Applied Energy 92 (2012) 733–738

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Applied Energy

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Anaerobic digestibility of Scenedesmus obliquus and Phaeodactylum tricornutumunder mesophilic and thermophilic conditions

Carlos Zamalloa, Nico Boon, Willy Verstraete ⇑Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent, Belgium

a r t i c l e i n f o

Article history:Received 4 April 2011Received in revised form 9 August 2011Accepted 10 August 2011Available online 17 September 2011

Keywords:Anaerobic digestionMicroalgaePhotobioreactorBiomethane potential (BMP)Biogas

0306-2619/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.apenergy.2011.08.017

⇑ Corresponding author. Address: Ghent Universityneering, Laboratory of Microbial Ecology and Technol653, B-9000 Gent, Belgium. Tel.: +32 (0) 9 264 59 76

E-mail address: Willy.Verstraete@UGent.be (W. VeURL: http://www.LabMET.UGent.be (W. Verstraete

a b s t r a c t

Two types of non-axenic algal cultures, one dominated by the freshwater microalgae Scenedesmus obli-quus and the other by the marine microalgae Phaeodactylum tricornutum, were cultivated in two typesof simple photobioreactor systems. The production rates, expressed on dry matter (DM) basis, were inthe order of 0.12 and 0.18 g DM L�1 d�1 for S. obliquus and P. tricornutum respectively. The biogas poten-tial of algal biomass was assessed by performing standardized batch digestion as well as digestion in ahybrid flow-through reactor (combining a sludge blanket and a carrier bed), the latter under mesophilicand thermophilic conditions. Biomethane potential assays revealed the ultimate methane yield (B0) of P.tricornutum biomass to be about a factor of 1.5 higher than that of S. obliquus biomass, i.e. 0.36 and0.24 L CH4 g�1 volatile solids (VS) added respectively. For S. obliquus biomass, the hybrid flow-throughreactor tests operated at volumetric organic loading rate (Bv) of 2.8 gVS L�1 d�1 indicated low conversionefficiencies ranging between 26–31% at a hydraulic retention time (HRT) of 2.2 days for mesophilic andthermophilic conditions respectively. When digesting P. tricornutum at a Bv of 1.9 gVS L�1 d�1 at eithermesophilic or thermophilic conditions and at an HRT of 2.2 days, an overall conversion efficiency of about50% was obtained. This work indicated that the hydrolysis of the algae cells is limiting the anaerobic pro-cessing of intensively grown S. obliquus and P. tricornutum biomass.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Biogas production from biomass is attracting increased atten-tion worldwide. Biogas can be made from a wide range of biomasscrops, as well as from a wide range of solid or liquid residues. How-ever, the main problems of the use of photosynthetic terrestrialcrops are their availability and the fact that they compete with ara-ble land for food crops [1,2]. Thus, alternative photosyntheticorganisms, for example microalgae, could be a good alternativeas biomass feedstock for energy generation. The advantages of mic-roalgae biomass over terrestrial crops include: high areal produc-tivities, the use of non-productive and/or non-arable land, avariety of water sources can be used (fresh, saline, brackish orwastewater) and herbicides or pesticides are no longer required[1,3]. However, the major disadvantage is that the cost to growand to dewater the biomass is not competitive with other kindsof biomass [4].

Microalgae have the potential to be used as a source of oil toproduce biofuel and as a source of biomass to produce biogas

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, Faculty of Bioscience Engi-ogy (LabMET), Coupure Links; fax: +32 (0) 9 264 62 48.rstraete).).

through anaerobic digestion [2,5,6]. The anaerobic digestion (AD)process converts organic material into biogas, mainly containingmethane and carbon dioxide, which could be used for energy pro-duction. Additionally, microalgae seem to be a good candidate forbiogas production as they contain relatively high levels of lipid,starch and protein (up to 70%, 50% and 50% on dry weight, respec-tively) while lignin is absent [2,5,7]. Although the use of microal-gae as biomass source for anaerobic digestion has been studiedbefore [8–10], the majority of research was performed using a con-tinuous stirred-tank reactor arrangement for continuous produc-tion of biogas from algae. High rate anaerobic reactors, such asupflow anaerobic sludge blanket (UASB), anaerobic filter (AF) andanaerobic membrane bioreactor (AnMBR), were proposed as analternative to directly digest microalgae biomass without the needto completely dewater the biomass [11]. By using these reactors,the cost associated with dewatering can be decreased and the solidretention time can be increased.

