Bioresource Technology 174 (2014) 60–66
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Adaptability of growth and nutrient uptake potential of Chlorella sorokiniana with variable nutrient loading Amritanshu Shriwastav, Sanjay Kumar Gupta, Faiz Ahmad Ansari, Ismail Rawat, Faizal Bux ⇑ Institute for Water and Wastewater Technology, Durban University of Technology, PO Box 1334, Durban 4000, South Africa
h i g h l i g h t s C. sorokiniana can adapt N and P uptakes according to levels in external medium. It can maintain uniform growth rates and productivity despite variable uptakes. Evidences of stresses were observed on quantum efficiencies and chlorophyll. Increased nitrite excretion was observed with high nitrate levels in the feed.
a r t i c l e
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Article history: Received 29 July 2014 Received in revised form 29 September 2014 Accepted 30 September 2014 Available online 7 October 2014 Keywords: Chlorella sorokiniana Nutrient uptake potential N/P ratio Quantum efficiencies Biomass productivity
a b s t r a c t Chlorella sorokiniana can sustain growth in conditions hostile to other species, and possesses good nutrient removal and lipid accumulation potentials. However, the effects of variable nutrient levels (N and P) in wastewaters on growth, productivity, and nutrient uptake by C. sorokiniana have not been studied in detail. This study demonstrates the ability of this alga to sustain uniform growth and productivity, while regulating the relative nutrient uptake in accordance to their availability in the bulk medium. These results highlight the potential of C. sorokiniana as a suitable candidate for fulfilling the coupled objectives of nutrient removal and biomass production for bio-fuel with wastewaters having great variability in nutrient levels. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Anthropogenic activities often result in increased concentrations of nitrogen (N) and phosphorus (P) in natural water bodies. Accumulation of these nutrients is undesirable and may lead to eutrophication (Schindler et al., 2008). Effective removal of these nutrients from wastewater is complex and expensive using conventional methods. The ability of algae to take up these nutrients for growth has invited much interest in developing algal-based technologies for their removal (Cai et al., 2013). In addition, the potential of algae to accumulate lipids has led recent efforts to cultivate algae on nutrient-rich wastewaters and harvesting the biomass for biofuel production (Li et al., 2011). Nutrient concentrations vary substantially in wastewater; therefore an algal species should be able to effectively cope up with such variability in influent without any detrimental effects on its growth and productivity if used as an efficient treatment option.
⇑ Corresponding author. Tel.: +27 31 373 2346; fax: +27 31 373 2777. E-mail address: [email protected] (F. Bux). http://dx.doi.org/10.1016/j.biortech.2014.09.149 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
For example, the applicability of Chlorella vulgaris for removing nutrients from wastewaters with highly variable nutrient concentrations has been investigated by many researchers (Table 1). The variability in nutrient levels also affects the lipid content and overall lipid productivity of microalgae. Nutrient starvation has been reported to increase the lipid accumulation in the algal cells. Zhang et al. (2013) observed significant lipid accumulation in Chlorella sorokiniana during N-starvation periods. Similar findings in C. sorokiniana have also been reported with different N sources supplementing wastewater (Ramanna et al., 2014). Lipid productivity has been identified as the key factor in comparison to the lipid content for selecting algal species for the objective of lipid production (Griffiths and Harrison, 2009). They also observed lipid productivity to correlate well with biomass productivity rather than lipid content only. Hence, biomass productivity is an important parameter which governs the nutrient removal from the growth media as well as the lipid productivity for an algal species. This highlights the importance of selection of an algal species which can maintain its growth and productivity in order to achieve the cumulative objectives of nutrient removal from wastewater and lipid production for biofuel purposes.
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A. Shriwastav et al. / Bioresource Technology 174 (2014) 60–66 Table 1 Variability in nutrient levels of different wastewaters treated with Chlorella vulgaris. Type of wastewater
Influent N (mg L1)
Influent P (mg L1)
N/P (molar ratio)
Specific growth rate (day1)
N-removal (%)
P-removal (%)
References
Settled piggery wastewater Domestic wastewater Pre-treated piggery wastewater Municipal wastewater Enriched urban wastewater (PO4)3
341 47.04 512 25.5 405
419 7.36 57 2.8 9.7
1.80 14.15 19.89 20.17 92.45
0.264 N/A 0.08 0.641 0.72
N/A 36 2.75 95.9 N/A
N/A 61 8.84 94.4 58a
Travieso et al. (2006) Mahapatra et al. (2013) Ji et al. (2013) Ryu et al. (2014) Ruiz et al. (2011)
N/A: data not available. a Removal efficiency calculated from graph.
The ability of an algal species to thrive in conditions hostile to other species, whilst effectively removing nutrients and accumulating sufficient lipids for further application, potentially makes it suitable for these objectives to be achieved concurrently. C. sorokiniana has demonstrated good nutrient removal capability (Kim et al., 2013b; Ogbonna et al., 2000) as well as good lipid accumulation potential (Qiao and Wang, 2009; Zheng et al., 2013). In addition, de-Bashan et al. (2008) reported the ability of this thermo-tolerant alga to grow in wastewaters hostile to other algal species. Griffiths and Harrison (2009) have also compared the lipid productivity amongst 55 algal species and identified C. sorokiniana as a potential candidate for achieving higher lipid productivity. These properties make C. sorokiniana a potential candidate for sustainable wastewater treatment and biomass production for biofuel productions. Since, the growth and biomass productivity govern both nutrient removal and lipid productivity of an algal species, it is important to investigate the effects of variability in nutrient levels on these parameters. Though C. sorokiniana has been identified and reported earlier as a suitable candidate for achieving nutrient removal as well as lipid production, the effects of variability in nutrient levels of different wastewaters on the growth and productivity of C. sorokiniana, relative uptakes of nutrients, and the impacts on the physiological conditions have not been studied in detail (Kim et al., 2013a; Ramanna et al., 2014). The objective of this study was to investigate the effects of variability in nutrient levels on growth and nutrient uptake of C. sorokiniana as well as effects of cultivation conditions on the physiology of C. sorokiniana. The ability of this alga to sustain uniform growth and productivity while regulating the relative nutrient uptake in accordance to their availability is demonstrated. 2. Methods 2.1. Algae culture Water samples were collected from a pond in Durban region, KwaZulu-Natal, South Africa. These samples were used for isolating the pure strain of C. sorokiniana (genbank accession number: AB731602.1) by subsequent subculturing using the streak plate method (Ramanna et al., 2014). Stock culture was maintained in BG11 media with 16:8 h of light–dark cycle with Gro-Lux lamps at 80 lmol m2 s1. An orbital shaker with 80 rpm (OrbiShakeShaker, Labotec, South Africa) was used to maintain culture in turbulent conditions at 22 ± 2 °C. Culture was maintained under axenic conditions by routine subculturing and microscopic observation under Nikon eclipse 80i microscope (Nikon, USA). 2.2. Experimental details The growth and nutrient uptake potential of C. sorokiniana at different nitrogen and phosphorus levels was observed for 10 days using nutrient starved cells. Starved cells were used to avoid the
effects of internally stored nutrients on growth and uptake that would result in erroneous conclusions. To obtain nutrient starved cells, 20 mL of the stock culture was centrifuged at 2000g for 15 min (Heraeus multifuge 4KR, USA). The supernatant was discarded and 20 mL of the ultrapure water (Aqua MAX Ultra 370, Younglin, Korea) was added to the algal cell pellet in the same tube and cells were agitated using a vortex mixer (VM-300, Gemmy, Taiwan) for 2 min. Culture was again centrifuged and the supernatant was discarded. Similar washing with ultrapure water was repeated two more times, after which cells were re-suspended in 20 mL of ultrapure water. Algal cells from stock were again withdrawn and washed in similar manner, all these washed cells were pooled. The pooled cells were kept under constant illumination of 80 lmol m2 s1 for 3 days on an orbital shaker. These conditions resulted in nutrient starved cells which were then used for inoculation in the experiments (Kaya and Picard, 1995). The growth and nutrient uptake experiments were conducted at five different nutrient levels as presented in Table 2. Rationale behind these nutrient levels was to analyze the effects of variation in one nutrient (either N or P) while keeping other nutrient in sufficient concentrations so as to not be rate limiting. Since BG11 media is a high strength media, it was diluted with ultrapure water (1/200 BG11) to lower the inherent nitrate and phosphate levels. This diluted BG11 media was spiked with different concentrations of nitrate (as NaNO3) and phosphate (as K2HPO4) to achieve required nutrient (N and P) levels in each experiment for C. sorokiniana (Table 2). Availability of other micronutrients in the diluted media to support the algal growth for the experimental duration in the present study was established independently so that the growth of C. sorokiniana was controlled by N or P only in the experiments conducted. These modified BG11 media for each of the five sets of experiments were prepared, their pH adjusted to 7 with 0.1 M HCl/KOH, and then autoclaved. In each set of experiments, 1.48 L of corresponding autoclaved media was used in a 2 L flask (henceforth referred as reactor). These reactors were inoculated with 20 mL of nutrient starved C. sorokiniana cells. The total working volume in the reactors at the start of experiments was 1.5 L and initial dry weight biomass was 20 mg L1. Reactors were cotton plugged and continuously aerated using portable air pumps to maintain sufficient CO2 supply as well as mixing of the culture. All reactors were sampled before the start of the experiments for initial values of parameters, and then placed under constant illumination of 80 lmol m2 s1. These illumination conditions were maintained continuously for 6 days, so that the effects of variability in nutrient levels and any detrimental effect on the culture physiology could be observed in growth favouring conditions without intermittent dark phase. After 6 days of continuous light, reactors were further maintained in dark condition for next 4 days to analyze the culture behavior and the recovery potential from any stress experienced. Temperature in reactors was maintained at 22 ± 2 °C. 70 mL aliquots were withdrawn from each reactor daily and analyzed for various parameters.
