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Long-term anaerobic digestion of microalgaegrown in HRAP for wastewater treatment. Effect ofmicrowave pretreatment

Fabiana Passos a, Mariona Hernandez-Marine b, Joan Garcıa a,Ivet Ferrer a,*aGEMMA e Group of Environmental Engineering and Microbiology, Department of Hydraulic, Maritime and

Environmental Engineering, Universitat Politecnica de Catalunya$BarcelonaTech, c/Jordi Girona 1-3, Building D1,

E-08034 Barcelona, SpainbUniversitat de Barcelona, Av. Joan XXIII s/n, E-08028 Barcelona, Spain

a r t i c l e i n f o

Article history:

Received 22 July 2013

Received in revised form

4 October 2013

Accepted 7 October 2013

Available online 17 October 2013

Keywords:

Algae

Bioenergy

Biogas

High rate algal pond

Hydrolysis

Methane

* Corresponding author. Tel.: þ34 934016463;E-mail address: ivet.ferrer@upc.edu (I. Fe

0043-1354/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.watres.2013.10.013

a b s t r a c t

This paper describes the anaerobic digestion of microalgal biomass from high rate ponds in

continuous anaerobic reactors for biogas production. With hydraulic retention times (HRT)

of 15 and 20 days, the volatile solids (VS) removal did not exceed 30%, and the methane

production rate ranged between 0.12 and 0.14 L CH4/L day. To improve process perfor-

mance, microwave irradiation at 900 W for 3 min (specific energy w70,000 kJ/kg VS) was

applied as a pretreatment step. The VS removal increased to 40 and 45% at 15 and 20 days

HRT, respectively. Consequently, the methane production rate increased to 0.16 and 0.20 L

CH4/L day at 15 and 20 days HRT, respectively. Microscopic analysis confirmed cell wall

damage, although generally without lysis, after irradiating microalgal biomass. However,

the energy consumption was much higher than the extra energy production of the process.

Indeed, microalgal biomass should not only be thickened but also dewatered if microwave

irradiation was to be applied as a pretreatment to anaerobic digestion for biogas

production.

ª 2013 Elsevier Ltd. All rights reserved.

1. Introduction energy consumption (Metcalf and Eddy, 2003). Thus, reducing

Combining wastewater treatment and bioenergy production

is already a well-known concept, developed inmany full-scale

facilities where sludge is digested to produce biogas and

electricity. This may cover around 50% of the electricity de-

mand in conventional activated sludge wastewater treatment

plants (WWTP). Themost energy intensive process is aeration

of the biological reactor, which accounts for 55% of the total

fax: þ34 934017357.rrer).

ier Ltd. All rights reserved

aeration requirements can contribute towards achieving en-

ergy sufficient WWTP. In this sense, natural treatment sys-

tems such as ponds have been developed, especially for small

communities. High rate algal ponds (HRAP) are shallow race-

way reactors, where microalgae and bacteria grow in symbi-

osis. In these systems, organic matter is degraded by

heterotrophic bacteria, which consume oxygen provided by

microalgal photosynthesis and, therefore, no aeration is

.

wat e r r e s e a r c h 4 9 ( 2 0 1 4 ) 3 5 1e3 5 9352

needed. Although the efficiency of microalgal systems for

wastewater treatment has been extensively studied, little is

known on the reuse of harvested biomass for bioenergy

purposes.

Lately, many studies have been focused on the conversion

of pure microalgae cultures to biodiesel due to their fast

growth rate and great capacity of accumulating carbon-rich

lipids. Inasmuch, in economic terms, this process seems not

to be feasible if compared to fossil fuel or biodiesel production

from other agricultural crops (Sialve et al., 2009). On the other

hand, anaerobic digestion for biogas production is a more

straight-forward technology, which was first studied by

Golueke et al., in 1957. The authors concluded that biomass

digestion was likely to be hampered by ammonium toxicity,

high pH values and/or resistance of microalgae cell wall.

Indeed, the methane yield of several microalgae species

(0.10e0.30 L CH4/g VS) (Table 1), is relatively low if compared to

other organic substrates, such as agricultural waste (up to

0.53 L CH4/g VS) (Gunnaseelan, 1997). In order to enhance the

rate and extent of microalgae hydrolysis, pretreatment tech-

niques have been applied. Recent studies have shown a pos-

itive effect on microalgae solubilisation and methane yield in

biochemical methane potential (BMP) tests after thermal and

microwave pretreatment (Gonzalez-Fernandez et al., 2012;

Passos et al., 2013a,b).

