microalgae conversion to biogas: thermal pretreatment contribution on net energy production

Microalgae Conversion to Biogas: Thermal PretreatmentContribution on Net Energy ProductionFabiana Passos† and Ivet Ferrer*,†

†GEMMA, Group of Environmental Engineering and Microbiology, Department of Hydraulic, Maritime and EnvironmentalEngineering, Universitat Politecnica de Catalunya·BarcelonaTech, c/Jordi Girona 1-3, Building D1, E-08034, Barcelona, Spain

ABSTRACT: Microalgal biomass harvested from wastewater treatmenthigh rate algal ponds may be valorised through anaerobic digestionproducing biogas. However, microalgae anaerobic biodegradability islimited by their complex cell wall structure. Thus, pretreatment techniquesare being investigated to improve microalgae methane yield. In the currentstudy, thermal pretreatment at relatively low temperatures of 75−95 °Cwas effective at enhancing microalgae anaerobic biodegradability;increasing the methane yield by 70% in respect to nonpretreated biomass.Microscopic images showed how the pretreatment damaged microalgaecells, enhancing subsequent anaerobic digestion. Indeed, digestate imagesshowed how after pretreatment only species with resistant cell walls, suchas diatoms, continued to be present. Energy balances based on lab-scalereactors performance at 20 days HRT, shifted from neutral to positive(energy gain around 2.7 GJ/d) after thermal pretreatment. In contrast with electricity consuming pretreatment methods, such asmicrowave irradiation, thermal pretreatment of microalgae seems to be scalable.

■ INTRODUCTION

High rate algal ponds (HRAP) for urban wastewater treatmenthave been studied since the 1950s.1 This technology is basedon a symbiotic relationship between microalgae and hetero-trophic bacteria, where bacteria degrade organic matterconsuming oxygen generated by microalgae photosynthesis(Figure 1). HRAP has been proved efficient in removingorganic matter and nutrients from contaminated effluents.2

Moreover, HRAP may be a cost-effective alternative in respectto activated sludge systems, since in this case no external inputof aeration is required.3 In fact, these systems may convertwastewater treatment plants (WWTP) into energy producers.4

The microalgae-bacterial biomass produced in such systemsmay be valorised through anaerobic digestion producing biogas.This process is already well-known for sewage sludge treatmentin conventional WWTP. Nevertheless, the anaerobic digestionof microalgal biomass has shown a slow biodegradability,reaching methane yields of 0.05−0.15 L CH4/g VS whenreactors are operated at HRT below 20 days.5 These values arelow in respect to other organic substrates, such as starch andsugar crops (e.g., corn 0.18−0.41 L CH4/g VS and potatoes0.43 L CH4/g VS),6 or primary sludge (0.31 L CH4/g VS).7

Indeed, microalgae methane yield is more similar to wasteactivated sludge (0.13−0.14 L CH4/g VS).8

Pretreatment techniques have been investigated to enhancebiomass hydrolysis rate and to increase both bioavailability andbiodegradability of macromolecules for anaerobic digestion. Inthis way, molecules that cannot be degraded inside microalgaecomplex cell wall, after pretreatment are more readily digestedand converted to methane. Pretreatment methods, such asmicrowave, ultrasound and thermal hydrolysis have alreadybeen proved efficient in batch and continuous reactors,increasing between 60 and 108% microalgae methaneyield.9−11 However, methods requiring electricity may not beviable in full-scale facilities, at least when biomass is notpreviously dewatered.11 Indeed, pretreatments with lowelectricity input should be prioritized. Thermal pretreatmentat relatively low temperatures (<100 °C) enhanced microalgae

Received: February 27, 2014Revised: May 6, 2014Accepted: May 13, 2014Published: May 13, 2014

Figure 1. Symbiosis between microalgae and bacteria in HRAP forwastewater treatment.

Article

pubs.acs.org/est

© 2014 American Chemical Society 7171 dx.doi.org/10.1021/es500982v | Environ. Sci. Technol. 2014, 48, 7171−7178

pubs.acs.org/est

solubilization and methane yield in biochemical methanepotential (BMP) tests,12 suggesting the potential to reach apositive energy balance in continuous reactors.In the current study, microalgal biomass harvested from

HRAP treating domestic wastewater was digested in continuousreactors at 15 and 20 days HRT. In each period, thermalpretreatment at low temperature was evaluated. Microscopicimages were analyzed for providing an insight into microalgalbiomass characteristics after pretreatment and also afteranaerobic digestion. Finally, an energy assessment was carriedout to elucidate the applicability of this technology at full-scale.

