Algal biofuel production coupled bioremediation of biomass power plant wastes based on Chlorella sp. C2 cultivation

Applied Energy 211 (2018) 296–305

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Algal biofuel production coupled bioremediation of biomass power plant wastes based on Chlorella sp. C2 cultivation Hui Chena, Jie Wanga,b, Yanli Zhenga,b, Jiao Zhana, Chenliu Hea, Qiang Wangc,

MARK

⁎

a

Key Laboratory of Algal Biology, Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan, Hubei 430072, China University of the Chinese Academy of Sciences, Beijing 100039, China c State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan, Hubei 430072, China b

H I G H L I G H T S cultivation is a costly process due to large nutrient substance needed. • Microalgal from biomass power plants can serve as nutrients for Chlorella cultivation. • Wastes cultures produce more lipids and exhibited higher growth rates. • Chlorella residual medium is almost nutrient-free and suitable for recycling. • The • An technical strategy for biofuel production coupled bioremediation is proposed.

A R T I C L E I N F O

A B S T R A C T

Keywords: Biomass power plant ash Biofuel Biological DeNOx Chlorella sp. C2 CO2 bioremediation

Microalgae have reported to be one of the most promising feedstock for biofuel production. However, microalgal cultivation for biofuel production is a costly process due to the large amounts of water, inorganic nutrients (mainly N and phosphate (P)), and CO2 needed. In this study, we evaluated whether the nutrient-rich ash and flue gas generated in biomass power plants could serve as a nutrient source for Chlorella sp. C2 cultivation to produce biolipids in a cost-efficient manner. When ash was incorporated in the culture medium and photosynthesis was enhanced by CO2 from flue gas, Chlorella cultures produced a lipid productivity of 99.11 mg L−1 d−1 and a biomass productivity of 0.31 g L−1 d−1, which are 39% and 35% more than the control cultures grown in BG11 medium. Additionally, the cultures reduced the nitrogen oxide (NOx) present in the flue gas and sequestered CO2, with a maximum ash denutrition rate of 13.33 g L−1 d−1, a NOx reduction (DeNOx) efficiency of ∼ 100%, and a CO2 sequestration rate of 0.46 g L−1 d−1. The residual medium was almost nutrient-free and suitable for recycling for continuous microalgal cultivation or farmland watering, or safely disposed off. Based on these results, we propose a technical strategy for biomass power plants in which the industrial wastes released during power generation nourish the microorganisms used to produce biofuel. Implementation of this strategy would enable carbon negative bioenergy production and impart significant environmental benefits.

1. Introduction The burning of fossil fuels has contributed to global climate change, environmental pollution, health problems, and an energy crisis associated with the irreversible depletion of traditional sources of fossil fuels [1]. Many countries are thus striving to develop renewable energy sources [2,3]. Among the various potential sources of renewable

energy, biofuel, the fuel obtained from biomass (i.e., organic matter derived from plants, animals, and microorganisms), is of great interest and is expected to play a crucial role in the global energy infrastructure in the future [4–6]. Biomass power plants are emerging as important sources of heat and power [7]. Biomass fuels are considered renewable fuel sources and do not affect the overall balance of CO2 in the atmosphere [8]. Obtaining

Abbreviations: AL, actinic light; BPPA, biomass power plant ash; Car, carotenoids; Chl, chlorophyll; CLSM, confocal laser scanning microscopy; CR, consumption rate; DeNOx, reduce the NOx; DR, denutrition rate; FCM, flow cytometry; FGFS, flue gas fixed salts; FR, fixation rate; ML, measuring light; N, nitrogen; NO, nitric oxide; P, phosphate; PSII, photosystem II; SP, saturation pulse; TLC, thin layer chromatography ⁎ Corresponding author at: State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Chinese Academy of Sciences, 7 South Donghu Rd., Wuhan, Hubei Province 430072, China. E-mail address: [email protected] (Q. Wang). https://doi.org/10.1016/j.apenergy.2017.11.058 Received 17 August 2017; Received in revised form 7 November 2017; Accepted 8 November 2017 0306-2619/ © 2017 Elsevier Ltd. All rights reserved.