In this study, two types of microalgae cultures, respectivelydominated by Scenedesmus obliquus and Phaeodactylum tricornu-tum were cultivated in two types of photobioreactors to producealgae biomass. The biomethane potential (BMP) of the two typesof biomass, was determined by means of batch assays. As a refer-ence, the BMP of commercial capsules of the cyanobacteriumSpirulina platensis was also determined. Furthermore, we investi-gated the performance of digestion of algae biomass in hybrid

Table 1Characteristics of algae biomass used in the experiments.

Parameter Spirulinaplatensis(n = 6)

Scenedesmusobliquus(n = 20)

Phaeodactylumtricornutum(n = 20)

734 C. Zamalloa et al. / Applied Energy 92 (2012) 733–738

flow-through reactors at mesophilic and thermophilic conditionsat a moderate organic loading rate (Bv). This study is the first at-tempt to valorise by anaerobic digestion microalgae biomass up-concentrated to the 1.9–2.8 gVS L�1 d�1 range at a low hydraulicretention time (HRT).

Total solids, TS (%) w/w 96.4 ± 3.4 23.9 ± 5.1 16.6% ± 2.9Volatile solids, VS (%-TS) 93.3 ± 2.2 71.8 ± 10.9 82.7 ± 5.1Ash (%-TS) 6.7 ± 2.2 31.2 ± 22.3 17.3% ± 5.1Chemical oxygen demand,

COD (g O2 g�1 TS)1.1 ± 0.04 1.1 ± 0.3 1.3 ± 0.3

Total nitrogen, TN (mg g�1 TS) 106 ± 4 60 ± 28 79 ± 22Total phosphorous, TP

(mg g�1 TS)8.7 ± 3.7 3.6 ± 2.4 8.1 ± 2.9

COD/N ratio 10.3 ± 0.6 15.6 ± 7.4 13.4 ± 3.4COD/VS ratio 1.2 ± 0.04 1.3 ± 0.3 1.4 ± 0.2TS/VS 1.1 ± 0.003 1.4 ± 0.2 1.2 ± 0.1

2. Material and methods

2.1. Algae strains

Two algae strains were used in this study, a freshwater algaS. obliquus (UTEX 2630) and a marine alga P. tricornutum(CCAP1055/1) obtained from the culture collection of the Labora-tory of Protistology and Aquatic Ecology, Ghent University. Strainswere supplied solely with NaNO3–N and KH2PO4–P in the order of12.3 mg N L�1 and 1.1 mg P L�1. Micronutrients and vitamins werenot supplied. The marine strain was cultivated in artificial seawater (Instant Ocean, Spectrum Brands, Atlanta, US) at a concen-tration of 25 g L�1of salinity. In addition, commercial cyanobacte-rium S. platensis capsules (Deba Pharma, Belgium) were used forthe standard batch test as a benchmark. This strain was chosen be-cause it is the most commercially produced organism [12].

2.2. Photobioreactors (PBR) and operation of microalgae cultures

Two different PBR systems were used in order to produce suffi-cient fresh biomass for feeding the anaerobic digesters: a serpen-tine PBR for growing P. tricornutum and six bubble column PBRfor growing S. obliquus. Both were operated under non-axenic con-ditions. The serpentine PBR consisted of a glass container with a to-tal volume of 80 L (working volume of 65 L) and a transparent tubeof 19 mm diameter and a length of about 80 m. The broth wasrecirculated using a peristaltic pump (Flowtec, Italy) at a flow rateof 10 L min�1. The PBR were installed in a greenhouse at 25 ± 2 �Cwith a light intensity of approximately �100 lmol m�2 s�1 for P.tricornutum and �250 lmol m�2 s�1 for the S. obliquus strain atthe surface of the PBR. Light was provided by continuous (24/24)metal halide lamps (Philips HPI-T) for the S. obliquus and by fluore-scents (Sylvania F36 W/GRO) for P. tricornutum. The light intensity(400–700 nm) was measured by a Quantum PAR light meter (Spec-trum Technologies, Illinois, US). The pH of the serpentine PBR wascontrolled at 8 and regulated by on-demand automated CO2 injec-tion (2% v/v). The bubble column PBR had a diameter of 0.2 m, aheight of 1 m and a working volume of 25 L. This PBR was madeof transparent acrylic tube. The pH was not controlled and rangedaround 8–9. Mixing in both PBR was accomplished by bubbling airthrough diffusers at an aeration rate of about 0.5 vvm (volume gasper volume of mixed liquor per minute).

