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    psis micthane pefciencause onaerob

    hermophilic reactor produced 48% higher methane yield (220 L CH4 kg VS)(149 L CH kg1 VS). The thermophilic reactor was apparently inhibited due

    typically performed using solvents from dried or wet algalbiomass, the algal residue representing most of the total biomass.Utilizing this by-product (referred to as residue cake), in anenvironmentally sound way is essential to the sustainability and

    rtilizers or circu-tic hydrotg conceptus from alg

    idues (Frank et al., 2013). The advantages of AD are relsimple and mature technology and no need for high procesperature or pressure. The major challenges of AD are fugitiveane emissions, relatively long treatment time and possibleinstability of the AD process. Indeed, decreased methane yield oreven the failure of AD process may arise from inhibition, whichis caused by excess ammonia, high salt concentration, or difcultdegradability of algae (Yen and Brune, 2007; Sialve et al., 2009).

    An optimal substrate C/N ratio for AD of algae is suggested torange from 12 to 30 (Yen and Brune, 2007; Ehimen et al., 2011);

    Corresponding author. Tel.: +358 50 331 0308.E-mail addresses: viljami.kinnunen@tut. (H.V. Kinnunen), perttu.koskinen@

    nesteoil.com (P.E.P. Koskinen), jukka.rintala@tut. (J. Rintala).1 Tel.: +358 50 458 2917.

    Bioresource Technology 155 (2014) 314322

    Contents lists availab

    Bioresource T

    els2 Tel.: +358 40 506 2425.The biofuel industry seeks sustainable, new, non-food feedstocks.In recent years, microalgae have often been noted as a potentialfeedstock to produce biofuels. Prior to rening renewable dieselfrom algae, oil must be extracted from the algal cells. This is

    AD digestate, which can be further rened as felated back to algae cultivation. Recently, catalygasication (CHG) has shown another promisinduce fuel gas and recover nitrogen and phosphor0960-8524/$ - see front matter 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.12.115hermalto pro-al res-ativelys tem-meth-Keywords:Anaerobic digestionMicroalgal residuesAmmonia inhibitionBiogas

    4

    to ammonia with organic loading rate (OLR) of 2 kg VS m3 d1 (hydraulic retention time (HRT) 46 d),whereas the mesophilic reactor performed with OLR of 3 kg VS m3 d1 (HRT 30 d). Algal salt contentdid not inhibit digestion. Additional methane (1833% of primary digester yield) was produced during100 d post-digestion.

    2014 Elsevier Ltd. All rights reserved.

    1. Introduction

    Currently, vegetable oils, waste oils, and animal fat wastes areused as raw materials for production of bio-based diesel fuels.

    protability of algal biodiesel production (Chisti, 2007; Sialveet al., 2009). Anaerobic digestion (AD) is one means of recoveringenergy and nutrients from the residue cake. In addition to pro-duced methane gas, nutrients in the substrate are retained in theAvailable online 5 January 2014extracted residue cake, a tthan a mesophilic reactorMethane production from NannochloroWet extracted alga had superior biome Anaerobic digestion at 55 C was more Thermophilic process was inhibited be Salt from marine alga did not inhibit a

    a r t i c l e i n f o

    Article history:Received 8 November 2013Received in revised form 23 December 2013Accepted 27 December 2013roalga residue was studied.otential compared with dry extracted.t compared with digestion at 35 C.f ammonia with low loading.ic digestion.

    a b s t r a c t

    This paper studies methane production using a marine microalga, Nannochloropsis sp. residue frombiodiesel production. Residue cake from Nannochloropsis, oils wet-extracted, had a methane potentialof 482 L CH4 kg1 volatile solids (VS) in batch assays. However, when dry-extracted, the methanepotential of residue cake was only 194 L CH4 kg

    1 VS. In semi-continuous reactor trials with dry-1h i g h l i g h t sMesophilic and thermophilic anaerobic laof Nannochloropsis microalga residues

    H.V. Kinnunen a,, P.E.P. Koskinen b,1, J. Rintala a,2aDepartment of Chemistry and Bioengineering, Tampere University of Technology, P.O. BbResearch and Development, Neste Oil Corporation, P.O. Box 310, 06101 Porvoo, Finland

    journal homepage: www.oratory-scale digestion

    541, 33101 Tampere, Finland

    le at ScienceDirect

    echnology

    evier .com/locate /bior tech

  • e Telower values would make the process susceptible to ammonia inhi-bition. For algae, much lower C/N ratios of 5.310.2 (Elser et al.,2000; Yen and Brune, 2007; Ehimen et al., 2009) and 4.45.6 (Ehi-men et al., 2009; Park and Li, 2012) have been reported for freshbiomass and residue cakes, respectively.