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Table 2 Initial nutrient levels for batch experiments with C. sorokiniana.
a
Experimenta
1 NO ) 3 -N (mg L
1 NO ) 2 -N (mg L
1 PO3 ) 4 -P (mg L
N/P (molar ratio)
Nh-Ph Nm-Ph Nl-Ph Nh-Pm Nh-Pl
22.99 ± 0.10 7.31 ± 0.07 1.53 ± 0.26 24.59 ± 0.21 24.60 ± 0.29
0.15 ± 0.04 0.14 ± 0.10 0.42 ± 0.04 0.03 ± 0.01 0.01 ± 0.01
8.37 ± 0.15 8.42 ± 0.27 8.26 ± 0.13 2.23 ± 0.09 0.37 ± 0.00
6.12 ± 0.11: 1.96 ± 0.07: 0.52 ± 0.07: 24.43 ± 1.02: 146.22 ± 1.73:
1 1 1 1 1
Key to subscripts: h-high, m-medium, l-low.
2.3. Analytical methods
a
250
Dark
Light
Dark
Light
Dark
5 6 Days Nm-Ph
7
-1
Light
100
0 25
b -1
N - NO3 (mg L )
20
-
15 10 5 0 2.5
c
1.5
-
-1
2.0
1.0 0.5 0.0 10
d
6
-3
-1
8
4 2 0 0
1
2 Nh-Ph
3. Results and discussion
Dark
50
ð1Þ
where Fv is the variable fluorescence resulting due to maximum fluorescence Fm and minimum fluorescence Fo in a dark adapted sample. The pH was measured with a pH meter (Orion Dual Star, Thermo Scientific). The light intensity at the internal surface of the reactors was measured by a light meter (MT 940, Major Tech, South Africa). The nitrate, nitrite and orthophosphate in the sample were measured by Gallery™ Automated Photometric Analyzer (Thermo Scientific, USA). All measurements were done in triplicate.
Light
150
P - PO4 (mg L )
F v =F m ¼ ðF m F o Þ=F m
200
N - NO2 (mg L )
Dry weight biomass (mg L ) was measured gravimetrically using 10 mL of the sample, and specific growth rate l (day1) and biomass productivity Pr (mg L1 day1) were calculated. Chlorophyll-a (Chl-a, mg L1) was measured after extraction in 100% methanol (Porra et al., 1989). An aliquot of 5 mL of the sample was centrifuged at 2000g for 15 min and supernatant discarded. The pellet was suspended in 10 mL of boiling methanol at 65 °C and stored in dark for 24 h. Chl-a was thus extracted in methanol and the absorbance of methanol solution was measured at 652 nm, 665 nm, and 750 nm using a spectrophotometer (Spectroquant Pharo 300, Merck, Germany) with pure methanol as blank. Specific Chl-a (mg Chl-a g1 algae) was calculated after dividing Chl-a concentration by corresponding algal biomass. A Dual-PAM 100 Chlorophyll Fluorometer (Heinz WalzGmbh, Effeltrich, Germany) was used for non-invasive fluorescence measurements. The sample was dark adapted before measurements so as to have all Photo System-II (PS-II) reaction centers in the chlorophyll open. The quantum efficiency of PS-II charge separation (Fv/Fm) was calculated as per following equation (Ramanna et al., 2014):
Algae (mg L )
1
3
4
8
9
10
Nl-Ph
3.1. Effect of variable nutrient levels on algal growth
Fig. 1. Effects of variable nitrogen levels on the dynamics of C. sorokiniana growth. (a) Biomass, (b) bulk phase N-NO 3 concentration, (c) bulk phase N-NO2 concentration, (d) bulk phase P-PO3 4 concentration.
The effect of N-NO 3 concentration on algal growth was studied by comparing results of three experiments at different initial N-NO 3 levels, namely Nh-Ph, Nm-Ph, and Nl-Ph (Table 2). In the case of the high nitrogen level (Nh-Ph), growth continued over 6 days of the light period, and then declined in the dark period (Fig. 1a). For medium and low nitrogen levels (Nm-Ph, and Nl-Ph, respectively), growth continued for 5 and 4 days, respectively, during the light period. Thereafter, despite the availability of light, culture growth declined (Fig. 1a). Nitrate levels declined in the bulk phase due to uptake by C. sorokiniana for growth (Fig. 1b), and resulted in the depletion of N-NO 3 in the bulk phase at low and medium levels within one and 3 days, respectively, during the light period. These depletions at low and medium N-NO 3 levels occurred well before their corresponding growths ceased, which suggests that growth also depended on internally stored nitrogen (Droop, 1974). The growth of C. sorokiniana ceased when the cell pool was depleted.
It was observed that excessive nitrogen was stored in the cells due to luxury uptake of nitrate in the case of higher initial N-NO 3 (Nh-Ph) and was excreted as nitrite during the dark phase (Fig. 1c). P-PO3 4 uptake depended on the initial N-NO3 concentra tions (Fig. 1d) and was lowest at the low N-NO3 level (Nl-Ph) because algal growth was restricted by nitrogen limitation. Phosphorus uptake continued even after nitrogen was depleted from bulk medium, for example nitrogen depletion occurred within 1 or 3 days, respectively, for low (Nl-Ph) and medium (Nm-Ph) N-NO 3 concentrations, whereas phosphorus uptake continued for 4–8 days (Fig. 1b and d respectively). Other researchers have also observed luxury uptake of phosphorus (Powell et al., 2008). Marginal excretion of both nitrogen and phosphorus in the late dark phase is attributed to cell mineralization due to respiration in the dark.
A. Shriwastav et al. / Bioresource Technology 174 (2014) 60–66
Three independent experiments with different initial P-PO3 4 levels (Nh-Ph, Nh-Pm, and Nh-Pl; Table 2) were conducted to compare the effect of P-limitation on growth of C. sorokiniana. At all P-PO3 4 levels, the growth of algae continued for 6 days in the light period and no effect of P-limitation on the growth was observed at the selected concentrations (Fig. 2a). In the dark, there was a marginal decline in biomass due to respiration loss. Similarly, N-NO 3 uptake was comparable in all three cases, which suggests no P-limitation (Fig. 2b). Uptake of N-NO 3 in all cases (Nh-Ph, Nh-Pm, and Nh-Pl) resulted in substantial excretion of nitrite from the algal cells into the bulk medium, suggesting for luxury uptake in excess to their stoichiometric requirements (Fig. 2c). The stored nitrate in the cell was first reduced to nitrite with nitrate reductase enzyme, which was then reduced to ammonium by nitrite reductase enzyme to be used for cell synthesis. Any accumulation of nitrite in the cell due to imbalance of these two enzymatic activities resulted in nitrite excretion to the bulk medium (Flynn et al., 1997). Collos (1998) also observed release of nitrite in algal cultures. The uptake of P-PO3 4 by C. sorokiniana was governed by initial levels in the bulk phase, as at low and medium levels (Nh-Pl, and Nh-Pm) the uptake by algae depleted phosphorus in bulk medium within 1 or 2 days (Fig. 2d). At a high P-PO3 4 level (Nh-Ph), the uptake of phosphate continued for 6 days in the light phase. Marginal release of phosphorus was observed during late dark phase due to respirational loss of biomass.