2.3. Harvesting and biomass characterization

Fresh algae biomass was collected every 2 or 3 days, centrifugedwith a Westfalia separator (Model: OTC 3-03-107) operated at10 000 min�1 (about 12 300 g) and manually recovered from theinterior bowl. The concentrated biomass was fed every day to theanaerobic digestion reactors at the respective volumetric organicloading rates (Bv values). Tap water was used to re-suspend thebiomass. Characteristics of the algae biomass are presented inTable 1.

2.4. Determination of biomethane potential (BMP)

Biological methane potential assays were performed in tripli-cate according to Angelidaki et al. [13]. The batch experimentswere performed in 1.15 L glass bottles (Scott AG, Mainz, Germany)

with a working volume of 1 L. Substrate (microalgae biomass) andgranular anaerobic inoculum were added to each bottle. The sub-strate concentration based on volatile solids content (VS) was2 gVS L�1. The inoculum to substrate ratio of 3:1 was used basedon VS content. The inoculum used came from a full-scale anaerobicdigester treating potato-processing wastewater (Mydibel, Bel-gium). The specific methanogenic activity (SMA) of the inoculumwas determined using acetate with a start concentration, ex-pressed on chemical oxygen demand (COD) basis, of 2 gCOD L�1.The SMA value obtained was 0.3 gCOD–CH4 g�1 VSS d�1. The pre-pared bottles were incubated at 33 ± 2 �C. The experiments wereconducted in triplicate for 30 days. The amount of biogas producedwas determined by water displacement of a saturated solution ofhydrochloric acid at pH 2 in calibrated glass cylinders. Assays withinoculum only were used as a control. The methane produced fromthe inoculum (from the control bottles) was subtracted from thesample assays. The biogas values presented are expressed for stan-dard temperature and pressure (STP) conditions (0 �C, 1 atm).Methane production rate constants (k) and ultimate methaneyields (B0) were estimated assuming that degradation of each sam-ple followed a first-order rate of decay [13,14]. Therefore, the pro-duction of methane was assumed to follow the following equation:

B ¼ B0ð1� e�ktÞ ð1Þ

where B is the cumulative methane yield at time t. The values of kand B0 were estimated using a nonlinear regression algorithm avail-able in SigmaPlot 11 (Systat Software Inc.California, US).

2.5. Hybrid flow-through systems

Four lab scale hybrid flow-through reactors were studied; MR1,MR2, TR1, and TR2. MR1 digested S. obliquus biomass while MR2digested P. tricornutum biomass, both operated at a mesophilictemperature (33 ± 2 �C). TR1 digested S. obliquus biomass, whereasTR2 digested P. tricornutum biomass, both operated under thermo-philic conditions (54 ± 2 �C). Both the specific mesophilic (33 �C)and the specific thermophilic (54 �C) temperatures were chosenbecause they are considered the most commonly used in large-scale digesters in the authors’ experience. Biomass was fed to thereactors on a daily basis. Each reactor consisted of one cylindricaltube with a diameter of 5 cm plus a three-phase separator in theupper part (Fig. 1). The total volume of the reactors was 2.3 L witha working volume of 2 L. An upflow velocity of around 1 m h�1 wasprovided continuously by a recirculating pump (Watson Marlow313S and 505S; Watson-Marlow Inc., Massachusetts, US). The bio-gas production was monitored by means of the liquid displace-ment method in airtight calibrated vessels of 10 L (see Fig. 1).The liquid inside the vessel contained water at pH = 2 to preventCO2 from dissolving. To enhance the retention of the algae biomass

Biogas

EffluentInfluent

Influent pump

Recyclepump

Water seal

Biogas collection

Flow throughreactor

Carrier rings(K1, AnoxKaldnes,

600 m2 m-3)

Fig. 1. Scheme of lab-scale experimental set-up of the hybrid flow-though reactor.

