Evaluation of an Oil-Producing Green Alga Chlorella sp. C2 for

Aug 8, 2014 - Microalgae: Antiquity to era of integrated technology. Akash Patel , Bharat Gami , Pankaj Patel , Beena Patel. Renewable and Sustainable...
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Evaluation of an Oil-Producing Green Alga Chlorella sp. C2 for Biological DeNOx of Industrial Flue Gases Xin Zhang,†,‡ Hui Chen,† Weixian Chen,†,‡ Yaqin Qiao,†,‡ Chenliu He,† and Qiang Wang*,† †

Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, Hubei China University of Chinese Academy of Sciences, Beijing 100094, China

‡

S Supporting Information *

ABSTRACT: NOx, a significant portion of fossil fuel flue gases, are among the most serious environmental issues in the world and must be removed in an additional costly gas treatment step. This study evaluated the growth of the green alga Chlorella sp. C2 under a nitrite-simulated NOx environment and the removal rates of actual flue gas fixed salts (FGFSs) from Sinopec’s Shijiazhuang refinery along with lipid production. The results showed that nitrite levels lower than 176.5 mM had no significant adverse effects on the cell growth and photosynthesis of Chlorella sp. C2, demonstrating that this green alga could utilize nitrite and NOx as a nitrogen source. High concentrations of nitrite (88.25−176.5 mM) also resulted in the accumulation of neutral lipids. A 60% nitrite removal efficiency was obtained together with the production of 33% algae lipids when cultured with FGFS. Notably, the presence of nitrate in the FGFS medium significantly enhanced the nitrite removal capability, biomass and lipid production. Thus, this study may provide a new insight into the economically viable application of microalgae in the synergistic combination of biological DeNOx of industrial flue gases and biodiesel production.

1. INTRODUCTION

The conventional NOx treatments, physicochemical DeNOx methods, are expensive and produce secondary wastes that often require further treatment.11 Because nitrogen (N) is one of the basic elements for algal production, notably, NOx can serve as a N source for microalgae and can be metabolized by microalgae.12 Thus, a biological DeNOx (bio-DeNOx) method that uses microalgae may be noteworthy for flue gas treatment to reduce NOx emissions and merit further studies. For NOx-removal by microalgae, NOx in flue gas are first dissolved in an aqueous phase, after which NOx are oxidized and assimilated by the algal cells. However, NO, which is the main component of NOx, is sparingly soluble in water, and the dissolution of NO into the microbial culture is the rate-limiting step for NO removal.13 In addition to enhancing the dissolution of NO in water, the initial fixation of massive NO or NOx into water and then the cultivation of algal cells by using fixed nutrients is a possibly effective way of improving NO or NOx removal efficiency. In general, the main composition of NOx emitted from incineration processes is NO and the proportion could be up to 95%.6 As the main component of NOx aqueous solutions, nitrite is the commonly used material to simulate the dissolved nutrients of NOx in water. By using nitrite, the aim of this study was to test the effects of NOx on Chlorella sp. C2, an oil-

The extensive utilization of fossil fuels has led to global climate change, environmental pollution, health problems, and an energy crisis, which is associated with the irreversible depletion of traditional sources of fossil fuels.1 After the combustion of fossil fuels, flue gases can contain hundreds of different compounds, such as CO2, nitrogen oxides (NOx), sulfur oxides (SOx), H2O, O2, N2, unburned carbohydrates (CxHy), CO, heavy metals, halogen acids and particulate matter (PM).2,3 Nitrogen oxides, which compose a significant portion of fossil fuel flue gases, have been restricted by legislation and must be removed in an additional costly gas treatment step.2,4 Flue gas contains different NOx species, including NO, NO2, N2O, N2O2, N2O3, N2O4, and NO3.5 Nitric oxide is the major constituent of NOx in fossil fuel flue gas, and NO emitted into the atmosphere can be slowly oxidized to NO2 by oxygen in the air.6 Nitric oxide and NO2 are NOx of environmental concern, both of which could lead to photochemical smog, acid rain formation and tropospheric ozone formation in urban air.7 In addition, exposure to NO and NO2 has been associated with the increased risk of illness.8,9 Nitrogen oxides emission is currently one of the most serious environmental issues in the world, particularly in present-day China. Due to fast economic development at the sacrifice of the environment, from 2006 to 2009, it was estimated that NOx emissions in China maintained a mean annual growth rate of 6.7%,10 which has resulted in the PM2.5 and the air pollution index breaking records in many of China’s cities since 2012. © 2014 American Chemical Society

