Effect of organic carbon and nutrient supplementation on the digestate-grown microalga, Parachlorella kessleri

Bioresource Technology 294 (2019) 122232

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Effect of organic carbon and nutrient supplementation on the digestategrown microalga, Parachlorella kessleri

T

Eleni Koutraa, Alexandros Kopsahelisa, Manolia Maltezoua, George Grammatikopoulosb, ⁎ Michael Kornarosa, a b

Laboratory of Biochemical Engineering & Environmental Technology (LBEET), Department of Chemical Engineering, University of Patras, 26504 Patras, Greece Laboratory of Plant Physiology, Department of Biology, University of Patras, 26504 Patras, Greece

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Agro-waste digestate Parachlorella kessleri Nutrient enrichment Cheese whey FAMEs

Digested effluents are usually deprived of the appropriate levels of organic carbon or macro- and micro-nutrients to effectively sustain microalgal growth. In this regard, Parachlorella kessleri was cultivated in an agro-waste digestate supplemented with different glucose concentrations, magnesium and trace metals and alternatively with cheese whey (CW), with view to enriching digestate with organic and inorganic constituents and decreasing freshwater demand. Between the conditions tested, CW addition resulted in the highest biomass concentration, 2.68 g L−1 within 18 days of cultivation. Chlorophyll content significantly decreased under 5 g L−1 glucose addition, in contrast to MgSO4 co-addition and CW supplementation. The latter also induced high photosynthetic activity and better-preserved vitality of the photosynthetic apparatus, compared to sole glucose addition. Concerning lipid accumulation, in the presence of high glucose concentration, % of total fatty acids decreased, and the saturated fraction increased over polyunsaturated fatty acids (PUFAs).

1. Introduction

et al., 2017). However, despite their great potential, up to date microalgae have been used mainly for added-value products and several challenges, including amelioration of biomass and lipid yield, processing cost reduction, and up-scale, should be met until complete exploitation of the entire range of biomass components becomes feasible (Barsanti and Gualtieri, 2018). Under this scope, gaseous and liquid

Over the past decades, a great amount of scientific effort and technological advancements have been focused on microalgal upstream and down-stream processes with view to materialize environmentally friendly and sustainable, large-scale applications (Lam

⁎

Corresponding author. E-mail address: [email protected] (M. Kornaros).

https://doi.org/10.1016/j.biortech.2019.122232 Received 9 August 2019; Received in revised form 28 September 2019; Accepted 30 September 2019 Available online 03 October 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.

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waste streams that are vastly available can be exploited in microalgal cultivation, providing a culture medium rich in inorganic and organic carbon, as well as macronutrients and trace elements, in order not only to fulfill environmental challenges but also to decrease microalgae production cost and diminish competition for freshwater (Salama et al., 2017). Currently, the effluents from anaerobic digesters, digestates, have been in the limelight as alternative substrates for microalgae cultivation, with view to applying an appropriate management practice in future biogas plants and producing microalgal biomass, which depending on its components could be integrated into a biorefinery approach for production of biofuels and various added-value products (Koutra et al., 2018a; Theuerl et al., 2019; Zhu et al., 2016). By the end of 2017, the number of biogas production plants in Europe reached 17,783, with total Installed Electric Capacity of 10,532 MW (EBA, 2018) and annual digestate production of 20 m3 per KW of IEC (Plana and Noche, 2016). Despite the promising results already demonstrated, several challenges hinder microalgal performance in digested effluents, including limited light transmittance, low C/N ratio, ammonia toxicity, insufficient concentration of fundamental nutrients and presence of inhibitors (Xia and Murphy, 2016). That being the case, different strategies are usually followed in order to enhance culture performance and biomass potential, such as dilution with water or other effluents, enrichment of digestates with appropriate macronutrients, trace elements and carbon, mainly in the form of CO2 from biogas, co-cultivation with bacteria, fungi and yeasts, as well as pre-treatment of digestate usually through nitrification, ammonia stripping or struvite precipitation (Gao et al., 2018; Jiang et al., 2018b, 2018a; Marazzi et al., 2017; Posadas et al., 2016; Praveen et al., 2018; Qin et al., 2018; Wang et al., 2019; Zhou et al., 2018). Due to the presence of organic carbon in digested effluents, with COD concentration reaching up to 6.9 g L−1 (Xia and Murphy, 2016), microalgal growth is based on mixotrophy, which is generally far more effective than autotrophic and heterotrophic metabolism (Cheirsilp and Torpee, 2012). However, the recalcitrant organic fraction, along with a low C/N ratio both hamper biomass and lipid production; therefore organic carbon supplementation can significantly increase culture outcomes. Heterotrophic metabolism of Chlorella vulgaris was supported only after glucose or Na-acetate addition in municipal wastewater, resulting in high biomass production and enhanced ammonium removal (Perez-Garcia et al., 2011). Similarly, digested effluents were supplemented with 2 g L−1 glycerol at the late exponential phase of C. vulgaris grown under mixotrophic conditions, reaching the highest biomass and lipid productivity of 0.064 g L−1 d−1 and 20.4 mg L−1 d−1, respectively, compared to other cultivation modes and different carbon feeding strategies (Ge et al., 2018). Also, biomass concentration of Chlorella pyrenoidosa in digested starch wastewater reached 4.03 g L−1 at lab-scale and 3.23 g L−1 outdoors, after the addition of 0.8% glucose, while fructose and galactose could also be used as external carbon sources (Tan et al., 2018a). However, the addition of pure monosaccharides is not considered as a sustainable way to increase biomass yield. Instead, the use of wastewater from food industry lacking growth inhibitors, such as cheese whey, might be a promising option (Girard et al., 2014; Lu et al., 2017). Besides insufficient carbon for microalgal cultivation, digestates might be also poor in appropriate nutrients. Magnesium limitation was observed during cultivation of Scenedesmus sp. in swine digestate, thus Mg2+ addition was needed to achieve almost complete nitrogen removal and a biomass yield of at least 0.33 g L−1, compared to 0.14 g L−1 in plain swine digestate (Bjornsson et al., 2013). Alternatively, increased biomass production and nutrient uptake were observed after enrichment of swine digestate with lake water or digested microalgal biomass (Bjornsson et al., 2013). Furthermore, phosphorus (P) has been previously demonstrated as the limiting nutrient in microalgal cultures based on digestates, as in case of C. pyrenoidosa which biomass concentration in digested swine manure reached 4.81 g L−1