C. Zamalloa et al. / Applied Energy 92 (2012) 733–738 735

particles, 150 g of plastic carrier rings (model K1, AnoxKaldnes,Sweden) were added to each reactor as an anaerobic filter. The fourreactors were inoculated with 500 mL (10 gVSS L�1) of granularseed sludge harvested from a full-scale anaerobic digester treatingpotato processing wastewater (Mydibel, Belgium) which served assludge blanket. This inoculum is the same as used in the BMP testsand its methane activity was measured by means of the SMA test.The adaptation of sludge to thermophilic conditions was facilitatedby cross-inoculation during the start-up with non-granular ther-mophilic sludge from an anaerobic reactor digesting a combinationof waste vegetables (Westvleteren, Belgium). The pH was con-trolled to be around 7–7.5 by supplying a buffer (1 g NaHCO3 g�1

COD influent). During the continuous operation, the reactors re-ceived only fresh non-pretreated microalgae biomass and the bio-mass concentrate was directly fed into the reactor. pH,temperature, chemical oxygen demand (COD), volatile fatty acids(VFAs) and biogas production were monitored continuously andreported at standard temperature (0 �C) and pressure (760 mmHg) (STP).

Only during the start-up period (20 days) the flow-throughreactors were fed with acetate and glycerol (50:50 on COD basis)as carbon source. The Bv was increased stepwise by increasingthe COD concentration of the influent from 1 up to 8 gCOD L�1 d�1

for the thermophilic and the mesophilic reactors. The start-upphase ensured that the reactor would be allowed to efficiently con-vert the Bv implemented. Results from the start-up period are notdiscussed.

2.6. Analytical procedures

Liquid samples were taken daily or every second day from thePBR and anaerobic reactors. The n-values reported in the resultssection refers to the number of samples analyzed through time. Al-gal productivity was determined on the basis of dry matter analy-sis (gDM L�1 d�1). The content of DM was determined by drying

the sample at 105 �C for 24 h. For P. tricornutum samples, the saltwas washed out and the DM was determined according to Zhuand Lee [15]. Total solids (TS), volatile solids (VS), volatile sus-pended solids (VSS), total Kjeldhal nitrogen (TKN), total ammonianitrogen (TAN), chemical oxygen demand (COD), total COD, solubleCOD, and pH were determined according to Standard Methods[16]. Volatile fatty acids (VFA) were, after extraction in diethylether, analyzed with a flame ionization detector (FID) gas chro-matograph (GC-2014, Shimadzu). Detection limit for VFA analysiswas 2 mg L�1. Gas chromatography (GC-14B, Shimadzu) with anFID was used for biogas analysis.

2.7. Statistical analysis

Analysis of variance (ANOVA) was used to test the significanceof differences between two or more groups of experimental results.Significant differences were reported at a of 0.05.

3. Results

3.1. PBR systems and algae productivity

The PBR were operated in a semi-continuous mode. The bubblecolumn PBR was used to grow S. obliquus and the serpentine PBR togrow P. tricornutum. The average volumetric productivity of thebubble column was 0.12 ± 0.06 gDM L�1 d�1 (n = 10) with an aver-age cell concentration of 0.86 ± 0.15 gDM L�1. The serpentine PBRhad a volumetric productivity of about 0.18 ± 0.06 gDM L�1 d�1

(n = 10) with a maximum average cell concentration of0.83 ± 0.25 gDM L�1.