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2. Materials and methods

energy from biomass could reduce the dependency on fossil fuel and energy import, reduce damage to the environment, achieve a zero carbon footprint, and enhance the use of the byproducts of energy production [9]. However, one of the problems associated with biomass combustion in biomass power plants is the ash and flue gas (mainly consisting of CO2 and nitrogen oxides (NOx)) generated during biomass combustion [10–14], and the environmental management of these pollutants, which can contaminate ecosystems [15–17]. When nitrogen (N) and carbon (C) were oxidized into NOx and CO2 in flue gas during the biomass combustion, while there are still significant amount of other elements such as Mg, K, and Ca, etc. left over in the ash, both the flue gas and the ash are considered contaminants and need to be further treated before disposal, thus there is an urgent need to develop sustainable technical strategies for dealing with these byproducts, as the volume of industrial wastes and thus the cost for disposal are increasing. Several technologies, including physical and chemical absorption, cryogenic distillation, and membrane separation, are used to capture CO2 [18], which is then transported and stored in geological formations. However, these procedures should only be considered as shortterm solutions because they are energy consuming and the captured CO2 needs to be disposed off. Microalgae are the fastest growing photosynthesizing organisms known and, in addition to consuming CO2 and N-based compounds, are one of the most important producers of oxygen on earth [19,20]. Microalgal biomass contains approximately 50% C by dry weight [21]. During the production of 100 tons of microalgal biomass using natural or artificial light, approximately 180 tons of CO2 can be fixed [22]. The ability microalgae to fix CO2 by photosynthesis is 10–50 times that plants [23]. Conventional NOx treatments, i.e., physicochemical DeNOx methods, are expensive and produce secondary wastes that often require further treatment [24]. NOx can serve as a N source for microalgae and can be metabolized by microalgae. Some green algae can utilize NOx as a nitrogen source for biofuel production [25,26]. In addition, biomass ash may be a nutrient source for algal cultivation, but very few studies have evaluated the effects of biomass ash on algal growth [27,28]. Biodiesel is recognized as an ideal renewable energy carrier [29]. Given their rapid growth and ability to convert solar energy into chemical energy via CO2 fixation, microalgae have been considered one of the most promising sources of oil for the production of biodiesel [30]. However, algal biodiesel are not produced commercially, as algal oil is more expensive than fossil fuel [31–33]. Microalgal cultivation is expensive due to the large amounts of water and inorganic nutrients (mainly N, phosphate (P), and CO2) needed [4]. One possible way to reduce these costs would be to use the ash, CO2, and NOx byproducts of biomass power plants to nourish the microalgae in the system [4,34]. We previously demonstrated the feasibility of coupling Chlorellabased production of microalgae-based lipids with the efficient biological DeNOx of flue gas [25,26,35]. In this study, we aimed to establish an integrated technique for algal-based lipid production and bioremediation of biomass power plant wastes (i.e., ash and flue gas). When biomass power plant ash (BPPA) and flue gas are used as the nutrient source, Chlorella sp. C2 cultures not only survive and grow, but produce increased levels of lipids. Our study provides an economically viable technical strategy for algal biofuel production coupled bioremediation of biomass power plant waste.

2.1. Microalgal culture Chlorella sp. C2 used in this study was isolated and autotrophically cultured in BG11 medium as described previously [36]. When the optical density at 700 nm (OD700) reached around 0.8, Chlorella sp. C2 was inoculated into a 1 L Erlenmeyer flask containing 500 mL BG11 medium at 25 °C with continuous illumination of 70 μmol m−2 s−1 and continuously bubbled with filtered air, the initial OD700 is 0.05. For culturing in BPPA media, Chlorella sp. C2 was harvested at the mid-logarithmic growth phase (OD700 = 0.8) by centrifuging at 3000 g for 3 min. The algal pellets were washed twice with N-free BG11 medium and then re-suspended to an OD700 of 0.2 in either regular BG11 or BPPA media in a column photobioreactor with a 750 mL working volume. Chlorella sp. C2 was inoculated at 28 °C with continuous illumination of 115 ± 3 μmol m−2 s−1 and continuously bubbled with filtered air and 3% CO2 derived from biomass power plant flue gas. 2.2. Preparation of BPPA media Eighty grams of biomass power plant ash, the burning wastes of biomass power generation by combustion of majorly rice straw, from the biomass power plant of SUNSHINE KAIDI NEW ENERGY GROUP CO. LTD was lixiviated with 1 L distilled water with a stirring speed of 150 rpm for 5 h. Then the leaching liquor was filtrated and sterilized using a 0.45 μm filter membrane. The main nutrient components (Mg, K, Ca, P, and Fe) in the leaching liquor were detected using Inductively Coupled Plasma-optical emission spectroscopy (ICP-OES, OPTIMA 8000DV, PekinElmer, USA). The nitrate and nitrite concentrations were detected using ion chromatography [37]. According to the main nutrient elements in leaching liquor (Table 1) and BG11 medium, 0.153 mM K2HPO4 was replenished in leaching liquor to be BPPA1 medium, 17.65 mM NaNO3 was replenished in BPPA1 medium to be BPPA2 medium, and NOx (flue gas fixed salts, FGFS) was replenished in BPPA1 medium to be BPPA3 medium (Table 2). 2.3. Preparation of CO2 and NOx CO2 was separated from flue gas emitted from the biomass power plant of SUNSHINE KAIDI NEW ENERGY GROUP CO. LTD and stored in a compressed gas vessel. Nitric oxide (NO), which is the main component of NOx, is sparingly soluble in water, and the dissolution of NO into the microbial culture medium is the rate-limiting step for NO removal. The initial fixation of massive NOx into water and the subsequent cultivation of algal cells using fixed nutrients is possibly an effective way to improve NOx removal efficiency [26]. After desulfurization and dust removal, NOx in flue gas from the biomass power plant was fixed into FGFS as described by Zhang et al. [26] and Chen et al. [25]. The final concentrations of NO2− and NO3− in the FGFS were 16.77 and 0.88 mM, respectively. 2.4. Algal growth analysis Cell growth was monitored by measuring OD700 and the biomass production. OD700 was measured every 24 h with five biological replicates. Biomass production was measured every 24 h with five biological replicates. Biomass collected at a known volume was precisely weighed after 24 h of freeze-drying [26]. Maximum biomass