a
200
Light
Dark
Light
Dark
-1
Algae (mg L )
250
150 100 50 0 30
-1
N - NO3 (mg L )
b
The effect of variable nutrient loading on growth and productivity of C. sorokiniana is presented in Table 3. The duration of the exponential growth phase in each experiment varied from 6 days, when nitrogen and phosphorus were both in high concentration (Nh-Ph), to 4 days when nitrogen was limiting (Nl-Ph). C. sorokiniana achieved similar growth rates during the exponential phase in all experiments (Table 3). This implied that the culture had the ability to sustain sufficiently uniform growth under diverse nutrient levels until one of the nutrients became rate limiting. In addition, the productivity of the algal biomass was also comparable at all nutrient levels. These growth rates and productivity values are consistent with those obtained by Samorì et al. (2013) at similar light intensities for Desmodesmus communis. Kim et al. (2013b) observed a growth rate of 0.24 day1 under continuous illumination of 60 lE m2 s1 for C. sorokiniana, which is also consistent with our data. Similarly, de-Bashan et al. (2008) reported growth rates ranging from 0.15 day1 to 0.30 day1 for C. sorokiniana at 28 °C and 60 lmol m2 s1. These results provide evidence of the ability of C. sorokiniana to adapt to variable nutrient loading and to maintain uniform growth and productivity. This ability of C. sorokiniana to maintain steady growth rates is possibly due to sufficient accumulation of N and P in the cell even at lower levels to assist growth. The specific nutrient uptake rates during exponential growth of C. sorokiniana are expressed in Table 3. The lowest N-uptake rate was observed for low N loading (Nl-Ph) as 4.91 ± 1.24 mg N g1 algae day1 while the lowest P-uptake rate was 0.61 ± 0.03 mg P g1 algae day1 for Nh-Pl. Total nutrient accumulation in C. sorokiniana biomass during exponential growth is presented in Fig. 3. As expected, the highest and lowest accumulations of both N and P were observed when their respective levels in the bulk phase were high or low. Even the lowest accumulations of N (19.63 ± 4.97 mg N g1 algae for Nl-Ph) and P (3.03 ± 0.14 mg P g1 algae for Nh-Pl) were in excess of their minimum contents required below which the algal growth ceases, i.e. 7.2 mg N g1 algae and 1 mg P g1 algae (Ambrose et al., 2006; Chapra et al., 2007). In addition, Droop (1974) identified that algal growth rates are governed by both internal and external cellular nutrient levels. Despite these levels varying, C. sorokiniana maintained uniform growth during exponential phase until such internal pools were exhausted causing growth to cease.
-
20
63
10
3.2. Effect of variable nutrient levels on culture physiology
0 3 -1
N - NO2 (mg L )
c
Light
Dark
Light
Dark
5 6 Days Nh-Pm
7
-
2
0 10 8 6
-3
-1
P - PO4 (mg L )
d
1
4 2 0 0
1
2 Nh-Ph
3
4
8
9
10
Nh-Pl
Fig. 2. Effects of variable phosphorus levels on the dynamics of C. sorokiniana growth. (a) Biomass, (b) bulk phase N-NO 3 concentration, (c) bulk phase N-NO2 concentration, (d) bulk phase P-PO3 4 concentration.
The maximum quantum efficiency (Fv/Fm) of reaction centers in PS-II of chlorophyll represents the physiological state of the culture and the nutrient stresses (White et al., 2011). A Fv/Fm value of less than 0.5 implies the culture is in a stressed state; and this value declines with increasing stress level of the culture (Ramanna et al., 2014). These quantum efficiencies were calculated for all five nutrient levels throughout the course of the experiments (Fig. 4a). Since all experiments were conducted with nutrient-starved cells to avoid the effect of internally stored nutrients on growth and uptake of nutrients, these cells were in a stressed condition based on the evidence of the initial Fv/Fm value for each experiment being less than 0.3. With the addition of nutrients in each experiment, the nutrient stress in the culture was reduced as evident by the increasing quantum efficiencies in the early light periods. Increase in Fv/Fm values was observed for 2 days only in cultures with the lowest initial nitrogen input (experiment Nl-Ph), after which these efficiencies declined to very low levels. This indicates that as the nitrogen became limiting, the culture became nutrient stressed. Nutrient stress affected the culture physiology and effects were visible in terms of low growth of C. sorokiniana (Fig. 1a). At other nutrient levels, quantum efficiencies increased for 3 days. For medium nitrogen levels (Nm-Ph), nutrient stress was evident after 3 days whereby Fv/Fm started to decline appreciably. At higher
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Table 3 Specific growth rates, biomass productivities, and specific nutrient uptake rates of C. sorokiniana at different nutrient levels. Experiment Nh-Ph Nm-Ph Nl-Ph Nh-Pm Nh-Pl
Exponential growth phase (day)
l (day1)a
Pr (mg L1 day1)b
Specific N uptake rate (mg N g1 algae day1)c
Specific P uptake rate (mg P g1 algae day1)c
0–6 0–5 0–4 0–5 0–5
0.35 ± 0.07 0.35 ± 0.04 0.33 ± 0.06 0.28 ± 0.07 0.29 ± 0.01
28.25 ± 5.70 24.00 ± 2.83 21.25 ± 14.97 23.30 ± 9.19 24.60 ± 5.83
16.63 ± 0.57 12.09 ± 0.35 4.91 ± 1.24 29.29 ± 2.37 26.74 ± 1.40
4.88 ± 0.37 6.70 ± 0.63 5.12 ± 0.98 3.83 ± 0.34 0.61 ± 0.03
a
l is the growth rate in exponential growth phase.
b
Pr is the productivity at the end of exponential growth. Specific nutrient uptake rates are calculated for exponential growth phase.
c
0.8
180
a
a
140
0.6
Light
Dark
Light
Dark
Light
Dark
120
Fv / Fm
Total Accumulated N -1 (mg N g algae)
160
100 80
0.4
60
0.2
40 20
0.0
0 Nh-Ph
Nm-Ph
Nl-Ph
Nh-Pm
Nh-Pl
b 30
20
4
2
10
0 0 Nh-Ph
Nm-Ph
Nl-Ph
Nh-Pm
Nh-Pl
Fig. 3. Effect of variable nutrient loading on total nutrient accumulation during exponential growth phase by C. sorokiniana. (a) N-accumulation, (b) Paccumulation.
nitrogen levels (Nh-Ph, Nh-Pm, and Nh-Pl), the quantum efficiencies reached values above 0.5 after 3 days and remained sufficiently high for the duration of the light period. The results indicated a healthier physiological state of C. sorokiniana during these nutrient levels. This was also evident with the growth data of these experiments (Fig. 2a). Quantum efficiencies in all experiments declined during the latter part of the continued dark period, which suggested the stressed state of the culture in the absence of the light. Chl-a content was also monitored to gain insight into the physiological status of the algal cells since stress due to nutrient limitation are also reflected in Chl-a content. For example, Zhang et al. (2013) observed a marked reduction in the Chl-a content of N-starved cells of C. sorokiniana. Fig. 4b presents the evolution of Chl-a under different nutrient loading. At all levels of initial P-PO3 with high N-NO 4 3 (Nh-Ph, Nh-Pm, and Nh-Pl), the Chl-a concentration increased during the light period (Fig. 4b). In comparison, there was a marked reduction in Chl-a at low initial N-NO 3 levels. At medium N-NO3 levels, Chl-a reached a plateau by the fourth day and then declined. This is in agreement with growth data of the same experiment. Specific Chl-a content of the cell declined at medium and low nitrogen levels, suggesting a stressed culture due to N-limitation (Fig. 4c). In contrast, cells retained their Chl-a content at all phosphorus levels, which implied no growth limitation at these levels. These results agree with growth data where the limiting effects of phosphorus were
Specific Chlorophyll-a Content -1 (mg Chl-a g Algae)
Total Accumulated P -1 (mg P g algae)
b
6
-1
Chlorophyll-a (mg L )
40
c
50 40 30 20 10 0 0
1
2
3
Nh-Ph Nh-Pm
4
5 Days Nm-Ph
6
7
8
9
10
Nl-Ph
Nh-Pl
Fig. 4. Effects of variable nutrient loading on the culture physiology of C. sorokiniana growth. (a) Quantum efficiency of PS-II reaction centers, (b) chlorophyll-a, (c) specific chl-a content.
not observed. Hill and Fanta (2009) also did not observe growth limitation and reduction in Chl-a content of a mixed algal culture at 0.3 mg L1 dissolved phosphorus. At low N levels (experiment Nl-Ph) a marked reduction in the Chl-a content was observed, while the algal growth continued till day four. With the low N levels, C. sorokiniana cells experienced visible nutrient stress after day two (Fig. 4a). With the onset of such stress, the cells of C. sorokiniana regulate their photosynthesis by varying Chl-a content as a mechanism to counter these stresses (Zhang et al., 2013). However, with increasing stress levels, the Chl-a were irreversibly damaged and thus reduced (Fig. 4b). As the Chl-a concentration decreased to low levels, photosynthesis and hence the formation of new cells was affected. This resulted in the decline of algal growth after day four (Fig. 1a).