3.2. Batch biogas production and methane yields

The cumulative methane yield after 30 days of incubation was0.21 ± 0.03 L CH4 g�1VS added for S. obliquus biomass and0.35 ± 0.03 LCH4 g�1VS added for P. tricornutum biomass. Maxi-mum substrate utilization rates occurred during the first days ofdigestion, more specifically the first 10 days for S. obliquus and S.platensis biomass (about 0.15 L CH4 g�1VS added) and the first6 days for P. tricornutum biomass (about 0.25 L CH4 g�1VS added).The ultimate methane yields (B0) and the methane production rateconstants (k) were estimated. The B0 estimated for S. obliquus bio-mass was 0.24 ± 0.03 L CH4 g�1VS added (R2 = 0.96) and for P. tri-cornutum biomass 0.36 ± 0.03 L CH4 g�1VS added (R2 = 0.97). Thek values for S. obliquus and P. tricornutum biomass were0.11 ± 0.01 day�1 and 0.14 ± 0.02 day�1 respectively. The cyano-bacterium S. platensis biomass had a methane yield of0.28 ± 0.008 L CH4 g�1VS added for 30 days of incubation. The esti-mated B0 was 0.35 ± 0.03 L CH4 g�1VS added (R2 = 0.99) and the kwas 0.06 ± 0.01 day�1.

3.3. Hybrid flow-through reactors

3.3.1. Performance of reactorsThe volumetric loading rates (Bv), expressed in total VS, applied

to the hybrid flow-through reactors attained average values of2.7 ± 0.7 gVS L�1 d�1for MR1 and 2.8 ± 0.6 gVS L�1 d�1for TR1digesting S. obliquus biomass; 1.9 ± 0.5 gVS L�1 d�1for MR2 and2.0 ± 0.7 gVS L�1 d�1for TR2 digesting P. tricornutum biomass (Ta-ble 2). The algae biomass productivity not only influenced the vol-umetric loading rates (Bv) applied to the reactors but also causedsome limited variations in the loading rates. The concentrationsin the effluent of soluble COD remained low in all reactors rangingfrom 241 to 372 mg COD L�1 (Table 3). However, the total solids(TS) and total COD in the effluent of the reactors remained high

Table 2Operative conditions and summary of the result obtained in the flow-through reactors.

Parameter MR1a (n = 40) TR1b (n = 40) MR2c (n = 40) TR2d (n = 40)

Operating temperature (�C) 33 ± 2 54 ± 2 33 ± 2 54 ± 2pH 7.2 ± 0.1 7.4 ± 0.1 7.3 ± 0.2 7.4 ± 0.2Organic loading rate, Bv (gVS L�1 d�1) 2.7 ± 0.7 2.8 ± 0.6 1.9 ± 0.5 2.0 ± 0.7Hydraulic retention time, HRT (days) 2.2 ± 0.4 2.2 ± 0.3 2.1 ± 0.3 2.3 ± 0.6Biogas production rate (L L�1 d�1) 0.4 ± 0.2 0.6 ± 0.2 0.8 ± 0.3 0.8 ± 0.3% Methane in biogas (%) 74.3 ± 2.5 77.1 ± 3.9 75.1 ± 8.9 78.6 ± 5.0

a Scenedesmus obliquus biomass, at mesophilic conditions.b Scenedesmus obliquus biomass, at thermophilic conditions.c Phaeodactylum tricornutum biomass, at mesophilic conditions.d Phaeodactylum tricornutum biomass, at thermophilic conditions.

Table 3Characteristics of the effluents of the flow through reactors.

Parameter MR1a (n = 20) TR1b (n = 20) MR2c (n = 20) TR2d (n = 20)

Total solids, TS (g L�1) 6.9 ± 4.1 6.2 ± 2.3 9.3 ± 4.1 5.0 ± 1.0Total chemical oxygen demand, Total COD (g L�1) 2.9 ± 3.3 4.2 ± 2.6 4.8 ± 2.6 3.1 ± 1.0Soluble chemical oxygen demand, Soluble COD (mg L�1) 273 ± 17 292 ± 103 241 ± 118 372 ± 123Volatile fatty acids, VFA (mg L�1)e <2 <2 <2 20 ± 53Total ammonium nitrogen, TAN (gNH4-N L�1) 84 ± 39 142 ± 39 119 ± 56 131 ± 45Total Kjeldahl nitrogen, TKN (mg L�1) 330 ± 136 266 ± 196 594 ± 220 354 ± 142Total phosphate, PO4-P (mg L�1) 46.6 ± 24 32 ± 10 40 ± 25 35 ± 22

a Scenedesmus obliquus biomass, at mesophilic conditions.b Scenedesmus obliquus biomass, at thermophilic conditions.c Phaeodactylum tricornutum biomass, at mesophilic conditions.d Phaeodactylum tricornutum biomass, at thermophilic conditions.e GC detection limit 2 mg L�1.

