anaerobic digestion of microalgal biomass with ultrasonic disintegration

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Anaerobic digestion of microalgal biomass with ultrasonicdisintegration

Ki Young Park a,b,*, Jihyang Kweon b, Phrompol Chantrasakdakul a, Kwanyong Lee a,Ho Young Cha a

aDepartment of Civil Environmental System Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, South KoreabDepartment of Environmental Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul, South Korea

a r t i c l e i n f o

Article history:Received 28 December 2012Received in revised form29 March 2013Accepted 31 March 2013Available online xxx

Keywords:Anaerobic digestionChlorella vulgarisDisintegrationMicroalgaeSpecific methane production

* Corresponding author. Department of Civil EnviroKonkuk University, 120 Neungdong-ro, Gwangjin-gu,2 450 3736.

E-mail addresses: [email protected], [email protected] (J. Kweon),(P. Chantrasakdakul), [email protected] (K. L(H.Y. Cha).

0964-8305/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2013.03.035

Please cite this article in press as: Park, K.YBiodeterioration & Biodegradation (2013), h

a b s t r a c t

The use of photosynthetic microalgae for nutrient removal and biofuel production has been widelydiscussed. Anaerobic digestion of waste microalgal biomass to produce biogas is a promising technologyfor bioenergy production. However, the methane yield from this anaerobic process was limited becauseof the hard cell wall of Chlorella vulgaris. The use of ultrasound has proven to be successful at improvingthe disintegration and anaerobic biodegradability of Chlorella vulgaris. Ultrasonic pretreatment in therange of 5e200 J ml�1 was applied to waste microalgal biomass, which was then used for batchdigestion. Ultrasound techniques were successful and showed higher soluble COD at higher appliedenergy. During batch digestion, cell disintegration due to ultrasound increased in terms of specific biogasproduction and the degradation rate. Compared to the untreated sample, the specific biogas productionwas increased in the ultrasound-treated sample by 90% at an energy dose of 200 J ml�1. For the dis-integrated samples, volatile solids reduction was also increased according to the energy input anddegradation. These results indicate that the hydrolysis of microalgal cells is the rate-limiting step in theanaerobic digestion of microalgal biomass.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Wastewater treatment using microalgae has been investigatedsince the 1950s (Oswald and Gotaas, 1957). Wastewater containingthe biological wastes of animals impregnated with abundantinorganic nitrogen and phosphorus is one of the most significantcauses of eutrophication in water bodies. The use of microalgae hasalso attracted attention because microalgae have the ability toremove CO2 and NOx during their growth (Park et al., 2009).

However, the process of disposing the residual biomass isdifficult even after beneficial lipid extraction, since the defattedbiomass still contain higher level of organic matter. Anaerobicdigestion can be an answer to this problem because this biotech-nological process can mineralize algal waste (Olguín, 2003).

nmental System Engineering,Seoul, South Korea. Tel.: þ82

[email protected] (K.Y. Park),[email protected]

ee), [email protected]

All rights reserved.

., et al., Anaerobic digestionttp://dx.doi.org/10.1016/j.ibio

Anaerobic digestion processes have the ability to recover energyfrom various waste sources such as methane. Co-digestion of algaewith waste activated sludge improves the reduction of volatilesolids (Yuan et al., 2012). Sialve et al. (2009) proposed that theanaerobic digestion of algae might be the optimal strategy for theenergetic recovery of cell biomass when the lipid composition inthe algal cell does not exceed 40%.Waste-algal biomass, although, isknown to be more difficult to digest than wastewater sludgebecause of the very high resistance of the algal cell wall tobiodegradation. Golueke et al. (1957) noted that following diges-tion, the biosolids developed a noticeable green color. This indi-cated the survival of chlorophyll, which is an intracellular material,and suggested that cellular lysis was not completed during diges-tion. It is therefore likely that any reduced total biogas production isassociated with overall substrate degradability rather than thecomposition of the algae. Therefore, pretreatment or disintegrationof the algal biomass is needed.

