mL. V2 = volume of irradiated solution taken for analysis, 10 mL. V3 = final volume of taken solution after dilution, 17 mL. NL = Avogadro number, 6.023  1023. t = irradiation time, s. f = quantum yield for Fe+2, 1.203. L = path length of cuvette, 1 cm. e = molar extinction coefficient of Fe+2 complex, 1.11 104 L mol1 cm1. Actinometry experiments were conducted at various applied light intensities. The average D/t value can be calculated by linear correlation between corresponding absorbance and time data for applied light intensity (see Eq. (1)). Assuming that all incident photons in the actinometry range (253–430 nm) contributed to the photochemical reaction resulting in the formation of Fe2+ ions, the rate of photon incidence (na) in the actinometry range (253– 430 nm) can be calculated using Eq. (1). The main light absorbing pigments present in green microalgae are chlorophyll-a, chlorophyll-b and carotenoids, which absorb photons in the visible (400–700 nm) range (Nelson and Cox, 2009). The rate of photon incidence and corresponding incident light intensity (I0) in the visible range (400–700 nm) can be calculated using actinometry data and the emission spectra of the light sources using the relationship below:

This suspension was illuminated (4  14 W CFLs; Philips, India) for 18 h followed by 6 h in dark conditions for one week. After this, 500 mL of the suspension was again filtered through GF/C filter and re-suspended in 25 L fresh MSM, before being maintained again in alternate light–dark conditions as before. After four such cycles, the biomass collected on the filter consisted only of algae and free from any bacteria or protozoa, as confirmed through microscopic examination (EC LUMAM-RPO, LOMO PLC, Russia) as well as crosscheck of microbial growth by streaking on agar plate in dark. The algal culture thus obtained was maintained for further experiments in four cotton plugged reactors under alternate light and dark conditions. A new stock culture was prepared every two weeks during the study period by transferring 10 mL of an existing culture to fresh MSM kept in a reactor. The older cultures were gradually discarded as new cultures were prepared. Regular checks for axenic conditions were performed by microscopic examination as well as checking microbial growth by streaking on agar plate in dark. Samples from the stock algal culture were sent to National Botanical Research Institute (NBRI), Lucknow, India for identification of algae species. Two species of algae, Chlorella vulgaris in abundance, and Chlamydomonas reinhardtii in lesser number were identified in the stock culture. Fig. 2 presents the microscopic slide of these species.

Photon incidence rate in actinometry range Fraction of photons in the actinometry range Photon incidence rate in visible range ¼ Fraction of photons in the visible range

Two illumination strategies were used in the first phase of batch experiments. In some experiments, a continuous light period of six days was provided followed by dark period of three days. In other experiments, alternate light and dark periods of 12 h each were provided for the experimental duration of 5 days. Researchers (Ogbonna and Tanaka, 2000; Ratchford and Fallowfield, 2003) have reported light inhibition of algal growth to occur at light intensities in excess to 300 mmol m2 s1. Hence, three light intensities were selected for these experiments such that behavior of algae at high intensity of 347 mmol m2 s1 for light inhibited growth, at medium intensity of 146 mmol m2 s1 for sustainable algal growth, and at low intensity of 14 mmol m2 s1 for light limited growth could be investigated with their subsequent recovery potential (Table 1). Nutrients, i.e., N, P, and inorganic carbon were provided in excess during these experiments. The second phase experiments were carried out under intermittent illumination at light intensities of 146 mmol m2 s1 or 14 mmol m2 s1 and under various nutrient-limited conditions. Since secondary treated domestic effluent retains sufficient levels of N (35–50 mg L1)

(2)

These calculations are summarized in Table 1. These calculations assume that the cumulative fractional photon distribution is the same for all light sources of a particular type and this distribution does not change with time. 2.4. Preparation of stock algae culture Wastewater was collected from an oxidation pond at IIT Kanpur, India. Immediately after collection, 500 mL of the wastewater was filtered (GF/C filter of 1.2 mm pore size; Whatman, UK) and the biomass collected on filter paper was re-suspended in 25 L mineral salt medium (MSM) (Muñoz et al., 2005) in a 30 L plastic container (diameter: 0.4 m; depth: 0.2 m; working liquid volume: 25 L) without any additional organic carbon source or external air supply, while cultivation temperature was maintained at 30  5  C.