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3.3. Effect of variable nutrient levels on relative uptake potential of N and P The average molar ratio of N and P in algae is assumed to follow the Redfield ratio of 16N:1P (Redfield, 1958), though many studies have found the evidence of deviations from this ratio (Hulatt et al., 2012; Klausmeier et al., 2008). The initial nutrient levels in all experiments varied thus providing different N/P molar ratio in each case. Experiment Nl-Ph had the lowest initial N/P ratio of 0.52 ± 0.07: 1, while the highest initial N/P of 146.22 ± 1.73: 1 was observed for Nh-Pl (Table 2). The nutrient content in the algal cells mainly depends on its uptake from the medium. Therefore, the relative uptake was calculated in all experiments for the light period of 6 days. The lowest N/P molar ratio of uptake was observed as 2.12 ± 0.51: 1 for Nl-Ph, while the highest N/P of such uptake was calculated as 106.05 ± 1.74: 1 for Nh-Pl. These uptakes followed the initial ratio of nutrients in the medium for other experiments also (Fig. 5). The dependence of nutrient uptakes on their external concentration was also demonstrated for Neochloris oleoabundans (Wang and Lan, 2011). Similar trends of flexible nutrient uptakes as per the N/P in surrounding medium have also been observed for Scenedesmus sp. (Xin et al., 2010). This suggests that C. sorokiniana has the ability to regulate its nutrient uptake potential in accordance to the external nutrient levels, while maintaining uniform growth and productivity till either N or P becomes rate limiting. The effects of such variable uptakes on the nutrient removal efficiency are presented in Fig. 6. 73.07 ± 0.61% N and 59.27 ± 4.12% P were removed at high nitrogen and phosphorus levels (experiment Nh-Ph). As the initial N-NO 3 concentration was reduced with high P-PO3 levels, the N-removal efficiency 4 increased (97.11 ± 2.65% for Nm-Ph and 83.57 ± 17.55% for Nl-Ph). The decline in the efficiency for Nl-Ph was due to very high initial
160 Initial nutrient loading Relative uptake by algae
N/P Molar Ratio
140 120 100 30 20 10 0
Nh-Ph
Nm-Ph
Nl-Ph
Nh-Pm
Nh-Pl
Fig. 5. Dependence of relative uptake of N and P by C. sorokiniana on their initial levels in the medium.
% N Removal
100
a
80 60 40 20 0 Nh-Ph
Nm-Ph
Nl-Ph
Nh-Pm
Nh-Pl
Nh-Ph
Nm-Ph
Nl-Ph
Nh-Pm
Nh-Pl
120 100 % P Removal
The effect of nutrient starvation and the induced physiological stresses on lipid production and accumulation in algal cells is another important aspect. The accumulated nutrient levels during exponential growth varied with different cultivation conditions (Fig. 3). The variability in nutrient accumulations resulted in cells experiencing different nutrient stresses. The cultivation of C. sorokiniana at low and medium N levels (Nl-Ph and Nm-Ph respectively) resulted in increased stress levels in the cells (Fig. 4a) in comparison to other conditions. Many researchers have investigated the role of stresses on the lipid accumulation potential of various algal species and established the increase in lipid content with such stresses (Xin et al., 2010; Zhang et al., 2013), since algae regulate their utilization of assimilated carbon to synthesize lipids rather than glucose as a future reserve under stressed conditions (Ramanna et al., 2014; Xin et al., 2010). In similar manner, these stresses in this study also would invariably increase the lipid accumulation in the cells as observed for C. sorokiniana by many other researchers (Ramanna et al., 2014; Zhang et al., 2013). This suggests for higher lipid accumulations in the stressed cells. In addition, the productivity and growth rates were uniformly maintained across all experimental conditions during exponential growth (Table 3). Hence, the lipid productivity from these stressed cells would be higher, since the lipid productivity was found to depend on biomass productivity (Griffiths and Harrison, 2009). However, Griffiths and Harrison (2009) reported C. sorokiniana to still accumulate sufficient lipids (18% of dry weight) during nutrient replete cultivation. Hence, sufficiently high lipid productivity could still be achieved with the nutrient replete cultivation in addition to the nutrient limited cultivation conditions of this study. These results support the role of C. sorokiniana as a suitable candidate for nutrient removal and production of lipids under highly variable cultivation conditions.
b
80 60 40 20 0
Fig. 6. Effect of variable nutrient loading on nutrient removal efficiency of C. sorokiniana. (a) N-removal efficiency, (b) P-removal efficiency.
N/P molar ratio (146.22:1). Similar decline in nitrogen removal efficiency was also observed for Scenedesmus sp. when N/P molar ratio exceeded 20:1 (Xin et al., 2010). The P-removal efficiency declined to 54.01 ± 4.64% and 20.62 ± 3.75% for Nm-Ph and Nl-Ph, respectively. Similarly, when initial phosphorus levels were lowered while maintaining high nitrogen concentration, 91.76 ± 5.63% P-removal was achieved for Nh-Pm and complete P-removal for Nh-Pl, with marginal decline in N-removal efficiencies (75.35 ± 1.08% for Nh-Pm and 72.53 ± 1.47% for Nh-Pl). Since C. sorokiniana regulated the relative uptake of nitrogen and phosphorus in accordance to their levels in the surrounding media, these regulations affected the treatment efficiencies as observed in these experiments. 4. Conclusion This study demonstrated the ability of C. sorokiniana to regulate its nutrient (N and P) uptake in accordance with the available nutrient levels in the surroundings without any detrimental effect on growth or productivity until either nutrient became rate limiting. This flexibility, in addition to the ability of sustaining hostile growth conditions better than most of other algal species, makes C. sorokiniana a suitable candidate for fulfilling the coupled objectives of nutrient removal and biomass production for biofuel with great variability in nutrient levels of wastewaters efficiently.
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Acknowledgement The authors hereby acknowledge the National Research Foundation and Durban University of Technology for providing financial assistance.
References Ambrose, R.B., Martin, J.L., Wool, T.A., 2006. WASP7 Benthic Algae – Model Theory and User’s Guide. Environmental Protection Agency, USA. Cai, T., Park, S.Y., Li, Y., 2013. Nutrient recovery from wastewater streams by microalgae: status and prospects. Renew. Sust. Energy Rev. 19, 360–369. Chapra, S.C., Pelletier, G.J., Tao, H., 2007. QUAL2K: a modeling framework for simulating river and stream water quality, version 2.07. Environmental Protection Agency, USA. Collos, Y., 1998. Nitrate uptake, nitrite release and uptake, and new production estimates. Mar. Ecol. Prog. Ser. 171, 293–301. de-Bashan, L.E., Trejo, A., Huss, V.A.R., Hernandez, J.-P., Bashan, Y., 2008. Chlorella sorokiniana UTEX 2805, a heat and intense sunlight-tolerant microalga with potential for removing ammonium from wastewater. Bioresour. Technol. 99 (11), 4980–4989. Droop, M.R., 1974. The nutrient status of algal cells in continuous culture. J. Mar. Biol. Assoc. U.K. 54 (04), 825–855. Flynn, K.J., Fasham, M.J.R., Hipkin, C.R., 1997. Modelling the interactions between ammonium and nitrate uptake in marine phytoplankton. Philos. Trans. R. Soc. B 352 (1361), 1625–1645. Griffiths, M., Harrison, S.L., 2009. Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J. Appl. Phycol. 21 (5), 493–507. Hill, W.R., Fanta, S.E., 2009. Quantifying phosphorus and light effects in stream algae. Limnol. Oceanogr. 54 (1), 368–380. Hulatt, C., Lakaniemi, A.-M., Puhakka, J., Thomas, D., 2012. Energy demands of nitrogen supply in mass cultivation of two commercially important microalgal species, Chlorella vulgaris and Dunaliella tertiolecta. Bioenergy Res. 5 (3), 669– 684. Ji, M.-K., Kim, H.-C., Sapireddy, V., Yun, H.-S., Abou-Shanab, R.I., Choi, J., Lee, W., Timmes, T., Inamuddin, Jeon, B.-H., 2013. Simultaneous nutrient removal and lipid production from pretreated piggery wastewater by Chlorella vulgaris YSW04. Appl. Microbiol. Biotechnol. 97 (6), 2701–2710. Kaya, V.M., Picard, G., 1995. The viability of Scenedesmus bicellularis cells immobilized on alginate screens following nutrient starvation in air at 100% relative humidity. Biotechnol. Bioeng. 46 (5), 459–464. Kim, S., Lee, Y., Hwang, S.-J., 2013a. Removal of nitrogen and phosphorus by Chlorella sorokiniana cultured heterotrophically in ammonia and nitrate. Int. Biodeterior. Biodegrad. 85, 511–516. Kim, S., Park, J.-E., Cho, Y.-B., Hwang, S.-J., 2013b. Growth rate, organic carbon and nutrient removal rates of Chlorella sorokiniana in autotrophic, heterotrophic and mixotrophic conditions. Bioresour. Technol. 144, 8–13. Klausmeier, C.A., Litchman, E., Daufresne, T., Levin, S.A., 2008. Phytoplankton stoichiometry. Ecol. Res. 23 (3), 479–485. Li, Y., Zhou, W., Hu, B., Min, M., Chen, P., Ruan, R.R., 2011. Integration of algae cultivation as biodiesel production feedstock with municipal wastewater treatment: strains screening and significance evaluation of environmental factors. Bioresour. Technol. 102 (23), 10861–10867.