    Avoiding fresh water consumption may be crucial for algal bio-fuel sustainability (Pate et al., 2011). When cultivated in marinewater, algal biomass and also algal residues contain sea salt, whichis known to have an inhibitive effect on AD (Lefebvre et al., 2007;Chen et al., 2008). Indeed, salt concentration of marine-cultivatedDunaliella tertiolecta has been found to inhibit AD (Lakaniemiet al., 2011). However AD has been demonstrated with marine algaTetraselmis and saline wastewaters (Asinari Di San Marzano et al.,1982; Lefebvre et al., 2007). Not only affecting on AD process, saltcould also limit the use of digestate as a plant fertilizer. As lowsodium concentration as 500 mg L1 in digested municipal solidwaste can be phytotoxic (McLachlan et al., 2004). For comparison,the sodium concentration of sea water is approximately11000 mg L1.

    Numerous studies on AD of algae have been completed, butexperimental investigations into methane production from algalresidues are scarce. Methane production from algae in continuousor semi-continuous reactors has been studied using an organicloading rate (OLR) between 0.01 (De Schamphelaire and Verstraete,2009) and 6 kg volatile solids (VS) m3 d1 (Yen and Brune, 2007;Park and Li, 2012). The OLRs at the high end of the range were re-ported to lead to overloading of the process (Yen and Brune, 2007;Ehimen et al., 2009; Park and Li, 2012). Hydraulic retention time(HRT) is an important factor, since algae degrade slowly. Basedon the literature and experiments with semi-continuous com-pletely stirred tank reactors (CSTR), Ras et al. (2011) found thatmethane yield from algae increased with increasing HRT, up toan HRT of 30 days. Beyond this HRT, the methane yield did not in-crease, meaning either that these algae are not further degradableor that degradation needs considerably more time. AD of algae in athermophilic process has been reported to produce higher meth-ane yields than in mesophilic digestion (Golueke et al., 1957;Zamalloa et al., 2012). However, this may be species-related, asnoted by Zamalloa et al. (2012), who found no difference in biogasyields between thermophilic and mesophilic processes whendigesting marine alga Phaeodactylum tricornutum, but about 24%greater biogas yield from freshwater alga Scenedesmus obliquus ina thermophilic process.

    Ehimen et al. (2009, 2011) studied methane production poten-tials and digestion of fresh water Chlorella residue after oils wereextracted, using batch assays and semi-continuously fed reactors.These authors concluded that HRT and OLR are signicant variablesaffecting methane production. They also found that some solvents,like the chloroform used for extraction, may inhibit the anaerobicprocess. Park and Li (2012) reported that co-digestion of Nanno-chloropsis salina residues with grease waste increased both meth-ane yield and allowed higher OLR compared to digestion of thesesubstrates alone. Recently, Keymer et al. (2013) found that theoil extraction process itself can increase methane yield from Scene-desmus-dominated algal biomass.

    The objective of this study was to evaluate the feasibility of asemi-continuous AD process in recovering energy, in the form ofmethane, from a marine green microalga, Nannochloropsis sp. resi-due cake. Two Nannochloropsis residue cakes were tested in biom-ethane potential batch assays, one dry-extracted (referred to asN1) and the other wet-extracted (referred to as N2). Dry-extractedresidue cake (N1) alone was further studied in mesophilic andthermophilic CSTRs. OLRs were increased and HRTs decreased

    H.V. Kinnunen et al. / Bioresourcstepwise during the 5-month reactor trial to determine feasibleloading potentials and to monitor the role of ammonia and/or saltinhibition.2. Methods

    2.1. Feeds and inocula

    Nannochloropsis sp. residue cake was used after extraction ofoils as a feed in AD. The algal biomass was cultivated in open mar-ine water ponds (at Israel), mixed constantly with paddle wheels.Algal biomass may contain small traces of other algae species.Fig. 1 shows a ow chart of materials and processes used in thisstudy. Methane production potential was determined in batch as-says from two residue cakes, oils extracted by dry (N1) or wetextraction (N2), but only N1 was used in semi-continuous reactortrials. Part of both N1 and N2 biomasses was rinsed to reduce thesalt concentration.

    2.1.1. Oil extraction and rinsingExtraction of oils from air-spray dried algal biomass (N1) was

    performed via methanol and hexane extraction. Algal biomasswas rst treated with methanol (10 kg MeOH per 1 kg algal bio-mass as dry matter) in a column, and methanol was circulatedthrough it with six separate solvent batches (600 L h1 1 h eachrinse) at room temperature. After this, biomass was treated 4 timeswith technical grade hexane (6 kg hexane per 1 kg algal biomass asdry matter) at room temperature. Then, biomass was separated byltration and solvent was removed under a hood and nally withvacuum over distillation in laboratory at temperature lessthan 100 C. In wet extraction (N2), ethanol was rst added towet algal biomass (15% dry matter content, 23 kg EtOH per 1 kgalgal biomass as dry matter) and part of the

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