2.5. Batch experiments

Table 1 Calculation of photon incidence rate in visible range in algal photo bioreactors for different light intensities. Applied light intensity (W)

Fraction of photons in 253– Photon incidence rate in 253– 430 nm range (from Fig. 1b) 400 nm range (by actinometry) Quanta s1

56 (4  14 W CFL) 200 (2  100 W CFL) 300 (3  100 W CFL) 400 (4  100 W CFL)

0.127

2.07  1016

0.973

1.58  1017

14

0.096

1.66  1017

0.984

1.70  1018

146

0.096

2.78  1017

0.984

2.85  1018

246

0.096

3.93  1017

0.984

4.03  1018

347

a

Fraction of photons in 400– Photon incidence rate in 700 nm range (from Fig. 1b) visible range (400– 700 nm) (using Eq. (2))a Quanta s1

Incident light intensity in visible range (400–700 nm) (I0)b mmol m2 s1

Rate of total photon incident in a sample volume of 270 mL. For the reactor configuration used in this study, volume to surface area ratio was calculated to be 14.01 L m-2. I0 values (in mmol m2 s1) were calculated after conversion of photon incidence rate in total 270 mL sample volume to unit sample volume of 1 L and then multiplying with volume to surface area ratio. b

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Fig. 2. Microscopic slide for Chlamydomonas reinhardtii and Chlorella vulgaris.

and P (6–10 mg L1) (Arceivala and Asolekar, 2007; Metcalf and Eddy et al., 1998), nutrient levels in second phase experiments were compared with these levels, and were termed as high if in the same range, and low if sufficiently lower than these levels. High light intensity of 347 mmol m2 s1 was not utilized since the objective of second phase experiments was to investigate the nutrient uptake behavior of algae within sustainable illumination regime without any light inhibition effect. The operating condition for the first and second phase experiments have been summarized in Tables 2a and 2b, respectively. The media used in these experiments consisted of a mixture containing 20 mL of MSM, 508 mg Na2CO3 and 504 mg NaHCO3 (as inorganic carbon source), which was diluted to 1000 mL. Potassium nitrate (KNO3), potassium dihydrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4) were added to the media to achieve the desired N and P concentrations. The media was then autoclaved and its pH adjusted to 7 by addition of HCl or KOH as necessary. Seeding of the reactors was done as follows: a 20 mL aliquot of the algal solution was taken from the stock culture and centrifuged (Biofuge Stratos, Heraeus, Germany) at 3000  g (Lau et al., 1995) for 15 min. The supernatant was discarded and the centrifuged algal mass was re-suspended in 20 mL of 1 M phosphate buffer, agitated for 2 min on vortex mixer (Vortex-2, Genie, Scientific Industries Inc., USA) and again centrifuged. The centrifuged algal mass was washed as above for two more cycles with 20 mL of de-ionized water. Finally the algae were re-suspended in 20 mL sterilized media and added to a sterilized BOD bottle. Further

250 mL of sterilized media was added to the bottle. The bottle was kept in the light chamber in illuminated conditions (4  14 W CFL) and with gentle mixing for 12 h for acclimatization. Finally, 20 mL aliquot of the algal suspension from this bottle was transferred to reactors (in duplicate) and further 250 mL of the media added to each reactor. These reactors were immediately put in light chamber and illuminated with light of the desired intensity. Nutrient blanks with only media but no added algae were also maintained in the light chamber. All reactors were loosely plugged with cotton wool, such that transfer of gases between the reactor and the atmosphere was not hampered during the experiments. Reactors were sampled as required for analysis of various parameters. All measurements were made in duplicate. 2.6. Semi-batch experiments Photo-bioreactors were operated in semi-batch mode over a period of 113 days. Reactor startup in all cases was similar to that described earlier. However in this case, certain amount of fully mixed reactor content was periodically removed from each reactor and replaced with an equal amount of fresh media. The main objective of these semi-batch experiments was to investigate the long term growth and recovery potential of algae with sustainable growth conditions. Applied light intensities of 146 mmol m2 s1 (200 W source, @2  100 W CFL) and 246 mmol m2 s1 (300 W source, @3  100 W CFL) were used during these experiments. Intensities of 347 mmol m2 s1 and 14 mmol m2 s1 were not investigated as they result in light inhibited and light limited

Table 2a Operating conditions for batch algal experiments for effect of illumination strategies with excess nutrients. Experiment no.