Mahapatra, D., Chanakya, H.N., Ramachandra, T.V., 2013. Treatment efficacy of algae-based sewage treatment plants. Environ. Monit. Assess. 185 (9), 7145– 7164. Ogbonna, J.C., Yoshizawa, H., Tanaka, H., 2000. Treatment of high strength organic wastewater by a mixed culture of photosynthetic microorganisms. J. Appl. Phycol. 12 (3–5), 277–284. Porra, R.J., Thompson, W.A., Kriedemann, P.E., 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. BBA Bioenergetics 975 (3), 384–394. Powell, N., Shilton, A.N., Pratt, S., Chisti, Y., 2008. Factors influencing luxury uptake of phosphorus by microalgae in waste stabilization ponds. Environ. Sci. Technol. 42 (16), 5958–5962. Qiao, H., Wang, G., 2009. Effect of carbon source on growth and lipid accumulation in Chlorella sorokiniana GXNN01. Chin. J. Oceanol. Limnol. 27 (4), 762–768. Ramanna, L., Guldhe, A., Rawat, I., Bux, F., 2014. The optimization of biomass and lipid yields of Chlorella sorokiniana when using wastewater supplemented with different nitrogen sources. Bioresour. Technol. 168, 127–135. Redfield, A.C., 1958. The biological control of chemical factors in the environment. Am. Sci. 46 (3), 230A (pp. 205–221). Ruiz, J., Ãlvarez, P., Arbib, Z., Garrido, C., Barragán, J., Perales, J.A., 2011. Effect of nitrogen and phosphorus concentration on their removal kinetic in treated urban wastewater by Chlorella vulgaris. Int. J. Phytorem. 13 (9), 884–896. Ryu, B.-G., Kim, E., Kim, H.-S., Kim, J., Choi, Y.-E., Yang, J.-W., 2014. Simultaneous treatment of municipal wastewater and biodiesel production by cultivation of Chlorella vulgaris with indigenous wastewater bacteria. Biotechnol. Bioprocess Eng. 19 (2), 201–210. Samorì, G., Samorì, C., Guerrini, F., Pistocchi, R., 2013. Growth and nitrogen removal capacity of Desmodesmus communis and of a natural microalgae consortium in a batch culture system in view of urban wastewater treatment: Part I. Water Res. 47 (2), 791–801. Schindler, D.W., Hecky, R.E., Findlay, D.L., Stainton, M.P., Parker, B.R., Paterson, M.J., Beaty, K.G., Lyng, M., Kasian, S.E.M., 2008. Eutrophication of lakes cannot be controlled by reducing nitrogen input: results of a 37-year whole-ecosystem experiment. Proc. Natl. Acad. Sci. U.S.A. 105 (32), 11254–11258. Travieso, L., Benítez, F., Sánchez, E., Borja, R., Martín, A., Colmenarejo, M.F., 2006. Batch mixed culture of Chlorella vulgaris using settled and diluted piggery waste. Ecol. Eng. 28 (2), 158–165. Wang, B., Lan, C.Q., 2011. Biomass production and nitrogen and phosphorus removal by the green alga Neochloris oleoabundans in simulated wastewater and secondary municipal wastewater effluent. Bioresour. Technol. 102 (10), 5639– 5644. White, S., Anandraj, A., Bux, F., 2011. PAM fluorometry as a tool to assess microalgal nutrient stress and monitor cellular neutral lipids. Bioresour. Technol. 102 (2), 1675–1682. Xin, L., Hong-ying, H., Ke, G., Ying-xue, S., 2010. Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp. Bioresour. Technol. 101 (14), 5494–5500. Zhang, Y.-M., Chen, H., He, C.-L., Wang, Q., 2013. Nitrogen starvation induced oxidative stress in an oil-producing green alga Chlorella sorokiniana C3. PLoS One 8 (7), e69225. Zheng, Y., Li, T., Yu, X., Bates, P.D., Dong, T., Chen, S., 2013. High-density fed-batch culture of a thermotolerant microalga Chlorella sorokiniana for biofuel production. Appl. Energy 108, 281–287.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Adaptability of growth and nutrient uptake potential of Chlorella sorokiniana with variable nutrient loading Amritanshu Shriwastav, Sanjay Kumar Gupta, Faiz Ahmad Ansari, Ismail Rawat, Faizal Bux ⇑ Institute for Water and Wastewater Technology, Durban University of Technology, PO Box 1334, Durban 4000, South Africa
h i g h l i g h t s C. sorokiniana can adapt N and P uptakes according to levels in external medium. It can maintain uniform growth rates and productivity despite variable uptakes. Evidences of stresses were observed on quantum efficiencies and chlorophyll. Increased nitrite excretion was observed with high nitrate levels in the feed.
a r t i c l e
i n f o
Article history: Received 29 July 2014 Received in revised form 29 September 2014 Accepted 30 September 2014 Available online 7 October 2014 Keywords: Chlorella sorokiniana Nutrient uptake potential N/P ratio Quantum efficiencies Biomass productivity
a b s t r a c t Chlorella sorokiniana can sustain growth in conditions hostile to other species, and possesses good nutrient removal and lipid accumulation potentials. However, the effects of variable nutrient levels (N and P) in wastewaters on growth, productivity, and nutrient uptake by C. sorokiniana have not been studied in detail. This study demonstrates the ability of this alga to sustain uniform growth and productivity, while regulating the relative nutrient uptake in accordance to their availability in the bulk medium. These results highlight the potential of C. sorokiniana as a suitable candidate for fulfilling the coupled objectives of nutrient removal and biomass production for bio-fuel with wastewaters having great variability in nutrient levels. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Anthropogenic activities often result in increased concentrations of nitrogen (N) and phosphorus (P) in natural water bodies. Accumulation of these nutrients is undesirable and may lead to eutrophication (Schindler et al., 2008). Effective removal of these nutrients from wastewater is complex and expensive using conventional methods. The ability of algae to take up these nutrients for growth has invited much interest in developing algal-based technologies for their removal (Cai et al., 2013). In addition, the potential of algae to accumulate lipids has led recent efforts to cultivate algae on nutrient-rich wastewaters and harvesting the biomass for biofuel production (Li et al., 2011). Nutrient concentrations vary substantially in wastewater; therefore an algal species should be able to effectively cope up with such variability in influent without any detrimental effects on its growth and productivity if used as an efficient treatment option.
⇑ Corresponding author. Tel.: +27 31 373 2346; fax: +27 31 373 2777. E-mail address: [email protected] (F. Bux). http://dx.doi.org/10.1016/j.biortech.2014.09.149 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
For example, the applicability of Chlorella vulgaris for removing nutrients from wastewaters with highly variable nutrient concentrations has been investigated by many researchers (Table 1). The variability in nutrient levels also affects the lipid content and overall lipid productivity of microalgae. Nutrient starvation has been reported to increase the lipid accumulation in the algal cells. Zhang et al. (2013) observed significant lipid accumulation in Chlorella sorokiniana during N-starvation periods. Similar findings in C. sorokiniana have also been reported with different N sources supplementing wastewater (Ramanna et al., 2014). Lipid productivity has been identified as the key factor in comparison to the lipid content for selecting algal species for the objective of lipid production (Griffiths and Harrison, 2009). They also observed lipid productivity to correlate well with biomass productivity rather than lipid content only. Hence, biomass productivity is an important parameter which governs the nutrient removal from the growth media as well as the lipid productivity for an algal species. This highlights the importance of selection of an algal species which can maintain its growth and productivity in order to achieve the cumulative objectives of nutrient removal from wastewater and lipid production for biofuel purposes.
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A. Shriwastav et al. / Bioresource Technology 174 (2014) 60–66 Table 1 Variability in nutrient levels of different wastewaters treated with Chlorella vulgaris. Type of wastewater
Influent N (mg L1)
Influent P (mg L1)
N/P (molar ratio)
Specific growth rate (day1)
N-removal (%)
P-removal (%)
References
Settled piggery wastewater Domestic wastewater Pre-treated piggery wastewater Municipal wastewater Enriched urban wastewater (PO4)3
341 47.04 512 25.5 405
419 7.36 57 2.8 9.7
1.80 14.15 19.89 20.17 92.45
0.264 N/A 0.08 0.641 0.72
N/A 36 2.75 95.9 N/A
N/A 61 8.84 94.4 58a
Travieso et al. (2006) Mahapatra et al. (2013) Ji et al. (2013) Ryu et al. (2014) Ruiz et al. (2011)
N/A: data not available. a Removal efficiency calculated from graph.