Received: Revised: Accepted: Published: 10497

March 21, 2014 July 1, 2014 August 8, 2014 August 8, 2014 dx.doi.org/10.1021/es5013824 | Environ. Sci. Technol. 2014, 48, 10497−10504

Environmental Science & Technology

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generation of transmission micrographs for visualization of nonfluorescent protoplast structures was achieved using the manufacturer’s filter settings. The specific experimental operation processes are previously described by Zhang et al.15 2.3.3. Flow Cytometry (FCM) Analysis. Samples stained with Bodipy 505/515 were analyzed on a board using a FACS Aria flow cytometer (Becton Dickinson, San Jose, CA) equipped with a laser emitting at 488 nm and an optical filter FL1 (530/ 30 nm).15 The collected data were analyzed using FlowJo software (Tree Star, San Carlos, CA). 2.4. Preparation of FGFSs. Actual flue gases from the caprolactam production plant of Sinopec’s Shijiazhuang Refining & Chemical Company, with and without desulfite treatment, were fixed with NaOH solution and detected using ion chromatography.19 The molar ratios of the main components of the fixed salts, sodium nitrite, sodium nitrate and sodium sulfate were 19:1:11 and 19:1:nd (not detected) before and after desulfite treatment, respectively. The two different types of salts were then added in N- medium to obtain FGFS 1 and 2 media, in which the final concentrations of sodium nitrite, sodium nitrate and sodium sulfate were 88.25, 4.64, and 0 mM (FGFS 1), and 88.25, 4.64, and 50.95 mM (FGFS 2), respectively. 2.5. Lab Scale Cultivation. Lab scale cultivation was performed in a 3 L column light bioreactor. Chlorella sp. C2 in the exponential phase was inoculated into 2 L sterilized BG11 medium at 25 °C with continuous illumination of 70 μmol m−2 s−1 and continuously bubbled with filtered air; the initial OD700 was 0.05. Cells in the mid logarithmic growth phase (OD700 approximately 0.8) were harvested by centrifugation at 2000g for 3 min at 25 °C. The algae pellets were washed and resuspended in regular BG11 medium, 5× NO2− medium, FGFS 1 and FGFS 2 media to OD700 0.2. Then, the cultures continued to be cultivated under the same growth conditions. 2.6. Nitrogen Removal Analysis and Total Lipid Analysis. Cells in the stationary phase were harvested by centrifugation and dried using a freeze-dryer. After centrifugation, the residual nitrate and nitrite contents in the supernatant were detected using ion chromatography.19 The total N contents in lyophilized materials were determined using the Kjeldahl method according to Matejovic20 The total lipid content of microalgae was extracted from 1 g lyophilized material according to the method by Zhang et al.15 and gravimetrically quantified.21 2.7. Statistical Analyses. Each result shown is the mean of at least three biological replicates. The statistical analysis of the data was performed using the program SPSS-13, and significance was determined at 95% or 99% confidence limits.

producing microalga isolated from the wild. The dose effects of nitrite on the growth of Chlorella sp. C2 and on its photosynthetic parameters were also observed. In particular, by using actual flue gas fixed salts (FGFSs) from the caprolactam production plant of Sinopec’s Shijiazhuang Refining & Chemical Company, the FGFS removal rates and lipid production by Chlorella sp. C2 were tested in a 3-L reactor column to evaluate the application of Chlorella sp. C2 in bioDeNOx. The results indicated that Chlorella sp. C2 could be a good option for NOx removal because this alga could survive and thrive using high concentrations of nitrite as a N source, along with stimulated level of lipid accumulation. It is hoped that the results of the present research will provide a useful reference for the usage and environmental control of NOx using microalgae.