■ EXPERIMENTAL SECTION

Microalgal Biomass. Microalgal biomass was grown in apilot WWTP composed by a screening pretreatment, followedby a primary settler, a HRAP and a secondary settler. This pilotWWTP was located in the facilities of the UniversitatPolitecnica de Catalunya (Barcelona, Spain). Urban wastewaterwas pumped from the municipal sewer to a tank (1.2 m3).Screening pretreatment was followed by gravity settlers with auseful volume of 7 L and a HRT of 0.9 h. The primary effluentwas pumped to the HRAP, which consisted of a PVC racewaypond with a paddle wheel for continuous mixed liquor stirring.The pilot HRAP had a useful volume of 0.47 m3, a surface areaof 1.5 m2 and a water depth of 0.3 m, it was operated with aHRT of 8 days, and received a surface loading rate of 24 gCOD/m2day and 4 g NH4−N/m2day from the primaryeffluent. Microalgal biomass was harvested from HRAP bygravity in the secondary settlers with a useful volume of 9 L anda HRT of 9 h. Subsequently, biomass was thickened in Imhoffcones for 24 h to increase the total solid (TS) concentration to2.0−2.5% (w/w).Thermal Pretreatment. Thermal pretreatment conditions

were 75 and 95 °C and 10 h of exposure time, according to ourprevious study on microalgae solubilization and digestion inBMP tests.12 Glass bottles with a liquid volume of 150 mL andtotal volume 250 mL were placed in an incubator at constanttemperature (75 or 95 °C) under continuous stirring for 10 h.Pretreated biomass was cooled at room temperature and storedat 4 °C until use.Continuous Anaerobic Digestion. Influence of pretreat-

ment on microalgae anaerobic digestion performance wasmonitored using two lab-scale reactors (2 L), with a usefulvolume of 1.5 L. In this manner, control and pretreated biomasswere simultaneously investigated. Reactors were operatedunder mesophilic conditions (37 ± 1 °C) by implementingan electric heating cover (Selecta, Spain). Constant mixing wasprovided by a magnetic stirrer (Thermo Scientific). Reactorswere operated on a daily feeding basis, where same volume waspurged from and added to digesters using plastic syringes (50mL). Biogas production was measured by water displacementand methane content was periodically analyzed by gaschromatography.The following experimental conditions were studied in three

different periods: Period I (15 days HRT and pretreatment at95 °C); Period II (20 days HRT and pretreatment at 95 °C)and Period III (20 days HRT and pretreatment at 75 °C). Ineach period, reactors were considered to be under steady-stateafter three complete HRT; they were then monitored over aperiod of at least two complete HRT. For Periods I, II, and III,the total experimental duration was 93, 113, and 118 days,respectively.

Analytical Methods. Physical-chemical parameters of theinfluent and effluent of both reactors were determined asfollows. Temperature was monitored daily. pH was neithercontrolled nor regulated, but determined twice a week with aCrison Portable 506 pH-meter. The concentration of TS,volatile solids (VS), ammonium (N-NH4), and total Kjeldhalnitrogen (TKN) were determined according to StandardMethods13 on a weekly basis. Soluble samples for N-NH4and volatile fatty acids (VFA) analyses were obtained bycentrifugation (UNICEN20, 4200 rpm, 8 min, 20 °C) andfiltration (glass fiber filter 47 mm). VFA were measured weeklyby gas chromatography (GC) (Agilent Technologies 7820A),as described by Passos et al.12 The methane content in biogaswas measured twice a week with a GC (Trace GC ThermoFinnigan) equipped with a thermal conductivity detector,according to Passos et al.12

Microalgae species identification was carried out periodicallyusing specific literature.14,15 Microscope images were used toprovide qualitative information on the effect of thermalpretreatment on microalgae cells and anaerobic digestion. Tothis end, samples of untreated and pretreated microalgalbiomass, before and after anaerobic digestion were analyzedonce the reactor was under steady-state for the optimalconditions found in this study (Period III: HRT 20 days andthermal pretreatment at 75 °C). The optical microscope(Aixoplan Zeiss, Germany) used was equipped with a camaraMRc5, using the software Axioplan LE. For transmissionelectron microscopy (TEM), samples were fixed and stained asdescribed previously,11 and examined using a JEOL 1010 TEMat 100 kV accelerating voltage.

Statistical Analysis. The effect of thermal pretreatment onthe methane production rate and yield was determined by theANOVA test using R 3.0.1 software (The R Foundation forStatistical Computing). ρ = 0.01 was set as the level of statisticalsignificance.