Different disintegration techniques have been successfullyapplied to activated and primary sludge to enhance methane pro-ductivities (Zhang et al., 2008) These disintegrationmethods can beclassified as: mechanical (ultrasonic, lysisecentrifuge, liquid shear,collision plate, grinding, etc.), biological (enzyme), thermal

of microalgal biomass with ultrasonic disintegration, Internationald.2013.03.035

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K.Y. Park et al. / International Biodeterioration & Biodegradation xxx (2013) 1e52

hydrolysis (heat, microwave) and chemical (oxidation, alkalitreatments, etc.) (Müller et al., 2009). Sludge disintegrationessentially disrupts the sludge/flocs in the aqueous phase and en-hances the solubility of the sludge particles. The dissolved com-ponents can be readily degraded and utilized as a substrate in thebiological process, thereby resulting in increased bioavailability(Park et al., 2004; Lee et al., 2005). In the case of methanefermentation of solid organic materials such as microbial cells,methane yield is significantly affected by the mass transfer in eachbiological step as well as by food availability (Izumi et al., 2010).These disintegrationmethods can also be used for microalgae. Chenand Oswald (1998) studied thermal pretreatments for an algalbiomass that was produced in sewage treatment ponds. Theyachieved a 33% improvement in methane production under theoptimal condition for anaerobic digestion at 100 �C over 8 h.However, little information is available regarding the disintegrationof microalgal biomass in anaerobic digestion.

The overall objectives of this study were to investigate thedisintegration and digestion of microalgae single cells that aregrown in swine wastewater. To investigate algal growth, swinewastewater was used as a growth medium. The harvested algaewere co-digested with sewage sludge at varying algae and sludgeratios and methane yield from anaerobic digestion was evaluated.Ultrasound as a pretreatment has been applied to microalgae todisrupt algal cell material and to enhance methane productivity.

2. Materials and methods

2.1. Cultivation of Chlorella vulgaris

The unicellular microalga Chlorella vulgaris (UTEX 265) wasused. C. vulgaris microalgae are eukaryotic photosynthetic micro-organisms that grow rapidly due to their simple structure (Li et al.,2008). C. vulgaris was cultured in sterilized modified Bold’s basalmedium prior to treatment in wastewater (Nichols, 1973). Theinocula used for initial loading were adjusted to 5 � 105 cell ml�1.The size of the C. vulgaris cells were determined to be375.9� 149.7 mm3 in size. This experiment used swine wastewaterfrom an aeration tank of an animal wastewater treatment plant inHongseong, South Korea. In order to prevent from bacterialcontamination, wastewater was filtered through a GF/C filter andthen autoclaved. After filtration and autoclave, a fraction of SS, COD,TOC and TP was slightly reduced. The characteristics of swinewastewater are: pH 7.8; SS 2.0 mg l�1; COD 55.0 mg l�1; T-N 5.3 mgl�1; NH4eN mg l�1; NO3eN mg l�1; and T-P 1.3 mg l�1. The growthof C. vulgaris cell was expressed as dry cell (DC) weight per liter ofculture suspension. The DC weight was evaluated by drying cells at85 �C for 24 h after filtration through a GF/C filter. All the otheranalyses were performed basically following standard methods(APHA, 2005).

2.2. Disintegration

Cell disruption of C. vulgaris was performed by using an ultra-sonic homogenizer model STH-750S (Sonictopia, Korea). The unithas a maximum power output of 750 W and can be operated at aconstant frequency of 20 kHz and a constant power input of 150W.It is equipped with a horn (20 � 123 mm diameter). Based on theliterature (Pérez-Elvira et al., 2009), ultrasonic waves have beenoperated at frequencies in the range of 5e200 J ml�1 with 100ml ofC. vulgaris. The ultrasonic dose is related to the amount of energysupplied per unit volume of substrate and is expressed as J ml�1 orkJ ml�1. However, it does not depend on the total solid concen-tration. The ultrasonic dose cannot be used to compare the sub-strate with different total solid content. As long as the total solid

Please cite this article in press as: Park, K.Y., et al., Anaerobic digestionBiodeterioration & Biodegradation (2013), http://dx.doi.org/10.1016/j.ibio

content remains constant, the ultrasound density is a practicalmethod of expressing the energy input for the disintegration ofsludge on a volume basis.