2. MATERIALS AND METHODS 2.1. Growth Conditions and Nitrite Treatment. The Nsufficient (17.65 mM) medium used was full-strength BG11 medium.14 The N-deficient medium (N-) was BG11 without NaNO3. Chlorella sp. C2 was cultured as previously described.15 For the simulated NOx treatment, cells at the mid logarithmic growth phase (OD700 approximately 0.8) were harvested, washed and resuspended in regular BG11 as the control or in N- medium in the presence of sodium nitrite at various concentrations to OD700 0.3. The corresponding nitrite concentrations were 17.65, 35.3, 88.25, and 176.5 mM (1× , 2× , 5× , and 10× NO2−), respectively. 2.2. Photosynthesis Analysis. 2.2.1. Pigment Quantification. Pigments were extracted with 100% methanol and spectrophotometrically quantified according to Lichtenthaler16 by using the formula as shown below. chlorophylla(Chla)(μg mL−1) = 16.72A 665.2 − 9.16A 652.4; chlorophyllb(Chlb)(μg mL−1) = 34.09A 652.4 − 15.28A 665.2 total chlorophylls(Chla + b)(μg mL−1) = 1.44A 665.2 + 24.93A 652.4 total carotenoids(Car)(μg mL−1) = (1000A470 − 1.63Chla − 104.96Chlb)/221.

2.2.2. Photosynthetic Oxygen Evolution and Dark Respiration Rates. Rates of steady state photosynthetic oxygen evolution and respiration were measured as described by Zhang et al.15 2.2.3. Chl Fluorescence Analysis. Chl fluorescence was measured as described by Zhang et al.15 2.2.4. 77K Fluorescence. Thylakoid membrane preparation and the 77K fluorescence emission spectra were performed as previously described.17 2.3. Lipid Extraction and Analysis. Total lipids were extracted from lyophilized material according to the method by Zhang et al.15 2.3.1. Thin Layer Chromatography (TLC) Analysis of Lipids. The TLC analysis of lipid extracts from whole cells was performed according to Reiser and Somerville18 with minor modifications as described by Zhang et al.15 2.3.2. Confocal Laser Scanning Microscopy (CLSM) Analysis. The microscopic analysis of the cells was performed using a confocal scanner (Zeiss LSM 710 NLO). The

3. RESULTS 3.1. Nitrite Utilization by Chlorella sp. C2. To evaluate the dose effects of nitrite on Chlorella sp. C2 growth, various concentrations of sodium nitrite were added into regular BG11 or N- media, and cell growth rates were followed (Figure 1). All N treatments showed much faster cell growth rates compared with the N- culture (Figure 1A and B). When grown with 1× , 2×, and 5× NO2− in both regular BG11 media (Figure 1B) and in N- media (Figure 1A), although growth rate was decreased slightly with increasing concentrations of NO2−, the green alga showed similar growth rates to the BG11 control, indicating that the addition of nitrite up to 88.25 mM did not cause significant negative effects on the growth of Chlorella sp. C2. When treated with 10× NO2− (176.5 mM nitrite), although 10498

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a, Chl b, and Chl a+b contents could be detected between the regular BG11 grown and the nitrite treatments (1×, 2×, 5×, and 10× NO2− medium) at day 3, and only the cell Car contents in 5× and 10× NO2− media were significantly reduced. Moreover, at day 7, 12, 15, and 18, all of the cell Chl a, Chl b, Chl a+b, and Car contents among the regular BG11 grown and the nitrite treatments had no significant differences (SI Figure S2). Similarly, the Chl a/b and Car/Chl a+b also had no significant changes in all treatments compared with the regular BG11 medium, which indicated that nitrite had no significant damaging effect on photosynthetic pigments in Chlorella sp. C2. To further investigate the variation in the photosynthesis of Chlorella sp. C2 when nitrite was used as a sole N source, the steady state photosynthetic rate and dark respiration rate of cells were examined. Compared with the regular BG11 medium cultured cells, the steady state photosynthetic rate of cells cultured with 1× NO2− medium significantly increased (Figure 2) at day 3, then kept decreasing with increased nitrite

Figure 1. Cell growth of Chlorella sp. C2 treated by NaNO2 at various concentrations. Cultures were treated by NaNO2 with N- medium (A) and BG11 medium (B) background, with BG11, 10 × NaNO3, N-, and 10 × NaCl as control experiments. All data points in the current and following figures and tables represent the means of three replicated studies in each independent culture, with the SD of the means (*, p < 0.05; **, p < 0.01).