The ability of an algal species to thrive in conditions hostile to other species, whilst effectively removing nutrients and accumulating sufficient lipids for further application, potentially makes it suitable for these objectives to be achieved concurrently. C. sorokiniana has demonstrated good nutrient removal capability (Kim et al., 2013b; Ogbonna et al., 2000) as well as good lipid accumulation potential (Qiao and Wang, 2009; Zheng et al., 2013). In addition, de-Bashan et al. (2008) reported the ability of this thermo-tolerant alga to grow in wastewaters hostile to other algal species. Griffiths and Harrison (2009) have also compared the lipid productivity amongst 55 algal species and identified C. sorokiniana as a potential candidate for achieving higher lipid productivity. These properties make C. sorokiniana a potential candidate for sustainable wastewater treatment and biomass production for biofuel productions. Since, the growth and biomass productivity govern both nutrient removal and lipid productivity of an algal species, it is important to investigate the effects of variability in nutrient levels on these parameters. Though C. sorokiniana has been identified and reported earlier as a suitable candidate for achieving nutrient removal as well as lipid production, the effects of variability in nutrient levels of different wastewaters on the growth and productivity of C. sorokiniana, relative uptakes of nutrients, and the impacts on the physiological conditions have not been studied in detail (Kim et al., 2013a; Ramanna et al., 2014). The objective of this study was to investigate the effects of variability in nutrient levels on growth and nutrient uptake of C. sorokiniana as well as effects of cultivation conditions on the physiology of C. sorokiniana. The ability of this alga to sustain uniform growth and productivity while regulating the relative nutrient uptake in accordance to their availability is demonstrated. 2. Methods 2.1. Algae culture Water samples were collected from a pond in Durban region, KwaZulu-Natal, South Africa. These samples were used for isolating the pure strain of C. sorokiniana (genbank accession number: AB731602.1) by subsequent subculturing using the streak plate method (Ramanna et al., 2014). Stock culture was maintained in BG11 media with 16:8 h of light–dark cycle with Gro-Lux lamps at 80 lmol m2 s1. An orbital shaker with 80 rpm (OrbiShakeShaker, Labotec, South Africa) was used to maintain culture in turbulent conditions at 22 ± 2 °C. Culture was maintained under axenic conditions by routine subculturing and microscopic observation under Nikon eclipse 80i microscope (Nikon, USA). 2.2. Experimental details The growth and nutrient uptake potential of C. sorokiniana at different nitrogen and phosphorus levels was observed for 10 days using nutrient starved cells. Starved cells were used to avoid the
effects of internally stored nutrients on growth and uptake that would result in erroneous conclusions. To obtain nutrient starved cells, 20 mL of the stock culture was centrifuged at 2000g for 15 min (Heraeus multifuge 4KR, USA). The supernatant was discarded and 20 mL of the ultrapure water (Aqua MAX Ultra 370, Younglin, Korea) was added to the algal cell pellet in the same tube and cells were agitated using a vortex mixer (VM-300, Gemmy, Taiwan) for 2 min. Culture was again centrifuged and the supernatant was discarded. Similar washing with ultrapure water was repeated two more times, after which cells were re-suspended in 20 mL of ultrapure water. Algal cells from stock were again withdrawn and washed in similar manner, all these washed cells were pooled. The pooled cells were kept under constant illumination of 80 lmol m2 s1 for 3 days on an orbital shaker. These conditions resulted in nutrient starved cells which were then used for inoculation in the experiments (Kaya and Picard, 1995). The growth and nutrient uptake experiments were conducted at five different nutrient levels as presented in Table 2. Rationale behind these nutrient levels was to analyze the effects of variation in one nutrient (either N or P) while keeping other nutrient in sufficient concentrations so as to not be rate limiting. Since BG11 media is a high strength media, it was diluted with ultrapure water (1/200 BG11) to lower the inherent nitrate and phosphate levels. This diluted BG11 media was spiked with different concentrations of nitrate (as NaNO3) and phosphate (as K2HPO4) to achieve required nutrient (N and P) levels in each experiment for C. sorokiniana (Table 2). Availability of other micronutrients in the diluted media to support the algal growth for the experimental duration in the present study was established independently so that the growth of C. sorokiniana was controlled by N or P only in the experiments conducted. These modified BG11 media for each of the five sets of experiments were prepared, their pH adjusted to 7 with 0.1 M HCl/KOH, and then autoclaved. In each set of experiments, 1.48 L of corresponding autoclaved media was used in a 2 L flask (henceforth referred as reactor). These reactors were inoculated with 20 mL of nutrient starved C. sorokiniana cells. The total working volume in the reactors at the start of experiments was 1.5 L and initial dry weight biomass was 20 mg L1. Reactors were cotton plugged and continuously aerated using portable air pumps to maintain sufficient CO2 supply as well as mixing of the culture. All reactors were sampled before the start of the experiments for initial values of parameters, and then placed under constant illumination of 80 lmol m2 s1. These illumination conditions were maintained continuously for 6 days, so that the effects of variability in nutrient levels and any detrimental effect on the culture physiology could be observed in growth favouring conditions without intermittent dark phase. After 6 days of continuous light, reactors were further maintained in dark condition for next 4 days to analyze the culture behavior and the recovery potential from any stress experienced. Temperature in reactors was maintained at 22 ± 2 °C. 70 mL aliquots were withdrawn from each reactor daily and analyzed for various parameters.
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Table 2 Initial nutrient levels for batch experiments with C. sorokiniana.
a
Experimenta
1 NO ) 3 -N (mg L
1 NO ) 2 -N (mg L
1 PO3 ) 4 -P (mg L
N/P (molar ratio)
Nh-Ph Nm-Ph Nl-Ph Nh-Pm Nh-Pl
22.99 ± 0.10 7.31 ± 0.07 1.53 ± 0.26 24.59 ± 0.21 24.60 ± 0.29
0.15 ± 0.04 0.14 ± 0.10 0.42 ± 0.04 0.03 ± 0.01 0.01 ± 0.01
8.37 ± 0.15 8.42 ± 0.27 8.26 ± 0.13 2.23 ± 0.09 0.37 ± 0.00
6.12 ± 0.11: 1.96 ± 0.07: 0.52 ± 0.07: 24.43 ± 1.02: 146.22 ± 1.73:
1 1 1 1 1
Key to subscripts: h-high, m-medium, l-low.
2.3. Analytical methods
a
250
Dark
Light
Dark
Light
Dark
5 6 Days Nm-Ph
7
-1
Light
100
0 25
b -1
N - NO3 (mg L )
20
-
15 10 5 0 2.5
c
1.5
-
-1
2.0
1.0 0.5 0.0 10
d
6
-3
-1
8
4 2 0 0
1
2 Nh-Ph
3. Results and discussion
Dark
50
ð1Þ
where Fv is the variable fluorescence resulting due to maximum fluorescence Fm and minimum fluorescence Fo in a dark adapted sample. The pH was measured with a pH meter (Orion Dual Star, Thermo Scientific). The light intensity at the internal surface of the reactors was measured by a light meter (MT 940, Major Tech, South Africa). The nitrate, nitrite and orthophosphate in the sample were measured by Gallery™ Automated Photometric Analyzer (Thermo Scientific, USA). All measurements were done in triplicate.
Light
150
P - PO4 (mg L )
F v =F m ¼ ðF m F o Þ=F m
200
N - NO2 (mg L )
Dry weight biomass (mg L ) was measured gravimetrically using 10 mL of the sample, and specific growth rate l (day1) and biomass productivity Pr (mg L1 day1) were calculated. Chlorophyll-a (Chl-a, mg L1) was measured after extraction in 100% methanol (Porra et al., 1989). An aliquot of 5 mL of the sample was centrifuged at 2000g for 15 min and supernatant discarded. The pellet was suspended in 10 mL of boiling methanol at 65 °C and stored in dark for 24 h. Chl-a was thus extracted in methanol and the absorbance of methanol solution was measured at 652 nm, 665 nm, and 750 nm using a spectrophotometer (Spectroquant Pharo 300, Merck, Germany) with pure methanol as blank. Specific Chl-a (mg Chl-a g1 algae) was calculated after dividing Chl-a concentration by corresponding algal biomass. A Dual-PAM 100 Chlorophyll Fluorometer (Heinz WalzGmbh, Effeltrich, Germany) was used for non-invasive fluorescence measurements. The sample was dark adapted before measurements so as to have all Photo System-II (PS-II) reaction centers in the chlorophyll open. The quantum efficiency of PS-II charge separation (Fv/Fm) was calculated as per following equation (Ramanna et al., 2014):
Algae (mg L )
1
3
4
8
9
10
Nl-Ph
3.1. Effect of variable nutrient levels on algal growth
Fig. 1. Effects of variable nitrogen levels on the dynamics of C. sorokiniana growth. (a) Biomass, (b) bulk phase N-NO 3 concentration, (c) bulk phase N-NO2 concentration, (d) bulk phase P-PO3 4 concentration.
The effect of N-NO 3 concentration on algal growth was studied by comparing results of three experiments at different initial N-NO 3 levels, namely Nh-Ph, Nm-Ph, and Nl-Ph (Table 2). In the case of the high nitrogen level (Nh-Ph), growth continued over 6 days of the light period, and then declined in the dark period (Fig. 1a). For medium and low nitrogen levels (Nm-Ph, and Nl-Ph, respectively), growth continued for 5 and 4 days, respectively, during the light period. Thereafter, despite the availability of light, culture growth declined (Fig. 1a). Nitrate levels declined in the bulk phase due to uptake by C. sorokiniana for growth (Fig. 1b), and resulted in the depletion of N-NO 3 in the bulk phase at low and medium levels within one and 3 days, respectively, during the light period. These depletions at low and medium N-NO 3 levels occurred well before their corresponding growths ceased, which suggests that growth also depended on internally stored nitrogen (Droop, 1974). The growth of C. sorokiniana ceased when the cell pool was depleted.