Table 1 Major nutrient elements in leaching liquor of biomass power plant ash. Mg (mM)

K (mM)

Ca (mM)

P (mM)

Fe (mM)

NO3− (mM)

NO2− (mM)

3.284 ± 0.328

2.668 ± 0.297

17.749 ± 1.774

0.022 ± 0.002

0.055 ± 0.006

0

0

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Table 2 The removal efficiency of the main nutrient elements in BPPA by algal cultivation. Medium

Mg (mM)

K (mM)

Ca (mM)

P (mM)

Fe (mM)

NO3- (mM)

NO2- (mM)

BG11

Initial concentration (mM) Residual concentration (mM) Removal efficiency (%)

0.623 ± 0.002 0.314 ± 0.005 49.54 ± 0.83B

0.351 ± 0.005 0.121 ± 0.017 65.59 ± 4.82A

0.245 ± 0.004 0.177 ± 0.001 27.75 ± 0.56C

0.175 ± 0.009 0.006 ± 0.000 96.65 ± 1.48A

0.012 ± 0.001 0.005 ± 0.000 59.03 ± 1.01B

17.65 ± 0.01 0.096 ± 0.004 99.46 ± 0.02A

0 0.007 ± 0.000 –-

BPPA1

Initial concentration (mM) Residual concentration (mM) Removal efficiency (%)

3.284 ± 0.328 0.742 ± 0.178 77.41 ± 5.47A

2.974 ± 0.297 0.847 ± 0.223 71.53 ± 7.51A

17.75 ± 1.77 4.001 ± 0.661 77.46 ± 3.73A

0.175 ± 0.002 0.006 ± 0.000 96.62 ± 1.24A

0.055 ± 0.006 0.006 ± 0.000 89.05 ± 5.61A

0 0.071 ± 0.004 –-

0 0.007 ± 0.000 –-

BPPA2

Initial concentration (mM) Residual concentration (mM) Removal efficiency (%)

3.284 ± 0.328 0.691 ± 0.085 78.95 ± 2.60A

2.974 ± 0.297 0.975 ± 0.153 67.21 ± 5.14A

17.75 ± 1.77 3.228 ± 0.176 81.81 ± 0.99A

0.175 ± 0.002 0.005 ± 0.000 97.20 ± 1.42A

0.055 ± 0.006 0.006 ± 0.000 88.51 ± 3.12A

17.65 ± 0.01 0.135 ± 0.013 99.23 ± 0.08A

0 0.013 ± 0.001 –-

BPPA3

Initial concentration (mM) Residual concentration (mM) Removal efficiency (%)

3.284 ± 0.328 2.961 ± 0.138 9.84 ± 0.41C

2.974 ± 0.297 1.280 ± 0.158 56.96 ± 1.13B

17.75 ± 1.77 8.474 ± 0.224 52.26 ± 1.26A

0.175 ± 0.002 0.068 ± 0.003 61.35 ± 1.56B

0.055 ± 0.006 0.004 ± 0.000 92.72 ± 0.84A

0.883 ± 0.005 0.170 ± 0.014 80.71 ± 1.61B

16.77 ± 0.02 0.021 ± 0.002 99.88 ± 0.01

All data points in the current and following tables represent the means of five replicated studies in each independent culture, with the SD of the means (p < 0.05 or p < 0.01). A, B, C show significant differences in same item and among groups are represented by different superscripts (p < 0.05).

productivity was calculated using Eq. (1), where N1 and N2 are defined as the biomass concentration at time 1 (t1) and time 2 (t2), respectively. In this study, t1 and t2 were 0 d (origin of cultivation) and 7 d (peak of cultivation), respectively.

(530/30 nm) [39]. The collected data were analyzed using FlowJo software (Tree Star, San Carlos, CA, USA). FCM analysis was carried out at 5 d and 10 d after culture with five biological replicates.

Biomass productivity (g·L−1·d−1) = (N2−N1)/(t2−t1)

2.7.3. Thin layer chromatography (TLC) analysis TLC analysis was performed according to Reiser and Somerville [40] with minor modifications as described by Zhang et al. [39]. TAGs were separated by developing the plates in hexane-ethyl ether-acetic acid (70:30:1, v/v/v). Samples were visualized by exposure to iodine vapor for approximately 10 min. Three microliters of each sample was used for TLC analysis. Glyceryl trioleate (3 μL, 10 mg mL−1) was used as a reference substance for TAGs. TLC analysis was carried out at 5 d and 10 d after culture with five biological replicates.

(1)

2.5. Pigment quantification Pigments were extracted in 100% methanol. The concentrations were spectrophotometrically determined and calculated using the formula developed by Lichtenthaler [38] as shown in Zhang et al. [39]. Pigments were measured at 2 d, 4 d and 6 d after culture with five biological replicates.

2.7.4. Total lipid analysis and fatty acid characterization The total lipid content of microalgae was extracted from 0.2 g lyophilized material and gravimetrically quantified [41]. The overall lipid productivity was calculated according to Eq. (2) below, whereas the biomass productivity was obtained from Eq. (1). Furthermore, 0.2 g lyophilized material was subjected to fatty acid characterization [25]. Total lipid analysis and fatty acid characterization were carried out at 7 d (peak of cultivation) after culture with five biological replicates.