In particular, microwave irradiation has been applied to

enhance solubilisation and biogas production of several

organic wastes (Toreci et al., 2009; Passos et al., 2013a). This

pretreatment works by leading water to a boiling state

through electromagnetic energy. The process polarizes mac-

romolecules, causing changes in the secondary and tertiary

structure of proteins and cell hydrolysis (Park et al., 2010).

Previous studies with waste activated sludge showed that

microwave irradiation increased the biogas yield by 24% in

continuous reactors operated at 10 days HRT (Toreci et al.,

2009). Regarding microalgae, our previous study was focused

on biomass solubilisation and methane yield in BMP tests

under different microwave pretreatment conditions. The re-

sults showed that the main parameter influencing biomass

solubilisation was the applied specific energy, regardless of

the output power and exposure time. Biomass solubilisation

and biogas yield showed a linear correlation, reaching the

highest biogas yield after microwave irradiation at 900 W for

3 min (78% increase in respect to untreated microalgae)

Table 1 e Continuous anaerobic digestion of microalgae under

Microalgae HRT (days) OLR (g VS/L

Scenedesmus sp. and Chlorella sp. 30 1.5

Tetraselmis sp. 14 2.0

Spirulina maxima 8, 12 and 16 1.0

Chlorella sp. and Scenedesmus sp. 10 2.0 and 6.

Chlorella vulgaris 16 and 28 1.0

Scenedesmus sp. 23 1.0a

Microalgal biomass 15 and 20 1.0 and 0.

a Data expressed as g COD/L$day.b Data expressed as L CH4/g COD.

(Passos et al., 2013a). Consequently, this pretreatment is here

evaluated in continuous reactors, operating at an HRT of 15

and 20 days.

Up to date, little research has focused on the anaerobic

digestion ofmicroalgae grown inwastewater; the sole study in

continuous anaerobic reactors following a pretreatment step

being that of Chen and Oswald (1998). So, to our knowledge,

only thermo-chemical pretreatment has been evaluated in

continuous mode. The aim of this research was, firstly, to

examine the anaerobic digestion of microalgal biomass in

continuous lab-scale reactors operated with an HRT of 15 and

20 days; and secondly, to evaluate the microwave pretreat-

ment effect in terms of cell disruption and biogas production.

Optic and transmission electron microscopic (TEM) images

were analysed to investigate microalgae cell wall integrity

after pretreatment. Finally, the energy balance of the process

was calculated to attest the viability of full-scale application.

2. Material and methods

2.1. Microalgal biomass characteristics

Microalgal biomass was grown in a pilot high rate algal pond

(HRAP) used for secondary treatment of domestic wastewater.

The primary treatment was composed of a primary settler.

The HRAP had a useful volume of 470 L andwas operated with

an HRT of 8 days. Average organic and nutrient loading rates

were 3 g COD/m2$day and 60 mg NeNH4þ/m2$day, respec-

tively. A full description of the HRAP operation can be found in

Passos et al. (2013a). Microalgal biomass was harvested in a

secondary settler with a nominal volume of 0.01 m3 (0.16 days

HRT) and an average recovery efficiency of 60%. In order to

increase the solids concentration of harvested biomass, it was

further thickened by gravity in Imhoff cones for 24 h and

stored at 4 �C.Due to the characteristics of the system, an open HRAP fed

with primary treated wastewater; not only microalgae, but a

mixed culture of different microorganisms was present. Ac-

cording to previous research, though, microalgae consist of

approximately 90% of the total biomass (Garcıa et al., 2006).

Therefore, in this study we refer to the microalgal-bacterial

biomass grown in the HRAP as microalgal biomass.

mesophilic conditions (without pretreatment).

$day) Methane yield(L CH4/g VS)

Reference

0.25 Golueke et al., 1957

0.31 San Marzano et al., 1982

0.09e0.15 Samson and Leduy, 1982

0 0.09e0.14 Yen and Brune, 2007

0.15 and 0.24 Ras et al., 2011

0.08b Gonzalez-Fernandez et al., 2012

75 0.13 and 0.17 This study

Table 2eAverage feed and digestedmicroalgal biomass characteristics, with andwithoutmicrowave pretreatment prior toanaerobic digestion at 15 and 20 days HRT.