after P addition and lipid accumulation increased from 15.6% to 19.5% (Cheng et al., 2015). Also, P supplementation of Nannochloropsis gaditana cultures in 20% digested activated sludge resulted in biomass productivity of 0.33 g L−1 d−1 and significantly increased nitrogen uptake from 51% to 85% (Sepúlveda et al., 2015). In this regard, the purpose of the present study was to investigate the performance of P. kessleri under organic carbon and nutrient enrichment of digested agro-waste. To this end, the addition of different concentrations of glucose in the cultures and acclimatization of P. kessleri to organic carbon prior to cultivation were first examined. Subsequently, co-addition of organic carbon and magnesium or trace metals was tested, followed by dilution of digestate with cheese whey (CW), with view to providing microalgae with the appropriate elements, decreasing freshwater demand and investigating an environmentally friendly process for digestate valorization. 2. Materials and methods 2.1. Agro-waste digestate Digestate was produced by a two-stage anaerobic reactor, operated at lab-scale under mesophilic conditions, that was treating a mixture of different agro-wastes, including end-of-life (expired) dairy products, swine-, cow- and chicken manure, cheese whey and slaughterhouse wastes. The digested effluent was collected and pre-treated in order to remove solid particles, through consecutive steps of centrifugation at 4500 rpm and filtration with Whatman glass microfiber filters (GF/F), and was kept in the freezer, at −18 °C. Based on its main physicochemical characteristics (g L−1), Chemical Oxygen Demand, COD = 10.693 ± 1.29, Total Carbohydrates (in equiv. glucose) = 0.434 ± 0.03, Total Kjeldahl Nitrogen, TKN = 3.873 ± 0.53, NH3-N = 3.668 ± 0.87, Total phosphorus, Total-P = 0.142 ± 0.01, digestate was diluted at 10% v/v, with three times distilled water, prior to use as a culture medium for microalgae, as previously described (Koutra et al., 2017). 2.2. Microalgal cultures and experimental conditions The microalgal species used in this study was Parachlorella kessleri, belonging to Chlorophyta (green algae), that has been previously described as a promising microalga in terms of biomass and lipid production, as well as valorization of digested effluents (Koutra et al., 2018b; Li et al., 2013). P. kessleri (211–11 g) was obtained from the SAG Culture Collection (University of Göttingen) and was aseptically preserved in BG-11 medium (73816, Sigma-Aldrich), supplemented with trace metal solution (Mix A5 with Co 92949, Sigma-Aldrich). Microalgal cultures were kept at room temperature, under continuous fluorescent light of about 25 μmol photons m−2 s−1. Prior to experimentation different types of pre-cultures were produced, depending on the aims of the study. In particular, pre-cultures were obtained from cultivation in BG-11 supplemented with trace metals, at 25 °C, under constant illumination of 200 μmol photons m−2 s−1 provided by white fluorescent lamps placed below the cultures and aeration of about 0.5 L min−1. In order to test the effect of acclimatization of P. kessleri on an organic carbon source, 5 g L−1 glucose was added in the BG-11 medium. After seven days of cultivation, the produced biomass was recovered through centrifugation at 4500 rpm, after which the supernatant was discarded and biomass was concentrated, and inoculated into photo-bioreactors (PBRs) after determining chlorophyll concentration. For the present study, cylindrical, continuously stirred glass (Schott) PBRs, with working volume of 1L, were operated at batch mode for 18 days. LED stripes around the PBRs provided constant illumination of 370 μmol photons m−2 s−1. Temperature was kept at 25 ± 2 °C and pH at 8 ± 0.5 (close to the original pH value of digestate that is equal to 8.6) with the addition of HCl 1 N, through an automatic controller (sc200, Hach). Cultures were constantly aerated 2

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with 0.5 L min−1 and agitated with a magnetic stirrer, at 250 rpm. In the first place, P. kessleri grown in BG-11 medium (autotrophic inoculum) was cultivated in 10% (v/v) digestate, as well as in digestate supplemented with 0.5 and 5 g L−1 glucose, respectively, as presented in the Graphical Abstract. The same experiments were repeated after pre-cultivation in BG-11 medium supplemented with 5 g L−1 glucose (mixotrophic inoculum), in order to test possible acclimatization effects. Subsequently, co-addition of 5 g L−1 glucose and 0.5 g L−1 MgSO4, as well as 5 g L−1 glucose and trace metal solution (Mix A5 with Co) were examined for macro or micro-nutrients limitation, respectively. Lastly, 5 g L−1 glucose addition was substituted with equivalent addition of cheese whey, based on its carbohydrate content in equivalent glucose (39 g L−1). All the scenarios tested were carried out in duplicate. At specific time intervals, samples were taken from the PBRs and biomass concentration and chlorophyll content were determined. Chl a fluorescence emission was analyzed through JIP-test and O2 evolution rate was also measured. Remediation of digestate was assessed through determination of COD, carbohydrates, NH3-N and TP throughout cultivation and lipid content of the produced biomass was analyzed at the end of the experiments.

the liquid fraction after biomass removal through filtration (Whatman, GF/F). COD, TKN, NH3-N and Total- P were measured according to Standard Methods (APHA, 1995). Carbohydrates were determined in equivalent glucose according to Joseffson (1983). Concentration of Mg2+ was measured by Atomic Absorption Spectrophotometry (Perkin Elmer, AAnalyst 300). 2.6. Fatty acid methyl esters (FAMEs) analysis For determination of the lipid content and FA profile of the biomass produced, microalgae were harvested at the end of the experiments through centrifugation, and biomass was washed through resuspension in distilled water followed by centrifugation and discard of the supernatant, and then was freeze-dried (Telstar, LyoQuest). Subsequently, FAs were converted into FAMEs following one-step in-situ transesterification, as previously described (Levine et al., 2010). The produced FAMEs were analyzed on a GC (Agilent Technologies, 7890A) equipped with a flame ionization detector (FID) and a capillary column (DB–WAX, 10 m × 0.1 mm × 0.1 μm). FAME Reference Standard (FAMQ-005, AccuStandard) and Internal standard (C17:0, Sigma) were used. Briefly, oven temperature was increased from 40 °C, held for 0.5 min, to 195 °C at a rate of 25 °C min−1, from 195 °C to 205 °C at a rate of 3 °C min−1 and from 205 °C to 230 °C, held for 4 min, at a rate of 8 °C min−1. Helium was used as carrier gas, with an average velocity of 30.34 cm s−1 and both injector and detector temperature was set at 250 °C. Final lipid concentration was calculated by multiplying final biomass concentration (mg DW L−1) with % lipids (% FAs per DW, w/ w) determined at the end of each experiment.

2.3. Determination of microalgal growth Microalgal biomass concentration was measured as dry cell weight (DW), according to Standard Methods for estimation of Total Suspended Solids (TSS) (APHA, 1995). Specific growth rate (μ, day−1) and maximum specific growth rate (μmax, day−1) related to the exponential growth phase, were estimated by the slope of the logarithmic plot of DW versus cultivation time, after subtraction of DW values recorded in control experiments (without inoculation).