Chlorophyll a (Chl a) (μg ml−1) = 16.72 A665.2 − 9.16 A652.4; Chlorophyll b (Chl b) (μg ml−1) = 34.09 A652.4 −15.28 A665.2; Total carotenoids (Car) (μg ml−1) = (1000 A470 − 1.63 Chl a − 104.96 Chl b)/221. 2.6. Chlorophyll fluorescence Chlorophyll fluorescence was measured as described [39] at 2 d, 4 d, and 6 d after culture with five biological replicates.

Lipid productivity (g·L−1·d−1) = Biomass productivity ∗Lipid content(%) (2)

2.7. Lipid extraction and analysis Total lipids were extracted from lyophilized material according to the method by Zhang et al. [39].

2.8. Nutrients analysis At the end of cultivation (10 d), 1 L cultures were harvested by centrifugation at 8000g for 10 min. The total organic nitrogen contents in lyophilized biomass were determined using the Kjeldahl method according to Matejovic [42]. A 50 ml sample of supernatant was collected and stored at −20 °C for nutrient analysis to calculate the overall nutrients removal efficiency and removal rate using Eqs. (3) and (4), where Ci and Cf are defined as the total nutrients added in the cultivation process and the residual nutrients in the supernatant after harvesting at time i (ti) and time f (tf), respectively. In this study, ti and tf were 0 d (origin of cultivation) and 10 d (end of cultivation), respectively. Nitrogen fixation rate was calculated using Eq. (5), where U1 and U2 are defined as the nitrogen content at time 1 (t1) and time 2 (t2), respectively. In this study, t1 and t2 were 0 d (origin of cultivation) and 10 d (end of cultivation), respectively. CO2 fixation rate was calculated using Eq. (6), where the biomass productivity was obtained from Eq. (1).

2.7.1. Confocal laser scanning microscopy (CLSM) analysis Cells were examined by microscopy analysis using a confocal laser scanning microscope (Zeiss LSM 710 NLO) as previously described by Zhang et al. [39]. A lipophilic fluorescent dye, Bodipy 505/515 (4,4difluoro-1,3,5,7-tetramethyl-4-bora-3a, 4a-diaza-sindacene; Invitrogen Molecular Probes, Carlsbad, CA, USA), was used to stain the intracellular oil-containing organelles, known as lipid bodies, with a final labeling concentration of 1 μM and 0.1% DMSO (v/v). Bodipy fluorescence (green) was excited with an argon laser (488 nm) and detected at 505–515 nm. Autofluorescence (red) of algal chloroplasts was detected simultaneously at 650–700 nm. CLSM analysis was carried out at 5 d and 10 d after culture with five biological replicates. 2.7.2. 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, USA) equipped with a laser emitting at 488 nm and an optical filter FL1

Nutrients removal efficiency(%) = 100∗ (Ci−Cf )/Ci 298

(3)

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Nutrients removal rate(mg·L−1·d−1) = (Ci−Cf )/(t f −ti)

(4)

Nitrogen fixation rate(mg ·L−1·d−1) = (U2−U1)/(t2−t1)

(5)

CO2 fixation rate(g ·L−1·d−1) = Biomass productivity ∗1.83

(6)

and demonstrate that industrial waste residue from biomass gasification can be recycled to reduce the negative environmental impact of bioenergy production. By 6–7 days after the introduction of additional CO2, biomass accumulation increased for cells cultured in all BPPA media and in the BG11 control (Fig. 1). In our previous study, in which Chlorella sp. C2 cells were cultured in BG11 medium continuously supplemented with air, microalgal biomass plateaued at day 14 [26]. Compared with this study, the cells in the current study grew faster after 6–7 days of cultivation and the biomass accumulation peaked at day 7 with a similar density as observed at day 14 in the previous study. Thus, supplementing the growth medium with a C source (i.e., CO2 from flue gas) stimulates algal cell growth and proliferation. However, algal cells cultured in sterilized leaching liquor with or without an additional 17.65 mM N grew poorly or stagnated (Fig. 1, LL and LL + N), and were not used in our subsequent analyses.

2.9. Statistical analyses Each result shown is the mean of five biological replicates and standard deviation (SD). Statistical analysis of the data was performed using the software program SPSS-13 (IBM) and significance was determined at the 95% or 99% confidence limits. A t test was used to determine the means and SD of replicated studies. Significant differences between the control and test values were determined by using one-way ANOVA test, and differences were considered to be significant at p < 0.05 or p < 0.01.