Reactor Control Pretreated Control Pretreated

HRT (days) 15 15 20 20

Working conditions

OLR (g VS/L$day) 0.99 (0.05) 0.92 (0.17) 0.76 (0.22) 0.77 (0.24)

OLR (g COD/L$day) 1.47 (0.10) 1.35 (0.14) 1.08 (0.29) 1.05 (0.30)

Feed composition

pH 7.5 (0.4) 7.5 (0.5) 7.4 (0.5) 7.3 (0.6)

TS [% (w/w)] 2.40 (0.55) 2.47 (0.55) 2.62 (0.67) 2.65 (0.69)

VS [% (w/w)] 1.42 (0.34) 1.47 (0.35) 1.52 (0.44) 1.54 (0.45)

VS/TS (%) 59.0 (1.96) 59.5 (2.26) 57.6 (2.25) 58.1 (2.14)

COD (g O2/L) 21.4 (1.96) 21.7 (2.36) 22.0 (6.21) 22.5 (5.73)

CODs (mg O2/L) 80 (18) 1030 (165) 94 (12) 1452 (121)

TKN (g/L) 1.06 (0.33) 1.06 (0.33) 1.26 (0.38) 1.26 (0.38)

NeNH4þ (mg/L) 27.2 (14.0) 36.0 (13.3) 13.8 (6.3) 26.0 (15.5)

VFA (mg COD/L) 33 (11.8) 53 (35.4) 16 (10.7) 18 (4.3)

Effluent composition

pH 7.18 (0.11) 7.12 (0.31) 7.12 (0.04) 7.22 (0.06)

TS [% (w/w)] 1.95 (0.21) 1.95 (0.08) 2.20 (0.20) 2.18 (0.25)

VS [% (w/w)] 1.09 (0.12) 1.05 (0.18) 1.16 (0.20) 1.04 (0.25)

VS/TS (%) 56.2 (1.30) 53.8 (1.30) 52.7 (2.70) 47.7 (1.00)

COD (g O2/L) 15.0 (1.02) 13.8 (13.1) 15.2 (5.17) 13.1 (3.7)

CODs (mg O2/L) 304 (5.4) 446.8 (7.8) 336.2 (8.79) 517.5 (14.8)

TKN (g/L) 1.16 (0.08) 1.18 (0.09) 1.23 (0.20) 1.2 (0.22)

NeNH4þ (mg/L) 271.9 (23.3) 464.5 (53.7) 272.1 (36.3) 394.8 (68.1)

VFA (mg COD/L) 87 (30.3) 100 (37.8) 101 (35.1) 110 (29.6)

Removal efficiency

VS removal [% (w/w)] 28.3 (1.8) 38.7 (2.0) 29.4 (2.1) 45.1 (4.0)

COD removal [% (w/w)] 30 (7.4) 36 (6.8) 31 (4.4) 42 (8.2)

wat e r r e s e a r c h 4 9 ( 2 0 1 4 ) 3 5 1e3 5 9 353

2.2. Microwave pretreatment

Microwave irradiation was applied to microalgal biomass in a

household type microwave (Samsung M1914, 2450 MHz fre-

quency). Based on previous BMP tests, microwave pretreat-

ment conditions were 900 W of output power and 3.0 min of

exposure time (the applied specific energy was 110,200 kJ/kg

VS, Passos et al., 2013a). In this case, the applied specific en-

ergy varied according to the volatile solids (VS) concentration

in thickened microalgal biomass (Eq. (1)).

Specific energyðkJ=kg VSÞ ¼ ½PowerðWÞ � TimeðsÞ�=Organic weightðg VSÞ (1)

A volume of 150 mL of thickened microalgal biomass was

pretreated in 250 mL glass bottles. Afterwards, biomass was

cooled to room temperature and stored at 4 �C.

2.3. Mesophilic anaerobic digestion

Two lab-scale reactors (2 L), with a useful volume of 1.5 L, were

used to evaluate microalgae anaerobic digestion with (pre-

treated) and without (control) microwave pretreatment. Both

digesters were operated under mesophilic conditions (35 �C);temperature was maintained by means of an electric heating

cover (Selecta, Spain). Constant mixing was provided by a

magnetic stirrer (Thermo Scientific, Spain). Reactors were

supplied with an inlet, outlet, gas collector and temperature

sensor. They were operated on a continuous feeding basis: the

same volume was daily purged from and added to the

digesters, using plastic syringes (50 mL). Biogas production

was measured by water displacement. The methane content

was analysed by gas chromatography (GC Trace, Thermo

Finnigan) twice a week, following the procedure described by

Passos et al. (2013b).