3. Results and discussion

2.4. Chlorophyll determination, JIP-test and O2 evolution

3.1. Microalgal growth

Chlorophyll was extracted with 85% methanol, containing 1.5 mM sodium ascorbate, at 60 °C for 20 min. The extract was collected by 15 min centrifugation at 4500 rpm and then analyzed using a UV–Vis spectrophotometer (Cary 50, Varian) at 650 and 664 nm. The sum of chlorophyll a and b was calculated based on the following equation (Porra, 1990):

P. kessleri was cultivated in 10% (v/v) digestate for 18 days, without and after the addition of an organic carbon source and nutrients. As presented in Fig. 1a, in 10% digestate biomass concentration reached 0.86 g L−1, with a μmax of 0.54 d−1. Addition of 0.5 g L−1 glucose resulted in higher biomass concentration of 1.09 g L−1, while in case of higher glucose concentration (5 g L−1) P. kessleri reached 0.66 g L−1, with a reduced μmax of 0.39 d−1. Control cultures without inoculation, revealed the existence of indigenous microorganisms which however contribute to the total biomass concentration only within the first two cultivation days (Fig. 1b). As expected, the addition of 5 g L−1 glucose increased DW in the control culture up to day 2, since when biomass concentration started leveling off. In case P. kessleri was pre-cultured in BG-11 supplemented with 5 g L−1 glucose, higher biomass production was observed during cultivation in glucose-supplemented digestate instead of digestate alone (Fig. 1b), however the recorded values were in all cases lower than those of the autotrophic inoculum; thus an emphasis is given on those cultures for further investigation. For the autotrophic inoculum, between the two glucose concentrations tested, 0.5 g L−1 was more effective in terms of biomass production than the addition of 5 g L−1. Chandra et al. (2014) also observed higher biomass production in case of 0.5 g L−1 glucose addition in a microalgal consortium cultivated in a synthetic wastewater, compared to organic carbon levels up to 3 g L−1 which inhibited growth. In contrast, addition of glucose up to 10 g L−1 enhanced growth of two marine species, Chlorella sp. and Nannochloropsis sp., though neither culture could increase more with further glucose supplementation (Cheirsilp and Torpee, 2012). In terms of possible microalgal acclimatization, no advantage was observed in case of pre-cultivation with glucose. However, organic carbon acclimatization could be performed for a rather longer period of time, until stable characteristics of repeated cultures are observed, as described in the literature (Liu et al., 2008). Furthermore, concurrent addition of 5 g L−1 glucose and 0.5 g L−1 MgSO4 or trace elements were tested (Fig. 1a). In case of

Chl a + b (μg mL−1) = 22.73 A650 + 3.84 A664 Subsequently, % relative chl content (C/C0) was estimated at microalgal DW basis. Induction kinetics of prompt chlorophyll a fluorescence was recorded by a portable fluorimeter (Handy-PEA, Hansatech Instruments Ltd. King’s Lynn Norfolk, UK) after dark adaptation of samples for 15 min. Samples were illuminated with actinic light of 3000 μmol photons m−2 s−1 and the induced chl a fluorescence emission was analyzed through JIP-test (Strasser et al., 1995). Parameters deduced by the JIP-test analysis were, maximum quantum yield for primary photochemistry (Fv/Fm), quantum yield for reduction of end electron acceptors at the PSI acceptor side (φR0), effective antenna size of an active reaction center, RC, (ABS/RC), density of active reaction centers (RC/CS), effective dissipation in an active RC (DI0/RC) and performance index for energy conservation from photons absorbed by PSII antenna to the reduction of QB (PIABS). Concerning data analysis, average ± SD values were derived from ten measurements and two independent replicate experiments, respectively. The net photosynthetic rate (Pm) and the respiration rate (Rd) of cell suspensions were determined using a Hansatech Oxygraph (Hansatech Instruments, Norfolk, UK), at 25 °C with actinic light 1400 μmol m−2 s−1 of photosynthetically active radiation (PAR) and chlorophyll concentration of 5–10 μg ml−1. Each treatment was carried out in duplicate. 2.5. Organic carbon and nutrient removal efficiency Physico-chemical characteristics of digestate were determined in 3

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Cultivation time (d) Fig. 1. Microalgal growth, expressed as g dry cell weight L−1, under different cultivation conditions. In Fig. 1a, autotrophic inocula were used, while in Fig. 1b, glucose addition (0.5 and 5 g L−1) was tested after acclimatization of P. kessleri in 5 g L−1 glucose (mixotrophic inoculum). Furthermore, 10% (v/v) digestate and supplementation of 5 g L−1 glucose were incubated without prior inoculation (control tests) (b).

remained green with final concentration similar to 10% (v/v) digestate and 0.5 g L−1 glucose addition. In case of digestate supplementation with CW, the highest chl content was observed (Fig. 2a), with continuously increasing values after day 2, as a result of biomass production. In terms of biomass chl content, an increase in % chl content up to 1.7 times was observed upon inoculation of P. kessleri in 10% (v/v) digestate, followed by a gradual decrease (Fig. 2b). Similar behavior was also demonstrated in case of 0.5 g L−1 glucose addition, in contrast to 5 g L−1 glucose, as well as trace metals co-addition, where chl content totally diminished after day 6. However, such a decrease in pigment content was not observed when MgSO4 was added. Lastly, the presence of CW caused an initial decrease in biomass chl content, followed by a gradual increase throughout cultivation. Chl reduction with increasing levels of glucose has been previously observed, while cultures turned to yellowish as a result of organic carbon addition (Cheirsilp and Torpee, 2012; Cho et al., 2011). Similarly, mixotrophic and heterotrophic cultures of C. vulgaris turned to yellow in glycerol supplemented digestate, probably attributed to a decrease or degeneration of chloroplasts as a result of autotrophic metabolism limitation (Ge et al., 2018). Besides the effect of organic carbon on pigment content, less green cultures are common under nutrient depletion. Such chl decrease has been described in case of P. kessleri cultivated under limited concentration of major elements (N, P, S) and ions such as Mg2+, due to disturbances in anabolic reactions (Fernandes et al., 2013). Furthermore, Mg2+ deficiency is more severe under conditions that require increased chlorophyll synthesis, e.g. low light intensity (Jaime et al., 1998). However, based on the results of the present study, Mg2+ addition inhibited pigment reduction, even in the presence of organic carbon, where metabolism is not primarily dependent on photosynthetic pigments. Concerning CW addition, the presence of equivalent concentration of organic carbon, despite an initial decrease in chl content between days 0–2, resulted in gradual chl content increase rather than pigment depletion, that might be attributed to other organic and inorganic constituents present in CW. Similarly, chl content of mixotrophic cultures of C. vulgaris was higher in the presence of nonhydrolyzed CW, compared to glucose and galactose addition, 0.6% over 0.37%, respectively (Abreu et al., 2012).

