Bioresource Technology 138 (2013) 79–86

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Bioresource Technology

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Impact of low temperature pretreatment on the anaerobicdigestion of microalgal biomass

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.03.114

⇑ Corresponding author. Tel.: +34 934016463; fax: +34 934017357.E-mail address: ivet.ferrer@upc.edu (I. Ferrer).

Fabiana Passos, Joan García, Ivet Ferrer ⇑GEMMA – Group of Environmental Engineering and Microbiology, Department of Hydraulic, Maritime and Environmental Engineering, Universitat Politècnica de Catalunya,BarcelonaTech, c/ Jordi Girona 1-3, Building D1, E-08034 Barcelona, Spain

h i g h l i g h t s

�Microalgal biomass solubilisation was enhanced by low temperature pretreatment.� Pretreatment at 55–95 �C for 10 h improved the methane production rate and yield.� The energy balance of the pretreatment step showed the need for microalgae thickening.

a r t i c l e i n f o

Article history:Received 25 January 2013Received in revised form 14 March 2013Accepted 16 March 2013Available online 26 March 2013

Keywords:MicroalgaeBiogasBiofuelHigh rate algal pondWastewater

a b s t r a c t

The aim of this study was to investigate the effect of low temperature pretreatment on the anaerobicdigestion of microalgal biomass grown in wastewater. To this end, microalgae were pretreated at lowtemperatures (55, 75 and 95 �C) for 5, 10 and 15 h. Biomass solubilisation was enhanced with the pre-treatment temperature and exposure time up to 10 h. The methane yield was improved by 14%, 53%and 62% at 55, 75 and 95 �C, respectively; and was correlated with the solubilisation increase. The pre-treatment at 95 �C for 10 h increased VS solubilisation by 1188%, the initial methane production rateby 90% and final methane yield by 60% compared to untreated microalgae. With diluted biomass (�1%VS) positive energy balance was not likely to be attained. However, with concentrated biomass (>2%VS) energy requirements may be covered and even surplus energy generated.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

During the last decade, microalgae have drawn attention withinscientists and entrepreneurs as an alternative energy carrier to fos-sil fuels. This bioenergy feedstock may help improving fuel secu-rity, while reducing CO2 emissions. The production of microalgaefor biofuel purposes includes different products, such as hydrogen,hydrocarbons, bioethanol, biodiesel and methane. However, micro-algae cultivation still shows insufficient biomass productivity to befeasible in economic terms. For instance, microalgae conversioninto biodiesel is not yet optimised and does not reach competitiveprices if compared with commercial fossil fuels (Sialve et al., 2009).On the other hand, anaerobic digestion is a well-established andcomparably low-cost technology, already consolidated for organicresidues, such as sewage sludge. Moreover, some recent studieshave reported promising results using microalgae as anaerobicdigestion substrate (Alzate et al., 2012; González-Fernández

et al., 2012a,b). Up to date, however, the literature dealing on mic-roalgae biomethanation is still scarce.

In the field of sewage sludge, methanogenesis has been shownto be limited by the hydrolysis rate of organic matter (Batstoneet al., 2009; Vavilin et al., 2008). Regarding microalgae, the hydro-lysis has also been pointed out as the bottleneck of the anaerobicdigestion. The hemicellulosic composition of microalgae cell wallsmakes them resistant and pretreatment techniques are necessaryto improve the methane production potential (González-Fernán-dez et al., 2012a; Ras et al., 2011). Previous studies have shown alinear correlation between biomass solubilisation and biogas pro-duction after pretreatment (Bougrier et al., 2006; Passos et al.,2013). The thermal pretreatment has been applied for sewagesludge disintegration at temperatures from 50 to 270 �C (Carrèreet al., 2010). However, temperatures above 180 �C may lead tothe production of recalcitrant and/or inhibitory compounds, whichreduce biomass digestibility (Wilson and Novak, 2009). Pretreat-ments at low temperatures (<100 �C) reduce the energy demand,which can improve the energy balance and profitability of the sys-tem (Ferrer et al., 2008). Some authors suggest that this techniquecan be considered a biological process, since biomass solubilisation

Table 1Low temperature pretreatment of microalgae.

Microalgae Pretreatmentconditions

Anaerobic digestionconditions

Results References

Microalgal biomass grown in wastewater 100 �C8 h

Batch Methane production increase by33%*

Chen and Oswald (1998)

Scenedesmus biomass 90 �C3 h

Batch35 �C

Methane production increase by220%*

González-Fernández et al.(2012a)

Scenedesmus biomass 80 �C25 min

Batch35 �C

Methane production increase by57%*

González-Fernández et al.(2012b)

Scenedesmus and Clamydomonas biomass 55 �C12 and 24 h

Batch35 �C

Methane production decrease by4–8%*

Alzate et al. (2012)

Acutodesmus obliquus and Oocystis sp.biomass

55 �C12 and 24 h

Batch35 �C

Methane production decrease by3–13%*

Alzate et al. (2012)

Microspora biomass 55 �C12 and 24 h

Batch35 �C

Methane production increase by4–5%*

Alzate et al. (2012)

* Compared to control.

80 F. Passos et al. / Bioresource Technology 138 (2013) 79–86

occurs due to a higher activity of thermophilic and hyperthermo-philic bacteria populations (Alzate et al., 2012; Carrère et al.,2010). Therefore, low temperature pretreatment acts as a thermo-philic or hyperthermophilic predigestion step (Lu et al., 2008). Fur-thermore, exposure time rather than pretreatment temperatureseems to play a more important role for biomass solubilisationand methane production at this temperature range (Appels et al.,2010).