Both reactors had been in operation for one year when the

experiment was conducted. The process was initially start-up

by inoculating digested sludge from a municipal WWTP in

Barcelona, Spain, and feeding thickened microalgal biomass

from the HRAP. For studying the influence of the HRT on

microalgae anaerobic digestion, both reactors were operated

at two HRT: firstly at 15 days and secondly at 20 days. Ac-

cording to previous studies (Table 1), the optimal HRT seemed

to range between 20 and 30 days. Shorter HRT were here

defined (15e20 days) to verify if microwave pretreatment

enhanced the digestion rate so as to reduce the HRT without

hampering process performance, compared to the control

reactor with untreated microalgae. During the whole experi-

mental period, one reactor was fed with untreated biomass

(control) while the other one was fed with microwave pre-

treated biomass.

Digesters were assumed to be in steady-state after

completing three HRT, i.e. after 45 days in the first period (HRT

15 days) and 60 days in the second period (HRT 20 days). Af-

terwards, anaerobic digestion was evaluated by the process

performance over a period corresponding to more than two

HRT, 35 days for the first period (HRT 15 days) and 49 days for

the second period (HRT 20 days). Reactors operation condi-

tions are summarised in Table 2.

wat e r r e s e a r c h 4 9 ( 2 0 1 4 ) 3 5 1e3 5 9354

2.4. Analytical methods

Total solids (TS), VS, total Kjeldhal nitrogen (TKN), ammonium

nitrogen (NeNH4þ), chemical oxygen demand (COD) and sol-

uble chemical oxygen demand (CODs) were determined ac-

cording to Standard Methods (APHA-AWWA-WPCF, 1999). TS,

VS and pH were determined twice a week, while TKN, Ne

NH4þ, COD and CODs were determined once a week. Volatile

fatty acids (VFA) were measured weekly with a gas chro-

matograph (GC) equipped with a Thermal Conductivity De-

tector, following the procedure described by Passos et al.

(2013b).

2.5. Microscopic images

Microalgae species identification and cell wall integrity im-

ages were taken with an optic microscope (Aixoplan Zeiss,

Germany), equipped with a camera MRc5, using the software

Axioplan LE. Basic microalgae and cyanobacteria diversity

morphotypes were identified from classical specific literature

(Bourrelly, 1966; Komarek and Fott, 1983). During the whole

experimental period (7 months), the main species growing in

the system were Monoraphidium sp., Stigeoclonium sp., Scene-

desmus sp. and Nitzchia sp. Biomass was mostly flocculated,

although free microalgae cells were also present.

For transmission electron microscopy (TEM) images,

biomass was centrifuged at 2000 rpm for 5 min. Samples were

fixed in a mixture of 2% paraformaldehyde and 2.5% glutar-

aldehyde, in 0.1 M cacodylate buffer for 2e4 h, washed in this

buffer and then posfixed in 1% osmium tetroxide (Abed et al.,

2002). Samples were dehydrated by a graded acetone series

and embedded in Spurr’s resin (SigmaeAldrich) (Spurr, 1969).

Sections were stained with 2% uranyl acetate and lead citrate

and examined using a JEOL 1010 TEM (Jeol, Japan) at 100 kV

accelerating voltage.

2.6. Statistical analysis

The effect of microwave pretreatment on the methane pro-

duction and yield in continuous reactors was determined by

means of the ANOVA test using R 3.0.1 software. r ¼ 0.05 was

set as the level of statistical significance.

2.7. Energy assessment

The energy balance of microalgae anaerobic digestion was

based on Ferrer et al. (2009). Heat requirements for the control

reactor were calculated by the heat difference between

ambient temperature and mesophilic digestion temperature

(35 �C). Ambient temperature was assumed to be a typical

average value for theMediterranean region (20 �C). Heat losses

through the reactor walls and piping were not taken into ac-

count, since they only represent 2e8% of the total heat de-

mand (Zupancic and Ros, 2003). The heat input for the control

reactor was calculated from Eq. (2).

Ei;heat ¼ ½r Q gðTd� TaÞ�=V (2)

where: Ei,heat: input heat (kJ/d); r: density of microalgal

biomass (kg/m3); Q: microalgal biomass flow rate (m3/d); g:

specific heat of microalgal biomass (kJ/kg �C); Td: Anaerobic

digestion temperature (�C); Ta: Ambient temperature (�C); V:

reactor volume (m3).