MgSO4, a high growth rate was initially observed, while biomass concentration reached 1.18 g L−1 after just 6 days of cultivation. The higher values observed under MgSO4 supplementation indicate Mg2+ deficiency in the digestate used for cultivation of P. kessleri. Mg2+ has been long recognized as an essential metal for microalgal growth and photosynthesis, since it is a structural chlorophyll component. In this regard, C. vulgaris failed to grow when cultivated in a synthetic medium deprived of Mg2+ (Ayed et al., 2015). Magnesium supplementation was also critical for growth of Scenedesmus sp. in swine digestate (Bjornsson et al., 2013; Park et al., 2010). In contrast, co-addition of trace metal solution and 5 g L−1 glucose, failed to enhance culture performance, in accordance with previous results showing poor microalgal response to digestate supplementation with trace metals and vitamins, indicative of their sufficient concentration in the medium (Bjornsson et al., 2013). The already high concentration of metals in the present digestate (data not shown), might be an inhibitory factor in case of further increase. Lastly, instead of adding glucose and nutrients, the amount of water used for dilution of digestate was partially displaced with CW, by adding 128 mL of this industrial wastewater, in order to provide a culture medium with similar carbohydrates level as in case of 5 g L−1 glucose addition, as well as with extra macro- and micronutrients present in CW. CW addition was by far superior to all the other tested scenarios, with biomass concentration reaching 2.68 g L−1 within 18 days (Fig. 1a). The growth promoting effect of CW ingredients, including proteins, lipids, minerals such as Ca2+, phosphorus and vitamins, has been also demonstrated by Abreu et al., (2012), who observed the highest productivity of C. vulgaris, 0.75 g L−1 d−1, in hydrolyzed CW instead of sole sugars-added mixotrophic cultures, 0.46 g L−1 d−1. 3.2. Chlorophyll content Chlorophyll was measured in all experiments in which autotrophic P. kessleri inoculum was used (due to their higher biomass concentrations), and it was expressed both as chl concentration (mg L−1) and % relative chl content (C/C0), at a DW basis. Within the first two days of cultivation, an increase in chl concentration (mg L−1) was observed in all cultures, apart from that with the addition of CW (Fig. 2a). After day 2, in case of 10% (v/v) digestate and in the presence of 0.5 g L−1 glucose, chl concentration slightly increased, reaching 5 and 5.98 mg L−1 on day 18, respectively. In contrast, addition of 5 g L−1 glucose brought about a significant decrease in chl content after day 2, with chl values close to zero after day 9 and culture color turning to yellow. The same trend was also observed in case of trace metals addition. However, when 5 g L−1 glucose was accompanied by MgSO4 addition, culture

3.3. Chl a fluorescence analysis and O2 evolution rate Photosynthetic performance of P. kessleri in 10% (v/v) digestate and upon organic carbon and nutrient supplementation was investigated in terms of O2 evolution rate and induction kinetics of prompt chlorophyll fluorescence. Variation of net photosynthetic rate (Pm) and respiration 4

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photochemical activity (Giovanardi et al., 2017).

rate (Rd), as well as of the JIP-test parameters during different cultivation conditions and stages, depicted in Fig. 3, suggest that both external organic carbon and nutrient addition in digestate affects vitality of the photosynthetic apparatus and photosynthetic activity of P. kessleri. Addition of 5 g L−1 glucose caused suppression of photosynthesis but relatively high respiration until glucose consumption. Interestingly, among the treatments, supplementation with CW except of increased respiration rate until day 6, also induced comparatively high photosynthetic activity (Fig. 3a and b). In general, mixotrophy in green microalgae induces down-regulation of photosynthesis usually expressed as decrease of net photosynthetic rate, while at the same time, respiration rate and consequently growth rate and final biomass substantially increase (Liu et al., 2008, Oesterhelt et al., 2007). However, in some cases photosynthesis was also found to benefit from the addition of an external carbon source (Giovanardi et al., 2017). Concerning the maximum quantum yield of primary PSII photochemistry (Fv/Fm), generally used for photosynthetic performance monitoring, the values recorded upon inoculation were slightly higher in 10% (v/v) digestate supplemented with 5 g L−1 glucose, MgSO4, trace metals and CW. On day 6, Fv/Fm remained high in 10% digestate and in the presence of 0.5 g L−1 glucose. However, it should be mentioned that CW addition preserved Fv/Fm values higher than those observed in the presence of 5 g L−1 glucose, MgSO4, and trace metals in which Fv/Fm decreased to extremely low values. At the end of cultivation, decreasing trends reversed in all treatments (Fig. 3c). Analogous decreasing and reversed trends were observed for quantum yield with which electrons reduce the PSI end electron acceptors (Fig. 3d) and for density of active reaction centers (Fig. 3f). On the other hand, addition of 5 g L−1 glucose, MgSO4, and trace metals induced a steep increase in ABS/RC parameter (Fig. 3e) which in conjunction with RC/CS values suggests rather an inactivation of RCs than an increase of antenna size (Strasser et al., 1999). Suppression of photosynthetic apparatus properties (Fv/Fm, φR0, RC/CS) and activity (Pm) caused a considerable increase in dissipation of excess energy processes (DI0/RC) in the presence of 5 g L−1 glucose, MgSO4, and trace metals but not in CW treatment (Fig. 3g). Finally, the potential for energy conservation from excitons to the reduction of intersystem electron acceptors (PIABS) decreased in treatments with CW and high glucose concentration irrespectively of MgSO4 or trace metals presence but reached initial values up to day 18 (Fig. 3h). Recently, the direct influence of external carbon addition in chloroplast function and ultrastructure has been demonstrated (Valverde et al., 2005; Liu et al., 2008; Giovanardi et al., 2017). Preservation of PSII function under mixotrophic cultivation of the green alga Neochloris oleoabundans was attributed to reduced PSII degradation and to a down-regulated chlororespiratory electron recycling, expressed as increased PSII