Energy Assessment. The theoretical energy balance offull-scale reactors was estimated from experimental data,considering a flow rate of 100 m3/d and a useful volume of1500 m3 for 15 days HRT (Period I) and 2000 m3 for 20 daysHRT (Periods II and III). Electricity and heat requirements formicroalgal biomass pretreatment and anaerobic digestion werecalculated according to Ferrer et al.16 All parameters used forthe energy assessment are summarized in Table 1.For the control reactor, where no pretreatment was applied,

input heat was calculated as the energy required to heat influentbiomass from ambient temperature (Ta) to digestion temper-ature (Td), according to eq 1. The density (ρ) and specific heat(γ) of microalgal biomass were assumed to be the same as thoseof water, 1000 kg/m3 and 4.18 kJ/kg·°C, respectively. Heatlosses through the reactor wall were considered; the heattransfer coefficient (k) was assumed to be 1 W/m2·d.17 Thereactor wall surface area was calculated from the reactor usefulvolume, considering a 2:1 diameter to height ratio; while thereactor bottom and top were not accounted for.16,17

ρ γ= − + −E Q T T kA T T( ) ( )86.4i ,heat d a d a (1)

where Ei,heat: input heat (kJ/d); ρ: density (kg/m3); Q: flowrate (m3/d); γ: specific heat (kJ/kg·°C); Td: anaerobicdigestion temperature (37 °C); Ta: ambient temperature (20°C); k: heat transfer coefficient (W/m2·°C); A: surface area ofthe reactor wall (m2).For the pretreated reactor, input heat was calculated as the

energy required to heat influent biomass from ambient

Environmental Science & Technology Article

dx.doi.org/10.1021/es500982v | Environ. Sci. Technol. 2014, 48, 7171−71787172

temperature to pretreatment temperature (Tp). Since thereactor was operated under mesophilic conditions (37 °C), noextra energy was needed to heat biomass from pretreatment todigestion conditions. In fact, heat would be recovered whencooling down biomass from pretreatment temperature todigestion temperature by means of a heat exchanger, with an

efficiency ϕ of 85%.18 Therefore, input heat was calculated asthe energy required to increase influent biomass temperaturefrom ambient to pretreatment temperature, but subtracting theenergy recovered by cooling biomass from pretreatmenttemperature to digestion temperature (eq 2). Heat lossesthrough the reactor walls were also accounted for.

ρ γ ρ γ ϕ= − − −

+ −

E Q T T Q T T

kA T T

( ) ( )

( )86.4

i ,heat p a p d

d a (2)

where Ei,heat: input heat (kJ/d); ρ: density (kg/m3); Q: flow rate

(m3/d); γ: specific heat (kJ/kg·°C); Td: anaerobic digestiontemperature (37 °C); Ta: ambient temperature (20 °C); Tp:pretreatment temperature (75 or 95 °C); ϕ: heat recoveryefficiency; k: heat transfer coefficient (W/m2·°C); A: surfacearea of the reactor wall (m2).Furthermore, input electricity for anaerobic digestion was

estimated as the energy required for biomass pumping andreactor mixing, which were assumed to be 1800 kJ/m3 and 300kJ/m3

reactor·d, respectively.18 Input electricity was the same for

both control and pretreated reactors (eq 3).

θ ω= +E Q Vi ,electricity (3)

where Ei,electricity: input electricity (kJ/d); Q: flow rate (m3/d);θ: electricity consumption for pumping (kJ/m3); V: usefulvolume (m3); ω: electricity consumption for mixing (kJ/m3

reactor·d).The energy output of the process was calculated from the

methane production rate of each reactor (control andpretreated), according to eq 4. The lower heating value of

Table 1. Energy Assessment Parameters

parameter unit value reference

density of water (ρ) kg/m3 1000 17specific heat of water (γ) kJ/kg °C 4.18 17ambient temperature (Ta) °C 20 this studyanaerobic digestion temperature(Td)

°C 37 this study

petreatment temperature (Tp) °C 75; 95 this studyflow rate (Q) m3/d 100 this studyheat transfer coefficient (k) W/m2·°C 1 17heat recovery by heat exchanger(ϕ)

% 85 18

surface area of the reactor wall(A)

m2 384; 465 calculated

ueful volume (V) m3 1500;2000

calculated

electricity consumption forpumping (θ)

kJ/m3 1800 18

electricity consumption rate formixing (ω)

kJ/m3·d 300 18

lower heating value of methane(ξ)

kJ/m3 35 800 17

methane production rate (PCH4) m3CH4/m

3·d Table 3 this studyeergy conversion efficiency (η) % 90 this study

Table 2. Influent and Digested Microalgal Biomass Characteristics with and without Thermal Pretreatment at 75 and 95 °C,Mean Values (Standard Deviation)