Several studies have used a low temperature pretreatment priorto the anaerobic digestion of organic substrates. For instance, amethane production increase between 20% and 60% has been re-ported for sewage sludge (Appels et al., 2010; Climent et al.,2007; Ferrer et al., 2008; Gavala et al., 2003). In the case of micro-algae, only a few studies have been conducted so far (Table 1).Chen and Oswald (1998) compared the pretreatment of microalgaegrown in high rate algal ponds at different temperatures (60, 80and 100 �C), exposure times (1, 2 and 3 h), and solid concentrations(3%, 6% and 9%), with and without NaOH addition. They concludedthat temperature was the most important parameter influencingmicroalgae anaerobic digestion; obtaining the highest methaneproduction increase (33%) after the pretreatment at 100 �C for8 h. More recently, González-Fernández et al. (2012a) observedthat the pretreatment of Scenedesmus at 70 �C had little effect onthe final methane yield (85 mL/g COD), while at 90 �C the finalmethane yield was 2.2-fold (170 mL/g COD) that of the controlwith untreated biomass. On the other hand, Alzate et al. (2012)studied the anaerobic digestion of three different mixed microal-gae cultures pretreated at 55 �C for 12 and 24 h. For both Clamydo-monas and Scenedesmus biomass, and Acutodesmus obliquus andOocystis biomass, the methane yield decreased after the pretreat-ment in comparison with untreated microalgae; while for Micro-spora biomass the methane yield increased by 4% and 5% after 12and 24 h of pretreatment, respectively. The variety of literature re-sults calls for a systematic study on the combined effect of temper-ature and exposure time on microalgae anaerobic digestion.

Table 2Average characteristics of microalgal biomass (substrate) and digested sludge (inoculumconditions.

Parameter Microalgal biomass Inocu

pH 7.8 (0.3) 6.9 (0TS [% (w/w)] 2.04 (0.02) 3.4 (0VS [% (w/w)] 1.17 (0.01) 2.3 (0VS/TS (%) 57 (0.02) 68 (0COD (g/L) 17.9 (0.97) 20.6Lipids (%) 17.4 (2.7) –Proteins (%) 49.3 (8.4) –Carbohydrates (%) 19.5 (3.2) –

Biochemical methane potential (BMP) tests are widely used toevaluate the anaerobic biodegradability of organic substrates andto identify key variables influencing this parameter, i.e. digestiontemperature, effect of pretreatment techniques, codigestion of dif-ferent substrates, etc. In spite of this, a standard method on theanaerobic biodegradability assay is still missing. In this sense,experimental conditions such as the substrate/inoculum (S/I) ratioare yet to be defined. Theoretically, the inoculum only affects thekinetics of the process, i.e. accelerates digestion. However, experi-mental data show significant differences not only on the degrada-tion rate, but also on the methane yield depending on the S/I ratio(Raposo et al., 2011). Besides, the origin and characteristics of theinoculum may also have an influence on the results. Digestedsludge from running anaerobic reactors in municipal wastewatertreatment plant (WWTP) is commonly used when acclimated inoc-ulum is not available (Raposo et al., 2011).

The purpose of the present study was to evaluate the effect oflow temperature pretreatment on microalgal biomass solubilisa-tion and methane production, at a range of temperatures and expo-sure times. Firstly, BMP tests were adapted to microalgae digestionby optimising the S/I ratio. Secondly, the effect of exposure timewas investigated at different temperatures. Based on the results,BMP tests were carried out at 55, 75 and 95 �C, for exposure timesof 5, 10 and 15 h; and the pretreatment effect was compared interms of biomass solubilisation and methane yield.

2. Methods

2.1. Microalgal biomass

The experimental set-up was located at the laboratory of theGEMMA research group (Universitat Politècnica de Catalunya.BarcelonaTech, Spain). Microalgal–bacterial biomass was grownin a pilot high rate algal pond (HRAP) (0.5 m3; 1.5 m2) treating

) used for BMP tests under different S/I ratios and low temperature pretreatment

lum (S/I ratio BMP test) Inoculum (Pretreatment BMP test)

.2) 7.5 (0.4)

.01) 4.57 (0.04)

.01) 3.15 (0.03).01) 69 (0.04)(0.46) 25.5 (0.76)

–––

Digestion time (days)0 5 10 15 20 25 30

Biog

as y

ield

(mL/

g VS

)

0

50

100

150

200

250

300

0.5 g COD/g VS1.0 g COD/g VS2.0 g COD/g VS4.0 g COD/g VS

Fig. 1. Cumulative biogas yield with different substrate to inoculum (S/I) ratios.

F. Passos et al. / Bioresource Technology 138 (2013) 79–86 81

wastewater from a municipal sewer of Barcelona. The HRAP re-ceived the primary effluent of a settling tank and was used as sec-ondary treatment, with a hydraulic retention time (HRT) of8 days. Average surface loading rates were 24 g COD/m2day and4 g NH4-N/m2day. A detailed description of the wastewater treat-ment system operation and performance may be found elsewhere(García et al., 2006; Passos et al., 2013). Microalgal–bacterial bio-mass was harvested from secondary settlers (0.1 day HRT) andthickened by gravity in laboratory Imhoff cones at 4 �C for 24 h.Average characteristics of harvested microalgae are summarisedin Table 2. It was mainly composed of Chlamydomonas and dia-toms Nitzchia, although other microalgae species like Chlorella,Ankistrodesmus, Monorraphidium and Scenedesmus were also pres-ent. Note that in HRAP microalgae coexist with bacteria, but mostof the total biomass (90%) belongs to microalgae (García et al.,2006).