Figure 2. Variations in steady-state oxygen evolution and in dark respiration in Chlorella sp. C2 after 3 days (72 h) of treatment with NaNO2 at various concentrations.

significantly inhibited, the Chlorella sp. C2 cells remained able to steadily grow at a relatively fast rate (Figure 1A and B). Because all N sources were supplied as sodium salts, to avoid the ionic effect, cell growth rates were also followed with 10 times sodium chloride (10× NaCl). The results showed that, when grown with 10× NaCl, the culture technically did not grow at all and died within 2 days, either in N- (Figure 1A) or regular BG11 media (Figure 1B), indicating that a high Na+ level had a toxic effect on cell growth. As controls, cells grown with both 10× NaNO3 (Figure 1A) and 10× NaNO2 (Figure 1A and B) showed much faster growth rates, suggesting that high level nitrogen, particularly nitrate, had no significant ionic toxic effect on Chlorella sp. C2. To rule out possible microbial contaminations, the cell cultures at different time points were randomly sampled and spread on regular BG11 solid plate, and no microbial contamination could be detected, even after more than 10 days of incubation (SI Figure S1). 3.2. Photosynthesis of Chlorella sp. C2 under Nitrite Treatments. As could be seen in Figure 1A, the differences in cell growth between regular BG11 and the tests could be well distinguished at day 3; thus, further tests using nitrite as a sole N source were mainly performed at day 3. While four other time points (day 7, 12, 15, and 18) were also sampled and tested and the results were shown as SI accordingly. To evaluate the variation in Chlorella sp. C2 photosynthesis, pigment contents were spectrophotometrically determined. As shown in SI Table S1, no significant differences in the cell Chl

concentration, and finally sharply dropped (47%) in cells cultured with 10× NO2− medium. Concerning the dark respiration rate, a similar trend was observed but to a much smaller extent, and the decrease was minor even when the nitrite level was up to 10× NO2− (Figure 2). When extended to 7, 12, 15, and 18 days, although both the steady state photosynthetic rate and the dark respiration rate in cells cultured with 10× NO2− medium were significantly increased compared with the regular BG11 medium cultured cells, there was no significant differences between the regular BG11 grown cells and the cells in 1× , 2× , and 5× NO2− medium (SI Figure S3), demonstrating that up to 5× NO2− had no adverse effect on both photosynthesis and respiration. Chlorophyll fluorescence has long been considered one the most sensitive and noninvasive tools to investigate stress responses of photosynthesis under unfavorable conditions.22 To gain greater insight into the photosynthetic activities of Chlorella sp. C2 when nitrite was used as a sole N source, various parameters of PSII activity were determined using a Dual-PAM-100 Chl fluorometer. As shown in Figure 3, although decreasing slightly at 5× and 10× NO2−, the maximum quantum yield of PSII (Fv/Fm) was unaffected by the nitrite treatments. However, the significantly decreased level of the effective quantum yields of PSII (Fv’/Fm’), the increased level of PSII excitation pressure (1-qL) and the damaging nonphotochemical quenching (Y(NO)) at 2×, 5×, 10499