736 C. Zamalloa et al. / Applied Energy 92 (2012) 733–738

and variable. The TS in the effluent of MR1 and TR1were6.9 ± 4.1 g L�1 and 6.2 ± 2.3 g L�1 respectively. For the effluent ofMR2 and TR2, TS were 9.3 ± 4.1 g L�1 and 5.0 ± 1.0 g L�1 respec-tively. The total COD in the effluent of MR1 and TR1 was2.9 ± 3.3 g L�1 and 4.2 ± 2.6 g L�1 respectively and in the effluentof MR2 and TR2 4.8 ± 2.6 g L�1 and 3.1 ± 1.0 g L�1 respectively.The residual short chain fatty acid (VFA) concentrations in thereactors were low. The reactors MR1, TR1 and MR2 had concentra-tions of VFA below detection limit (<2 mg L�1) and reactor TR2contained only 20 ± 53 mg L�1. The low concentration of VFA indi-cated a good stability of the process. The total ammonium nitrogen(TAN) concentration in the effluent of MR1 was significantly(P < 0.05) lower than in the effluent of TR1. TAN concentrationswere 84 ± 39 and 142 ± 39 mg NH4-N L�1 for MR1 and TR1 respec-tively. The TAN concentrations for MR2 and TR2 were not signifi-cantly different, i.e. 119 ± 56 mg NH4-N L�1 for MR2 and131 ± 45 mg NH4-N L�1 for TR2. The average HRT was 2.2 days forall reactors (Table 2). The pH values were on average 7.2 ± 0.1 forMR1, 7.4 ± 0.1 for TR1, 7.3 ± 0.2 for MR2 and 7.4 ± 0.2 for TR2respectively (Table 2).

3.3.2. Biogas productionThe biogas production rates of MR1 and TR1 digesting S. obli-

quus biomass were low, i.e. 0.4 ± 0.2 L L�1 d�1and0.6 ± 0.2 L L�1 d�1 respectively (Table 2). The methane concentra-tion in the biogas was not significantly different, i.e. on average74.3 ± 2.5% for MR1 and 77.1 ± 3.9% for TR1 respectively. The meth-ane yield was estimated to be 0.13 ± 0.05 L CH4 g�1VS for MR1 and0.17 ± 0.08 L CH4 g�1VS for TR1. The biogas production rates ofMR2 and TR2 digesting P. tricornutum biomass were around0.8 ± 0.3 L L�1 d�1 in both reactors (Table 2). The methane concen-tration in the biogas was not significantly different, i.e. on average75.1 ± 8.9% for MR2 and 78.6 ± 5.0% for TR2. The estimated meth-ane yields were in the same order, i.e. at 0.27 ± 0.09 L CH4 g�1VSfor MR2 and 0.29 ± 0.11 L CH4 g�1VS for TR2. Somewhat lower

biomass conversion efficiencies of MR1 relative to TR1 were ob-served (P < 0.05), i.e. 26 ± 11% and 31 ± 11% respectively. MR2and TR2 on the other hand had conversion efficiencies which werenot significantly different, i.e. of 52 ± 20% and 55 ± 15%respectively.

4. Discussion

4.1. Biomass productivity and composition

The microalgae volumetric productivities differed significantly(P < 0.05) between growth units. The reason for this might be thatthe two systems were used to grow different microalgae strainsand were operated under different conditions such as differentPAR light intensity irradiations. From the operational point of view,the bubble column PBR were easier to operate. A study using CO2

from the air in a tubular PBR cultivating S. obliquus reported pro-ductivities between 0.09–0.14 gDM L�1 d�1 [6]. For P. tricornutum,productivities were reported ranging from 0.003–1.9 gDM L�1 d�1

for different PBR arrangements [17]. Overall, the productivitiesachieved in this study were within the ranges previously reported.