2.2. BMP test to optimise the S/I ratio

In order to determine the optimum balance between substrateand inoculum for microalgae anaerobic digestion, preliminaryBMP tests were conducted with four S/I ratios: 0.5, 1, 2 and4 g COD substrate/g VS inoculum. The inoculum was mesophilicdigested sludge from a full-scale anaerobic reactor located in amunicipal WWTP near Barcelona (Spain). Average characteristicsof the inoculum are shown in Table 2. BMP tests were carriedout in serum bottles (160 mL), with a liquid volume of 100 mLand a headspace volume of 60 mL. The bottles were filled withthe corresponding amount of substrate and inoculum, flushedwith Helium gas, sealed with butyl rubber stoppers and incubatedat 35 �C until biogas production ceased. A blank treatment withonly inoculum was used to quantify the amount of methane pro-duced by endogenous respiration. Each S/I ratio and blank testwere performed in triplicate. Accumulated biogas productionwas calculated according to the pressure increase in the

Table 3Anaerobic digestion under different S/I ratios.

S/I ratio (g COD/g VS) Initial methane production rate(mL CH4/g VS d)

Final methan(mL CH4/g VS

0.5 90.4 (5.94) 156.0 (2.15)1 85.9 (4.66) 158.5 (2.19)2 72.0 (0.81) 153.8 (1.85)4 56.5 (1.05) 143.7 (0.21)

headspace volume, which was periodically measured with anelectronic manometer (Greisinger GMH 3151). The net biogasproduction was calculated by subtracting the blank to each S/I ra-tio results. The methane content in biogas was periodically ana-lysed by gas chromatography.

2.3. Thermal pretreatment and biomass solubilisation

The thermal pretreatment of microalgal biomass was carriedout in glass bottles with a total volume of 250 mL and liquid vol-ume of 150 mL, which were placed in an incubator under continu-ous stirring, at 55, 75 or 95 �C for 5, 10 and 15 h (in triplicate).Pretreatment exposure times were based on a preliminary solubili-sation experiment, in which the following exposure times wereevaluated: 1, 2, 3, 4, 5, 6, 8, 10, 12, 18 and 24 h, at 55 and 95 �C.The solubilisation degree was calculated by soluble to total volatilesolids ratio (VSs/VS) and by the increase in this ratio as comparedto untreated biomass (Eq. (1)), where sub-indexes refer to pre-treated (p) and untreated (o) biomass.

S ð%Þ ¼ ½ðVSs=VSÞp � ðVSs=VSÞo�=ðVSs=VSÞo � 100 ð1Þ

The response surface methodology was used to explore the rela-tion between independent variables (temperature and exposuretime) and the response variable (biomass solubilisation). In orderto express the influence of temperature and time on biomass solu-bilisation, a second-degree polynomial was adjusted with R soft-ware (Eq. (2)). The quadratic model was subsequently simplifieduntil all coefficients had a p-value below 0.05.

S ¼ b0 þ b1Xtemp þ b2Xtime þ b11X2temp þ b22X2

time

þ b12XtempXtime ð2Þ

2.4. BMP test with pretreated biomass

BMP tests were used to compare the anaerobic biodegradabil-ity of pretreated and untreated microalgal biomass. The selectedS/I ratio was 0.5 g COD/g VS, corresponding to 28 g of microalgae(substrate) and 32 g of sludge (inoculum) per bottle. Serum bot-tles (160 mL) were then filled with distilled water up to100 mL, flushed with Helium gas, sealed with butyl rubber stop-pers and incubated at 35 �C until biogas production ceased. Ablank treatment with only inoculum was used to quantify theamount of methane produced by endogenous respiration. Eachpretreatment was performed in duplicate, whereas the control(untreated biomass) and blank (inoculum) were performed intriplicate. Average characteristics of the substrate and inoculumare summarised in Table 2. The net biogas production was calcu-lated by subtracting the blank results to each pretreatment. Themethane content in biogas was periodically analysed by gaschromatography.

The anaerobic biodegradability of biomass was deduced fromthe net methane production (mL CH4) and the theoretical methaneyield under standard conditions (350 mL CH4/g CODremoved), withrespect to the initial COD of each trial (Eq. (3)).

e yield)

Methane content in biogas(% CH4)

Anaerobic biodegradability(%)

68.2 (1.21) 53.2 (2.14)68.3 (0.93) 54.0 (0.75)69.1 (1.03) 52.4 (0.63)68.7 (0.78) 49.0 (0.07)

Exposure time (hours)0 5 10 15 20 25 30

Solu

bilis

atio

n ra

tio (V

S S/VS

)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Thermal pretreatment at 55 ºCThermal pretreatment at 95 ºC

Fig. 2. Microalgal biomass solubilisation over 24 h of thermal pretreatment at 55and 95 �C.

Fig. 3. Response surface plot of microalgal biomass solubilisation versus indepen-dent pretreatment variables (exposure time and temperature).