The soluble COD (SCOD) release was used as a direct mea-surement of C. vulgaris cell disintegration. When C. vulgaris cellwas sonicated, the intracellular enzymes and organalles ofC. vulgaris were released into the aqueous phase. An increase inSCOD after ultrasonic disintegration was an indication of theefficiency of disintegration of the C. vulgaris cells. SCOD releasedfrom the C. vulgaris was determined for each operating conditionas per the HACH method 8000 by using a UV/VIS spectropho-tometer (Hach, USA). The samples were centrifuged at 4000 rpmfor 5 min and the supernatant was filtered through a 0.45 mmpore size membrane filter and then used for the analysis ofSCOD.

The sludge disintegration efficiency was represented by thedegree of disintegration, which was calculated by using Equation(1) (Zhang et al., 2008):

Degree of Disintegration ¼ ðSCOD� SCOD0ÞðTCOD0� SCODÞ (1)

where SCOD0 is the SCOD of the sludge sample before the disin-tegration treatment (mg l�1).

2.3. Biochemical methane potential

Subsequent batch digestion was undertaken in a series ofbiochemical methane potential (BMP) assays by incubatingwastewater inoculatedwith anaerobic bacteria for a period of about25 d based on Angelidaki et al. (2009). Batch anaerobic digestionwas performed in 150 ml serum bottles with a working volume of100 ml. The batch digestion was carried out in duplicate. The datafor both cumulative gas generation and volatile solids reduction(VSR) were obtained during the digestion.

Two rounds of anaerobic digestion batch experiments wereconducted under mesophilic conditions. The first round focused onthe digestibility of C. vulgaris and this was compared with sewageas a co-digestion feedstock. For digestibility testing, different vol-umes of C. vulgaris biomass were added to each digestion bottle,and this led to volatile mass compositions of C. vulgaris in the di-gesters at 0% algae and 100% sludge (sludge alone), 50% algae and50% sludge (algae þ sludge), and 100% algae and 0% sludge (algaealone). In the second round, disintegrated C. vulgaris was fed tobatch digesters. Gas production was measured daily by inserting aneedle that was attached to a frictionless syringe through theseptum and allowing the headspace to equilibrate with atmo-spheric pressure. The compositions of the headspaces wereanalyzed via gas chromatography (HP 5890, PA, USA) by a thermalconductivity detector (TCD) and helium as the carrier gas inaccordance with Martín-González et al. (2010). The oven, injector,and detector temperatures were 70 �C, 150 �C, and 180 �C,respectively.

3. Results and discussion

3.1. Growth of C. vulgaris

Batch experiments were conducted to study the growthC. vulgaris in raw swine wastewater and nitrified secondaryeffluent. The logistic growth model equation was used in this studyas follows (Jiménez-Perez et al., 2004):


800Sludge+Algae

Sludge alone

K.Y. Park et al. / International Biodeterioration & Biodegradation xxx (2013) 1e5 3

y ¼ y0 þA� �

4mm�� (2)

Elapsed time (h)

0 100 200 300 400 500 600 700

Cu

mu

la

tive g

as

p

ro

du

ctio

n (m

l)

0

200

400

600

Algae alone

Fig. 2. Biogas production from the co-digestion of algae and sludge.

1þ expA

ðl� tÞ þ 2

where y ¼ algae population (cells ml�1), l ¼ lag-phase time (h),y0 ¼ initial population (cells m�1), mm ¼ specific growth rate (h�1),and A ¼ asymptote of growth (cells ml�1).

