Coupling algal biomass production and anaerobic digestion: Production assessment of some native temperate and tropical microalgae

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<ul><li><p></p><p>b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 4 ) 1e6Available online at wScienceDirect</p><p>http: / /www.elsevier .com/locate/biombioeShort CommunicationCoupling algal biomass production and anaerobicdigestion: Production assessment of some nativetemperate and tropical microalgaeEric Fouilland a,b,*, Christophe Vasseur a,b, Christophe Leboulanger a,b,Emilie Le Floc'h a,b,c, Claire Carre a, Bruno Marty d, Jean-Philippe Steyer e,Bruno Sialve e</p><p>a Ecologie des Systemes Marins cotiers UMR 5119 ECOSYM (Universite Montpellier 2, CNRS, IRD, IFREMER,</p><p>Universite Montpellier 1), Universite Montpellier 2, Place E. Bataillon, CC093, 34095 Montpellier Cedex 5, Franceb Ecologie des Systemes Marins cotiers UMR 5119 ECOSYM (Universite Montpellier 2, CNRS, IRD, IFREMER,</p><p>Universite Montpellier 1), Station Mediterraneenne de l'Environnement Littoral, 2 Rue des Chantiers, 34200 Sete,Francec Centre d'ecologie marine experimentale MEDIMEER (Mediterranean Center for Marine Ecosystem ExperimentalResearch) UMS 3301 (Universite Montpellier 2, CNRS), Station Mediterraneenne de l'Environnement Littoral, 2 Ruedes Chantiers, 34200 Sete, Franced Naskeo Environnement, Avenue des Etangs, Narbonne F-11100, Francee INRA, UR050, Laboratoire de Biotechnologie de l'Environnement, Avenue des Etangs, Narbonne F-11100, Francea r t i c l e i n f o</p><p>Article history:</p><p>Received 10 January 2014</p><p>Received in revised form</p><p>19 June 2014</p><p>Accepted 22 August 2014</p><p>Available online xxx</p><p>Keywords:</p><p>Microalgae</p><p>Digestates</p><p>Wastewaters</p><p>Extreme natural environments</p><p>Chlorophyta</p><p>Cyanobacteria* Corresponding author. Ecologie des SystemUniversite Montpellier 1), Station Mediterran</p><p>E-mail address: eric.fouilland@univ-mon</p><p>Please cite this article in press as: Fouillaassessment of some native temperatej.biombioe.2014.08.027</p><p> 2014 Elsevier Ltd. All rights resea b s t r a c t</p><p>Coupling algal biomass production and anaerobic digestion is one of the most promising</p><p>bioprocesses for economically viable algal production. This study assesses the production</p><p>rates of some native microalgae growing in media supplemented with algal digestate,</p><p>urban wastewater or digested sludge. Native microalgal populations isolated from</p><p>temperate freshwaters (Scenedesmus spp.) and marine ecosystems (Nannochloris spp.) had</p><p>the highest potential production rates (about 100 mg DW L1 d1) with algal digestate at</p><p>about 20% loading ratio. However, no growth was measured for Nannochloris spp., when the</p><p>ammonium concentration exceeded 100 mg L1 although Scenedesmus spp. appeared to be</p><p>tolerant to higher NH4 concentrations. Very low production rates, or no growth, were</p><p>measured when microalgae isolated from high salinity waters (Dunaliella salina, Lyngbya</p><p>aestuarii) were used, suggesting that populations well adapted to extreme environmental</p><p>conditions are not suitable candidates for growing on wastewater or anaerobic digestate.</p><p> 2014 Elsevier Ltd. All rights Marins cotiers UMR 5eenne de l' (E. Fouilland).</p><p>nd E, et al., Coupling aland tropical microalga</p><p>027rved.119 ECOSYM (Universite Montpellier 2, CNRS, IRD, IFREMER,nt Littoral, 2 Rue des Chantiers, 34200 Sete, France.