Initial nitrate (mg L1 as N)

Initial phosphate (mg L1 as P)

I0 (mmol m2 s1)

Illumination strategy

1. 2. 3. 4. 5. 6.

21.6 25.9 26.5 27.3 23.3 22.6

4.1 4.4 7.5 23.6 16.4 16.4

347 146 14 347 146 14

Continuous Continuous Continuous Intermittent Intermittent Intermittent

Note: all continuous experiments over a period of 6 days. All intermittent experiments with alternating 12 h light and dark periods for 5 days.

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Table 2b Operating conditions for batch algal experiments for effect of nutrients limitation under intermittent illumination. Experiment no.

Initial nitrate (mg L1 as N)

Initial phosphate (mg L1 as P)

I0 (mmol m2 s1)

Remarks

7. 8. 9. 10. 11. 12.

24.3 22.4 3.7 3.7 3.6 3.7

1.00 2.2 16.8 16.5 0.6 2.2

146 14 146 14 146 14

N N N N N N

high; P low high; P low low; P high low; P high low; P low low; P low

Note: all experiments with alternating 12 h light and dark periods for 5 days.

algal-growth respectively. Instead, algal-growth at light intensity of 246 mmol m2 s1 was chosen to further investigate the upper limits of light intensity for sustainable growth. Table 3 presents the operational details of semi batch reactor. Reactors were sampled as required for analysis of various parameters, of which algal Sp. Chl-a content is of relevance to this study. Measurements were made in duplicate.

2.7. Analytical techniques Chlorophyll-a concentration was measured using a procedure described in literature (Porra et al., 1989; Sartory and Grobbelaar, 1984). This method involved chlorophyll-a measurement using a spectrophotometer (Helios Epsilon, Thermo Scientific, USA) and a quartz cuvette of 1 cm path length after extraction in methanol.

Table 3 Operating conditions for semi batch experiments. Days

0–3 3–11 11–18 18–23 23–26 26–28 28–31 31–39 39–48 48–50 50–51 51–54.67 54.67–56 56–57 57–58 58–59 59–65 65–65.67 65.67–69 69–70 70–71 71–72.5 72.5–73 73–78 78–79 79–80 80–81.5 81.5–84 84–88 88–90 90–91 91–91.5 91.5–92 92–93 93–93.5 93.5–94 94–96 96–98.5 98.5–101.5 101.5–103 103–107 107–111.67 111.67–112 112–113

Feed characteristics Nitrate, mg L1 (as N)

Phosphate, mg L1 (as P)

3.39 3.39 3.39 3.39 3.39 3.39 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 6.77 13.55 13.55 27.10 31.61 6.77 6.77 6.77 6.77 9.03 9.03 9.03 9.03 9.03 11.29 11.29 9.03 6.77 6.77 6.77

1.63 1.63 1.63 1.63 1.63 1.63 1.63 1.63 1.63 1.63 1.63 1.63 2.45 1.63 1.63 1.63 1.63 1.63 1.63 1.63 1.63 1.63 1.63 1.63 1.63 3.26 3.26 6.53 6.53 1.63 1.63 1.63 1.63 2.02 2.02 2.02 2.02 2.02 2.28 3.26 2.45 1.63 1.63 1.63

HRT, days

Light/dark period

Incidence photon rate, mmol m2 s1

Batch 3 3 2 1 2 2 1 1 2 1 0.5 0.5 1.5 4 1 1 1 1 3 2 1 1 0.5 1.5 1 1 1 2 1 0.5 0.75 1 0.5 0.75 1 2 1 1 1 1 0.5 1 0.5

12 h/12 h 12 h/12 h 8 h/16 h 12 h/12 h 12 h/12 h 12 h/12 h 12 h/12 h 12 h/12 h 6 h/6 h 6 h/6 h 6 h/6 h 7 h/1 h 7 h/1 h 6 h/6 h 6 h/6 h 6 h/6 h 5 h/3 h 4 h/4 h 2 h-D/4 h-L/2 h-D 6 h/6 h 6 h/6 h 6 h/6 h 2 h-D/4 h-L/2 h-D 6 h/2 h 6 h/6 h 6 h/6 h 9 h/3 h 9 h/3 h 9 h/3 h 6 h/6 h 6 h/2 h 9 h/3 h 6 h/6 h 6 h/2 h 6 h/6 h 6 h/6 h 6 h/6 h 6 h/6 h 6 h/6 h 6 h/6 h 6 h/6 h 6 h/2 h 6 h/6 h 6 h/2 h