3.2. Photosynthesis of Chlorella grown in BPPA media increased in the presence of supplementary CO2

3. Results

As shown in Fig. 1, cell growth in regular BG11 and BPPA media plateaued after 6–7 days; thus, we analyzed photosynthesis prior to the sixth day of culture. To compare the photosynthetic activity of Chlorella sp. C2 grown in the different media, we analyzed the pigment contents by spectrophotometry. Although the chlorophyll (Chl) a content of the BPPA cultures was significantly lower at 2 d than that of the BG11 control, the Chl a contents of cells grown in the BPPA media increased over time and the differences among the four cultures diminished (Fig. 2A), indicating that Chlorella sp. C2 gradually adapted to the culture environment. Similarly, differences in Chl b and carotenoid contents amongst cells grown in BG11 and those cultured in the BPPA media diminished over time (Fig. 2B and C), indicating that BPPA did not affect the synthesis or activity of photosynthetic pigments in Chlorella sp. C2. Absorption spectrum analysis showed that all algal cells cultured in BG11 and BPPA media peaked at 685 nm, which is the typical absorption peak of chlorophyll (Fig. S1). Compared with the BG11 control, no significant difference was found in the height of the 685 nm peak derived from cells cultured in BPPA2 medium. Although the 685 nm peak of cells cultured in BPPA1 was lower than that of the control at 2 d, the subsequent recovery of the peak height to the same level as that of the control suggests that the cells had adapted to their environment. By contrast, we observed a decrease in the height of the 685 nm peak for algae cultured in BPPA3 medium, suggesting that these cells were stressed. Chlorophyll fluorescence has long been considered one of the most sensitive, non-invasive tools used to investigate stress responses of photosynthesis under unfavorable conditions [43]. We next examined various parameters of photosystem II (PSII) to further investigate the photosynthetic activities of Chlorella sp. C2 grown in BPPA media. As shown in Fig. 3, no significant differences in the maximum quantum yield of PSII (Fv/Fm) and the effective quantum yield of PSII (Fv’/Fm’) were detected among cells grown in BG11, BPPA1, and BPPA2 media, especially during the exponential growth phase, indicating that the photosynthetic activity of Chlorella sp. C2 was unaffected by BPPA1 and BPPA2 media. However, the significant decrease in Fv/Fm and Fv’/Fm’ (one-way ANOVA test, p < 0.05), as well as the increased level of PSII excitation pressure (1-qL) and the damaging non-photochemical quenching (Y(NO)), in cells grown in BPPA3 medium indicated that photosynthetic activity was inhibited and that the photosynthetic apparatus might thus be damaged. Similarly, the fluorescence emission spectra, which primarily originate from PSII and represent PSII content, showed that cells grown in BPPA3 medium had a lower 685 nm peak than did cells grown in any of the other media at 6 days of culture (Fig. S2). These results also indicate that PSII was damaged in cells grown in BPPA3 medium. Together, our results showed that cells cultured in BPPA media

3.1. Nutrients from BPPA can be used for Chlorella cultivation Amongst the main nutrients present in BG11 medium, which is commonly used to culture Chlorella, we found that Mg, K, Ca, P, Fe, and N were also present in the leaching liquor (see Materials and Methods) from BPPA, as determined by ICP-OES and ion chromatography (Table 1). Most of these elements were present at much higher concentrations in the leaching liquor than in the BG11 medium. As N and P, two essential nutrients for cell growth, were below the detectable limit in the BPPA media, the concentrations of these elements may need to be adjusted for optimal algal culture. We evaluated the abilities of three variations of BPPA medium (BPPA1–3; Table 2; Materials and Method) to support Chlorella growth. Chlorella sp. C2 exhibited similar growth rates when grown in BPPA1 medium and the BG11 control, indicating that the leaching liquor derived from BPPA and containing additional P did not negatively impact Chlorella sp. C2 growth (Fig. 1). Algal cells cultured in BPPA2 medium grew more rapidly than those cultured in BG11 medium, and exhibited 35% greater biomass productivity (Table 3). When cultured in BPPA3 medium, in which NOx was used as the N source, cell growth was slower than in the BG11 control (Fig. 1), resulting in a 9% decrease in biomass productivity (Table 3). These results indicate that microalgae can use the residual nutrients in BPPA for biomass accumulation,

Fig. 1. Cell growth of Chlorella sp. C2 cultured in BPPA media. BG11 (1), BG11 control; BPPA1 (2), leaching liquor from biomass power plant ash added 0.153 mM K2HPO4; BPPA2 (3), BPPA1 added 17.65 mM NaNO3; BPPA3 (4), BPPA1 added NOx; LL (5), leaching liquor; LL + N (6) leaching liquor with additional 17.65 mM NaNO3. All data points in the current and following figures represent the means of five replicated studies in each independent culture, with the SD of the means (p < 0.05 or p < 0.01).

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Table 3 Biomass and lipid production analysis of algae cultured using BPPA. Parameters

BG11

BPPA1

BPPA2

BPPA3

Organic nitrogen

Percentage in biomass (%)

7.00 ± 0.11

6.59 ± 0.16

6.36 ± 0.25

6.38 ± 0.66

Biomass

Dry weight (g L−1)

1.63 ± 0.16B

1.67 ± 0.28B

2.15 ± 0.28A

1.49 ± 0.21B

Biomass productivity (g L−1 d−1)