82 F. Passos et al. / Bioresource Technology 138 (2013) 79–86

Biodegradability ð%Þ ¼ ½ðCH4production ðmLÞ� Blank CH4 production ðmLÞÞ=350ðmL=gCODremovedÞ�=gCODinitial � 100 ð3Þ

2.5. Analytical methods

All analyses were triplicated and the results are given as meanvalues. Microalgal biomass and sewage sludge were characterisedby the concentration of total solids (TS), volatile solids (VS) andchemical oxygen demand (COD), following the procedure de-scribed in the standard methods (APHA, AWWA, WPCF, 1999).The lipid content of biomass was determined by the Soxhletextraction method (APHA, AWWA, WPCF, 1999). The total Kjeldahlnitrogen (TKN) to protein conversion factor was 5.95, according toLópez et al. (2010). Carbohydrates were determined by phenol–sulphuric acid method, after acid hydrolysis and measured byspectrophotometry (Spectronic Genesys 8) (Dubois et al., 1956).Microalgae identification was carried out by microscopic examina-tion (Nikon Optiphot-pol, Japan) and determined from specific lit-erature (Bourrelly, 1966; Palmer, 1962).

The methane content in biogas was measured with a gas chro-matograph (GC) (Trace GC Thermo Finnigan) equipped with a Ther-mal Conductivity Detector, by injecting gas samples into a packedcolumn (Hayesep 3 m 1/8 in. 100/120). The carrier gas was Heliumin split less mode (column flow: 19 mL/min). The oven temperaturewas 35 �C with a retention time of 1.5 min. Injector and detectortemperatures were 150 and 250 �C, respectively. The system wascalibrated with methane (50% CH4) and carbon dioxide (50% CO2).

Volatile fatty acids (VFA) were analysed in soluble phase usingGC (Argilent Technologies 7820A). Soluble samples were obtained

Table 4Microalgal biomass solubilisation after the low temperature pretreatment.

Trial Temperature(�C)

Exposure time(h)

VSs/VSratio

VSs/VS increase(%)

Control – – 0.016 –T1 55 5 0.071 330T2 55 10 0.083 402T3 55 15 0.083 400T4 75 5 0.133 704T5 75 10 0.192 1058T6 75 15 0.190 1048T7 95 5 0.179 983T8 95 10 0.212 1184T9 95 15 0.213 1188

after biomass centrifugation (UNICEN20, 4200 rpm, 8 min, 20 �C),followed by filtration and sulphuric acid and diisopropyl etheraddition. The chromatograph was equipped with a capillary col-umn (DB-FFAP Agilent 30 m � 0.25 mm � 0.25 lm) and a flameionisation detector (FID). Helium was used as carrier gas, with asplit ratio of 13 (column flow: 3.5 mL/min). The temperatures ofthe injector and detector were 200 and 300 �C, respectively. Thesystem was calibrated with dilutions of commercial VFA (acetic,propionic, iso-butyric, n-butyric, iso-valeric and n-valeric acids)(Scharlau, Spain) with concentrations in the range of 0–1000 mg/L. The detection limit of VFA analysis was 5 mg/L.

3. Results and discussion

3.1. Influence of the S/I ratio on BMP tests

In this study, preliminary BMP tests aimed at determining theoptimum S/I ratio between microalgal biomass and digested sludgefrom a municipal WWTP. BMP tests with S/I ratios of 0.5, 1, 2 and 4were compared over an incubation period of 26 days (Fig. 1). Theinitial methane production rate, final methane yield and anaerobicbiodegradability are summarised in Table 3. As can be seen, the fi-nal methane yield was similar with the lowest S/I ratios of 0.5 and1 g COD/g VS, 156.0 and 158.5 mL CH4/g VS, respectively; and de-creased with the highest S/I ratios of 2 and 4 g COD/g VS to 153.8and 143.7 mL CH4/g VS, respectively. The lowest S/I ratios also fa-voured the kinetics of the process: the highest initial methane pro-duction rate was attained with 0.5 g COD/g VS (90.4 mL CH4/g VS d), followed by 1.0 g COD/g VS (85.9 mL CH4/g VS d).

Cho et al. (2005) indicated that S/I ratios between 0.4 and0.6 g COD/g VS were most appropriate for specific methanogenicactivity assays, since low concentrations of inoculum increasedthe time required for complete digestion. Alzate et al. (2012) as-sessed the effect of the S/I ratio (0.5, 1.0 and 3.0 g VS/g VS) onthe digestion of three different microalgae mixtures, observingthe highest methane production and anaerobic biodegradabilitywith 0.5 g VS/g VS. Thus, the selected S/I ratio for BMP with micro-algal biomass was 0.5 g COD/g VS.

3.2. Microalgal biomass solubilisation: effect of pretreatmenttemperature and exposure time

A preliminary thermal pretreatment experiment was carried outto evaluate the effect of exposure time on microalgal biomass solu-bilisation at the lowest (55 �C) and highest (95 �C) temperature.

Digestion time (days)0 10 20 30 40 50

Met

hane

yie

ld (m

L C

H4/g

VS)

0

50

100

150

200

Control 55 ºCT1 5h 55ºCT2 10h 55ºCT3 15h 55ºC

Digestion time (days)0 10 20 30 40 50

Met

hane

yie

ld (m

L C

H4/g

VS)

0

50

100

150

200

Control 75 ºCT4 5h 75 ºCT5 10h 75 ºCT6 15h 75 ºC

Digestion time (days)0 10 20 30 40 50

Met

hane

yie

ld (m

L C

H4/g

VS)

0

20

40

60

80

100

120

140

160

180

200

Control 95 ºCT7 5h 95 ºCT8 10h 95 ºCT9 15h 95 ºC

(a)

(b)

(c)

Fig. 4. Cumulative methane yield with microalgal biomass pretreated at 55 �C (a);75 �C (b) and 95 �C (c).