3.4. Digestate remediation Organic carbon and nutrient removal was between the primary purposes of the present study. As presented in Fig. 4a, without the addition of glucose or CW, concentration of carbohydrates was rather low and no significant variation was observed throughout cultivation. In case of 0.5 g L−1 glucose addition, initial concentration increased to 420 mg L−1, with 89.5% decrease within the first 2 days of cultivation. In case of 5 g L−1 glucose or CW addition, carbohydrates increased by about 10 times and total reduction surpassed 96% in all cases. However, removal rate was higher in case of glucose and MgSO4 co-addition, as well as for CW, as it took only 4 days for almost complete carbohydrates removal, while for glucose-trace metals co-addition and 5 g L−1 glucose alone, 9 and 12 days were needed, respectively. In the control culture, under 10% (v/v) digestate and 5 g L−1 glucose without microalgae, 47% removal was observed, indicating that other microorganisms contribute to total carbohydrates consumption; however microalgae were responsible for more than half of the uptake observed. Similarly, in case of COD (Fig. 4b), 50.3 and 60% removal was observed in 10% (v/v) digestate and after the addition of 0.5 g L−1 glucose, respectively. However, 50% COD was also removed in the control culture (10% digestate without inoculation), and thus, in this case, the organic fraction might not contribute significantly to microalgal growth. When organic carbon increased by 5 g L−1, % COD removal substantially increased, with the lowest uptake of 78% observed in case of glucose and trace metals co-addition, while % removal in the control culture was only 40%. Co-cultivation of C. vulgaris and fungi (Ganoderma lucidum) in digestate with 55% CO2 resulted in 68.3% COD removal (Zhou et al., 2018). When CW was added instead of glucose, COD concentration surpassed that of pure saccharide addition, due to the presence of other organic constituents in CW and total removal reached 90.6%, with 75% consumption within 4 days of cultivation. However, the overall amount of COD could not be removed from the cultures, probably due to the presence of recalcitrant molecules in digestate, as well as microalgal excretions and products of cell lysis, which can also increase COD, as previously described (Wang et al., 2019). In case of NH3-N (Fig. 4c), total removal in 10% (v/v) digestate and after 0.5 g L−1 glucose addition was 45% and 60%, respectively. Under increased organic carbon levels, 78% removal was observed, however about half of the NH3-N concentration was also removed in the control cultures. Therefore, nitrogen is not only consumed by P. kessleri, but other microorganisms, as well as ammonia-stripping due to alkaline pH (equal to 8 ± 0.5), contribute to total % removal. In the presence of 5

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Tan et al. (2018b) observed 87.7% and 87.2% ammonium and phosphorus decrease, respectively, however volatilization of ammonia and phosphorus precipitation had a significant contribution to % total removal, as evaluated in terms of N and P balance in the system. In total however, nutrient removal from digestate in the present study was incomplete, in accordance with previous findings where Scenedesmus sp. growth in digestate terminated due to other limiting factors than N and P depletion (Bjornsson et al., 2013). Concerning magnesium concentration in the cultures and between the different forms it can exist (dissolved, adsorbed or accumulated in the cells) (Ayed et al., 2015), only dissolved Mg2+ was determined. Based on the data presented in Table 1, in digestate alone Mg2+ concentration was rather low and 55%

MgSO4, nitrogen removal exceeded 90%, while addition of CW resulted in total removal of 80.5%. Additionally, total phosphorus removal varied from 55.5 to 81.9% between the tested conditions (Fig. 4d). As can be seen, a relatively high TP percentage was also removed in the control cultures, however % removal was higher in the presence of P. kessleri. Compared to plain digestate, organic carbon addition enhanced TP removal, while in case of CW the culture was significantly enriched with phosphorus, possibly contributing to the highest biomass yield observed. Despite the substantially higher initial TP concentration (53.9 mg L−1) in the CW case, 91.7% of the TP was successfully removed by the microalgal culture. During outdoor cultivation of C. pyrenoidosa in starch digestate supplemented with alcohol wastewater, 6

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Cultivation time (d) Fig. 4. Variation of Carbohydrates (a), Chemical Oxygen Demand, COD (b), NH3-N (c) and Total-P concentration (d) of digestate, during 18 days of microalgal cultivation. Table 1 Variation of Mg2+ concentration on days 0, 6 and 18 of P. kessleri cultivation in digestate alone, after glucose and MgSO4 co-addition and upon cheese whey (CW) supplementation. Cultivation day

10% (v/v) digestate

10% (v/v) digestate + 5 g L−1 glucose + MgSO4

10% (v/v) digestate + CW (equiv. to 5 g L−1 glucose)

Mg2+ Concentration (mg L−1)

0 6 18

0.68 ± 0.01 0.25 ± 0.07 0.30 ± 0.05

43.40 ± 0.85 26.85 ± 3.18 37.96 ± 2.21

6.70 ± 0.59 5.11 ± 0.78 6.34 ± 0.74

% Removal Uptake (mg L−1) Uptake (mg g−1 DW)

0–18

55.35 ± 8.84 0.37 ± 0.07 0.47 ± 0.08

12.47 ± 6.79 5.44 ± 3.05 9.11 ± 0.67

5.44 ± 2.73 0.36 ± 0.15 0.27 ± 0.09

mainly suitable either for biodiesel production or dietary purposes in case of accumulated PUFAs. To this end, % lipid concentration of the produced biomass was determined, along with FA profile. As presented in Table 2, P. kessleri cultivated in 10% digestate was characterized by 14.3% FAs at a DW basis, while the percentage slightly increased with the addition of 0.5 g L−1 glucose. In contrast, high levels of glucose caused a reduction of % FAs below 5%. Similar behavior was also observed in case of the mixotrophic inoculum of P. kessleri. Despite the fact that high C/N ratio can increase lipid production (Cho et al., 2011), organic carbon in the present study decreased microalgal lipids, in agreement with Cheirsilp and Torpee (2012) who observed lower % lipids in marine microalgae with increasing concentration of glucose up to 4 g L−1. Furthermore, % lipid content failed to substantially increase with MgSO4 addition, whilst glucose and trace metals resulted in the lowest % FAs observed. In case of CW, high organic loading also caused a decrease in % FAs, however the percentage of 8.6%, was higher than in case of the addition of pure glucose. Also, in terms of final lipid

total removal was observed within 18 days. After MgSO4 addition, % removal was lower but in terms of magnesium uptake, expressed both as mg L−1 and mg g−1 DW, higher values were recorded. In case of CW addition, Mg2+ concentration was between the previous values, however % removal and uptake was minimum. In C. vulgaris cultures supplemented with Mg2+, total removal was observed in case of low initial levels, in contrast to concentrations up to 46 mg L−1 which inhibited further consumption (Ayed et al., 2015). Interestingly, similar to the present results, Kimura et al. (2019) observed high levels of remaining Mg2+ during cultivation of C. sorokiniana in macrophytes digestate and higher uptake under increasing Mg2+ concentration, suggesting that Mg2+ was only partially available for microalgae, possibly due to the presence of organic molecules that might link Mg2+.

3.5. Lipid composition Microalgae have been long recognized as a valuable source of lipids, 7

8

n.d.: not detected.