0.23 ± 0.02B

0.24 ± 0.04B

0.31 ± 0.04

C14:0 C14:1 C16:0 C16:1 C18:0 C18:1 C20:0 C20:1 C20:2 C20:4

4.20 ± 0.61C 0.04 ± 0.00B 31.87 ± 2.46B 4.05 ± 0.05B 2.58 ± 0.71B 27.49 ± 0.51B 0.62 ± 0.04B 0.06 ± 0.00C – –

3.27 ± 0.67C 0.03 ± 0.00B 23.17 ± 1.60B 3.07 ± 0.34C 1.43 ± 0.02B 51.57 ± 5.64A 0.21 ± 0.01C 0.02 ± 0.00D – –

7.18 ± 0.24A 0.11 ± 0.01A 49.57 ± 3.69A 5.92 ± 0.25A 3.29 ± 0.21A 1.46 ± 0.07C 0.72 ± 0.03A 0.20 ± 0.01A – 0.10 ± 0.00

Main fatty acid composition (as a percentage of total fatty acids, %)

A

0.25 ± 0.03B 5.37 ± 0.10B 0.04 ± 0.00B 58.19 ± 1.52A 2.96 ± 0.26C 3.89 ± 0.76A 1.21 ± 0.13C 0.66 ± 0.02B 0.14 ± 0.00B 0.57 ± 0.03 0.08 ± 0.00

A, B, C, D show significant differences in same item and among groups are represented by different superscripts (p < 0.05).

Chlorella cells cultured in the BPPA media exhibited a higher level of lipids, lipid productivity, bio-DeNOx efficiency, CO2 fixation rate (FR), and nutrient consumption rate (CR) than did control cells cultured in BG11 (Table 3 and 4). These results demonstrate that Chlorella can be used for algal biofuel production coupled to the bioremediation of biomass power plant wastes, including ash and the NOx and CO2 present in industrial flue gases. In addition, the concentration of nutrients in the residual BPPA media (with an ash denutrition rate (DR) of 11.43–13.33 g L−1 d−1) was much lower than the discharge standard of pollutants for urban wastewater treatment plants of China (GB189182002). Thus, by using the integrated strategy described here, the residual BPPA media are nutrient-free and can be either recycled for algal cultivation or farmland watering, or safely disposed off (Table 4).

maintained a high photosynthetic capacity. Different degrees of adverse effects on photosynthesis were observed when N was limited (BPPA1) or when N was supplied as NOx (BPPA3), but the cells progressively adapted to the culture environment and maintained rapid growth rates during the exponential growth phase, which suggests that the adverse effects on photosynthesis were compensated by supplementation with CO2 derived from the flue gas. 3.3. Chlorella can be used for algal biofuel production coupled to BPPA and flue gas bioremediation To investigate the potential value of this strategy, we analyzed the total lipid contents of the biomass and the lipid productivity obtained from Chlorella sp. C2 cultured in BPPA media (Fig. 4). Although the total lipid contents of biomass in BPPA media was slightly greater than that in BG11 (Fig. 4 A), the lipid productivity in BPPA2 and BPPA3 media was significantly greater (one-way ANOVA test, p < 0.05) (Fig. 4 B), with a maximum productivity of 99.11 ± 6.83 (mg L−1 d−1), which is 39% more than that observed for algae cultured in BG11 medium. Neutral lipids are the main component in lipids for biodiesel production. We further quantified the intracellular neutral lipid content of microalgae grown in the four different media using FCM (Fig. 5A and B), CLSM (Fig. 5C), and TLC (Fig. 5D). Cells grown in BPPA media, especially BPPA3, accumulated over 3 times more neutral lipids than those grown in BG11 (Fig. 5). Thus, using nutrient-rich ash and NOx as the main nutrient elements and N source, the N enrichment culture not only stimulates biomass accumulation but also significantly induces neutral lipid accumulation in Chlorella sp. C2. Additionally, lipid production increased in cells grown in the BPPA media, resulting in a greater proportion of lipids in the biomass (Table 3). The main fatty acid components in the cultures were C14 to C20, with C16 and C18 accounting for over 60% of the fatty acids present (Table 3). The level of nutrient elements remaining in the media after cultivation was detected to determine whether the nutrients in BPPA media could be effectively assimilated by Chlorella sp. C2. Compared with the BG11 control, the removal efficiency of most of the nutrients in BPPA media by Chlorella sp. C2 improved to different degrees (Table 2), indicating the potential application of Chlorella sp. C2 in the reutilization of BPPA for bioremediation. However, in BPPA3 medium, the utilization efficiency of Mg, a key structural element in chlorophyll, was significantly lower (one-way ANOVA test, p < 0.05), which may be related to the inhibition of photosynthetic efficiency observed in the cells cultured in BPPA3 medium. Nevertheless, an excellent NO2- removal efficiency of 99.88 ± 0.01% was obtained (Table 2), indicating that Chlorella sp. C2 is suitable for bioremediation of BPPA in combination with bio-DeNOx.