period I period II period III

parameter control pretreatment control pretreatment control pretreatment

Operation Conditionspretreatment temperature (°C) - 95 - 95 - 75HRT (days) 15 15 20 20 20 20OLR (kg VS/m3d)) 0.88 (0.17) 0.89 (0.28) 0.72 (0.18) 0.73 (0.21) 0.71 (0.04) 0.68 (0.10)OLR (kg COD/m3d) 1.28 (0.24) 1.28 (0.40) 1.04 (0.26) 1.06 (0.31) 1.05 (0.08) 1.06 (0.08)Influent CompositionpH 7.5 (0.3) 7.6 (0.4) 7.3 (0.4) 7.3 (0.6) 7.4 (0.3) 7.4 (0.4)TS [% (w/w)] 2.63 (0.33) 2.59 (0.18) 2.12 (0.42) 2.12 (0.48) 2.14 (0.25) 2.16 (0.26)VS [% (w/w)] 1.24 (0.16) 1.12 (0.10) 1.17 (0.21) 1.14 (0.22) 1.33 (0.16) 1.35 (0.16)VS/TS (%) 50.41 (3.70) 50.4 (2.17) 59.2 (2.62) 52.5 (1.55) 62.2 (1.55) 62.4 (0.28)COD (g/L) 19.86 (4.14) 19.95 (6.21) 19.50 (4.89) 19.87 (5.81) 19.88 (1.55) 19.85 (1.57)TKN (g/L) 1.10 (0.36) 1.06 (0.41) 0.75 (0.32) 0.75 (0.29) 0.87 (0.25) 0.87 (0.25)N-NH4 (mg/L) 10.60 (4.05) 19.81 (3.20) 12.46 (0.92) 24.95 (3.22) 8.10 (0.80) 12.35 (1.62)VFA (mg COD/L) 0 152.48 (45.11) 0 168.33 (35.29) 20.25 (14.9) 100.31 (55.9)Effluent CompositionpH 7.1 (0.3) 7.0 (0.5) 7.6 (0.4) 7.6 (0.6) 7.5 (0.3) 7.6 (0.4)TS [% (w/w)] 2.22 (0.43) 2.16 (0.48) 2.12 (0.60) 2.05 (0.59) 2.11 (0.31) 1.98 (0.27)VS [% (w/w)] 1.10 (0.32) 0.94 (0.30) 0.98 (0.14) 0.75 (0.23) 1.12 (0.14) 0.95 (0.10)VS/TS (%) 47.93 (6.82) 43.19 (1.78) 46.37 (4.86) 41.08 (5.47) 53.22 (2.63) 50.98 (2.12)COD (g/L) 15.46 (0.36) 13.51 (0.93) 12.41 (0.52) 10.40 (1.74) 11.85 (0.71) 10.59 (0.48)TKN (g/L) 1.07 (0.06) 1.06 (0.14) 0.99 (0.09) 0.98 (0.19) 1.08 (0.03) 1.13 (0.08)N-NH4 (mg/L) 264.0 (12.27) 293.2 (23.55) 205.0 (19.00) 360.0 (23.00) 218.0 (9.54) 323.0 (17.15)VFA (mg COD/L) 0 0 0 51.5 (27.57) 55.6 (13.24) 150.2 (58.61)Removal EfficiencyVS removal (%) 21.3 (2.8) 23.1 (2.0) 29.4 (2.1) 55.1 (4.0) 35.7 (4.1) 52.3 (3.8)COD removal (%) 20.1 (4.1) 29.3 (8.2) 31.4 (6.2) 44.7 (5.4) 34.2 (4.1) 48.7 (4.6)



methane (ξ) was assumed to be 35 800 kJ/m3 CH4.17 An

efficiency of 90% on energy conversion was considered (η).

ξ η=E P Vo CH4 (4)

where Eo: output energy (kJ/d); PCH4: methane production rate(m3CH4/m

3·d); ξ: lower heating value of methane (kJ/m3CH4); V: useful volume (m3); η: energy conversionefficiency.Finally, results were expressed as energy balance (ΔE) and

energy ratio (Eo/Ei) for both reactors (control and pretreated)in each studied period. The energy balance was calculated asthe difference between the energy output and energy input(heat and electricity) (eq 5), while the energy ratio wascalculated from the energy output over the energy input (heatand electricity) (eq 6).