For the pretreated digester, the energy required for mi-

crowave irradiation was based on the applied specific energy,

according to Eq. (3). In this case, there was no energy input to

reach mesophilic conditions, since microalgal biomass was at

a higher temperature after microwave irradiation (around

95 �C).

Ei;pretreatment ¼ ½Specific energy OLR V�=V (3)

where: Ei,pretreatment: input energy for pretreatment (kJ/d);

Specific energy: average specific energy applied by microwave

pretreatment (kJ/kg VS); OLR: organic loading rate (g VS/m3d);

V: reactor volume (m3).

The electricity input for microalgal biomass pumping and

reactor mixing were estimated as in Eq. (4) (Lu et al., 2008).

Ei;electricity ¼ ½Q qþ Vu�=V (4)

where: Ei,electricity: input electricity (kJ/d); Q: microalgal

biomass flow rate (m3/d); q: electricity consumption for

pumping (kJ/m3); V: reactor volume (m3); u: electricity con-

sumption for reactor mixing (kJ/m3d).

Therefore, the total energy input for the control digester

was the sum of input heat (Ei,heat) and input electricity

(Ei,electricity); whereas for the pretreated digester, it was the

sum of input energy for pretreatment (Ei,pretreatment) and

input electricity (Ei,electricity).

The energy output was expressed by the methane pro-

duction rate for both digesters, according to Eq. (5). The lower

heating value of methane (x) was 35,800 kJ/m3 (Metcalf and

Eddy, 2003).

Eo ¼ PCH4x (5)

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

(m3 CH4/m3d); x: lower heating value of methane (kJ/m3 CH4).

Finally, the energy ratio (Ei/Eo) was calculated to evaluate

the control (Eq. (6)) and the pretreatment (Eq. (7)) reactor

viability.

Eo=Ei;control ¼ ½PCH4xV��½ðr Q gðTd� TaÞÞ þ ðQ qþ VuÞ� (6)

Eo=Ei;pretreatment ¼ ½PCH4xV��½ðSpecific energy OLR VÞ

þ ðQ qþ VuÞ� (7)

3. Results and discussion

3.1. Anaerobic digestion of microalgal biomass

With the aim of improving process performance, the anaer-

obic digestion of microalgal biomass was studied at an HRT of

15 and 20 days (Tables 2 and 3). Consequently, the organic

loading rate (OLR) decreased from approximately 1.0 g VS/

L$day during the first period (15 days HRT) to approximately

0.75 g VS/L$day during the second period (20 days HRT).

Average concentration of organic matter was 1.42 and 1.52%

VS in the influent and 1.09 and 1.16% VS in the effluent at 15

and 20 days HRT, respectively. Regardless of the HRT, the VS

removal remained the same (28e29%) during the whole

Digestion time (days)

0 15 30 100 120 140

Met

hane

yie

ld (m

L C

H4/g

VS)

0.0

0.1

0.2

0.3

0.4

Control Pretreated

syad02TRHsyad51TRH

Fig. 1 e Average methane yield obtained from untreated

(control) and microwave pretreated microalgal biomass

anaerobic digestion (n [ 5).

wat e r r e s e a r c h 4 9 ( 2 0 1 4 ) 3 5 1e3 5 9 355

experimental period. As a result, the methane production rate

was also similar, i.e. 0.12 L CH4/L$day (15 days HRT) and 0.14 L

CH4/L$day (20 days HRT).

Microalgal biomass achieved amethane yield of 0.13 L CH4/

g VS at 15 days HRT and 0.17 L CH4/g VS at 20 days HRT (Fig. 1).

Previous studies reported amethane yield of 0.24e0.25 L CH4/g

VS at 30 days HRT, digesting microalgal biomass from

wastewater treatment HRAP in continuous reactors (Golueke

et al., 1957; Chen and Oswald, 1998); which is 47 and 92%

higher than our results at 20 and 15 days HRT, respectively.

Similarly, the anaerobic digestion of a pure Chlorella vulgaris

culture achieved 0.24 L CH4/g VSS and 51% COD removal at 28

days HRT (Ras et al., 2011). A BMP test with microalgae grown

in wastewater, mainly Chlorella vulgaris and Scenedesmus obli-

quus, attained a methane yield of 0.13 L CH4/g COD (Gonzalez-

Fernandez et al., 2011b). As evidenced by the results of

continuous and batch anaerobic reactors, there is a high

variability on the methane yield among microalgae species

(Mussgnug et al., 2010; Gonzalez-Fernandez et al., 2011). Such

a high variability may be explained by the macromolecular

composition and cell wall characteristics of such species.