146 146 146 146 146 146 146 146 146 146 146 146 146 146 146 146 246 246 246 246 246 246 246 246 246 246 246 246 347 246 246 246 246 246 246 246 246 246 246 246 246 246 246 246

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parameters. Nitrate, nitrite, and phosphate were analyzed using an Ion Chromatograph (882Compact IC Plus Anion, Metrohm, Switzerland) which was fitted with a 20 mL injection loop and Anion Column (Metrosep A Supp5 250/4.0) with Magic Net 1.1 software for analysis.

The method used above was validated by determining the absorbance of known amounts of chlorophyll-a standard (Sigma–Aldrich, USA) dissolved in methanol. pH and temperature were measured in-situ by inserting a probe (pH 200 with Senso Direct Probe, Orbeco Hellige, USA) into the reactors. Algal biomass was determined gravimetrically. For this purpose, a known volume of the algal suspension was filtered through a pre-dried and pre-weighed filter paper (0.22 mm pore size; Millipore Corp., USA) using a syringe filter assembly. The weight of the filter paper was measured post-filtration and after drying at 105  C for 24 hours in an oven (Mahendra Scientific Instruments, Kanpur India). Weighing was done using an analytical balance (AB 135-S; METTLER TOLEDO, USA). The filtrate obtained during above filtration was used for subsequent analysis of other

3. Results 3.1. Effect of illumination regime on algal growth 3.1.1. Continuous illumination When operated under continuous illumination at the highest light intensity of 347 mmol m2 s1, algal growth ceased after two days (see Fig. 3a) and went into a decline. The specific chlorophyll-a

250

a -1 Algal Biomass, mg L

200

150

100 I0 = 347 µmol m-2 s-1 I0 = 146 µmol m-2 s-1 I0 = 14 µmol m-2 s-1

50

0 3 Days

15 10

20 15 10 5 0

9

6

3

0

0

5

c

9

20

6

25

mg Chl-a g-1 Algae

25

5

30

b

-1

mg Chl-a g Algae

30

4

6

2

3

1

0

0

Days

Days

d

25 20

-1

mg Chl-a g Algae

30

15 10

9

6

3

0

0

5

Days Fig. 3. Effect of continuous illumination on algal growth at various light intensities (a); and on specific algal chlorophyll-a content at I0 = 347 mmol m2 s1 (b), 146 mmol m2 s1 (c), 14 mmol m2 s1 (d).

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(Sp. Chl-a) content of algal cells declined drastically from approximately 25 mg g1 to less than 5 mg g1 (see Fig. 3b) over the first three days of illumination. This was accompanied by a visible discoloration of the algal culture from its initial green color to a yellow-green color. After illumination for 6 days, the algal culture was maintained in dark conditions for 3 days. However, this did not result in any increase in the Sp. Chl-a content (see Fig. 3b) which indicates irreversible damage to algae cells due to light. Operation under continuous illumination at lower light intensities of 146 mmol m2 s1 and 14 mmol m2 s1 resulted in continued algal growth (see Fig. 3a) over the entire 6-day period. Sp. Chl-a content of algal cells also declined in these cases (see Fig. 3c and d, respectively). However,

220

in these cases, maintenance of the culture under dark conditions for a further 3 days resulted in partial restoration of the Sp. Chl-a content. 3.1.2. Intermittent illumination When operated under intermittent illumination condition at a light intensity of 347 mmol m2 s1, the algal growth ceased after 4 days (see Fig. 4a). However growth at light intensities of 146 mmol m2 s1 and 14 mmol m2 s1 continued throughout the experimental period of 5 days (see Fig. 4a), with the growth being faster at the higher light intensity. In all cases, the Sp. Chl-a content of algal cells declined (see Fig. 4b–d) after the first cycle of illumination but recovered after the second cycle. However at light