F. Passos et al. / Bioresource Technology 138 (2013) 79–86 83

Biomass solubilisation over a period of 24 h is shown in Fig. 2. At55 �C, the solubilisation ratio increased from 0.03 to 0.15 VSs/VSwithin 4 h, reaching an asymptote (0.16 VSs/VS) after 8 h. At95 �C, the solubilisation ratio increased rapidly (from 0.02 to0.15 VSs/VS) within the first 3 h, and then more slowly reachingthe maximum (0.24 VSs/VS) after 18 h.

Based on these results, the influence of both pretreatment tem-perature and exposure time was systematically studied by combin-ing three pretreatment temperatures (55, 75 and 95 �C) with threeexposure times (5, 10 and 15 h) (Table 4). For better analysing theinfluence of independent pretreatment variables on microalgalbiomass solubilisation, the following polynomial equation was ad-justed (Eq. (6)).

S ¼ �5:318� 10�1 þ 1:478� 10�2Xtemp þ 3:411� 10�3Xtime

� 7:807� X2temp ð4Þ

In this equation all coefficients are significant (p-value < 0.05).The model significantly describes the experimental response (R2

0.95, F-statistic 33.06, p-value 0.001) and the response surface plotfor microalgal biomass solubilisation is shown in Fig. 3. Accordingto the results, temperature seems the most influencing parameteron biomass solubilisation (Fig. 3).

Indeed, the preliminary experiment already evidenced that bio-mass solubilisation was higher at 95 �C than 55 �C, 13% after 8 hand 34% after 18 h of pretreatment (Fig. 2). This suggests that thepretreatment at the highest temperature (95 �C) succeeded in sol-ubilising compounds that remained intact when a lower tempera-ture was applied (55 �C). Still, the pretreatment at 70–90 �C wasnot likely to disintegrate microalgae cell wall, composed of cellu-lose and hemicellulose which are not disintegrated at this temper-ature range (Hendriks and Zeeman, 2009).

Previous studies have suggested that the main parameter influ-encing the thermal pretreatment is temperature; exposure timehaving less impact on microalgal biomass (Chen and Oswald,1998) and sewage sludge (Carrère et al., 2008) solubilisation. How-ever, in the specific case of low temperature pretreatment, timeseems to play an important role (Appels et al., 2010). The prelimin-ary experiment showed how biomass solubilisation increased dur-ing the first 4 h at 55 �C (4.8-fold) and 3 h at 95 �C (4.6-fold). Theresults are in accordance with those reported by González-Fernán-dez et al. (2012a); the pretreatment of Scenedesmus biomass at90 �C for 3 h increased soluble organic matter by 4.4-fold. How-ever, these authors did not assess longer exposure times. In ourstudy, longer pretreatment times were required to obtain the max-imum solubilisation increase, 6.0-fold at 55 �C and 8.4-fold at95 �C. Experiments with microalgae pretreated at 55, 75 and95 �C, confirmed that biomass solubilisation increased with theexposure time from 5 to 10 h (17% at 55 �C, 44% at 75 �C and 18%at 95 �C), whereas differences between 10 and 15 h were almostnegligible. Therefore, exposure time enhanced biomass solubilisa-tion mostly until 10 h.

On the whole, the solubilisation increase seemed more relevantwith temperature than with exposure time. For instance, after 10 hthe solubilisation ratio was 130% higher at 75 compared to 55 �C,and 155% higher at 95 compared to 55 �C. The maximum biomasssolubilisation was achieved within 10 h at 95 �C.

3.3. Methane production potential of pretreated biomass

The net biogas production over an incubation period of43 days is shown in Fig. 4. Compared to untreated biomass, allpretreatment conditions enhanced the initial methane productionrate (34–90% increase) and final methane yield (12–61% increase)(Table 5). The highest final methane yield was obtained withT8 � T9 at 95 �C (170–169 mL CH4/g VS, respectively), followedby T5 � T6 at 75 �C (155–160 mL CH4/g VS, respectively) andT2 � T3 at 55 �C (125–127 mL CH4/g VS, respectively). Thus, thedifference between an exposure time of 10 and 15 h was negligi-ble for all pretreatment temperatures; in accordance with bio-mass solubilisation results. At 75–95 �C, the final methane yieldwas 16–33% lower with an exposure time of 5 h compared to10–15 h. However, at 55 �C the final methane yield was similarfor all exposure times (124–127 mL CH4/g VS). Indeed, the lowestfinal methane yield was obtained at 55 �C (124–127 mL CH4/g VS); only 13% higher than the control with untreated microal-gae (111 mL CH4/g VS). Here again the results are in accordancewith the solubilisation degree, which was much lower at 55 �C(3 to 4-fold increase) than at 75–95 �C (7 to 12-fold increase).

Table 5Anaerobic digestion of pretreated microalgal biomass.