</p><p>gal biomass production and anaerobic digestion: Productione, Biomass and Bioenergy (2014),</p><p></p></li><li><p>b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 4 ) 1e621. Introduction</p><p>Since thepublicationof thepaperbyOswaldandGotaas [1], the</p><p>combination ofwastewater treatment andmicroalgal biomass</p><p>productionhasbeen tested successfully inmanycountries and</p><p>shown to be an efficient means of removing nitrogen (N),</p><p>phosphorus (P) and toxic metallic or organic micropollutants</p><p>[2]. Processing wastewater grown algal biomass by lipid</p><p>transesterification, carbohydrate fermentation or anaerobic</p><p>digestion was recently proposed for sustainable production of</p><p>biofuels [3e5]. Coupling algal biomass production and anaer-</p><p>obic digestion could be the most promising bioprocess as</p><p>anaerobic digestion of a wide variety of microalgae seems</p><p>achievable [6,7]. Anaerobic digestion can convert the whole of</p><p>themicroalgae biomass or organic residue into energy (biogas)</p><p>and fertilizer (digestate). This process can, therefore, provide a</p><p>sustainable source for the energy required from the cultivation</p><p>step through to extraction as well as the nutrients required for</p><p>growth [8]. At lab and pilot scales, the amount of microalgal</p><p>biomass used to feed the digester (loading rate) has been re-</p><p>ported from 0.28 to 22 g VS per liter and awide range of species</p><p>and operating conditions has been used successfully for</p><p>anaerobic digestionwithmethane yields from70 to 587mlCH4</p><p>g VS1 [9]. The methane produced can be used directly for en-ergy production or indirectly for innovative chemical applica-</p><p>tions (e.g. graphene synthesis, [10]).</p><p>A range of valuable microalgae (e.g. Scenedesmus sp.,</p><p>Botryococcus sp., Spirulina sp.) has been reported to growwell in</p><p>urban, industrial and agricultural wastewaters [4,5]. The use</p><p>of native species from both freshwater and marine ecosys-</p><p>tems has recently been tested for local bioresource production</p><p>[11], including the production of lipids [12,13], basedmainly on</p><p>the adaptation of indigenous organisms to local biotic and</p><p>abiotic conditions to limit potential invasive or noxiousFig. 1 e Production rates of microalgae growing on urban,</p><p>industrial or agricultural wastewaters in raceways</p><p>reported in the literature.</p><p>Please cite this article in press as: Fouilland E, et al., Coupling aassessment of some native temperate and tropical microalgaj.biombioe.2014.08.027behavior. It can reasonably be assumed that native species,</p><p>selected from highly productive natural systems, would also</p><p>be good candidates for developing a bioprocess requiring a</p><p>high biomass production rate with nutrient loading from</p><p>treated or untreated wastewater. This is supported by the</p><p>production rates of mixtures of isolated native microalgae,</p><p>being higher than the rates measured for commonly culti-</p><p>vated microalgae species [4] in open ponds (Fig. 1).</p><p>In order to validate this assumption, we tested several</p><p>consortia from temperate and tropical systems considered to</p><p>be naturally very productive (up to 0.6 g L1 of biomass in dryweight). Each consortiumwas dominated by a population of a</p><p>single microalgal species or genus. These microalgae were all</p><p>supplied with wastewater and digestate containing different</p><p>concentrations of ammonium (NH4) and phosphate (PO4</p><p>3).The growth rate, biomass production and production rate</p><p>were measured in batch lab cultures for different loading ra-</p><p>tios for each of the wastewater and digestate sources.2. Methods</p><p>2.1. Selection of microalgal populations</p><p>The microalgal populations studied were obtained from</p><p>samples from diverse ecosystems and were acclimated to</p><p>laboratory culture conditions. The acclimation process led to</p><p>the dominance of a single homogeneous population. We refer</p><p>to i) a group of species (spp.) when no microscopic discrimi-</p><p>nation between species within one genus was possible, and ii)</p><p>a true species when only one taxon was identified in the</p><p>acclimated cultures.