a

200

Algal Biomass, mg L-1

180 160 140 120 100 80 60

I0 = 347 µmol m-2 s-1

40

I0 = 146 µmol m-2 s-1 I0 = 14 µmol m-2 s-1

20 0 3 Days

50

40 30 20

4.5

3.5

4.5

0

2.5

10

Days

d

50 40 30 20

4.5

2.5

0

1.5

10 0.0 0.5

mg Chl-a g-1 Algae

Days 60

3.5

1.5

0

2.5

10

6

c mg Chl-a g-1 Algae

20

0.0 0.5

mg Chl-a g-1 Algae

30

5

50

b 40

4

1.5

2

0.0 0.5

1

3.5

0

Days Fig. 4. Effect of intermittent illumination on algal growth at various light intensities (a); and on specific algal chlorophyll-a content at I0 = 347 mmol m2 s1 (b), 146 mmol m2 s1 (c), 14 mmol m2 s1 (d).

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intensities of 347 mmol m2 s1 and 146 mmol m2 s1 the Sp. Chla content showed a continuing declining trend after the third, fourth and fifth cycles of illumination. This rate of decline was more severe at higher light intensity as compared to that at the lower light intensity (compare Fig. 4b and c). In case of the lowest light intensity of 14 mmol m2 s1, the Sp. Chl-a content increased after the second cycle of illumination (see Fig. 4d) and then remained stabilized at approximately 45 mg g1 over the third, fourth and fifth cycles.

209

3.2. Effect of nutrient limitation on algal growth

3.2.2. Algal growth at low N and high P The experiments shown in Fig. 6 were also carried out at two light intensities, but low initial N concentrations and high P concentrations. In this case, algal growth continued through the entire experimental duration of 5 days (see Fig. 6a) in the reactor exposed to lower light intensity. However, evidence of algal growth impairment was seen in the reactor exposed to higher light intensity from the fourth day onwards (see Fig. 6a). Corresponding N uptake data is shown in Fig. 6b. Comparing Fig. 6a and b, it is evident that even though dissolved N in bulk phase declined to low levels only after two days in both cases, algal growth continued unimpaired even after the second day.

3.2.1. Algal growth at high N and low P The experiments shown in Fig. 5 were carried out at two light intensities and with low initial P concentrations, but high N concentrations. Algal growth continued in both the experiments through the entire experimental duration of 5 days (see Fig. 5a). Algal growth was more in the reactor exposed to higher intensity of light. Corresponding P uptake data is shown in Fig. 5b. Comparing Fig. 5a and b, it is evident that algal growth continued even after dissolved P in the media was reduced to low levels.

3.2.3. Algal growth at low N and low P In the next set of experiments shown in Fig. 7, both initial nitrate and phosphate concentrations were kept low. In the reactor operated at the higher light intensity, the dissolved N in the bulk phase declined to very low levels within two days of reactor operation (see Fig. 7b), while the dissolved P in the bulk phase declined to very low values after the first day of reactor operation itself (see Fig. 7c). However, algal growth continued unimpeded up

a

250

Algal Biomass, mg L-1

-2

-1

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to the fourth day of reactor operation (see Fig. 7a). In case of the reactor operated at lower light intensity, algal growth (see Fig. 7a) continued unimpeded for the entire duration of experiments despite the nitrate levels (see Fig. 7b) in the reactor falling to very low values by the second day. 4. Discussion 4.1. Illumination strategies for long term sustainable growth of algae Algal growth at the highest light intensity of 347 mmol m2 s1 under continuous illumination is clearly unsustainable, since algal growth in this case ceased and declined after two days of illumination (see Fig. 3a). This is attributable to the drastic loss in algal Sp. Chl-a content (see Fig. 3b). Bonente et al. (2012) also observed such loss at higher light intensities and attributed it to excessive light stress. Such stresses occur when the extra energy associated with high light intensity cannot be dissipated as fluorescence and heat, and starts to affect the photo synthesis itself (Wilhelm and Jakob, 2011). Such high light intensity might breach the light saturation limit of algae and thus put it under high light stress. This clearly makes light intensity of 347 mmol m2 s1 unsustainable. However, under continuous illumination at lower light intensities of 146 and 14 mmol m2 s1, cessation of algal growth was not observed during the experimental duration