Sialve et al. (2009) estimated the theoretical microalgae

methane yield as 0.48e0.80 L CH4/g VS; however experimental

results have so far been limited to 0.05e0.31 L CH4/g VS

(Gonzalez-Fernandez et al., 2011). This occurs because most

microalgae cell wall is composed of organic compounds with

low biodegradability, such as cellulose and hemicellulose.

Table 3 e Methane production with untreated (control) and preat 15 and 20 days HRT.

Reactor Control

HRT (days) 15

Methane production rate [L CH4/L$day] 0.12 (0.03)

Methane yield [L CH4/g VS] 0.13 (0.02)

Methane yield [L CH4/g COD] 0.09 (0.02)

Methane content [% CH4] 68.5 (1.70)

a Stand for significantly higher values between paired columns (r ¼ 0.05

Our study looked at the anaerobic digestion of a mixed

culture of microalgae grown in wastewater, wheremicroalgae

species were neither defined nor controlled. The macromo-

lecular composition was in average 49% proteins, 17% lipids

and 20% carbohydrates (Passos et al., 2013a). Indeed, the high

TKN of the substrate (1.0e1.2 g TKN/L) was explained by the

high protein content of microalgal biomass. The main issue

regarding the degradation of proteins is the increase of

ammonium (NeNH4þ) concentration, which could become

toxic to methanogens. Previous studies with lipid extracted

microalgal biomass have not shown any toxicity at ammo-

nium concentrations of 800e4300 mg Ne NH4þ/L (Ehimen

et al., 2011). In our case, ammonium concentrations were in

average 270 mg NeNH4þ/L at both HRT (Table 2), far below

toxicity values of 4000e6000 mg NeNH4þ/L (Koster and

Lettinga, 1988). Besides, no VFA accumulation was observed.

Total VFA values of the digestatewere around 90 and 100mg/L

15 and 20 days HRT, respectively (Table 2).

On thewhole,microalgae anaerobic digestion seemed to be

limited by the hydrolysis, since the VS removal remained low

regardless of the HRT. In order to enhance the rate and extent

of methane production in anaerobic reactors, two approaches

could then be undertaken: 1) to continue increasing the HRT or

2) to apply a pretreatment technique in order to enhance

biomass solubilisation. For instance, digesting Chlorella sp. the

COD removalwas improved from 33% to 51% by increasing the

HRT from 16 to 28 days (Ras et al., 2011). In the present study,

the second approach was considered, with the aim of

improving process performance without increasing the

reactor volume and capital cost.

3.2. Anaerobic digestion after microalgae microwavepretreatment

Previous research on microalgae anaerobic digestion in BMP

tests, showed positive effects of microwave pretreatment on

the methane production rate (27e75% increase) and methane

yield (12e78% increase) (Passos et al., 2013a). Microalgae sol-

ubilisation was increased by microwave irradiation and

therefore, organic matter was more accessible to anaerobic

bacteria. The optimal pretreatment condition (900 W for

3 min) was subsequently evaluated in continuous reactors at

15 and 20 days HRT.

Compared to the control at 15 days HRT, the methane

production rate increased by 33% (from 0.12 to 0.16 L CH4/

L$day) and themethane yield by 30% (from 0.13 to 0.17 L CH4/g

VS) after microwave pretreatment (Table 3, Fig. 1). Indeed, the

treated microalgal biomass in anaerobic digesters operated

Pretreated Control Pretreated

15 20 20

0.16 (0.05)a 0.14 (0.06) 0.20 (0.04)a

0.17 (0.04)a 0.17 (0.03) 0.27 (0.05)a

0.14 (0.03)a 0.12 (0.02) 0.17 (0.04)a

69.3 (1.17) 68.1 (0.86) 68.5 (0.56)

).