It was observed that excessive nitrogen was stored in the cells due to luxury uptake of nitrate in the case of higher initial N-NO 3 (Nh-Ph) and was excreted as nitrite during the dark phase (Fig. 1c). P-PO3 4 uptake depended on the initial N-NO3 concentra tions (Fig. 1d) and was lowest at the low N-NO3 level (Nl-Ph) because algal growth was restricted by nitrogen limitation. Phosphorus uptake continued even after nitrogen was depleted from bulk medium, for example nitrogen depletion occurred within 1 or 3 days, respectively, for low (Nl-Ph) and medium (Nm-Ph) N-NO 3 concentrations, whereas phosphorus uptake continued for 4–8 days (Fig. 1b and d respectively). Other researchers have also observed luxury uptake of phosphorus (Powell et al., 2008). Marginal excretion of both nitrogen and phosphorus in the late dark phase is attributed to cell mineralization due to respiration in the dark.
A. Shriwastav et al. / Bioresource Technology 174 (2014) 60–66
Three independent experiments with different initial P-PO3 4 levels (Nh-Ph, Nh-Pm, and Nh-Pl; Table 2) were conducted to compare the effect of P-limitation on growth of C. sorokiniana. At all P-PO3 4 levels, the growth of algae continued for 6 days in the light period and no effect of P-limitation on the growth was observed at the selected concentrations (Fig. 2a). In the dark, there was a marginal decline in biomass due to respiration loss. Similarly, N-NO 3 uptake was comparable in all three cases, which suggests no P-limitation (Fig. 2b). Uptake of N-NO 3 in all cases (Nh-Ph, Nh-Pm, and Nh-Pl) resulted in substantial excretion of nitrite from the algal cells into the bulk medium, suggesting for luxury uptake in excess to their stoichiometric requirements (Fig. 2c). The stored nitrate in the cell was first reduced to nitrite with nitrate reductase enzyme, which was then reduced to ammonium by nitrite reductase enzyme to be used for cell synthesis. Any accumulation of nitrite in the cell due to imbalance of these two enzymatic activities resulted in nitrite excretion to the bulk medium (Flynn et al., 1997). Collos (1998) also observed release of nitrite in algal cultures. The uptake of P-PO3 4 by C. sorokiniana was governed by initial levels in the bulk phase, as at low and medium levels (Nh-Pl, and Nh-Pm) the uptake by algae depleted phosphorus in bulk medium within 1 or 2 days (Fig. 2d). At a high P-PO3 4 level (Nh-Ph), the uptake of phosphate continued for 6 days in the light phase. Marginal release of phosphorus was observed during late dark phase due to respirational loss of biomass.
a
200
Light
Dark
Light
Dark
-1
Algae (mg L )
250
150 100 50 0 30
-1
N - NO3 (mg L )
b
The effect of variable nutrient loading on growth and productivity of C. sorokiniana is presented in Table 3. The duration of the exponential growth phase in each experiment varied from 6 days, when nitrogen and phosphorus were both in high concentration (Nh-Ph), to 4 days when nitrogen was limiting (Nl-Ph). C. sorokiniana achieved similar growth rates during the exponential phase in all experiments (Table 3). This implied that the culture had the ability to sustain sufficiently uniform growth under diverse nutrient levels until one of the nutrients became rate limiting. In addition, the productivity of the algal biomass was also comparable at all nutrient levels. These growth rates and productivity values are consistent with those obtained by Samorì et al. (2013) at similar light intensities for Desmodesmus communis. Kim et al. (2013b) observed a growth rate of 0.24 day1 under continuous illumination of 60 lE m2 s1 for C. sorokiniana, which is also consistent with our data. Similarly, de-Bashan et al. (2008) reported growth rates ranging from 0.15 day1 to 0.30 day1 for C. sorokiniana at 28 °C and 60 lmol m2 s1. These results provide evidence of the ability of C. sorokiniana to adapt to variable nutrient loading and to maintain uniform growth and productivity. This ability of C. sorokiniana to maintain steady growth rates is possibly due to sufficient accumulation of N and P in the cell even at lower levels to assist growth. The specific nutrient uptake rates during exponential growth of C. sorokiniana are expressed in Table 3. The lowest N-uptake rate was observed for low N loading (Nl-Ph) as 4.91 ± 1.24 mg N g1 algae day1 while the lowest P-uptake rate was 0.61 ± 0.03 mg P g1 algae day1 for Nh-Pl. Total nutrient accumulation in C. sorokiniana biomass during exponential growth is presented in Fig. 3. As expected, the highest and lowest accumulations of both N and P were observed when their respective levels in the bulk phase were high or low. Even the lowest accumulations of N (19.63 ± 4.97 mg N g1 algae for Nl-Ph) and P (3.03 ± 0.14 mg P g1 algae for Nh-Pl) were in excess of their minimum contents required below which the algal growth ceases, i.e. 7.2 mg N g1 algae and 1 mg P g1 algae (Ambrose et al., 2006; Chapra et al., 2007). In addition, Droop (1974) identified that algal growth rates are governed by both internal and external cellular nutrient levels. Despite these levels varying, C. sorokiniana maintained uniform growth during exponential phase until such internal pools were exhausted causing growth to cease.
-
20
63
10
3.2. Effect of variable nutrient levels on culture physiology
0 3 -1
N - NO2 (mg L )
c
Light
Dark
Light
Dark
5 6 Days Nh-Pm
7
-
2
0 10 8 6
-3
-1
P - PO4 (mg L )
d
1
4 2 0 0
1
2 Nh-Ph
3
4
8
9
10
Nh-Pl
Fig. 2. Effects of variable phosphorus levels on the dynamics of C. sorokiniana growth. (a) Biomass, (b) bulk phase N-NO 3 concentration, (c) bulk phase N-NO2 concentration, (d) bulk phase P-PO3 4 concentration.
The maximum quantum efficiency (Fv/Fm) of reaction centers in PS-II of chlorophyll represents the physiological state of the culture and the nutrient stresses (White et al., 2011). A Fv/Fm value of less than 0.5 implies the culture is in a stressed state; and this value declines with increasing stress level of the culture (Ramanna et al., 2014). These quantum efficiencies were calculated for all five nutrient levels throughout the course of the experiments (Fig. 4a). Since all experiments were conducted with nutrient-starved cells to avoid the effect of internally stored nutrients on growth and uptake of nutrients, these cells were in a stressed condition based on the evidence of the initial Fv/Fm value for each experiment being less than 0.3. With the addition of nutrients in each experiment, the nutrient stress in the culture was reduced as evident by the increasing quantum efficiencies in the early light periods. Increase in Fv/Fm values was observed for 2 days only in cultures with the lowest initial nitrogen input (experiment Nl-Ph), after which these efficiencies declined to very low levels. This indicates that as the nitrogen became limiting, the culture became nutrient stressed. Nutrient stress affected the culture physiology and effects were visible in terms of low growth of C. sorokiniana (Fig. 1a). At other nutrient levels, quantum efficiencies increased for 3 days. For medium nitrogen levels (Nm-Ph), nutrient stress was evident after 3 days whereby Fv/Fm started to decline appreciably. At higher
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Table 3 Specific growth rates, biomass productivities, and specific nutrient uptake rates of C. sorokiniana at different nutrient levels. Experiment Nh-Ph Nm-Ph Nl-Ph Nh-Pm Nh-Pl
Exponential growth phase (day)
l (day1)a
Pr (mg L1 day1)b
Specific N uptake rate (mg N g1 algae day1)c
Specific P uptake rate (mg P g1 algae day1)c
0–6 0–5 0–4 0–5 0–5
0.35 ± 0.07 0.35 ± 0.04 0.33 ± 0.06 0.28 ± 0.07 0.29 ± 0.01
28.25 ± 5.70 24.00 ± 2.83 21.25 ± 14.97 23.30 ± 9.19 24.60 ± 5.83
16.63 ± 0.57 12.09 ± 0.35 4.91 ± 1.24 29.29 ± 2.37 26.74 ± 1.40
4.88 ± 0.37 6.70 ± 0.63 5.12 ± 0.98 3.83 ± 0.34 0.61 ± 0.03
a
l is the growth rate in exponential growth phase.
b
Pr is the productivity at the end of exponential growth. Specific nutrient uptake rates are calculated for exponential growth phase.
c
0.8
180
a
a
140
0.6
Light
Dark
Light
Dark
Light
Dark
120
Fv / Fm
Total Accumulated N -1 (mg N g algae)
160
100 80
0.4
60
0.2
40 20
0.0
0 Nh-Ph
Nm-Ph
Nl-Ph
Nh-Pm
Nh-Pl
b 30
20
4
2
10
0 0 Nh-Ph
Nm-Ph
Nl-Ph
Nh-Pm
Nh-Pl
Fig. 3. Effect of variable nutrient loading on total nutrient accumulation during exponential growth phase by C. sorokiniana. (a) N-accumulation, (b) Paccumulation.