C10:0 C14:0 C14:1 C15:0 C15:1 C16:0 C16:1 C17:1 C18:0 C18:1 C18:2 C18:3n3 C20:1 C20:5n3 C22:1n9 Total FAs % Saturated FAs % MUFAs % PUFAs Final lipid concentration (mg L−1)

Fatty acids

n.d. 0.02 ± 0.03 0.01 ± 0.02 n.d. n.d. 3.09 ± 0.32 1.21 ± 0.30 0.76 ± 0.02 0.21 ± 0.10 5.37 ± 0.71 1.46 ± 0.11 1.65 ± 0.02 0.14 ± 0.05 0.38 ± 0.06 n.d. 14.30 ± 1.70 23.20 ± 0.06 52.21 ± 1.51 24.59 ± 1.58 121.64 ± 0.62

10% (v/v) digestate

Autotrophic inoculum

n.d. n.d. 0.05 ± 0.03 0.02 ± 0.03 n.d. 3.14 ± 0.53 1.26 ± 0.49 0.68 ± 0.12 0.33 ± 0.08 5.46 ± 1.24 1.50 ± 0.24 1.98 ± 0.13 0.19 ± 0.11 0.27 ± 0.00 n.d. 14.88 ± 2.94 23.54 ± 0.73 51.01 ± 3.28 25.45 ± 2.56 162.58 ± 30.9

10% (v/v) digestate + 0.5 g L−1 glucose

n.d. n.d. 0.27 ± 0.01 0.84 ± 0.37 0.31 ± 0.14 0.96 ± 0.44 0.28 ± 0.10 0.21 ± 0.16 n.d. 1.15 ± 0.62 0.09 ± 0.12 0.17 ± 0.10 n.d. 0.11 ± 0.08 0.17 ± 0.21 4.55 ± 1.33 41.14 ± 10.46 51.51 ± 5.94 7.35 ± 4.52 28.13 ± 4.85

10% (v/v) digestate + 5 g L−1 glucose

n.d. n.d. 0.13 ± 0.07 0.33 ± 0.47 n.d. 1.34 ± 0.27 0.11 ± 0.04 0.14 ± 0.19 0.02 ± 0.03 2.43 ± 0.60 0.39 ± 0.55 0.45 ± 0.47 n.d. 0.01 ± 0.01 n.d. 5.35 ± 1.10 33.68 ± 19.99 52.09 ± 3.55 14.23 ± 16.44 58.97 ± 29.2

10% (v/v) digestate + 5 g L−1 glucose + MgSO4 n.d. n.d. 0.41 ± 0.44 0.59 ± 0.37 0.18 ± 0.25 0.80 ± 0.78 n.d. n.d. n.d. 0.47 ± 0.36 n.d. n.d. n.d. 0.08 ± 0.12 n.d. 2.53 ± 0.19 54.37 ± 12.09 42.54 ± 16.47 3.09 ± 4.38 10.24 ± 0.12

10% (v/v) digestate + 5 g L−1 glucose + trace metals n.d. 0.04 ± 0.06 0.05 ± 0.07 0.08 ± 0.11 n.d. 1.79 ± 0.25 0.59 ± 0.09 0.53 ± 0.22 0.02 ± 0.03 2.80 ± 0.04 1.18 ± 0.00 1.35 ± 0.01 n.d. 0.14 ± 0.01 n.d. 8.56 ± 0.20 22.51 ± 0.68 46.33 ± 0.15 31.16 ± 0.83 229.6 ± 5.4

10% (v/v) digestate + CW (equiv. to 5 g L−1 glucose) n.d. 0.02 ± 0.02 n.d. 0.03 ± 0.04 n.d. 2.85 ± 0.44 0.63 ± 0.89 0.69 ± 0.11 0.21 ± 0.30 5.29 ± 0.69 1.29 ± 0.17 1.49 ± 0.23 0.12 ± 0.01 0.44 ± 0.10 n.d. 13.06 ± 2.73 23.71 ± 0.81 51.33 ± 2.11 24.95 ± 2.93 38.69 ± 28.0

10% (v/v) digestate

0.03 ± 0.04 n.d. 0.03 ± 0.05 n.d. n.d. 2.94 ± 0.77 0.95 ± 0.33 0.70 ± 0.19 0.18 ± 0.10 4.79 ± 0.74 1.45 ± 0.37 1.57 ± 0.49 0.16 ± 0.10 0.48 ± 0.01 n.d. 13.29 ± 3.17 23.55 ± 1.18 50.12 ± 1.32 26.33 ± 0.14 90.56 ± 12.5

10% (v/v) digestate + 0.5 g L−1 glucose

Mixotrophic inoculum

Table 2 Final lipid concentration expressed as mg L−1 and FA profile, expressed as % FAs per DW (w/w), of P. kessleri under organic carbon and nutrient supplementation of agro-waste digestate.

n.d. n.d. 0.21 ± 0.10 0.92 ± 0.23 0.22 ± 0.13 1.46 ± 0.77 0.39 ± 0.24 0.21 ± 0.16 0.12 ± 0.09 2.86 ± 1.63 0.31 ± 0.44 0.41 ± 0.32 0.06 ± 0.08 0.24 ± 0.26 n.d. 7.40 ± 3.74 23.55 ± 1.18 50.12 ± 1.32 26.33 ± 0.14 38.99 ± 4.05

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concentration, high biomass production in this case resulted in the highest lipid concentration of 229 mg L−1, between all the tested conditions. However, it is worth noting that excess macronutrients in the cultures did not stimulate lipid production, in accordance with literature, since lipids in P. kessleri usually reach up to 10% under optimal conditions (Fernandes et al., 2013). Another strategy that might have enhanced lipid accumulation would be addition of the organic carbon at the late exponential phase instead of the onset of cultivation, as previously described (Ge et al., 2018). Concerning FA profile, based on the FAMEs produced, C16:0, C18:1 and C18:3n3 were the most abundant FAs in 10% digestate, as well as after 0.5 g L−1 glucose addition. MUFAs accounted for more than half of the total FAs amount, while saturated FAs were almost 23%. However, while MUFAs remained at high percentage, saturated FAs increased over PUFAs in case of high organic carbon levels, similarly to previous findings (Chandra et al., 2014; Tan et al., 2018b).