despite consistent decline in algal Sp. Chl-a content (see Fig. 3c and d, respectively). It appears that algal growth cessation only occurs when algal Sp. Chl-a content falls to a very low value (ca. below 5 m g g1 algae) as observed during reactor operation at the highest, i.e., 347 mmol m2 s1 light intensity. Since Sp. Chl-a content did not decline so drastically during reactor operation at light intensities of 146 and 14 mmol m2 s1, algal growth cessation was not observed in these cases. However, since algal Sp. Chl-a content continued to decline consistently during these experiments (see Fig. 3c and d) it is probable that cessation of algal growth would have also occurred in these cases if the reactors were operated for a longer duration. Hence, it appears that algal photobioreactor operation under continuous illumination conditions is not sustainable in the long term in the range of light intensities employed in this study. However, as reported by some researchers (Aslan and Kapdan, 2006; Ogbonna et al., 2000; Wang et al., 2010), operation of algal reactors at continuous illumination conditions may well be possible at very low light intensities. Algal growth rates under such conditions are however likely to be severely limited by the lack of light availability. In general, sustainable algal growth under intermittent illumination conditions appears more likely, because any decline in Sp. Chl-a content during light periods can be partially or wholly restored during the intervening dark periods. Reactor operation at the highest light intensity of 347 mmol m2 s1 was however not

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sustainable even under intermittent illumination conditions, as seen by the decline in the algal growth rate from the fourth cycle onwards (see Fig. 4a). This is attributed to the fact that the loss in algal Sp. Chl-a content during the light periods could not be fully restored during the dark periods, resulting in a gradual decline of algal Sp. Chl-a content to levels which adversely impact algal growth (see Fig. 4b) and causes irreversible damage to the algal cells. However, similar decline in algal growth rate was not observed in the reactors operated at lower light intensities of 146 and 14 mmol m2 s1, respectively. This is attributable to the fact that algal Sp. Chl-a content in these reactors did not decline to unsustainable levels over the duration of reactor operation (see Fig. 4c and d). Reactor operation at a light intensity of 14 mmol m2 s1 under intermittent illumination conditions is clearly sustainable in the long-term, since the algal Sp. Chl-a content in such a reactor does not decline with continued reactor operation (see Fig. 4d). However, considering the decline in algal Sp. Chl-a content over the duration of reactor operation, similar conclusion cannot be readily drawn for reactor operation at a light intensity of 146 mmol m2 s1 (see Fig. 4c). In order to demonstrate the longterm sustainability of reactor operation at light intensities higher than 14 mmol m2 s1, reactors were successfully operated in the

semi-batch mode for 113 days at light intensities of 146 mmol m2 s1 and also 246 mmol m2 s1. In the latter case, Sp. Chl-a content of the algal biomass in the reactors was found to stabilize at
20 mg g1 when operated with a hydraulic retention time (HRT) of 12 h and under light intensity of 246 mmol m2 s1. It was thus apparent that reactors can be operated sustainably at light intensities up to 246 mmol m2 s1 under conditions of this study. This is in agreement with other studies where light inhibition of algal growth at intensities higher than 300 mmol m2 s1 was observed (Ogbonna and Tanaka, 2000; Ratchford and Fallowfield, 2003). 4.2. Addressing contradictions about nutrient uptake mechanisms by algae 4.2.1. Monod versus Droop Data presented in Figs. 5–7 clearly indicate that algal growth can continue even after the residual levels of N, or P, or both, have declined to very low levels in the bulk phase. This is probably because algae store the nutrients internally and hence may continue to grow using this internal store of nutrients even when nutrient is not available in the bulk phase. This clearly contradicts