Fig. 2 e Microscopic images of different microalgae species before (a, c, e, g) and after (b, d, f, h) microwave pretreatment.

wat e r r e s e a r c h 4 9 ( 2 0 1 4 ) 3 5 1e3 5 9356

VS removalwas in average 38%, whereas in the control reactor

it was in average 28%. Furthermore, the influent soluble COD

increased from 80 mg/L (control) to 1030 mg/L (pretreated),

almost 12-fold higher, indicating that the pretreatment was

successful at solubilising organic matter prior to anaerobic

digestion. When the HRT was raised to 20 days, the methane

production rate increased by 43% (from 0.14 to 0.20 L CH4/

L$day) and themethane yield by 58% (from 0.17 to 0.27 L CH4/g

VS) compared to the control reactor (Table 3, Fig. 1). Indeed,

the VS removal was also higher (45%) in the pretreated reactor

compared to the control reactor (29%)with untreated biomass.

Additionally, ammonium concentration was higher in the

pretreated reactor digestate (395 mg NeNH4þ/L), indicating

higher protein solubilisation. Note that these values are still

Fig. 3 e TEM images of Monoraphidium sp. before (a) and after (b) microwave pretreatment. Note that cell walls seem to be

intact, although organelles are damaged.

wat e r r e s e a r c h 4 9 ( 2 0 1 4 ) 3 5 1e3 5 9 357

below toxicity concentrations of 4000e6000 mg NeNH4þ/L

(Koster and Lettinga, 1988).

The statistical significance of experimental results was

verified with the ANOVA test, comparing the control and

pretreated reactor. For both HRT (15 and 20 days), themethane

yield and methane production rate were significantly higher

after the pretreatment step, whereas the methane content in

biogas was the same (around 68%) (Table 3).

In order to improve the kinetics of methane production

and/or methane yield, pretreatment techniques have long

been used to enhance the solubilisation of different organic

substrates prior to anaerobic digestion (Carrere et al., 2010).

However, literature on microalgae pretreatment and anaer-

obic digestion performance is still scarce. The sole study

dealing with continuous reactors following microalgae pre-

treatment was that of Chen and Oswald (1998). The authors

reported an increasedmethane yield (by 33%) aftermicroalgae

pretreatment at 100 �C for 8 h. In BMP tests, Schamphelaire

and Verstraete (2009) did not find any improvement on the

methane yield after pretreating algal biomass at 80 �C for 2.5 h;

while Gonzalez-Fernandez et al. (2012) found a similar

methane yield for Scenedesmus biomass without pretreatment

(76 L CH4/g COD) and after pretreatment at 70 �C (85 L CH4/g

COD), but 2.2-fold higher methane yield when it was pre-

treated at 90 �C. Microscopic analysis showed much higher

cell wall damage at the highest temperature. Similarly, low

temperature (55, 75 and 95 �C) pretreatment of microalgal

Table 4 e Parameters considered for the energy assessment.

Parameter Unit

Density of water (r) kg/m3

Specific heat of water (g) kJ/kg �CAmbient temperature (Ta) �CAnaerobic digestion temperature (Td) �CFlow rate (Q) m3/day

Reactor volume (V) m3

Organic loading rate (OLR) kg VS/m3 day

Specific energy kJ/kg VS

Energy consumption for pumping (q) kJ/m3

Energy consumption rate for stirring (u) kJ/m3 day

Lower heating value of methane (x) kJ/m3

Methane production rate (PCH4 ) m3CH4/m

3 day

biomass grown in HRAP improved the methane yield by 14%,

53% and 62%, respectively, after 15 h of exposure time (Passos

et al., 2013b). In the case of microwave irradiation, analogous

temperatures (95 �C) where reached with a much shorter

exposure time (3 min), leading to similar methane yield

enhancement (30e58%).

3.3. Microscopic analysis of microalgae cell wall

In order to evaluate the impact of microwave irradiation on

microalgae cell wall structure, microscopic images were

analysed. Hypothetically, pretreatment techniques have been

applied to break the cell wall and release intracellular com-

pounds to the soluble phase, in such away that cell disruption

would increase the concentration of readily available organic

matter and hydrolysis rate. Fig. 2 shows images of different

microalgae species before (Fig. 2a, c, e, and g) and after (Fig. 2b,

d, f and h) microwave pretreatment. Observation in the optic

microscope showed that microalgae were affected by micro-

wave irradiation, as indicated by the decrease in chlorophyll

pigmentation. However, it did not seem to induce complete

microalgae cell wall lysis, since some cells appeared to be still

intact after the pretreatment step. To further evaluate cell

wall integrity, TEM images were taken (Fig. 3); which evi-

denced that cells were stressed by microwave irradiation, as

organelles were affected and damaged beyond repair (Fig. 3).