The composition of the three types of biomass used in this studywas not significantly different regarding carbon content which isrepresented as COD/VS ratio (Table 1). Yet, the COD/N ratio wassignificantly lower (P < 0.05) for S. platensis, which indicates a highprotein content, i.e. 60% on the dry matter (obtained by multiply-ing the total nitrogen by the factor 6.25) which is close to valuesfrom literature, i.e. 40–60% [7]. The COD/N ratio of S. obliquusand that of P. tricornutum biomass were not significantly differentwhich leads to an estimated protein content between 30–50%. Inliterature, values of 10–45% are reported [18,19]. Furthermore,ash values were high for S. obliquus and P. tricornutum biomass,i.e. on average 31% and 17% respectively. However, high ash con-centrations have been reported for these strains before. For in-stance, values of ash have been reported as high as 34% in

C. Zamalloa et al. / Applied Energy 92 (2012) 733–738 737

Scenedesmus sp. and 15% in P. tricornutum biomass [18,19]. Ash con-tent in algae biomass is usually higher than in lignocellulosic feed-stocks, i.e. up to 50% higher for macroalgae and around 10% formicroalgae [20,21]. It is known that culturing conditions can affectthe composition of microalgae [22]. Therefore, fractions of lipids,carbohydrates and proteins can change. This work reports on fea-sible ways of producing two types of algae biomass, and on thedigestibility of the latter in order to obtain a first assessment of thisline of processing.

4.2. The influence of microalgae biomass on the biomethane potential

The digestibility of P. tricornutum biomass was about a factor of1.5 higher than that of S. obliquus biomass indicating that the for-mer has a higher potential for anaerobic digestion than its freshwa-ter counterpart. Golueke et al. [9] studied the batch anaerobicdigestion of Scenedesmus spp. and Chlorella spp at an initial Bv of1.4 gVS L�1 d�1. The methane yield obtained in their study was0.17 L CH4 g�1VS added for Scenedesmus spp. with a HRT of 30 daysand 0.32 L CH4 g�1VS added for Chlorella spp for the same HRT.Additionally, De Schamphelaire and Verstraete [8] performedbatch tests of a mixed fresh microalgae culture at an initial Bv of0.6 gVS L�1 d�1 achieving a methane yield of 0.31 L CH4 g�1VSadded for a 45-day HRT. In another study, Mussgnug et al. [23]reported a high B0 values of S. obliquus and Arthospira platensis (S. platensis) of 0.29 ± 0.01 L CH4 g�1VS added and 0.48 ±0.01 L CH4 g�1VS added respectively. These results confirm ourfindings that the biodegradability particularly depends on the al-gae species used. This was also suggested by Mussgnug et al.[23] who indicated that the suitability of microalgae biomass foranaerobic digestion is strongly dependent upon the species itselfrather than the organism classification. The microalgae cell wallcomposition is suggested as the main characteristic of its digestioncapabilities [9,23].

4.3. The influence of temperature on the anaerobic conversion ofmicroalgae biomass in hybrid flow-through reactors

The continuous hybrid flow-through reactors were operatedfor 40 days. The performance of the reactors depended signifi-cantly on the types of biomass and on the operational tempera-tures. Overall, the reactors digesting S. obliquus biomass hadlower performances than the reactors with P. tricornutum biomassas was expected from the results of the batch tests. The Bv andbiogas production rate fluctuations (expressed as standard devia-tion in Table 2) are high, but not unusual and still allow to deductimportant tendencies. Although the biomasses were not submit-ted to any specific pre-treatment, the shift from saline to non-sal-ine environment might influence the hydrolysis of P. tricornutumbiomass. For instance, Mussgnug et al. [23] found a quicker celldisintegration for the marine strain D. Salina than for the freshwa-ter strains in an anaerobic digester. The thermophilic conditionsresulted in a factor 1.3 higher biogas production rate than undermesophilic conditions for S. obliquus. The lower results of thedigestion of S. obliquus at a mesophilic temperature can be speciesrelated. It has been reported that this strain can grow at temper-atures up to 35 �C [24]. Thus, it can be expected the hydrolysis ofthis biomass is likely to be slower when subjected to mesophilicfermentation (33 �C) than at thermophilic fermentation (54 �C).For P. tricornutum on the other hand, the difference in terms ofbiogas production rate between operational temperatures wasnot significant. The higher conversions of P. tricornutum can beattributed to the fact that the marine biomass was removed froma high saline environment to a non-saline environment whichmight induce more rapid lysis of the cells. Salt generally hampersanaerobic digestion although adaptation can be achieved [25]. In