nitrogen levels (Nh-Ph, Nh-Pm, and Nh-Pl), the quantum efficiencies reached values above 0.5 after 3 days and remained sufficiently high for the duration of the light period. The results indicated a healthier physiological state of C. sorokiniana during these nutrient levels. This was also evident with the growth data of these experiments (Fig. 2a). Quantum efficiencies in all experiments declined during the latter part of the continued dark period, which suggested the stressed state of the culture in the absence of the light. Chl-a content was also monitored to gain insight into the physiological status of the algal cells since stress due to nutrient limitation are also reflected in Chl-a content. For example, Zhang et al. (2013) observed a marked reduction in the Chl-a content of N-starved cells of C. sorokiniana. Fig. 4b presents the evolution of Chl-a under different nutrient loading. At all levels of initial P-PO3 with high N-NO 4 3 (Nh-Ph, Nh-Pm, and Nh-Pl), the Chl-a concentration increased during the light period (Fig. 4b). In comparison, there was a marked reduction in Chl-a at low initial N-NO 3 levels. At medium N-NO3 levels, Chl-a reached a plateau by the fourth day and then declined. This is in agreement with growth data of the same experiment. Specific Chl-a content of the cell declined at medium and low nitrogen levels, suggesting a stressed culture due to N-limitation (Fig. 4c). In contrast, cells retained their Chl-a content at all phosphorus levels, which implied no growth limitation at these levels. These results agree with growth data where the limiting effects of phosphorus were
Specific Chlorophyll-a Content -1 (mg Chl-a g Algae)
Total Accumulated P -1 (mg P g algae)
b
6
-1
Chlorophyll-a (mg L )
40
c
50 40 30 20 10 0 0
1
2
3
Nh-Ph Nh-Pm
4
5 Days Nm-Ph
6
7
8
9
10
Nl-Ph
Nh-Pl
Fig. 4. Effects of variable nutrient loading on the culture physiology of C. sorokiniana growth. (a) Quantum efficiency of PS-II reaction centers, (b) chlorophyll-a, (c) specific chl-a content.
not observed. Hill and Fanta (2009) also did not observe growth limitation and reduction in Chl-a content of a mixed algal culture at 0.3 mg L1 dissolved phosphorus. At low N levels (experiment Nl-Ph) a marked reduction in the Chl-a content was observed, while the algal growth continued till day four. With the low N levels, C. sorokiniana cells experienced visible nutrient stress after day two (Fig. 4a). With the onset of such stress, the cells of C. sorokiniana regulate their photosynthesis by varying Chl-a content as a mechanism to counter these stresses (Zhang et al., 2013). However, with increasing stress levels, the Chl-a were irreversibly damaged and thus reduced (Fig. 4b). As the Chl-a concentration decreased to low levels, photosynthesis and hence the formation of new cells was affected. This resulted in the decline of algal growth after day four (Fig. 1a).
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3.3. Effect of variable nutrient levels on relative uptake potential of N and P The average molar ratio of N and P in algae is assumed to follow the Redfield ratio of 16N:1P (Redfield, 1958), though many studies have found the evidence of deviations from this ratio (Hulatt et al., 2012; Klausmeier et al., 2008). The initial nutrient levels in all experiments varied thus providing different N/P molar ratio in each case. Experiment Nl-Ph had the lowest initial N/P ratio of 0.52 ± 0.07: 1, while the highest initial N/P of 146.22 ± 1.73: 1 was observed for Nh-Pl (Table 2). The nutrient content in the algal cells mainly depends on its uptake from the medium. Therefore, the relative uptake was calculated in all experiments for the light period of 6 days. The lowest N/P molar ratio of uptake was observed as 2.12 ± 0.51: 1 for Nl-Ph, while the highest N/P of such uptake was calculated as 106.05 ± 1.74: 1 for Nh-Pl. These uptakes followed the initial ratio of nutrients in the medium for other experiments also (Fig. 5). The dependence of nutrient uptakes on their external concentration was also demonstrated for Neochloris oleoabundans (Wang and Lan, 2011). Similar trends of flexible nutrient uptakes as per the N/P in surrounding medium have also been observed for Scenedesmus sp. (Xin et al., 2010). This suggests that C. sorokiniana has the ability to regulate its nutrient uptake potential in accordance to the external nutrient levels, while maintaining uniform growth and productivity till either N or P becomes rate limiting. The effects of such variable uptakes on the nutrient removal efficiency are presented in Fig. 6. 73.07 ± 0.61% N and 59.27 ± 4.12% P were removed at high nitrogen and phosphorus levels (experiment Nh-Ph). As the initial N-NO 3 concentration was reduced with high P-PO3 levels, the N-removal efficiency 4 increased (97.11 ± 2.65% for Nm-Ph and 83.57 ± 17.55% for Nl-Ph). The decline in the efficiency for Nl-Ph was due to very high initial
160 Initial nutrient loading Relative uptake by algae
N/P Molar Ratio
140 120 100 30 20 10 0
Nh-Ph
Nm-Ph
Nl-Ph
Nh-Pm
Nh-Pl
Fig. 5. Dependence of relative uptake of N and P by C. sorokiniana on their initial levels in the medium.
% N Removal
100
a
80 60 40 20 0 Nh-Ph
Nm-Ph
Nl-Ph
Nh-Pm
Nh-Pl
Nh-Ph
Nm-Ph
Nl-Ph
Nh-Pm
Nh-Pl
120 100 % P Removal
The effect of nutrient starvation and the induced physiological stresses on lipid production and accumulation in algal cells is another important aspect. The accumulated nutrient levels during exponential growth varied with different cultivation conditions (Fig. 3). The variability in nutrient accumulations resulted in cells experiencing different nutrient stresses. The cultivation of C. sorokiniana at low and medium N levels (Nl-Ph and Nm-Ph respectively) resulted in increased stress levels in the cells (Fig. 4a) in comparison to other conditions. Many researchers have investigated the role of stresses on the lipid accumulation potential of various algal species and established the increase in lipid content with such stresses (Xin et al., 2010; Zhang et al., 2013), since algae regulate their utilization of assimilated carbon to synthesize lipids rather than glucose as a future reserve under stressed conditions (Ramanna et al., 2014; Xin et al., 2010). In similar manner, these stresses in this study also would invariably increase the lipid accumulation in the cells as observed for C. sorokiniana by many other researchers (Ramanna et al., 2014; Zhang et al., 2013). This suggests for higher lipid accumulations in the stressed cells. In addition, the productivity and growth rates were uniformly maintained across all experimental conditions during exponential growth (Table 3). Hence, the lipid productivity from these stressed cells would be higher, since the lipid productivity was found to depend on biomass productivity (Griffiths and Harrison, 2009). However, Griffiths and Harrison (2009) reported C. sorokiniana to still accumulate sufficient lipids (18% of dry weight) during nutrient replete cultivation. Hence, sufficiently high lipid productivity could still be achieved with the nutrient replete cultivation in addition to the nutrient limited cultivation conditions of this study. These results support the role of C. sorokiniana as a suitable candidate for nutrient removal and production of lipids under highly variable cultivation conditions.
b
80 60 40 20 0
Fig. 6. Effect of variable nutrient loading on nutrient removal efficiency of C. sorokiniana. (a) N-removal efficiency, (b) P-removal efficiency.
N/P molar ratio (146.22:1). Similar decline in nitrogen removal efficiency was also observed for Scenedesmus sp. when N/P molar ratio exceeded 20:1 (Xin et al., 2010). The P-removal efficiency declined to 54.01 ± 4.64% and 20.62 ± 3.75% for Nm-Ph and Nl-Ph, respectively. Similarly, when initial phosphorus levels were lowered while maintaining high nitrogen concentration, 91.76 ± 5.63% P-removal was achieved for Nh-Pm and complete P-removal for Nh-Pl, with marginal decline in N-removal efficiencies (75.35 ± 1.08% for Nh-Pm and 72.53 ± 1.47% for Nh-Pl). Since C. sorokiniana regulated the relative uptake of nitrogen and phosphorus in accordance to their levels in the surrounding media, these regulations affected the treatment efficiencies as observed in these experiments. 4. Conclusion This study demonstrated the ability of C. sorokiniana to regulate its nutrient (N and P) uptake in accordance with the available nutrient levels in the surroundings without any detrimental effect on growth or productivity until either nutrient became rate limiting. This flexibility, in addition to the ability of sustaining hostile growth conditions better than most of other algal species, makes C. sorokiniana a suitable candidate for fulfilling the coupled objectives of nutrient removal and biomass production for biofuel with great variability in nutrient levels of wastewaters efficiently.
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Acknowledgement The authors hereby acknowledge the National Research Foundation and Durban University of Technology for providing financial assistance.
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