</p><p>The population of the freshwater microalgae Scenedesmus</p><p>spp. (Fig. 2A) was originally isolated from a wastewater</p><p>treatment lagoon system near Meze, southern France</p><p>(432503400N, 33602700E). Prior to the experiment, Scenedesmusspp. was cultivated at 20 C under an average photosyntheticphoton flux density (PPFD) of 261 20 mE m2 s1 with a 12:12light:dark cycle in batch cultures using a modified Z8 medium</p><p>where ammonia (supplied asNH4Cl) replaced nitrate (supplied</p><p>as NaNO3) as the sole source of nitrogen and NaH2PO4 was</p><p>added as a source of phosphorus.</p><p>The population of the marine microalgae Nannochloris spp.</p><p>(Fig. 2B) was isolated from Thau Lagoon, southern France</p><p>(432405300N, 34101600E), one of the most intensive oysterfarming sites in France. Prior to the experiment, Nannochloris</p><p>spp. was cultivated under the same temperature and light</p><p>conditions as those for Scenedesmus spp. in batch cultures in</p><p>enriched seawater using a modified Conway medium where</p><p>ammonia (supplied as NH4Cl) replaced nitrate (supplied as</p><p>NaNO3) as the sole source of nitrogen and NaH2PO4 was added</p><p>as a source of phosphorus.</p><p>The population of halophile microalgae Dunaliella salina</p><p>(Fig. 2C) was isolated from temperate salterns in Gruissan,</p><p>southern France (430602700N, 30501100E). Prior to the experi-ment, D. salina was cultivated using the same modified Con-</p><p>way medium and conditions as for Nannochloris spp.</p><p>The saline cyanobacteria Lyngbya aestuarii (Fig. 2D) was</p><p>cultivated from a tropical saline lake (Dziani Dzaha, Mayotte,</p><p>France) in the Indian Ocean (12500 S, 45100 E) where primarylgal biomass production and anaerobic digestion: Productione, Biomass and Bioenergy (2014),</p><p></p></li><li><p>b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 4 ) 1e6 3production reached its highest natural levels (0.8 g Chloro-</p><p>phyll a L1, 20 g O2 m2 d1). This population was acclimated</p><p>at 35 C under an average PPFD of 261 20 mE m2 s1 with a12:12 light:dark cycle during a few days prior to the</p><p>experiment.</p><p>2.2. Growth and production rates of microalgae usingdifferent types of wastewater and digestate supplements</p><p>Urban wastewater was collected from the wastewater treat-</p><p>ment plant in Narbonne (southern France) a few days before</p><p>the experiment and contained 106mgNH4 L1 and 19mg PO4</p><p>3</p><p>L1. The concentrations of NH4 and PO4</p><p>3 were determined byionic chromatography (ICS 3000, Dionex, USA). The secondary</p><p>activated sludge was collected from the same wastewater</p><p>treatment plant and digested (1 m3 continuous digester under</p><p>mesophilic conditions with a loading rate of 1 g L1 d1).The freshwater microalgae Scenedesmus spp. isolated from</p><p>the wastewater treatment lagoon system near Meze was</p><p>cultivated in batch conditions using the modified Z8 medium</p><p>described above in a 56 m2 outdoor open pond at pilot scale.</p><p>The biomass was harvested by settling when the concentra-</p><p>tion reached 0.5 gDW L1. The concentrated biomass of Sce-nedesmus spp. was digested using a 2.5 L digester inmesophilic</p><p>conditions with a loading rate of 1 g L1 d1, prior to the</p><p>experiment presented here. The methane yield was about 143</p><p>(20) mL CH4 gVS1. The liquid phase of both digested sludge</p><p>and algal digestates was separated by centrifugation (5 min,</p><p>18,600 G, 5 C).The liquid digested sludge contained 1360 mg NH4</p><p> L1 and400 mg PO4</p><p>3 L1, while the liquid algal digestates containedbetween 630 and 960 mg NH4</p><p> L1 and 160 mg PO43 L1.Fig. 2 e Microalgae populations isolated from (A) wastewater tre</p><p>waters (Nanochloris spp), (C) Mediterranean salterns (Dunaliella</p><p>aestuarii).