Δ = − +E E E E( )i io ,heat ,electricity (5)

= +E E E E E/ /( )i i io o ,heat ,electricity (6)

■ RESULTS AND DISCUSSIONMicroalgae Anaerobic Digestion at 15 days HRT. In

the first experimental period, microalgal biomass anaerobicdigestion was evaluated at 15 days HRT (Table 2). During thisstage, average methane yield and methane production rate were0.10 L CH4/g VS and 0.09 L CH4/L·d, respectively. After

pretreatment at 95 °C for 10 h, microalgal biomass reached anaverage methane yield and methane production rate of 0.12 LCH4/g VS and 0.12 L CH4/L·d, respectively (Figure 2; Table3). Thus, methane production rate was significantly higher inthe pretreated reactor (33% increase), but not the methaneyield (20% increase). Poor results in terms of microalgaemethanisation with and without pretreatment suggested thatthe applied HRT was lower than required for microalgaeconversion to methane. In fact, volatile solid removal was only21% and 23% in the control and pretreated reactor,respectively.A low HRT may be preferred in full-scale systems design in

order to reduce the reactor volume. However, as for otherparticulate organic substrates (e.g., waste activated sludge andlignocellulosic biomass), longer HRTs seem to be necessary forattaining higher methane yields. This is mainly attributed torecalcitrant substances and to the nature of microalgae cell wall.In fact, BMP tests with different microalgae species have shownthat anaerobic digestion is strain-specific and it depends on thecomposition and biodegradability of the cell wall, which ismainly composed by cellulose, hemicellulose and pectin.5,19 Forinstance, the cellulosic content may hinder anaerobic bacterialattack, since it requires different enzymes for solubilization andit depends strongly on the inoculum source, biomassconcentration and cellulose bioavailability in the cellstructure.20 Some microalgae species, such as Chlorella sp.

Figure 2. Average methane yield (weekly values) of untreated (control) and thermally pretreated microalgal biomass for the three studied periods.

Table 3. Biogas Production from Microalgal Biomass with and without Thermal Pretreatment at 75 and 95 °C. Mean values(standard deviation)



Operation Conditionspretreatment temperature (°C) - 95 - 95 - 75HRT (days) 15 15 20 20 20 20Biogas Characteristicsmethane production rate (L CH4/L·d) 0.09 (0.04) 0.12 (0.05)a 0.12 (0.07) 0.21 (0.12)a 0.13 (0.09) 0.20 (0.10)a

methane yield (L CH4/g VS) 0.10 (0.03) 0.12 (0.04) 0.18 (0.05) 0.31 (0.08)a 0.18 (0.05) 0.30 (0.09)a

methane yield (L CH4/g COD) 0.07 (0.02) 0.08 (0.02) 0.13 (0.03) 0.22 (0.02)a 0.12 (0.03) 0.19 (0.05)a

methane content in biogas (% CH4) 67.8 (0.8) 68.1 (0.4) 67.9 (0.7) 68.2 (0.4) 68.3 (0.6) 68.1 (0.6)aStand for significantly higher values between paired columns (ρ = 0.01).



and Scenedesmus sp., are formed by a rigid cellulose-based cellwall, hampering the anaerobic digestion process.5 Otherspecies, such as Chlamydomonas sp., have a glycoprotein cellwall structure with lack of cellulose, thus they are more readilybiodegradable.In our study, microalgal biomass was grown in a wastewater

treatment HRAP and, therefore, it was composed by aspontaneous mixed culture of 90% microalgae and 10% bacteriapopulations.2 Microalgal biomass was mainly composed byStigeoclonium sp., Monorraphidium sp., and the diatoms Nitzchiasp. and Amphora sp., among other species of Chlorophytamicroalgae. In a minor extent, zooplankton, such as rotifers andprotozoos were also present. This biomass has positivecharacteristics, such as the tendency to form flocs, whicheases biomass harvesting by sedimentation. However, this alsoinduces the production of extracellular polymeric substances(EPS) forming these flocs and biofilms, which are particulateorganic compounds hampering biomass hydrolysis rate.So far, few studies have been carried out with microalgae

anaerobic digestion in continuous reactors. For instance,Scenedesmus biomass produced 0.08 L CH4/g COD at 23days HRT;9 while Chlorella vulgaris methane yield was 0.11 LCH4/g COD at 16 days HRT and 0.18 L CH4/g COD at 28days HRT (60% increase).21 Experimental results highlight howlong HRT could contribute improving anaerobic digestionperformance, in particular with pretreated microalgal biomass.11

Microalgae Anaerobic Digestion at 20 days HRT.During periods II and III, when microalgal biomass wasdigested at 20 days HRT, the methane yield and methaneproduction rate increased to 0.18 L CH4/g VS and 0.12−0.13 L