Trial Initial methane production rate(mL CH4/g VS d)

Final methane yield(mL CH4/g VS)

Methane content in biogas(% CH4)

Anaerobic biodegradability(%)

Control 55 �C 31.87 (0.40) 110.94 (1.59) 68.6 (1.19) 30.5 (0.44)T1 42.86 (0.87) 123.88 (4.26) 68.6 (0.93) 34.0 (1.17)T2 46.33 (0.58) 124.59 (3.26) 68.7 (1.03) 34.2 (0.90)T3 49.32 (0.47) 127.43 (1.59) 68.3 (1.05) 35.0 (0.44)

Control 75 �C 22.73 (1.86) 104.56 (12.62) 68.8 (0.34) 28.7 (2.47)T4 33.02 (0.58) 125.67 (2.67) 69.3 (0.73) 34.5 (0.76)T5 39.62 (1.55) 154.57 (6.77) 68.9 (1.23) 42.4 (1.86)T6 43.89 (0.67) 160.42 (0.67) 69.1 (0.78) 44.0 (0.18)

Control 95 �C 34.19 (2.96) 105.23 (8.72) 68.7 (0.42) 28.9 (2.39)T7 56.02 (0.62) 146.95 (4.01) 68.8 (0.85) 40.4 (1.10)T8 63.62 (1.80) 169.88 (3.68) 68.8 (0.96) 46.6 (1.01)T9 64.96 (0.48) 168.75 (1.92) 68.3 (0.96) 46.3 (0.53)

L/g

VS)

160

170

180

R2 = 0.89

84 F. Passos et al. / Bioresource Technology 138 (2013) 79–86

To summarise, no matter the exposure time, microalgal biomasspretreated at 55 �C showed the lowest methane productionpotential.

In a previous study with different mixed microalgae cultures,the biological pretreatment at 55 �C showed negligible methaneproduction enhancement (Alzate et al., 2012). Actually, for Scene-desmus and Clamydomonas biomass, as well as for Acutodesmusobliquus and Oocystis sp. biomass, a decrease on the final methaneyield was observed. For Microspora biomass, the methane yieldincreased by 4% and 5% after 12 and 24 h of pretreatment, respec-tively. This was attributed to the presence of bacteria excretinghydrolytic enzymes in the microalgal–bacterial consortium (Alz-ate et al., 2012). The effect of the low temperature pretreatmentat 70–90 �C on Scenedesmus biomass was evaluated by Gon-zález-Fernández et al. (2012a,b), concluding that all temperaturesincreased COD solubilisation. However, the methane yieldwas hardly improved by the 70 �C pretreatment (85–90 mLCH4/g COD, 9–12% increase), compared to the 80 �C pretreatment(129 mL CH4/g COD, 57% increase) and 90 �C pretreatment(170 mL/g COD, 2.2-fold increase). At 70 �C microscopic analysisshowed intact cells, consequently the COD solubilisation increasewas attributed to exopolymers (González-Fernández et al.,2012a).

Our results showed a linear correlation between the methaneyield and solubilisation degree (Fig. 5). In this way, the higherthe biomass solubilisation, the higher the methane production.Even if microalgal biomass pretreatment enhanced the methaneproduction, the anaerobic biodegradability was still low (<47%)(Table 5), indicating that most organic matter was not readilyavailable for digestion. According to Hendriks and Zeeman (2009)pretreatments at temperatures of 150–180 �C are capable of solu-bilising cellulose and hemicellulose, components of most microal-gae cell wall. Thus, future research should look at the effect of hightemperature pretreatment on microalgae solubilisation and anaer-obic digestion.

Solubilisation (% VSs/VS increase)

200 400 600 800 1000 1200 1400

Met

hane

yie

ld (

m

110

120

130

140

150

Fig. 5. Correlation between the methane yield and solubilisation increase after thelow temperature pretreatment of microalgal biomass.

3.4. Energy aspects

The energy balance of microalgal biomass pretreatment wasestimated to get an insight into the viability of full-scale imple-mentation. Compared to other pretreatment techniques, strengthof low temperature pretreatments (<100 �C) is the low energy de-mand, which may be supplied by means of a heater or a combinedheat and power unit fuelled with biogas, as in wastewater treat-ment plants. In this study, the energy balance of the pretreatmentwas defined by the energy input (Ei) to the energy output (Eo) ratio(Ei/Eo), as proposed by Ferrer et al. (2009, 2011).

The energy input was determined by the heat required to raisethe microalgae temperature (Tm) to the pretreatment temperature(Tp), considered as the main energy consumption in this case. Tm

and Tp were defined as 20 �C (i.e. ambient temperature) and 55,75 or 95 �C (depending on each pretreatment temperature),respectively. Heat losses were not accounted for because they haveshown to be negligible (2–8%) in insulated digesters (Zupancic andRoš, 2003). Microalgae specific density (q) and specific heat (c)were assumed to be those of water, 1 g/mL and 4.18 � 10�3 kJ/g �C, respectively. In practice, pretreated biomass (55, 75 and95 �C) would have to be cooled down before mesophilic digestion(35 �C). Thus, the pretreatment effluent would be used to pre-heatinfluent biomass, by means of a heat exchanger, with heat recoveryefficiency (U) of 85% (Lu et al., 2008). In order to normalise the re-sults, the energy input was divided by the volatile solids content inpretreated biomass (g VS). Therefore, the energy input of the lowtemperature pretreatment was calculated by the following equa-tion (Eq. (5)).

Ei ðkJ=g VSÞ ¼ ½qðg=mLÞ � V ðmLÞ � c ðkJ=g �CÞ � ðTp ð�CÞ� Tm ð�CÞÞ � ð1� /Þ�=g VS ð5Þ

The energy output was calculated from the difference betweenthe methane yield after each pretreatment and the control, sincethe energy required for the pretreatment should at least be coveredby the extra methane produced. Therefore, the energy output was

Table 6Energy ratio of microalgal biomass low temperature pretreatment step.