4. Discussion The astoundingly high cost of research and production may remain a bottleneck for further development of microalgal biofuel for a long time to come. Microalgal mass cultivation may be more environmentally sustainable, cost-effective, and profitable if combined with various processes such as bioremediation of flue gas, biomass ash, and wastewater treatments [4,44]. For example, Zhou et al. [44] introduced and analyzed a novel system for producing algal biofuels that synergistically integrates algal wastewater treatment with the production of biofuel co-products to provide a viable pathway to sustainable, carbon-neutral energy independence. Photoautotrophic cultivation may be a preferred method for effective bioremediation of inorganic nutrients from BPPA and CO2 and NOx from flue gases, which are lightdependent physiological processes. Many microalgal species achieve an average lipid productivity ranging from 20 to 100 mg L−1 d−1 via photoautotrophism [45–47]. For example, the lipid productivity of Chlorella pyrenoidosa reached 107 mg L−1 d−1 when cultured in the presence of 5% CO2 [48]. In this study, using the nutrients from BPPA for photoautotrophic cultivation, Chlorella sp. C2 achieved a maximum lipid productivity of 99.11 mg L−1 d−1 (Fig. 4), which is a high lipid yield compared with the average lipid productivity via photoautotrophism. Compared to BG11 medium, the concentration of P and N is low in leaching liquor of ash (Table 1). Although addition N and P need to be added into leaching liquor, the cost of algal cultivation using BPPA media is lower than regular algal cultivation using BG11 medium. Furthermore, N element could be acquired using NOx from flue gas. Methods that use nutrients derived from biomass power plant waste (ash, CO2, and NOx) to sustain the cultures in the system would reduce the cost of biolipid production (carbon negative bioenergy generation), and the resulting medium remaining after algal culture would meet safety standards for disposal (discharge standard of pollutants for urban 300

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Fig. 3. Chl fluorescence parameters of Chlorella sp. C2 grown in BPPA media. A, Fv/Fm; B, Fv′/Fm′; C, 1-qL; D, Y (NO).

Microalgal biolipid accumulation is influenced by culture conditions. Stress (e.g., N starvation) increases the lipid content and decreases the biomass [49,50], as high lipid content is often accompanied by weak or inhibited cell division [51]. We previously demonstrated that adding a trace amount of urea to the microalgal cultivation medium significantly increased neutral lipid production without inhibiting cell growth [52]. Thus, our findings suggest a single-step approach for boosting lipid production while maintaining cell growth. Furthermore, this method can be performed under outdoor cultivation conditions [52]. We previously showed that a high concentration NOx could induce neutral lipid accumulation in Chlorella sp. C2 with increasing biomass [26]. In the present study, using BPPA media as the main source of nutrients, the photosynthesis of algal cells increased in the presence of supplementary CO2 and C assimilation continued, so that some of the C fixed during photosynthesis was channelled into the biolipid synthesis pathway, resulting in a 35% increase in biomass productivity and a 39% increase in lipid productivity compared with cells grown in BG11 medium. A study by Razzak et al. [53] showed that biomass (0.9 g L−1) and productivity (0.118 g L−1 d−1) of Chlorella vulgaris cultured in a single reactor were greatest in the presence of 4 % CO2. The biomass productivity of 0.31 g L−1 d−1 observed under the conditions used in the present study, which resulted in high lipid productivity, is more than that of many Chlorella species under photoautotrophy (0.04–0.30 g L−1 d−1) [54–58]. These results suggest a feasible strategy for significantly improving the production of neutral lipids by microalgae with increased biomass, and reducing the cost of bioremediation of industrial waste and biodiesel production. The possibility of using microalgae as a robust organism for biological nutrient removal from industrial and domestic wastewater, flue gas, and waste residue has been of great interest [4,25,28,59]. In our previous studies, we achieved an overall DeNOx efficiency of 96% when we applied FGFS to the Chlorella sp. C2 culture medium, demonstrating the feasibility and practicality of efficient biological DeNOx by microalgae [25,26]. In one study, the cyanobacterium Spirulina platensis was cultured with simulated flue gas containing CO2 (107 mgC d−1) and NOx (20 mgN d−1) mixed with air in a mixer flask. A high abatement of CO2 (407 mg d–1), 90% removal of NOx, and a biomass production of 188.7 mg L–1 d–1 were achieved when sufficient levels of flue gas were used in a photoautotrophic fed-batch test [60]. Waste gas (i.e., flue gas) and biomass ash are the main waste products of biomass power plants.

Fig. 2. Variations in pigment contents in Chlorella sp. C2 grown in BPPA media. A, Chl a content; B, Chl b content; C, carotenoid content. The significance of the differences between the control (BG11) and other test values in the current and following figures was tested using one-way ANOVA. *, p < 0.05; **, p < 0.01.

wastewater treatment plants of China, GB18918-2002). The strategy in this study could also combine with the treatment of wastewater, which is the abundant sources P and N for algal cultivation and further reduce the cost. This would reduce the amount of freshwater needed and thus the volume of wastewater to be treated. More significantly, after the removal of nutrient element from ash and flue gas for algal cultivation, the remnants of ash and medium could be disposed off without polluting the environment. Thus, our study provide a strategy for bioremediating industrial waste emissions as well as an environmentallyfriendly and cost-effective solution to the challenge of producing biodiesel from microalgae. 301

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Fig. 4. Total lipid content and lipid productivity of Chlorella sp. C2 grown in BPPA media. 1, BG11; 2, BPPA1; 3, BPPA2; 4, BPPA3.

CO2 fixation rate of 0.46 g L−1 d−1, and a biomass productivity of 0.31 g L−1 d−1 were attained (Table 4), and the microalgal biomass with high lipid content thus produced could, in turn, be used for biofuel production. Several conventional technologies for CO2 sequestration are energy consuming and need to be further disposed off [18]. Conventional physico-chemical DeNOx methods, such as selective catalytic reduction (SCR) and selectivity catalytic oxidation (SCO), are either expensive and/or produce secondary wastes that require further treatment.