and fed-batch cultivation. Bioresour. Technol. 110, 510–516. https://doi.org/10. 1016/j.biortech.2012.01.125. European Biogas Association (EBA), 2018. EBA Statistical Report 2018. http://europeanbiogas.eu/. Fernandes, B., Teixeira, J., Dragone, G., Vicente, A.A., Kawano, S., Pr, P., 2013. Relationship between starch and lipid accumulation induced by nutrient depletion and replenishment in the microalga Parachlorella kessleri. Bioresour. Technol. 144, 268–274. https://doi.org/10.1016/j.biortech.2013.06.096. Gao, S., Hu, C., Sun, S., Xu, J., Zhao, Y., Zhang, H., 2018. Performance of piggery wastewater treatment and biogas upgrading by three microalgal cultivation technologies under different initial COD concentration. Energy 165, 360–369. https://doi.org/10. 1016/j.energy.2018.09.190. Ge, S., Qiu, S., Tremblay, D., Viner, K., Champagne, P., Jessop, P.G., 2018. Centrate wastewater treatment with Chlorella vulgaris: simultaneous enhancement of nutrient removal, biomass and lipid production. Chem. Eng. J. 342, 310–320. https://doi.org/ 10.1016/j.cej.2018.02.058. Giovanardi, M., Poggioli, M., Ferroni, L., Lespinasse, M., Baldisserotto, C., Aro, E., Pancaldi, S., 2017. Higher packing of thylakoid complexes ensures a preserved Photosystem II activity in mixotrophic Neochloris oleoabundans. Algal Res. 25, 322–332. https://doi.org/10.1016/j.algal.2017.05.020. Girard, J., Roy, M., Ben, M., Gagnon, J., Faucheux, N., Heitz, M., Tremblay, R., Deschênes, J., 2014. Mixotrophic cultivation of green microalgae Scenedesmus obliquus on cheese whey permeate for biodiesel production. Algal Res. 5, 241–248. https://doi.org/10.1016/j.algal.2014.03.002. Jaime, F., Dom, A., Garc, D., Lamela, T., Otero, A., 1998. Induction of astaxanthin accumulation by nitrogen and magnesium deficiencies in Haematococcus pluvialis. Biotechnol. Lett. 20, 623–626. Jiang, Y., Wang, H., Wang, W., Deng, L., Wang, W., 2018a. The optimization of microalgal culturing in liquid digestate after struvite precipitation using gray relational analysis. Environ. Sci. Pollut. Res. 25, 13048–13055. Jiang, Y., Wang, H., Zhao, C., Huang, F., Deng, L., 2018b. Establishment of stable microalgal-bacterial consortium in liquid digestate for nutrient removal and biomass accumulation. Bioresour. Technol. 268, 300–307. https://doi.org/10.1016/j. biortech.2018.07.142. Joseffson, B., 1983. Rapid spectrophotometric determination of total carbohydrates. In: Grasshoff, K., Ehrhardt, M., Kremling, K. (Eds.), Methods of Seawater Analysis. Verlag Chemie GmbH, Berlin, pp. 340–342. Kimura, S., Yamada, T., Ban, S., Koyama, M., Toda, T., 2019. Nutrient removal from anaerobic digestion effluents of aquatic macrophytes with the green alga, Chlorella sorokiniana. Biochem. Enj. J. 142, 170–177. https://doi.org/10.1016/j.bej.2018.12. 001. Koutra, E., Economou, C.N., Tsafrakidou, P., Kornaros, M., 2018a. Bio-based products from microalgae cultivated in digestates. Trends Biotechnol. 36, 819–833. https:// doi.org/10.1016/j.tibtech.2018.02.015. Koutra, E., Grammatikopoulos, G., Kornaros, M., 2018b. Selection of microalgae intended for valorization of digestate from agro-waste mixtures. Waste Manag. 73, 123–129. https://doi.org/10.1016/j.wasman.2017.12.030. Koutra, E., Grammatikopoulos, G., Kornaros, M., 2017. Microalgal post-treatment of anaerobically digested agro-industrial wastes for nutrient removal and lipids production. Bioresour. Technol. 224, 473–480. https://doi.org/10.1016/j.biortech. 2016.11.022. Lam, G.P., Vermuë, M.H., Eppink, M.H.M., Wijffels, R.H., Berg, C., Den, Van, 2017. Multiproduct Microalgae Biore fi neries : from concept towards reality. Trends Biotechnol. 36, 216–227. Levine, R.B., Costanza-robinson, M.S., Spatafora, G.A., 2010. Neochloris oleoabundans grown on anaerobically digested dairy manure for concomitant nutrient removal and biodiesel feedstock production. Biomass Bioenergy 35, 40–49. https://doi.org/10. 1016/j.biombioe.2010.08.035. Li, X., Přibyl, P., Bišová, K., Kawano, S., Cepák, V., Zachleder, V., Čížková, M., Brányiková, I., Vítová, M., 2013. The microalga Parachlorella kessleri–A novel highly efficient lipid producer. Biotechnol. Bioeng. 110, 97–107. https://doi.org/10.1002/ bit.24595. Liu, X., Duan, S., Li, A., Xu, N., 2008. Effects of organic carbon sources on growth, photosynthesis, and respiration of Phaeodactylum tricornutum. J. Appl. Phycol. 21, 239–246. https://doi.org/10.1007/s10811-008-9355-z. Lu, Q., Chen, P., Addy, M., Zhang, R., Deng, X., Ma, Y., Cheng, Y., Hussain, F., Chen, C., Liu, Y., Ruan, R., Paul, S., 2017. Carbon-dependent alleviation of ammonia toxicity for algae cultivation and associated mechanisms exploration. Bioresour. Technol. 249, 99–107. https://doi.org/10.1016/j.biortech.2017.09.175. Marazzi, F., Sambusiti, C., Monlau, F., Cecere, S.E., Scaglione, D., Barakat, A., Mezzanotte, V., Ficara, E., 2017. A novel option for reducing the optical density of liquid digestate to achieve a more productive microalgal culturing. Algal Res. 24, 19–28. https://doi.org/10.1016/j.algal.2017.03.014. Oesterhelt, C., Schmälzlin, E., Schmitt, J.M., Lokstein, H., 2007. Regulation of photosynthesis in the unicellular acidophilic red alga Galdieria sulphuraria †. Plant J. 51, 500–511. https://doi.org/10.1111/j.1365-313X.2007.03159.x. Park, J., Jin, H., Lim, B., Park, K., Lee, K., 2010. Bioresource Technology Ammonia removal from anaerobic digestion effluent of livestock waste using green alga Scenedesmus sp. Bioresour. Technol. 101, 8649–8657. https://doi.org/10.1016/j. biortech.2010.06.142. Perez-Garcia, O., Bashan, Y., Puente, M.E., 2011. Organic carbon supplementation of sterilized municipal wastewater is essential for heterotrophic growth and removing ammonium by the microalga Chlorella vulgaris. J. Phycol. 47, 190–199. https://doi. org/10.1111/j.1529-8817.2010.00934.x. Plana, P.V., Noche, B., 2016. A review of the current digestate distribution models: storage and transport. WIT Trans. Ecol. Environ. 202, 345–357.