Trial Energy output This study (1.17% VS) Case study (2% VS) Case study (3% VS)

Energy input Energy ratio Energy input Energy ratio Energy input Energy ratio

(kJ/gVS) (kJ/gVS) (Ei/Eo) (kJ/gVS) (Ei/Eo) (kJ/gVS) (Ei/Eo)

T1 0.5 19 4.0 1.1 2.4 0.7 1.6T2 0.5 1.9 3.8 1.1 2.2 0.7 1.5T3 0.6 1.9 3.2 1.1 1.9 0.7 1.2T4 0.8 2.9 3.9 1.7 2.3 1.1 1.5T5 1.8 2.9 1.6 1.7 1.0 1.1 0.6T6 2.0 2.9 1.5 1.7 0.9 1.1 0.6T7 1.5 4.0 2.7 2.4 1.6 1.6 1.0T8 2.3 4.0 1.7 2.4 1.0 1.6 0.7T9 2.3 4.0 1.8 2.4 1.0 1.6 0.7

Note: Values in bold indicate neutral or positive energy balances (Ei/Eo 6 1).

F. Passos et al. / Bioresource Technology 138 (2013) 79–86 85

calculated according to the following equation (Eq. (6)), whereDPCH4 is the methane yield increase after pretreatment (mL/g VS)and n is the lower heating value of methane (35,800 kJ/m3 CH4)(Metcalf and Eddy, 2003).

Eo ðkJ=g VSÞ ¼ ½DPCH4 ðmL=g VSÞ � n ðkJ=m3Þ�=106 ð6Þ

Energy ratios (Ei/Eo) are summarised in Table 6, where valuesbelow 1 indicate net energy production. According to this, the en-ergy input of the pretreatment step would be higher than the en-ergy output from the extra methane produced (1.5–4 Ei/Eo). Thisis attributed to two main reasons: (a) the low organic solids con-centration in microalgal biomass, and (b) the assessment of themethane production potential in batch tests.

Indeed, the microalgal biomass used in this study had a solidsconcentration around 2% TS and 1.2% VS; which is relatively lowas anaerobic digestion substrate. High water content in organicsubstrates has shown to be the main factor affecting excessive en-ergy consumption during the pretreatment step (Tang et al., 2010).The energy input was thus recalculated for scenarios with moreconcentrated microalgal biomass, 2% and 3% VS (Table 6). In bothcases the pretreatment at 75 and 95 �C for 10 and 15 h (T5, T6, T8

and T9) reached a neutral or positive energy balance (Ei/Eo 6 1).Furthermore, with 3% VS some 60–70% of the extra methane pro-duced would be consumed in the pretreatment step, while the restcould be used elsewhere. The results put forward the importanceof microalgae thickening to optimise biogas production.

Regarding BMP tests, the lack of acclimated inoculum mayunderestimate the methane production potential, hence the energyoutput. For a more realistic energy assessment, continuous reac-tors with acclimated inoculum should be used, which ought tobe addressed in future studies.

4. Conclusions

In this study, the effect of low temperature pretreatment onmicroalgae solubilisation and methane production was investi-gated. The pretreatment efficiency was mostly influenced by tem-perature, with optimum results at 75–95 �C with an exposure timeof 10 h. All pretreatment conditions increased the initial methaneproduction rate (34–90%) and final methane yield (12–61%) in re-spect to untreated biomass. With diluted biomass (�1% VS) posi-tive energy balance was not likely to be obtained. However, withconcentrated biomass (>3% VS) energy requirements may be cov-ered and even surplus energy generated.

Acknowledgements

This research was funded by the Spanish Ministry of Scienceand Innovation (Project BIOALGAS CTM2010-17846). Fabiana

Passos appreciates her PhD scholarship funded by the Coordinationfor the Improvement of Higher Level Personal (CAPES) from theBrazilian Ministry of Education.

References

Alzate, M.E., Muñoz, R., Rogalla, F., Fdz-Polanco, F., Pérez-Elvira, S.I., 2012.Biochemical methane potential of microalgae: influence of substrate toinoculum ratio, biomass concentration and pretreatment. Bioresour. Technol.123, 488–494.

APHA-AWWA-WPCF, 1999. Standard Methods for the Examination of Water andWastewater, 20th ed. Washington.

Appels, L., Degrève, J., Van der Bruggen, B., Van Impe, J., Dewil, R., 2010. Influence oflow temperature thermal pretreatment on sludge solubilization, heavy metalrelease and anaerobic digestion. Bioresour. Technol. 101, 5743–5748.

Batstone, D.J., Tait, S., Starrenburg, D., 2009. Estimation of hydrolysis parameters infull-scale anaerobic digesters. Biotechnol. Bioeng. 102, 1513–1520.

Bougrier, C., Albasi, C., Delgenès, J.P., Carrère, H., 2006. Effect of ultrasonic, thermaland ozone pretreatment on waste activated sludge solubilization and anaerobicdigestion. Chem. Eng. Process. 45, 711–718.

Bourrelly, P., 1966. Les algues d’eau douce. Tome I: Les algues vertes. Édition N.Boubée & Cie, Paris.

Carrère, H., Bougrier, C., Castets, D., Delgenès, J.P., 2008. Impact of initialbiodegradability on sludge anaerobic digestion enhancement by thermalpretreatment. J. Environ. Sci. Health, Part A 43, 1551–1555.