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concentrations tested. The results strongly correlate with the chlorophyll fluorescence tests, in that the energy transfer efficiency, that is, the photosynthesis efficiency, but not the photosynthesis capacity, was affected by high nitrite concentrations. 3.3. Neutral Lipid Accumulation in Chlorella sp. C2 Induced by High Concentrations of Nitrite. The abovementioned results demonstrated that Chlorella sp. C2 could grow relatively well with high concentrations of nitrite. In addition to the proven strong ability to use nitrite as a N source, could the oil-productive character of the green alga make the process more cost-effective? To determine the lipid accumulation, the intracellular neutral lipid levels in Chlorella sp. C2 were extracted and separated using TLC (Figure 4A), and then Bodipy 505/515 stained and visualized using both microscope for single cells (Figure 4B) and FCM for populations (Figure 4C). Figure 4 shows that after a 3 days treatment, compared with regular BG11 grown cells, the Nstarved cells accumulated much neutral lipids, as expected (Figure 4A−C). Those cells cultured with both 1× NO2− and 2× NO2− media only accumulated a minor amount of neutral lipids (Figure 4C), which was even under the detection levels of both TLC (Figure 4A) and microscopy (Figure 4B), whereas those cells cultured with 5× NO2− and 10 × NO2− media accumulated a significant amount of neutral lipids (Figure 4A− C), which also increased gradually with treatment time (SI Figure S6). These results indicated that, similar to N starvation, an enriched N source such as nitrite could also induce neutral lipid accumulation in Chlorella sp. C2, without sacrificing the accumulation of biomass (Figure 1). 3.4. Potentially Industrial Use for Bio-DeNOx by Chlorella sp. C2. By far, all the results indicated that when simulated with nitrite, the green alga Chlorella sp. C2 could be potentially used for flue gas bio-DeNOx, together with the production of the value-added byproduct, alga oil. As shown above, when cultured with 5× NO2− media, cells accumulated a significant amount of neutral lipids (Figure 4) and no significant decrease in biomass accumulation (Figure 1) and photosynthesis (Figure 2 and 3 and SI Figures S2−S5; Table 1 and SI Table S1) were observed, thus 5× NO2− (88.25 mM nitrite) was chosen as optimal concentration for further flue gas bio-DeNOx tests. To test the actual DeNOx capacity, flue gases emitted from the caprolactam production plant of Sinopec’s Shijiazhuang Refining & Chemical Company were alkalinefixed, as described in the Material and Methods, and the green alga, Chlorella sp. C2 was tested in a 3 L bioreactor. In the application of alga for removing NOx, biomass accumulation is always a key factor that must be considered. In the 3 L lab-scale photoautotrophic cultivation, when compared with the regular BG11 grown culture, the biomass of FGFS 1 grown culture slightly increased, although not significantly, whereas that of the FGFS 2 grown culture significantly decreased (Table 2). Interestingly, the total lipid accumulation in the FGFS media, as assessed by the gravimetrical method,21 showed the same trend (Table 2) as the biomass, and the total lipid accumulation of the FGFS 1 grown cells had a remarkable content of 33.3% (Table 2). To evaluate the NOx removing capacity of Chlorella sp. C2, the total organic N of cells cultured with BG11, 5× NO2−, FGFS 1, and FGFS 2 media were quantified using the Kjeldahl method.20 The residual N leftover in the growth mediums were measured using ion chromatography.6 Thus, the NOx removing capacities were correspondingly calculated. The total organic N

Figure 3. Chl fluorescence parameters of Chlorella sp. C2 after 3 days (72 h) of treatment with NaNO2 at various concentrations. Control (BG11) values were set to 100 for easy comparison.

and 10× NO2− indicated that photosynthesis efficiency was down-regulated. While at day 7, 12, 15, and 18, except the Fv/ Fm in cells cultured in 10× NO2− medium reduced compared with the regular BG11 medium cultured cells, the Fv/Fm was unaffected by the nitrite treatments in 1× , 2×, and 5× NO2− medium, and the effect of nitrite on Fv’/Fm’, 1-qL and Y(NO) were reduced with extended treatment time (SI Figure S4). These results suggested that although the photosynthesis efficiency did decreased by ∼20−40% with high concentration of nitrite treatments, the photosynthesis capacity, that is, the photosynthetic apparatus, was not damaged by up to 88.25 mM nitrite. The impact of nitrite on excitation energy transfer and distribution and the stoichiometry of photosystems were also diagnostically investigated using 77K fluorescence spectroscopy. Thylakoid membranes were isolated, and 77K fluorescence emission spectra were recorded in liquid nitrogen with excitation wavelengths at 435 nm to excite Chl. The typical emission spectra of the thylakoid membrane isolated from cells cultured in regular BG11 medium (SI Figure S5, straight line) showed a major peak at 723 nm (F723), which corresponded to PSI, and two smaller peaks at 687 nm (F687) and 697 nm (F697), which primarily originated from PSII. When cultured in medium with increasing concentrations of NO2−, both the amplitude and bandwidth of the PSI fluorescence (F723) simultaneously decreased (SI Figure S5), as well as the PSII bands (F687 and F697, SI Figure S5). Normally, the FPSI/FPSII ratio generally correlates well with the relative content of PSI and PSII.23 When excited at 435 nm, Table 1 shows that although slightly decreased, the FPSI/FPSII ratios were not significantly affected when cultured with the various nitrite Table 1. PSI:PSII Ratio of the Thylakoid Membrane in Chlorella sp. C2 after 3 Days (72 h) of Treatment with NaNO2 at Various Concentrations sample BG11 1× NO2− 2× NO2− 5× NO2− 10× NO2−