CH4/L·d, respectively (Figure 2; Table 3); with VS removalaround 30−35% (Table 2). This represents an increment of80% in respect to untreated biomass digested at 15 days HRT.However, this increment was even superior for the pretreatedreactor. During period II, when microalgal biomass waspretreated at 95 °C, the methane yield was 0.31 L CH4/gVS, 160% higher in respect to pretreated microalgae digested at15 days HRT. Since our previous study in BMP tests showedthat the methane yield increase was similar after 75 and 95 °C,and the former requires lower heat input, pretreatment at 75 °Cwas also evaluated in continuous reactors. Indeed, the methaneyield was 0.30 L CH4/g VS, 150% higher in respect topretreated microalgae digested at 15 days HRT. With a HRT of20 days, methane yield and methane production rate werestatistically higher after thermal pretreatment, with a methaneyield increase of 72 and 67% for pretreatment at 95 and 75 °C,respectively.Concerning the reactors operation, in both of them,

anaerobic digestion was fairly stable. Most importantly,ammonium (N-NH4) and VFA concentrations were alwayslower than toxicity values. N-NH4 concentration after anaerobicdigestion was between 200 and 360 mg/L, below toxicconcentrations of 1.7 g/L.10 Furthermore, VFA concentrationswere very low in all studied periods (0−150 mg COD/L).So far, only two studies on microalgae thermal pretreatment

and anaerobic digestion in continuous reactors have beenpublished.22,10 Both of them reported a positive effect of thethermal pretreatment on subsequent anaerobic digestion. Theformer pretreated microalgal biomass grown in wastewatertreatment HRAP at 100 °C for 8 h, increasing the methane

Figure 3. Optical microscope images of untreated (a and b) and pretreated microalgal biomass (c and d).



yield by 33% in respect to untreated microalgae.22 In the latterstudy, Nannochloropsis salina anaerobic digestion was improvedafter pretreatment at 100 and 120 °C for 2 h, increasing themethane yield from 0.13 to 0.27 L CH4/g VS (108%increase).10 In BMP tests, the methane yield of differentmicroalgae species was increased by 10−60% after pretreatmentat 110 °C for 15 min (from 0.20 to 0.27 to 0.22−0.41 L CH4/gVS)23 and by 220% after pretreatment at 90 °C for 2 h (from0.08 to 0.17 L CH4/g VS).9 Different experimental resultscould be explained by the pretreatment conditions and by thecharacteristics of studied microalgae and its cell wallcompounds bioavailability and biodegradability.

In order to understand how microalgae cells were affected bythe pretreatment step, samples of biomass were studied underoptical microscope before (Figure 3a,b) and after microalgaepretreatment under the optimum pretreatment condition (75°C) (Figure 3c,d). In Figure 3a, the filamentous green algaeStigeoclonium sp. is evidenced at the center of the image, whilein Figure 3b, several green microalgae species immersed in abiological floc are present, before thermal pretreatment. Afterpretreatment, biomass clearly showed lower pigmentation andmostly dead cells were present (Figure 3c,d). Stigeoclonium sp.(Figure 3c) and Amphora sp. (Figure 3d) had nonpigmentedretracted chloroplasts, due to the effect of heat during the

Figure 4. TEM images of the diatom cells Nitzschia sp. (a) and Amphora sp. (b) after thermal pretreatment.

Figure 5. Optical microscope images of digested biomass for the control reactor (a and b) and pretreated reactor (c and d).



pretreatment step. Nevertheless, most microalgae cell wallswere not disrupted. This means that, although microalgae cellswere damaged and dead after thermal pretreatment, they werenot fragmented as indicated by other authors.5 This fact wasalready evidenced in our previous study on microalgaemicrowave pretreatment11 and also for Nannochloropsis salinathermal pretreatment.10 In both cases, microalgae cell wallswere mostly intact, although with disintegrated and indistinctinternal organelles. Furthermore, in the case of diatomsNitzschia sp. and Amphora sp., transmission electronic micro-scopic (TEM) images showed that most cell structuresremained intact after pretreatment (Figure 4a); althoughsome of them appeared empty, which could indicate cell wallfissure (Figure 4b).Even if microalgae cell walls were not fragmented after

pretreatment, damaged organelles may be more readilydigestible. Indeed, optical microscope images of the controlreactor digestate showed the presence of Stigeoclonium sp. anddiatoms Nitzschia ap. and Amphora sp. (Figure 5a,b). Thissuggests that other green microalgae species (e.g., Monoraphi-dium sp.) were digested even without pretreatment. Thishypothesis is in accordance with the fact that microalgae cellwall characteristics are strain-specific and, therefore, mayrespond differently to bacteria attack. Nevertheless, opticalmicroscopic images of the pretreated reactor digestate showed amore degraded biomass (Figure 5c,d), possibly due to the celldamage caused by the thermal pretreatment (Figure 3c,d). InFigure 5c, Stigeoclonium sp. cell wall is partly disrupted. Figure5d shows digested biomass, where mainly diatoms could beidentified.In summary, experimental results suggest that the most easily

degradable microalgae (such as Monoraphidium sp.) weredigested even without pretreatment, while filamentous micro-algae species forming flocs (such as Stigeoclonium sp.) weredigested after thermal pretreatment and, finally, very rigid cellwalls, as those of diatoms which are made of nanopatternedsilica (SiO2),