Carrère, H., Dumas, C., Battimelli, A., Batstone, D.J., Delgenès, J.P., Steyer, J.P., Ferrer,I., 2010. Pretreatment methods to improve sludge anaerobic degradability: areview. J. Hazard. Mater. 183, 1–18.

Chen, P.H., Oswald, W.J., 1998. Thermochemical treatment for algal fermentation.Environ. Int. 24, 889–897.

Cho, Y.T., Young, J.C., Jordan, J.A., Moon, H.M., 2005. Factors affecting measurementof specific methanogenic activity. Water Sci. Technol. 52, 435–440.

Climent, M., Ferrer, I., Baeza, M.D., Artola, A., Vazquez, F., Font, X., 2007. Effects ofthermal and mechanical pretreatments of secondary sludge on biogasproduction under thermophilic conditions. Chem. Eng. J. 133, 335–342.

Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Clorometricmethod for determination of sugar and related substances. Anal. Chem. 28,350–356.

Ferrer, I., Ponsa, S., Vazquez, F., Font, X., 2008. Increasing biogas production bythermal (70 �C) sludge pretreatment prior to thermophilic anaerobic digestion.Biochem. Eng. 42, 186–192.

Ferrer, I., Serrano, E., Ponsá, S., Vázquez, F., Font, X., 2009. Enhancement ofthermophilic anaerobic digestion by 70 �C pretreatment: energy considerations.J. Residuals Sci. Technol. 59, 1777–1784.

Ferrer, I., Vázquez, F., Font, X., 2011. Comparison of the mesophilic and thermophilicanaerobic sludge digestion from an energy perspective. J. Residuals Sci. Technol.8, 81–87.

García, J., Lundquist, T., Green, B.F., Mujeriego, R., Hernández-Mariné, M., Oswald,W.J., 2006. Long term diurnal variations on contaminant removal in high rateponds treating urban wastewater. Bioresour. Technol. 97, 1709–1715.

Gavala, H.N., Yenal, Y., Skiadas, I.V., Westermann, P., Ahring, B.K., 2003. Mesoplilicand thermophilic anaerobic digestion of primary and secondary sludge. Effect ofpretreatment at elevated temperature. Water Res. 37, 4561–4572.

González-Fernández, C., Sialve, B., Bernet, N., Steyer, J.P., 2012a. Thermalpretreatment to improve methane production of Scenedesmus biomass.Biomass Bioenerg. 40, 105–111.

González-Fernández, C., Sialve, B., Bernet, N., Steyer, J.P., 2012b. Comparison ofultrasound and thermal pretreatment of Scenedesmus biomass on methaneproduction. Bioresour. Technol. 110, 610–616.

Hendriks, A.T.W.M., Zeeman, C., 2009. Pretreatments to enhance the digestibility oflignocellulosic biomass. Bioresour. Technol. 100, 10–18.

86 F. Passos et al. / Bioresource Technology 138 (2013) 79–86

López, C.V.G., Céron-García, M.C., Acién-Fernandéz, F.G., Segóvia-Bustos, C., Chisti,Y., Fernández-Sevilla, J.M., 2010. Protein measurements of microalgal andcyanobacterial biomass. Bioresour. Technol. 101, 7587–7591.

Lu, J., Gavala, H.N., Skiadas, I.V., Mladenovska, Z., Ahring, B.K., 2008. Improvinganaerobic sewage sludge digestion by implementation of a hyperthermophilicprehydrolisis step. J. Environ. Manage. 88, 881–889.

Metcalf, Eddy, 2003. Wastewater Engineering: Treatment and Reuse, fourth ed.McGraw-Hill Higher Education, Boston, USA.

Palmer, C.M., 1962. Algas en los abastecimientos de agua. Manual ilustrado acercade la identificación, importancia y control de las algas en los abastecimientos deagua. Editiorial Interamericana, Mexico.

Passos, F., Solé, M., García, J., Ferrer, I., 2013. Biogas production from microalgaegrown in wastewater: Effect of microwave pretreatment. Appl. Energy, http://dx.doi.org/10.1016/j.apenergy.2013.02.042.

Raposo, F., De La Rubia, M.A., Fernández-Cegrí, V., Borja, R., 2011. Anaerobicdigestion of solid organic substrates in batch mode: an overview relating tomethane yields and experimental procedures. Renew. Sust. Energ. Rev. 16, 861–877.

Ras, M., Lardon, L., Sialve, B., Bernet, N., Steyer, J.P., 2011. Experimental study on acoupled process of production and anaerobic digestion of Chlorella vulgaris.Bioresour. Technol. 102, 200–206.

Sialve, B., Bernet, N., Bernard, O., 2009. Anaerobic digestion of microalgae as anecessary step to make microalgal biodiesel sustainable. Biotechnol. Adv. 27,409–416.

Tang, B., Yu, L.F., Huang, S.S., Luo, J.Z., Zhuo, Y., 2010. Energy efficiency of pre-treating excess sewage sludge with microwave irradiation. Bioresour. Technol.101, 5092–5097.

Vavilin, V.A., Fernandez, B., Palatsi, J., Flotats, X., 2008. Hydrolysis kinetics inanaerobic degradation of particulate organic matter: an overview. WasteManage. 28, 939–951.

Wilson, C.A., Novak, J.T., 2009. Hydrolysis of macromolecular components ofprimary and secondary wastewater sludge by thermal hydrolytic pretreatment.Water Res. 43, 4489–4498.

Zupancic, G.D., Roš, M., 2003. Heat and energy requirements in thermophilicanaerobic sludge digestion. Renew. Energy 28, 2255–2267.