F723/F687 1.85 1.67 1.61 1.68 1.64

± ± ± ± ±

0.31 0.21 0.27 0.06 0.26 10500

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Figure 4. Lipid accumulation of Chlorella sp. C2 after 3 days (72 h) of treatment with NaNO2 at various concentrations. Lipid accumulation in N(a), BG11(b), 1× (c), 2× (d), 5 × (e), and 10× NO2−(f) medium analyzed by TLC (A), CLSM (B), and FCM (C). Asterisk symbol in A, glyceryl trioleate as loading standard.

Table 2. Biomass Accumulation of Chlorella sp. C2 after 10 Days Inoculation in a 3 L Column Light Bioreactor components (% of dry weight) medium

process type

BG11 5× NO2− FGFS 1 FGFS 2

batch batch batch batch

−1

dry weight (g L ) 0.931 0.734 0.948 0.806

± ± ± ±

organic nitrogen

0.023 0.007* 0.015 0.042*

7.540 7.887 7.887 7.844

± ± ± ±

total lipid

0.212 0.038 0.082 0.117

32.600 29.933 33.333 29.433

± ± ± ±

1.153 1.124 1.350 2.850*

The significance of the differences between the control (BG11) and test values were tested by using one-way ANOVA. *, p < 0.05 vs control.

Table 3. N Source Removal by Chlorella sp. C2 after 10 Days Inoculation in a 3 L Column Light Bioreactora initial conc. (mM)

a

medium

NO3−

BG11 5× NO2− FGFS 1 FGFS 2

17.65 4.64 4.64

NO2

−

SO42−

residual conc. (mM)

nitrogen removal efficiency (%)

−

NO2−

NO3−

0.69 ± 0.03 47.11 ± 0.53 35.19 ± 0.69 42.94 ± 0.59

36.14 ± 2.014

NO3

11.27 ± 0.36 88.25 88.25 88.25

50.95

2.47 ± 0.12 2.80 ± 0.10

46.68 ± 2.602 39.69 ± 2.212

NO2− 46.46 ± 0.607C 60.01 ± 0.784A 51.21 ± 0.669B

A, B, C shows significant differences among groups are represented by different superscripts (p < 0.01).

Thus, by using FGFSs in 3 L scale cultivation, Chlorella sp. C2 could rapidly grow to accumulate abundant biomass and fixed N by using nitrite as a N source. Moreover, along with the high removal efficiency of FGFSs, massive lipids, particularly neutral lipids, were also accumulated, indicating that Chlorella sp. C2 might be suitable for bio-DeNOx.

contents of the treatments increased slightly but were not significantly higher than that of the control (Table 2). As presented in Table 3, compared with the 36% nitrate removal capacity of the BG11 grown culture, the removal capacities of Chlorella sp. C2 for nitrite in 5× NO2−, FGFS 1, and FGFS 2 media were much higher and reached 46%, 60%, and 51%, respectively. Moreover, when considering the actual N concentration, the nitrite (5×) removal by Chlorella sp. C2 was 8 times (0.6/0.36 × 5) more than the nitrate (1×) removal. Interestingly, when compared with the simulative 5× NO2− medium, the nitrite removal efficiency of the nitrate containing treatments, that is, the FGFS 1 and FGFS 2 media growth cultures, were significantly higher (Table 3), indicating that the presence of nitrate could markedly enhance the removal capabilities of Chlorella sp. C2 for nitrite. However, the presence of sulfate in the medium caused an adverse effect and reduced the removal capabilities of Chlorella sp. C2 (Table 3).