24 were hardly digested even after the thermalpretreatment applied. These hypotheses are based onqualitative information, which ought to be complementedwith quantitative microscopic analyses in future research.Energy Assessment of Microalgae to Biogas Con-

version. The anaerobic digestion of microalgal biomass withand without thermal pretreatment was evaluated from anenergy perspective (Table 4). In period I (15 days HRT, 95°C), for both control and pretreated reactor, the energy inputwas higher than energy output generated from the producedmethane. This is due to the low methane production rate whenreactors were operated at a low HRT of 15 days. However,when reactors were operated at a HRT of 20 days (Periods IIand III), control reactor reached a neutral energy balance (∼0)and energy ratio (∼1), while pretreated reactors reachedpositive energy balances (>2 GJ/d) and energy ratios (>1)

(Table 3). The pretreatment produced 19 and 27% moreenergy than it consumed at 95 and 75 °C, respectively.So the best scenarios included microalgae thermal pretreat-

ment at 75−95 °C followed by anaerobic digestion at a HRT of20 days. In these cases, the net energy production was 2.12 and2.73 GJ/d after pretreatment at 95 and 75 °C, respectively.Since the methane production rate after pretreatment at 95 °Cis only slightly better than after pretreatment at 75 °C, andinput heat required for raising temperature up to 95 °C ishigher, the optimal scenario is the pretreatment at 75 °Cfollowed by anaerobic digestion at 20 days HRT. For attaininga neutral energy balance (Ei = Eo), the methane production rateshould be at least 0.16 m3 CH4/m

3d when applying thermalpretreatment at 75 °C, whereas it should be at least 0.18 m3

CH4/m3d when applying pretreatment at 95 °C.

Compared with other pretreatment techniques, low temper-ature pretreatment seems the most feasible method so far. Forinstance, the energy required for microwave pretreatment wasmuch higher than the extra energy generated from the process,due to the high microwave electricity demand.11 Thus, biomassshould be dewatered before undergoing microwave pretreat-ment. Likewise, after thermal pretreatment of dewateredNannochloropsis salina, the net energy gain would be 232kWh/m3 of electricity and 317 kWh/m3 of heat, considering aconsumption of 47 kWh/m3 for centrifugation.10 In our case,energy gain could be achieved even without previous biomassdewatering; although biomass dewatering may improve furtherthe net energy production. Based on the obtained results,thermal pretreatment at 75 °C seems applicable to full-scalefacilities treating wastewater in microalgae-based ponds. Thus,future pilot-scale experiments including 75 °C pretreatment arestrongly recommended.

■ AUTHOR INFORMATION

Corresponding Author*Phone: +34 934016463; fax: +34 934017357; e-mail: [email protected].

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This research was financially supported by the Spanish Ministryof Economy and Competitiveness (BIOALGAS Project,CTM2010-17846). Fabiana Passos appreciates her PhDscholarship funded by the Coordination for the Improvementof Higher Level Personal (CAPES) from the Brazilian Ministryof Education. We acknowledge Mariona Hernandez-Marine from the University of Barcelona and Joan Garcia from theUniversitat Politecnica de Catalunya for the valuable help onmicroalgae microscopic images and characterisation.

Table 4. Energy Assessment of Microalgal Biomass Anaerobic Digestion with and without Thermal Pretreatment



Ei,heat (GJ/d) 6.77 10.53 6.87 10.63 6.87 9.38Ei,electricity (GJ/d) 0.63 0.63 0.78 0.78 0.78 0.78Eo (GJ/d) 4.35 5.80 7.73 13.53 8.38 12.89ΔE (GJ/d) -3.05 -5.36 0.08 2.12 0.72 2.73Eo/Ei 0.59 0.52 1.01 1.19 1.09 1.27



mailto:[email protected]

mailto:[email protected]