Biomass ash from charcoal was used as a source of nutrients to cultivate Chlorella sp. and was deemed sufficient to maintain cell growth rates and biomass productivities [28]. Few studies have examined methods to bioremediate contamination caused by flue gas released from biomass power plants. In this study, we developed a sustainable and cyclic bioremediation method in which the wastes derived from biomass power plants, including the NOx and CO2 in flue gas and ash residues, are used as the source of N, C, and other main nutrients for microalgal cultivation. A maximum DeNOx efficiency of approximately 100%, a

Fig. 5. Neutral lipid accumulation of Chlorella sp. C2 grown in BPPA media.

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Table 4 Summary of the fixation rate (FR), consumption rate (CR), and denutrition rate (DR) in BPPA by algal cultivation. Parameters

BG11

BPPA1

BPPA2

BPPA3

Nitrogen FR (mg L−1 d−1) CO2 FR (g L−1 d−1)

35.09 ± 0.02B 0.42 ± 0.04B 1.06 ± 0.02B 1.28 ± 0.09B 0.39 ± 0.01C 0.75 ± 0.01A 0.056 ± 0.001C –

– 0.44 ± 0.07B 8.72 ± 0.62A 11.85 ± 1.24A 78.56 ± 3.78A 0.75 ± 0.01A 0.392 ± 0.025A 11.43

35.00 ± 0.02B 0.57 ± 0.07A 8.89 ± 0.29A 11.14 ± 0.85A 82.98 ± 1.00A 0.75 ± 0.01A 0.392 ± 0.014A 11.43

40.74 ± 0.01A 0.46 ± 0.06B 1.29 ± 0.05B 11.01 ± 0.22A 37.1 ± 0.89B 0.43 ± 0.01B 0.204 ± 0.002B 13.33

Nutrient element CR (mg L−1 d−1)

Ash DR (g L−1 d−1)

Mg K Ca P Fe

A, B, C show significant differences in same item and among groups are represented by different superscripts (p < 0.05).

negative biodiesel production, and the residual medium could be safely recycled for algal cultivation or crop watering. The resulting microalgal residue and straw derived from the harvested crops could also be directly used for biomass power generation. This strategy would reduce the cost of producing electrical power, bio-oils, and other valuable products derived from the cultivation of microalgae, and impart significant economic benefits. In addition, recycling the industrial waste products would minimize environmental pollution, imparting significant environmental benefits. Notably, flue gas bioremediation may alleviate current environmental problems, particularly the haze pollution in China and other developing countries, imparting significant social benefits.

Furthermore, these methods pose a hazard, due to the high temperatures used and the high flammability of the gas phase ammonia used in SCR [61–63]. To treat ash, many methods have been developed, including landfill and thermal plasma, which are expensive and complicated to operate [64]. Although the flue gas and ash should also be pretreated or separated before being added to the cultivation system for bioremediation, which adds processing costs, the strategy presented in this study would reduce the cost of the process overall due to bio-oils and other valuable products derived from the algae biomass, and the controllable operation and the safe and stable conditions of the algal cultivation system impart an economical, manageable and realizable method to dispose of industrial wastes. Notably, NO, which is the main component of NOx, is poorly soluble in water, thus the dissolution of NOx into the microalgal culture is the rate-limiting step for NO removal [65]. The removal capacities when directly using the NOx/NO are 50–90% in some studies [60,66–68]. However, in these studies, the removal rate of microalgae was dependent on the extremely low NOx flux and massive cell density, and resulted in very low NOx removal efficiency. By contrast, using prepared FGFS for algal cultivation is a truly feasible solution, which has been successfully achieved in our previous studies [25,26]. In this study, the feasibility of our approach is further demonstrated by combining with the feasible application of CO2 sequestration and ash reusing for algal cultivation, providing a new insight into a promising industrial strategy for economically viable bioremediation of biomass power plant wastes and carbon negative bioenergy production. In summary, we propose a technical strategy for reusing the contaminants present in the flue gas and ash released from biomass power plants to nourish the microalgae used for algal biofuel production (Fig. 6). Biomass power plants typically use agricultural residues for Cneutral power generation, while, in this study, we used flue gas and ash byproducts as the main nutrients for oil-producing microalgal cultivation. The oil extracted from the microalgal biomass was then used for C-

5. Conclusion A comprehensive technical strategy is provided and proved firstly, in which the wastes derived from biomass power plants, including the NOx and CO2 in flue gas and ash residues, are used as the source of N, C, and other key nutrients for microalgal cultivation. The prominent NOx removal and CO2 sequestration rates based on Chlorella culture are achieved along with considerable lipid productivity, indicating the economic feasibility of C negative bioenergy production with significant environmental benefits. Notes The authors declare no competing financial interests. Acknowledgements This work was supported jointly by the National Natural Science Foundation of China (31770128, 31700107), Hubei Provincial Natural Science Foundation of China (2017CFA021), the State Key Laboratory Fig. 6. A technical strategy for reusing the contaminants from biomass power plants to nourish the microalgae for algal biofuel production.

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