4. Conclusions Microalgal cultivation in digestates represents a promising alternative to traditional management of effluents from biogas plants, while concurrently valuable biomass is produced, that can be further valorized through a biorefinery approach. Organic carbon addition is appropriate for mixotrophic growth in digested effluents, however high glucose concentration was ineffective in terms of biomass production of P. kessleri and caused a significant decrease in chl content and % total FAs. In contrast, under CW supplementation both biomass and lipid concentration increased, however further investigation is needed until complete nutrient and COD removal from digestates, along with high lipid productivity are accomplished. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Financial support from the European Commission Project LIFE10 ENV/CY/000721 (DAIRIUS) “Sustainable management via energy exploitation of end-of-life dairy products in Cyprus” is gratefully acknowledged. References Abreu, A.P., Fernandes, B., Vicente, A.A., Teixeira, J., Dragone, G., 2012. Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source. Bioresour. Technol. 118, 61–66. https://doi.org/10.1016/j.biortech.2012.05.055. APHA AWWA WEF, 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed. American Public Health Association, Washington DC, USA. Ayed, H.B.A.B.A., Taidi, B., Ayadi, H., Pareau, D., Stambouli, M., 2015. Effect of magnesium ion concentration in autotrophic cultures of Chlorella vulgaris. Algal Res. 9, 291–296. https://doi.org/10.1016/j.algal.2015.03.021. Barsanti, L., Gualtieri, P., 2018. Is exploitation of microalgae economically and energetically sustainable ? Algal Res. 31, 107–115. https://doi.org/10.1016/j.algal. 2018.02.001. Bjornsson, W.J., Nicol, R.W., Dickinson, K.E., Mcginn, P.J., 2013. Anaerobic digestates are useful nutrient sources for microalgae cultivation : functional coupling of energy and biomass production. J. Appl. Phycol. 25, 1523–1528. https://doi.org/10.1007/ s10811-012-9968-0. Chandra, R., Rohit, M.V., Swamy, Y.V., Mohan, S.V., 2014. Regulatory function of organic carbon supplementation on biodiesel production during growth and nutrient stress phases of mixotrophic microalgae cultivation. Bioresour. Technol. https://doi.org/ 10.1016/j.biortech.2014.02.102. Cheng, J., Xu, J., Huang, Y., Li, Y., Zhou, J., Cen, K., 2015. Growth optimisation of microalga mutant at high CO2 concentration to purify undiluted anaerobic digestion effluent of swine manure. Bioresour. Technol. 177, 240–246. https://doi.org/10. 1016/j.biortech.2014.11.099. Cheirsilp, B., Torpee, S., 2012. Enhanced growth and lipid production of microalgae under mixotrophic culture condition: Effect of light intensity, glucose concentration

9

Bioresource Technology 294 (2019) 122232

E. Koutra, et al.

nitrogen sources on fatty acid contents and composition in the green microalga, Chlorella sp. 227. J. Microbiol. Biotechnol. 21, 1073–1080. https://doi.org/10.4014/ jmb.1103.03038. Tan, X., Zhao, X., Yang, L., Liao, J., Zhou, Y., 2018a. Enhanced biomass and lipid production for cultivating Chlorella pyrenoidosa in anaerobically digested starch wastewater using various carbon sources and up-scaling culture outdoors. Biochem. Eng. J. 135, 105–114. https://doi.org/10.1016/j.bej.2018.04.005. Tan, X., Zhao, X., Zhang, Y., Zhou, Y., Yang, L., Zhang, W., 2018b. Enhanced lipid and biomass production using alcohol wastewater as carbon source for Chlorella pyrenoidosa cultivation in anaerobically digested starch wastewater in outdoors. Bioresour. Technol. 247, 784–793. https://doi.org/10.1016/j.biortech.2017.09.152. Theuerl, S., Herrmann, C., Heiermann, M., Grundmann, P., Landwehr, N., Kreidenweis, U., Prochnow, A., 2019. The Future Agricultural Biogas Plant in Germany, doi: 10. 3390/en12030396. Valverde, F., Ortega, J.M., Losada, M., Serrano, A., 2005. Sugar-mediated transcriptional regulation of the Gap gene system and concerted photosystem II functional modulation in the microalga Scenedesmus vacuolatus. Planta 221, 937–952. https://doi. org/10.1007/s00425-005-1501-0. Wang, L., Addy, M., Liu, J., Nekich, C., Zhang, R., Peng, P., Cheng, Y., Cobb, K., Liu, Y., Wang, H., Ruan, R., 2019. Integrated process for anaerobically digested swine manure treatment 273, 506–514. doi: 10.1016/j.biortech.2018.11.050. Xia, A., Murphy, J.D., 2016. Microalgal Cultivation in Treating Liquid Digestate from Biogas Systems. Trends Biotechnol. 34, 264–275. https://doi.org/10.1016/j.tibtech. 2015.12.010. Zhou, K., Zhang, Y., Jia, X., 2018. Co-cultivation of fungal-microalgal strains in biogas slurry and biogas purification under different initial CO2 concentrations. Sci. Rep. 8, 1–12. https://doi.org/10.1038/s41598-018-26141-w. Zhu, L., Yan, C., Li, Z., 2016. Microalgal cultivation with biogas slurry for biofuel production. Bioresour. Technol. 220, 629–636. https://doi.org/10.1016/j.biortech. 2016.08.111.

Porra, R.J., 1990. A simple method for extracting chlorophylls from the recalcitrant alga Nannochloris atomus, without formation of spectroscopically-different magnesiumrhodochlorin derivatives. Biochimica Biophysica Acta (BBA) – Bioenergetics 1019, 137–141. Posadas, E., Szpak, D., Lombó, F., Domínguez, A., Díaz, I., Blanco, S., 2016. Feasibility study of biogas upgrading coupled with nutrient removal from anaerobic effluents using microalgae-based processes. J. Appl. Phycol. 2147–2157. https://doi.org/10. 1007/s10811-015-0758-3. Praveen, P., Guo, Y., Kang, H., Lefebvre, C., Loh, K., 2018. Enhancing microalgae cultivation in anaerobic digestate through nitrification. Chem. Eng. J. 354, 905–912. https://doi.org/10.1016/j.cej.2018.08.099. Qin, L., Liu, L., Wang, Z., Chen, W., Wei, D., 2018. Efficient resource recycling from liquid digestate by microalgae-yeast mixed culture and the assessment of key gene transcription related to nitrogen assimilation in microalgae. Bioresour. Technol. 264, 90–97. https://doi.org/10.1016/j.biortech.2018.05.061. Salama, E., Kurade, M.B., Abou-shanab, R.A.I., El-dalatony, M.M., 2017. Recent progress in microalgal biomass production coupled with wastewater treatment for biofuel generation. Renew. Sustain. Energy Rev. 79, 1189–1211. https://doi.org/10.1016/j. rser.2017.05.091. Sepúlveda, C., Acién, F.G., Gómez, C., Jiménez-ruíz, N., Riquelme, C., Molina-grima, E., 2015. Utilization of centrate for the production of the marine microalgae Nannochloropsis gaditana. Algal Res. 9, 107–116. https://doi.org/10.1016/j.algal. 2015.03.004. Strasser, R.J., Strivastava, A., Govindjee, 1995. Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. Photochem. Photobiol. 61, 32–42. https://doi. org/10.1111/j.1751-1097.1995.tb09240.x. Strasser, B.J., Dau, H., Heinze, I., Senger, H., 1999. Comparison of light induced and cell cycle dependent changes in the photosynthetic apparatus: a fluorescence induction study on the green alga Scenedesmus obliquus. Photosynth. Res. 60, 217–227. https:// doi.org/10.1023/A:1006247514691. Sunja, C., Lee, D., Luong, T.T., Park, S., Oh, Y., Lee, T., 2011. Effects of carbon and

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