The logistic growth model was fitted successfully to determinethe biokinetic parameters (Fig. 1). The curves showed a good fitbetween the experimental data and calculated values in all cases(R2 was between 0.994 and P was below 0.0001). The kinetic pa-rameters for C. vulgaris in the swine wastewater were calculated asbeing A ¼ 69.2, mm ¼ 0.6, and l ¼ 108.8, respectively. Based on anapproximate formula for algal biomass of CH1.7O0.4N0.15P0.0094(Oswald, 1988), the observed total nitrogen and phosphorusremoval could be accounted for via incorporation into the algalbiomass. The effect of nitrogen sources on the growth was moresignificant than the phosphorus concentration because growthcontinued even after depletion of the phosphorus.

3.2. Digestibility of algal biomass

Initial experiments investigated the co-digestion of C. vulgarisand sewage sludge. Microalgal cells were found in waste activatedsludge, and these could survive within bacterial cells. This resis-tance to attack manifests itself as a low degree of degradability inan anaerobic digester relative to the digestion of sewage sludge(Cecchi et al., 1996). Using methane content measurement and theGompertz equation (Zwietering et al., 1990), the biochemicalmethane potential was calculated. All gases were measured at35 �C. The gas volumewas corrected for background gas productionas well as standard temperature and pressure (STP) conditionsusing the following Equation (3).

V ¼ ðVs � VbÞ273

ð273þ 35Þ$29:2P

(3)

where V ¼ net gas produced at STP, Vs ¼ gas produced by sample at35 �C, Vb ¼ Gas produced by blank at 35 �C, P ¼ atmosphericpressure.

Graph fitting was performed to investigate the BMP. In thisstudy, we proposed to use the Gompertz equation as shown inEquation (4).

M ¼ P � exp�� exp

�Rm$eP

ðl� tÞ þ 1��

(4)

0

2

4

6

8

10

0

50

100

150

0 50 100 150 200 250 300

Nu

trie

nt (

mg

-N

l-1

an

d m

g-P

l-1)

C. v

ulg

aris

(m

g-D

C l

-1)

Time (h)

C. vulgaris

Nitrogen

Phosphorus

Fig. 1. Growth curve of the microalgae and nutrient uptake fitted using a logisticmodel.


where M ¼ cumulative methane production, l ¼ lag-phase time,P ¼ methane production potential, Rm ¼ methane production rate,and e ¼ exp(1).

It was clear that the set that contained algae alone was noteffectively digested compared to the sludge alone as shown inFig. 2. Table 1 summarizes the experimental conditions and thecorresponding methane conversion yield in this study and incomparison to the values reported in the literature. The biogas yieldfrom co-digestion varied from 240 to 440 ml g-VS�1 depending onthe species and culture conditions. In this study, the measured gasyield for algae alone, algae þ sludge, and sludge alone were 364,415, and 497 ml g-VS�1 added, respectively. The anaerobic biode-gradability of the microalgae showed that this was a slow andincomplete process and that methane production was lower thanthat achieved for sewage sludge. The methanogenic potential ofmicroalgae is related to their cellular composition, especially to thestructures of their cell walls. Some microalgae have strong cellwalls so that the biodegradable organic material inside the cell isnot available to the anaerobic bacteria.

The biomass of microalgae contains high amounts of lipids andproteins, and the resistance of the cell wall is one of the limitingfactors of cell digestibility (Sialve et al., 2009). Due to a thin trila-minar outer wall, many green microalgae have a very high resis-tance to chemical and enzymatic degradation based on theincorporation of insoluble, non-hydrolysable aliphatic bio-macromolecules called algeanans (Derenne et al., 1992). Thedigestion of pure algal biomass resulted in approximately 58% VS

Table 1Comparison of mesophilic digestion of microalgae and sewage sludge.