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the basic assumptions of Monod formulations where growth directly depends on nutrient levels in the bulk media and lends further support to Droop’s formulation. Further, some researchers (Droop, 1968; Leadbeater, 2006) have postulated that algal cells have evolved this mechanism for storing nutrients inside the cell for future use to deal with the variable nutrient levels in the environment. Storage of excess P as polyphosphate inside the algal cell, and utilization from this internal quota for cell growth during photosynthesis has been reported by some researchers (Bolier et al., 1992; Powell et al., 2008). Others have designated the maximum amount of a nutrient that can be stored in an algal cell as the ‘cell quota’ for the nutrient. Algal growth is believed to occur through consumption of nutrients from this stored pool. The consequent reduction in nutrient stored in the algal cell is replenished through uptake of nutrients from the bulk phase (Droop, 1968, 1974; Nambiar and Bokil, 1981). The average N:P ratio in algal cells is reported to be 16:1 (Redfield, 1958). While the rate of nutrient uptake by algae is loosely governed by this ratio, the actual uptake rate at a particular point of time may vary widely depending on prevailing physiological condition of the algal cells (Klausmeier et al., 2004a, 2008). Fig. 8 compares the actual N and P uptakes by algae with their stoichiometric requirements. The wide variation in uptakes is observed and in some cases, N and P uptakes are clearly in excess of their stoichiometric requirements. Such flexibility in the rate of nutrient uptake allows algae to deal with environmental fluctuations more effectively (Diehl et al., 2005). The uptake rate of N and P also depends on the relative abundance of these nutrients in bulk medium (Wang and Lan 2011; Xin et al., 2010), variations between algae species (Boelee et al., 2011; Cai et al., 2013; Hulatt et al., 2012; Tam and Wong, 1989; Zhang et al.,

Actual Nitrogen Uptake mg N mg-1 algae

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4.2.2. Law of the minimum versus multiplicative rule Fig. 9 summarizes algal growth at various levels of nutrient availability with intermittent illumination. At the lower light intensity of 14 mmol m2 s1, there is no marked difference in algal growth irrespective of the prevalent levels of nutrient availability (see Fig. 9a) in various reactors. This suggests that algal growth was limited by the availability of light as explained by Liebig’s law of the minimum (Droop, 1974; Klausmeier et al., 2004b) which states that at any instant, algal growth is only limited by the factor that has the maximum negative impact on growth. Such data cannot be explained with multiplicative rule. At the higher light intensity of 146 mmol m2 s1 (Fig. 9b) there was no marked difference in algal growth among the reactors up to four days of reactor operation. Beyond this time, growth was clearly impaired in reactors having low N concentrations. It can thus be concluded that the growth in all four reactors was controlled by light availability for the first four days of reactor operation. Beyond that, the growth in the reactors

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2008) and other operating factors, e.g., temperature and light availability (Richmond, 1992). It has also been reported that P uptake by algae from the bulk phase is largely independent of immediate growth requirements and depends on both internal storage as well as nutrient availability in the bulk phase (Bolier et al., 1992; Lyon and Woo, 1980). Similar observations of N uptake have been made by other researchers (Ambrose et al., 2006; Chapra et al., 2007). Despite such support for cell quota model of algal growth and nutrient uptake, Monod formulations are still used because of their simplicity, but at a risk of wrong representation of the real system. Under nutrient limiting conditions, Monod’s formulation is likely to under predict algal growth.

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with high N (Experiments 5 and 7) continued to be controlled by light availability, while in the reactors with low N (Experiments 9 and 11), the growth was limited by availability of N. The progression of algae in the latter reactors to the N limited state can be explained as follows. First, N uptake by algae resulted in the bulk N concentration being reduced to the very low values by the second day of reactor operation. Next, from the second to the fourth day, algal growth was maintained by utilization of N stored in the algal cells. Finally, from the fourth day onwards, the N stored in the algal cells was depleted and could not be replenished due to absence of N in the bulk phase, resulting in the algae showing growth impairment due to N limitation. It is also interesting to note that in the reactor with low P but high N (Experiment 7), growth continued to be governed by light availability and no symptom of growth impairment due to P limitation was evident. Evidently, the stored P in the algal cells of this reactor was not sufficiently depleted during the reactor operation period and supported the algal growth without P limitation. In comparison, Experiment 8 resulted in lower algal growth despite higher P uptake (Fig. 8b). This is due to the growth being limited by light despite the availability of both N and P. These data further support the Liebig’s law of minimum. Similarly Droop (1974) also observed better representation of experimental data for multiple nutrient limitation with Liebig’s law of minimum rather than multiplicative rule (Chapra et al., 2007; Droop, 1974; Klausmeier et al., 2004b). The