So, although the pretreatment was not responsible for cell

Value Reference

1000 Metcalf and Eddy, 2003

4.18 Metcalf and Eddy, 2003

20 This study

35 This study

0.75 � 10�4; 1.0 � 10�4 This study

1.5 � 10�3 This study

0.92; 0.77 This study

73,470; 70,130 This study

1800 Lu et al., 2008

300 Lu et al., 2008

35,800 Metcalf and Eddy, 2003

Table 3 This study

HRT (days)

0251

Ener

gy ra

tio (E

o/Ei)

0.0

0.5

1.0

1.5

2.0

Control reactorPretreated reactor

Fig. 4 e Energy ratio (Ei/Eo) for anaerobic digestion with and

without microwave pretreatment under the studied

conditions (1.5% VS). Note: Eo/Ei > 1 indicate net energy

production.

wat e r r e s e a r c h 4 9 ( 2 0 1 4 ) 3 5 1e3 5 9358

wall lysis, stressed cells may have been more susceptible to

bacteria attack, enhancing the anaerobic biodegradability of

pretreated microalgae.

To further evaluate the effect of pretreatment on micro-

algal biomass solubilisation and anaerobic digestion, micro-

scopic images of digested biomass ought to be analysed. In

this manner, it would be possible to elucidate whether pre-

treated cells were more accessible to methanogens, even if

cell walls were not lysed after the pretreatment step.

3.4. Energy considerations

For scaling up the pretreatmentmethod, the energy balance of

the process can be estimated from the energy output

(methane production) and energy input (heat and electricity).

All parameters used for calculations are summarised in Table

4. In Fig. 4, energy ratios (Eo/Ei) higher than 1 indicate net en-

ergy production. The control reactor with untreated micro-

algal biomass almost reached a positive energy balance (Eo/

Ei ¼ 0.80), with an HRT of 15 days. Increasing the HRT to 20

days (and methane production rate from 0.12 to 0.14 L CH4/

L$day) led to net energy production (0.71 kJ/L$day). On the

other hand, the pretreated reactor did not reach a positive

energy balance at any HRT, although the methane production

ratewas improved bymicrowave irradiation (to 0.16 and 0.20 L

CH4/L$day for HRT of 15 and 20 days, respectively). This sug-

gests that microwave pretreatment consumed more energy

than the extra energy it produced.

Previous studies on microwave pretreatment of different

substrates also found positive effects on the methane pro-

duction, although the energy consumption was always higher

than the energy production. Hu et al. (2012) calculated the

energy efficiency by the ratio Ei/Eo and reported ratios in the

range of 5e130, depending on the output power, exposure

time and VS concentration. Tang et al. (2010) regarded

biomass concentration as the most important parameter

affecting the energy efficiency of microwave pretreatment.

Consequently, the energy balance was recalculated

considering a higher solids concentration (>1.5%) in pre-

treated microalgal biomass; a VS concentration of 14% was

necessary to reach a positive energy balance. This means that

harvested microalgal biomass should not only be thickened

but also dewatered before microwave pretreatment. Actually,

this is also the case of sludge thermal hydrolysis, which has

long been applied at full-scale WWTP.

To summarise, microwave pretreatment was successful at

improving microalgae biomethanisation, however other pre-

treatment techniques should be investigated to improve the

methane yield with lower energy input. For instance, low

temperature pretreatment (<100 �C) could be a promising

alternative to enhance microalgae digestion with residual

heat from cogeneration engines fuelled by biogas.

4. Conclusions

The anaerobic digestion of microalgae grown in wastewater

HRAP was studied in continuous reactors. The methane yield

was improved by 30% when increasing the HRT from 15 to

20 days; however low VS removal indicated that hydrolysis

was limiting process performance. Microwave pretreatment

enhanced themethane yield by 30% at 15 days HRT and 58% at

20 days HRT. Microscopic images showed that biomass was

affected by microwave irradiation, yet not enough for com-

plete microalgae cell wall lysis. The main disadvantage of the

pretreatment technique was high energy consumption,

meaning that microalgal biomass thickening and dewatering

would be needed to reach a positive energy balance. Alterna-

tive less energy consuming pretreatment techniques ought to

be investigated.

Acknowledgements

Authors want to thank the Spanish Ministry of Economy and

Competitiveness for financial support to this project (BIO-

ALGAS CTM2010-17846). Fabiana Passos appreciates her PhD

scholarship funded by the Coordination for the Improvement

of Higher Level Personal (CAPES) from the BrazilianMinistry of

Education.

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