■ REFERENCES(1) Oswald, W. J.; Golueke, C. G. Biological transformation of solarenergy. Adv. Appl. Microbiol. 1960, 2, 223−262.(2) García, J.; Green, B. F.; Lundquist, T.; Mujeriego, R.; Hernandez-Marine, M.; Oswald, W. J. Long term diurnal variations in contaminantremoval in high rate ponds treating urban wastewater. Bioresour.Technol. 2006, 97 (14), 1709−1715.(3) Clarens, A. F.; Resurreccion, E. P.; White, M. A.; Colosi, L. M.Environmental life cycle comparison of alge and other bioenergyfeedstocks. Environ. Sci. Technol. 2010, 44, 1813−1819.(4) Menger-Krug, E.; Niederste-Hollenberg, J.; Hillenbrand, T.;Hiess, H. Integration of microalgae systems at municipal wastewatertreatment plants: Implication for energy and emission balances.Environ. Sci. Technol. 2012, 46, 11505−11514.(5) Gonzalez-Fernandez, C.; Sialve, B.; Bernet, N.; Steyer, J. P.Impact of microalgae characteristics on their conversion to biofuel.Part II: Focus on biomethane production. Biofuels, Bioprod. Biorefin.2011, 6, 205−218.(6) Frigon, J. C.; Guiot, S. R. Biomethane production from starch andlignocellulosic crops: A comparative review. Biofuels, Bioprod. Biorefin.2010, 4, 447−458.(7) Kepp, U.; Solheim, O. E. Thermodynamical Assessment of theDigestion Process. In 5th European Biosolids and Organic ResidualsConference, Wakefield, UK, 2000.(8) Bougrier, C.; Delgenes, J. P.; Carrere, H. Combination of thermaltreatments and anaerobic digestion to reduce sewage sludge quantityand improve biogas yield. Process Saf. Environ. Prot. 2006, 84, 280−284.(9) Gonzalez-Fernandez, C.; Sialve, B.; Bernet, N.; Steyer, J. P.Thermal pretreatment to improve methane production of Scenedesmusbiomass. Biomass Bioenerg. 2012, 40, 105−111.(10) Schwede, S.; Rehman, Z.-U.; Gerber, M.; Theiss, C.; Span, R.Effects of thermal pretreatment on anaerobic digestion of Nannoclor-opsis salina biomass. Bioresour. Technol. 2013, 143, 505−511.(11) Passos, F.; Hernandez-Marine, M.; García, J.; Ferrer, I. Long-term anaerobic digestion of microalgae grown in HRAP for wastewatertreatment. Effect of microwave pretreatment. Water Res. 2014, 49 (1),351−359.(12) Passos, F.; Sole, M.; García, J.; Ferrer, I. Impact of lowtemperature pretreatment on the anaerobic digestion of microalgalbiomass. Bioresour. Technol. 2013, 138, 79−86.(13) APHA-AWWA-WPCF. Standard Methods for the Examination ofWater and Wastewater. 20th ed.; Washington, 1999.(14) Palmer, C. M. Algas en los Abastecimientos de Agua. ManualIlustrado Acerca de la Identificacion, Importancia y Control de las Algas enlos Abastecimientos de Agua.; Editiorial Interamericana: Mexico, 1692.(15) Bourrelly, P. Les Algues d’eau Douce. Tome I: Les Algues Vertes.;Edition N. Boubee & Cie: Paris, 1966.(16) Ferrer, I.; Serrano, E.; Ponsa, S.; Vazquez, F.; Font, X.Enhancement of thermophilic anaerobic digestion by 70 °Cpretreatment: Energy considerations. J. Resid. Sci. Technol. 2009, 59,1777−1784.(17) Metcalf & Eddy, Tchobanoglous, G.; Burton, F. L.; Stensel, H.D. Wastewater Engineering, Treatment and Reuse, 4th ed.; McGraw HillEducation, 2003.(18) Lu, J.; Gavala, H. N.; Skiadas, I. V.; Mladenovska, Z.; Ahring, B.K. Improving anaerobic sewage sludge digestion by implementation ofa hyperthermophilic prehydrolisis step. J. Environ. Manag. 2008, 88,881−889.(19) Mussgnug, J. H.; Klassen, V.; Schluter, A.; Kruse, O. Microalgaeas substrates for fermentative biogas production in a combinedbiorefinery concept. J. Biotechnol. 2010, 150, 51−56.(20) Molinuevo-Salces, B.; Gomez, X.; Moran, A.; García-Gonzalez,M. C. Anaerobic co-digestion of livestock and vegetable processingwastes: Fibre degradation and digestate stability. Waste Manag. 2013,33 (6), 1332−1338.(21) Ras, M.; Lardon, L.; Sialve, B.; Bernet, N.; Steyer, J. P.Experimental study on a coupled process of production and anaerobicdigestion of Chlorella vulgaris. Bioresour. Technol. 2011, 102, 200−206.

(22) Chen, P. H.; Oswald, W. J. Thermochemical treatment for algalfermentation. Environ. Int. 1998, 24, 889−897.(23) Alzate, M. E.; Munoz, R.; Rogalla, F.; Fdz-Polanco, F.; Perez-Elvira, S. I. Biochemical methane potential of microalgae: Influence ofsubstrate to inoculum ratio, biomass concentration and pretreatment.Bioresour. Technol. 2012, 123, 488−494.(24) Sumper, M.; Brunner, E. Learning from diatoms: Nature’s toolsfor the production of nanostructured silica. Adv. Funct. Mater. 2006,16, 17−26.



microalgae conversion to biogas: thermal pretreatment contribution on net energy production

Documents