</p><p>Please cite this article in press as: Fouilland E, et al., Coupling alassessment of some native temperate and tropical microalgaj.biombioe.2014.08.027Each of the four microalgal populations was inoculated</p><p>into a 96 well microplate using 30 mL of parent culture (10% of</p><p>total well volume) and incubated with different volumes of</p><p>wastewater and digestates (from 0% to 50% of the total well</p><p>volume: loading ratios) in 6 replicates and topped up with</p><p>deionized water. For D. salina only, the experiment was also</p><p>performed with 35 gNaCl L1 in deionized water. The micro-plates were incubated for 7 days at a fixed temperature (21 Cfor temperate populations and 35 C for the tropical popula-tion) and the PPFD was maintained at an average of</p><p>261 20 mE m2 s1 with a 12:12 light:dark cycle using OSRAML18W/954 daylight fluorescent tubes. The absorbance of the</p><p>cultures was chosen as a non-destructive proxy for the</p><p>microalgal biomass. The chlorophyll a b peak absorptionwas targeted in order to reduce the interference caused by</p><p>adding the substrate and to improve the signal:noise ratio. A</p><p>wavelength of 650 nm (10 nm bandpass filter) was chosen,according to previous studies [14]. The linearity of the rela-</p><p>tionship between OD650 and the dry weight was checked. For</p><p>each algal population collected in the parent culture just</p><p>before the inoculation into the microplate, the microalgal</p><p>biomass was diluted with the culture media using from 6 to 8</p><p>different dilution rates ranging from 0 to 100% to determine</p><p>the relationship between OD650 and the dry weight of the</p><p>diluted microalgal biomass. During the microplate incubation</p><p>period, OD650 was measured daily using a Chameleon micro-</p><p>plate reader (Hidex, Turku, Finland) and corrected for the</p><p>absorbance of the digestate measured immediately after</p><p>microplate inoculation. The daily values of the corrected</p><p>OD650 were then converted into dry weight (mg DW L1) using</p><p>the linear relationship determined above. The Verlhurst</p><p>equation was fitted to the biomass measured for the differentatment lagoon system (Scenedesmus spp), (B) marine coastal</p><p>salina) and (D) tropical saline Dziani Dzaha lake (Lyngbya</p><p>gal biomass production and anaerobic digestion: Productione, Biomass and Bioenergy (2014),</p><p></p></li><li><p>Table 1 e Mean (avg) and standard deviation (std) of the maximum growth (d1) and production (mg DW L1) estimated from 6 replicates and derived production rates(mg DW L1 d1) for the four species with different loading ratios (0e50%) of untreated wastewater, organic waste sludge digestate and digestate from Scenedesmus spp.The values for Dunaliella salina in this table were obtained using salted (35 g NaCl L1) waste and digestates, as no growth was reported when using unsalted waste anddigestates. Significant differences between growth and production values measured with loading ratio of 0% of and those measured with a loading ratio greater than 0%are shaded (t-student test, p 0.05). NG: No Growth, NP: Not Performed.</p><p>Dilution(%)</p><p>Untreated wastewaters Digested sludges Digestates from Scenedesmus spp</p><p>Max growthrate (d1)</p><p>Max biomass(mgDW L1)</p><p>Max productionrate (mgDW L1 d1)</p><p>Max growthrate (d1)</p><p>Max biomass(mgDW L1)</p><p>Max productionrate (mgDW L1 d1)</p><p>Max growthrate (d1)</p><p>Max biomass(mgDW L1)</p><p>Maxproduction rate(mgDW L1 d1)avg std avg std avg std avg std avg std avg std</p><p>Scenedesmus spp 0 1.84 0.31 33 1 65 1.56 0.05 22 0 51 1.50 0.13 78 1 66</p><p>2.5 1.11 0.02 101 4 55 0.89 0.05 101 1 45 1.07 0.01 123 2 58</p><p>5 1.78 0.31 44 1 68 0.71 0.03 146 2 43 0.83 0.06 169 3 55</p><p>7.5 1.17 0.20 101 4 60 0.77 0.04 112 2 40 0.77 0.05 236 5 61</p><p>17.5 0.66 0.08 112 4 34 NG NG NG 0.73 0.03 485 9 99</p><p>25 0.93 0.21 55 2 36 NG NG NG 0.61 0.02 723 23 115</p><p>37.5 0.58 0.10 101 8 29 NG NG NG 0.54 0.03 508 27 76</p><p>50 0.79 0.098 180 9 52 NG NG NG 0.42 0.03 282 27 37</p><p>Nannochloris spp 0 0.49 0.05 159 10 27 0.69 0.10 28 1...</p></li></ul>


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