multiplicative rule underestimates the growth in presence of multiple nutrients at comparable levels of limiting factors, while law of minimum provides more realistic representation of growth. An acceptable growth representation is achieved by multiplicative rule only when there is vast difference in the corresponding limiting factors of multiple nutrients present and converges to law of minimum estimates. 5. Conclusion The present study involved batch and semi-batch experiments concerning algal growth and associated processes in algal photobioreactors under various illumination strategies and under various levels of nutrient availability. Based on the analysis of the results, certain important conclusions regarding the impact of illumination strategy and nutrient availability on algal growth in photo-bioreactors were inferred. - Algal photosynthesis at high light intensities results in a decline

in the Sp. Chl-a content of algal cells. In the present study, decline of algal Sp. Chl-a content to very low levels (ca. less than
5 mg g1 algae) indicated irreversible damage to algal cells. Low algal Sp. Chl-a content (ca. less than
20 mg g1 algae) was a strong indicator of light-induced stress on algal cells, leading to impairment of algal growth. However algal chlorophyll content

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in such cases could be restored through the provision of dark period. - For the algal culture used in this study, any illumination strategy which maintains the average algal Sp. Chl-a levels at values greater than
20 mg g1 algae was deemed suitable for algal growth without photo-inhibition. Hence algal growth under continuous illumination conditions was thought to be infeasible except under very low light intensity. Algal growth under intermittent illumination conditions (alternate 12-h light–dark periods) was successfully demonstrated up to a light intensity of 246 mmol m2 s1. - Contradictions in literature regarding nutrient uptake mechanisms were also addressed. Continued algal growth even in the near absence of nutrients in the bulk phase was clearly demonstrated through experiments. This implies the capability of algae to internally store nutrients and that algal growth is often driven by this stored nutrient pool as described by Droop’s cell quota formulation. Hence, modeling of algal growth using the Monod formulation, which does not account for internal storage of nutrients, is clearly unwarranted. The necessity of incorporating internal cell quota and luxury uptake for N and P for realistic description of algal growth dynamics was established. - Based on the analysis of experimental data presented in this paper, it was established that rate limitation on algal growth can be best described by the Liebig’s law of the minimum, which suggests that at any instant, algal growth is only limited by the factor that has the maximum negative impact on growth, and not with multiplicative rule. Finally, based on the results of this study it was concluded that sustainable algal growth in a photo-bioreactor requires an illumination strategy such that the average Sp. Chl-a content of the algal cells is always above a threshold. Further, algal growth should be described using the Droop’s formulation for nutrient uptake and Liebig’s law of minimum for describing the effect of growth limiting factors. The insights gained from this study will help in more realistic modeling of algal growth and associated processes in photo-bioreactors. Acknowledgement The authors hereby acknowledge the Indian Institute of Technology Kanpur for providing financial assistance to carry out the present work. References Ai, W., Guo, S., Qin, L., Tang, Y., 2008. Development of a ground-based space microalgae photo-bioreactor. Adv. Space Res. 41, 742–747. Ambrose, R.B., Martin, J.L., Wool, T.A., 2006. WASP7 Benthic Algae – Model Theory and User’s Guide. Environmental Protection Agency, USA. Arceivala, S.J., Asolekar, S.R., 2007. Wastewater Treatment for Pollution Control and Reuse, 3rd ed. Tata McGraw-Hill, New Delhi. Aslan, S., Kapdan, I.K., 2006. Batch kinetics of nitrogen and phosphorus removal from synthetic wastewater by algae. Ecol. Eng. 28, 64–70. Béchet, Q., Shilton, A., Guieysse, B., 2013. Modeling the effects of light and temperature on algae growth: state of the art and critical assessment for productivity prediction during outdoor cultivation. Biotechnol. Adv. 31, 1648–1663. Boelee, N.C., Temmink, H., Janssen, M., Buisman, C.J.N., Wijffels, R.H., 2011. Nitrogen and phosphorus removal from municipal wastewater effluent using microalgal biofilms. Water Res. 45, 5925–5933. Bolier, G., Koningh, M.C.J., Schmale, J.C., Donze, M., 1992. Differential luxury phosphate response of planktonic algae to phosphorus removal. Hydrobiologia 243–244, 113–118. Bonente, G., Pippa, S., Castellano, S., Bassi, R., Ballottari, M., 2012. Acclimation of Chlamydomonas reinhardtii to different growth irradiances. J. Biol. Chem. 287, 5833–5847.

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