4. DISCUSSION Because NO could be eliminated via microalgal assimilation and utilized preferentially as a N source for cell growth, the utilization of microalgae has recently been considered an attractive option to reduce NOx from flue gases.12,13 However, NOx in flue gases might affect the growth and biochemical composition of microalgae.24,25 In the present study, the adverse effect of nitrite on cell growth and photosynthesis of Chlorella sp. C2 could be ignored when 17.65−88.25 mM nitrite was utilized as the N source and only adverse effect was 10501

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Algal biomass can be utilized for renewable energy sources, such as hydrogen and biodiesel.15,30−32 In recent years, renewed interest in producing biodiesel from microalgae has arisen because microalgae can grow rapidly and convert solar energy into chemical energy via CO2 fixation and, thus, are now considered one of the most promising sources of oil for making biodiesel.33−35 However, the price of algae-based biodiesel remains much higher than normal diesel, which encumbers large-scale manufacturing and applications of algae-based biodiesel.33,36 Williams and Laurens37 noted that 30 and 50% of the algal primary product mass was lost on producing cell proteins and lipids, respectively, resulting in obtained yields that are 1/3−1/10 of the theoretical ones. Additionally, the increased lipid content reduces other valuable compounds in the biomass, and the “biofuel only” option is unlikely to be economically viable.37 Because the large consumption of water resources, inorganic nutrients (mainly N and phosphate) and CO2 are costly for microalgae cultivation,38,39 biodiesel production from microalgae can be more environmentally sustainable, cost-effective, and profitable if combined with processes such as wastewater and flue gas treatments.35,40 In the present study, although be slight or nonsignificant, there were some adverse effects on cell growth and photosynthesis efficiency (Figures 1−3 and SI Table S2−S5) with high concentrations of nitrite, which could stimulate the neutral lipid accumulation in Chlorella sp. C2 after 3 days of treatment, whereas simultaneously, no neutral lipid accumulation was detected in cells that used nitrate as a N source (Figure 4 and SI Figure S6). Meaningfully, unlike N starvation, which restrained the accumulation of biomass, Chlorella sp. C2 cells with high concentrations of nitrite remained able to steadily grow at a relatively fast rate (Figure 1), so enriched nitrite could induce neutral lipid accumulation in Chlorella sp. C2 with increasing biomass (Figure 4 and SI Figure S6). In a 3 L reactor column, the high lipid yield and biomass were also synchronously obtained in Chlorella sp. C2 cultivated in FGFS 1 medium (Table 2). All the results suggested that the synergistic combination of microalgae bio-DeNOx and biofuel production had a remarkable utilization potential for cost reduction in both flue gas bio-DeNOx and biodiesel production. In summary, this study suggests an obvious applicable value of Chlorella sp. C2 in NOx removal from industrial flue gases. Compared with the expensive cost of physicochemical DeNOx and biodiesel production, this study may also elucidate the synergistic combination of the cost-effective microalgae bioDeNOx of industrial flue gases and economically viable microalgae biodiesel production.