this work, however, examining the digestion under low salt stresswas preferred. In addition, the highest optimal temperature ofthis strain has been reported to be 25 �C [26]. The fact that P. tri-cornutum strains can grow optimally only up to 25 �C indicatesthat this strain can be sensitive to higher temperatures. The con-version efficiencies are reflected in the release of total ammoniumnitrogen (TAN) from the degradation of proteins from the micro-algae which is considered as one of the potential difficulties ofthe digestion of microalgae biomass with a high protein content.Comparing the TAN release in the reactors digesting S. obliquus, itwas found that the mesophilic reactor had a significantly lowerTAN concentration in the effluent than the thermophilic reactor.It is important to mention that TAN concentrations obtained inthese reactors are below inhibitory concentrations. Inhibitoryconcentrations are reported in literature from 1.7 g TAN L�1; notethat TAN inhibitory concentrations depend on the differences insubstrates, inocula, environmental conditions (temperature, pH),and acclimation periods [27,28]. Clearly these N-data show thatthis line of flow-through processing of algae biomass is notattaining sufficient hydrolysis of the algae cells. The latter is alsoevident from the high levels of residual suspended solids in theeffluent and the low biogas rates (Table 3).

Previous studies in continuous operations obtained comparablebiogas productions but for higher HRT. Golueke et al. [9] forinstance, reported a methane yield in a continuous operationof about 0.17 L CH4 g�1VS for mesophilic conditions and0.32 L CH4 g�1VS for thermophilic conditions for a 30 days HRT ata Bv of 1.4 gVS L�1 d�1 digesting Scenedesmus and Chlorella bio-mass. The biomass conversion efficiencies ranged between 40%and 76%. Samson and LeDuy [10] digested the cyanobacterium Spi-rulina maxima operating at an HRT of 33 days and a Bv of1 gVS L�1 d�1. They reported a methane yield of 0.24 L CH4 g�1VSat mesophilic conditions with a conversion efficiency ranging be-tween 68% and 72%. Ras et al. [29] performed a continuous diges-tion of Chlorella vulgaris biomass at mesophilic temperaturesobtaining a methane yields of 0.15 L CH4 g�1VS for 16 days ofHRT and 0.24 L CH4 g�1VS for 28 days HRT with conversion effi-ciency between 29% and 49% respectively at Bv of 1 gCOD L�1 d�1.

Finally, in these experiments the biomass was obtained by cen-trifugation which is not yet techno-economically feasible underfull scale conditions. The feasibility of the integration of algaegrowing followed by up-concentration and digestion in a high-ratereactor, still needs to be optimized.

5. Conclusions

Our results demonstrate that it is possible to attain conversionefficiencies ranging between 20% and 50% utilizing a hybrid flow-through anaerobic reactor with an HRT of 2.2 days. The stabilityof this process was demonstrated by the almost complete bio-methanation of the VFAs (Table 3). The digestion of microalgae bio-mass has been previously studied in conventional digesters whichwere operated as once-through completely mixed reactors [9,10].In these systems, the HRT is equal to the solids retention time(SRT). The utilization of the hybrid flow-through reactor in thisstudy, in which a granular sludge blanket and anaerobic filter iscombined, uncouple the HRT and SRT (SRT > HRT) by trappingthe solids and by developing microbial biomass on the surface ofthe carrier material. However, the overall results indicate thatthe algae biomass is not readily biodegradable under given diges-tion conditions. Hence, in terms of growing, harvesting and pro-cessing algae in a biorefinery concept, anaerobic digestion mustbe integrated in a process chain with either a thorough pre-treat-ment or post-treatment in order to recover the full energetic andchemical potential of the algae biomass.

738 C. Zamalloa et al. / Applied Energy 92 (2012) 733–738

Acknowledgments

This work was supported by the Institute for the Promotion ofInnovation by Science and Technology-Strategic Basic Research(IWT-SBO) Sunlight Project – Lipid-based, high value productsand renewable energy from microalgae Grant 80031 and GhentUniversity Grant 179I16D9W. We thank Suzanne Read for the use-ful suggestions and Jan Arends, David van der Ha and Sofie Van DenHende for critically reading the manuscript.

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