detected when the nitrite level increased up to 176.5 mM (Figure 1), and only the photosynthesis efficiency, but not the photosynthesis capacity, was affected (Figures 2 and 3 and SI Table S5). As nitrite was more easily and preferentially used than nitrate,12 nitrite at 17.65 mM may be even more conducive for photosynthesis of Chlorella sp. C2 than nitrate at the early stage of culture (Figure 2 and 3). Thus, the effect of nitrite on Chlorella sp. C2 was dose dependent and it could be used as the sole nitrogen source for cell growth at concentrations lower than 176.5 mM. The removal capacity of NOx from flue gases is a key indicator for evaluating the microalgae utilized in bio-DeNOx. In earlier studies, NOx or NO was used directly to cultivate microalgae. By using a marine microalga, strain NOA-113, Yoshihara et al.26 achieved a 50% NOx removal capacity when inoculated at 1.5 g L−1 cell dry weight with aeration of 300 ppm (v/v) NO and 15% (v/v) CO2 in N2 at a rate of 150 mL min−1 (0.0375 vvm). Nagase et al.27 obtained an NO removal yield of approximately 65% by using the unicellular microalga Dunaliella tertiolecta when a model flue gas (100 ppm (v/v) NO and 15% (v/v) CO2 in N2) at a flow rate of 150 mL min−1 (0.0375 vvm) was supplied to the algal culture at an initial cell density of 0.7 g L−1 cell dry weight. In a study by Van den Hende et al.,28 a removal efficiency of approximately 87% for NOx was obtained by using microalgal bacterial flocs for the treatment of sewage, combined with flue gas flow rates 0.6 L h−1 (0.0025 vvm) from a coal power plant. In another study, a Spirulina platensis culture was fed with CO2 (107 mgC d−1) and NOx (20 mgN d−1) mixed with air in a mixer flash, which simulates a flue gas, and a high abatement of CO2 (407 mg d−1) and 90.0% removal of NOx was achieved when opportune doses of flue gas were used in a fed-batch test performed at an initial cell concentration of 1.0 g L−1.4 However, in these studies, the removal capacity of microalgae was calculated according to the difference between the input and output of NOx or NO in bioreactor, and the medium dissolved part of NOx or NO was not taken into account, and the high NOx removal capacity dependent on the low NOx flux and massive cell density4,26−28 would keep it away from the industrial application. In the present study, actual flue gases were fixed to FGFSs, and the initial high nitrite content in mediums were set to avoid the unfavorable effect caused by low gas flux. And the actual consumed nitrite and/or nitrate in media were detected to calculate the real bio-DeNOx capacity of Chlorella sp. C2. As a result, 46% of nitrite was removed from the simulated 5× NO2− (88.25 mM) medium with an inoculated cell density of 0.07 g L−1 (0.2 of OD700) cell dry weight, and the coexistence of nitrate in the actual flue gases significantly improved the nitrite removal efficiency to 60% (Table 3), which provide Chlorella sp. C2 an obvious applicable value in flue gas bio-DeNOx. In many green algae, nitrate and nitrite can be used as N sources through N assimilation.29 Nitrate taken up by the cells is reduced to nitrite by nitrate reductase and then to ammonium by nitrite reductase. In the final step, ammonium is assimilated to form amino acids by glutamine synthetase and by glutamate synthetase.29 Using the Kjeldahl method, the accumulated organic N (amino acids) in algae cells can be detected; however, the inorganic N (nitrate, nitrite, and ammonium) that is absorbed and assimilated in cells cannot be detected, which explained why the removed N by Chlorella sp. C2 was greater than the assimilated organic N in the algal cells in this study (Table 2).

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ASSOCIATED CONTENT

S Supporting Information *

Table S1. Variations in pigment contents in Chlorella sp. C2 after 3 days (72 h) treated by NaNO2 at various concentrations. Figure S1. The detection of microbial contamination in the culture systems. Figure S2. Variations in pigment contents in Chlorella sp. C2 treated by NaNO2 at various concentrations. Figure S3. Variations in steady-state oxygen evolution and in dark respiration in Chlorella sp. C2 treated with NaNO2 at various concentrations. Figure S4. Chl fluorescence parameters of Chlorella sp. C2 treated with NaNO 2 at various concentrations. Figure S5. Steady-state fluorescence emission spectra at 77 K of intact cells of Chlorella sp. C2 after 3 days (72 h) of treatment with NaNO2 at various concentrations. Figure S6. Lipid accumulation of Chlorella sp. C2 treated with 10502

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NaNO2 at various concentrations. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-27-68780790; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported jointly by the National Program on Key Basic Research Project (2012CB224803), the Natural Science Foundation of Hubei Province of China (2013CFA109), the National Natural Science Foundation of China (31300030), Sinopec (S213049), and the Knowledge Innovation Program of the Chinese Academy of Sciences (Y35E05).

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ABBREVIATIONS bio-DeNOx biological DeNOx Chl chlorophyll CLSM confocal laser scanning microscopy DMSO dimethyl sulfoxide FCM flow cytometry FGFSs flue gas fixed salts N nitrogen NN-deficient NOx nitrogen oxides PM particulate matter SOx sulfur oxides TAGs triacylglycerols TLC thin layer chromatography

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