Substrate HRT (d) VSR (%) Biogas yield(ml g-VS�1)

Methane (%) Reference

Sewage sludge 11.7 26.1 236.9 61.4 Cecchi et al.(1996)Sewage sludge þ

Algae(40%)14.5 28.7 240 69.4

Raw sludge 30 58.5 567.4 62 Golueke et al.(1957)Algae(100%) 30 40 411.5 61.1

Sewage sludge 28 e 442.7 63 Chen (1987)Algae(100%) 28 e 417.7 73Sewage sludge þ

Algae(50%)28 e 290.5 64

Sewage sludge 25 49.1 498 65.1 This studyAlgae(100%) 25 58.0 366 62.5Sewage sludge þ

Algae(50%)25 54.2 420 63.2


Fig. 3. Microscopic photograph of the microalgae C. vulgaris (a) Before and (b) after sonication.

5000 J ml-1

K.Y. Park et al. / International Biodeterioration & Biodegradation xxx (2013) 1e54

reduction, which was greater than the 49% VS reduction observedfor sludge alone. These results imply that the co-digestion of algaeand sewage sludge increases the digestibility of algae with littleeffect on the anaerobic digesters that are used to treat sewagesludge.

3.3. Disintegration

Disintegration technologies have focused on wastewater sludgeto enhance anaerobic digestion. Sonication of C. vulgaris prior toanaerobic digestion significantly enhances the biodegradability ofthese cells. The important role of sonication in the disintegration ofwaste-algal biomass is the destruction of the cell wall. Sonicationcan also improve the digestibility of crude proteins, as shown by theresults of experiments on the digestibility of C. vulgaris in rats(Janczyk et al., 2007). The same effect has been observed whenusing high-pressure homogenization (Komaki et al., 1998). Disin-tegration has widely been examined based on visual observationsusing light and electron microscopes (Khanal et al., 2007). A light-basedmicrograph depicts qualitative information such as structuralchanges during the ultrasonic treatment. A microscopic photo-graph of microalgae C. vulgaris is shown in Fig. 3. During sonication,significant disruption of the microalgal cells was not observedwhereas destruction occurred in the bacterial cells. However, thedark color of C. vulgaris faded when treated with higher specificenergy doses.

Total solids (TS), volatile solids, and SCOD before and aftertreatment are also presented in Table 1.Many studies have pre-sented the SCOD increasewith respect to the duration of sonication,which makes it harder to compare one study with the other. This isbecause ultrasonic disintegration depends on several factors suchas the TS content, frequency, sludge type, ultrasonic density,

0

20

40

60

80

100

0

300

600

900

1200

1500

0 50 100 150 200

De

gre

e o

f d

isin

te

gra

tio

n (%

)

SC

OD

an

d T

S (m

g l

-1)

Energy (J ml-1

)

SCODTS Degree of disintegration

Fig. 4. Disintegration of microalgae using various sonication energies.


temperature, duration of sonication, and such information is notwell described in the literature (Khanal et al., 2007). Different initialconcentrations of algae were sonicated under different appliedenergies, and we measured the SCOD and calculated the degree ofdisintegration after each treatment (Fig. 4). Disintegration isdirectly proportional to the energy that is applied. An energy doseof 200 J ml�1 produced a degree of disintegration of 80%.

3.4. Digestion of disintegrated microalgae

Disintegration would likely be adopted for treating microalgae(Li et al., 2008), since the resistance of the cell wall of C. vulgaris isone of the limiting factors for cell digestibility. The pretreatment ordisintegration of waste biomass to increase the overall biodegrad-ability of the substrate has been the focus of many technologies. Byfar, the most common application of disintegration technologies islimited to waste activated sludge prior to digestion, as it iscommonly thought that waste activated sludge is significantly lessdegradable than primary sludge (Müller et al., 2009). The relativeimprovement by ultrasonic disintegration with respect to gasproduction from sludge varies from 0% to 60% (Palmowski et al.,2006). This variation is due to the relative importance of the 3main effects of disintegration treatment on sludge: particle sizereduction, which leads to increased surface area available for mi-crobial degradation (Müller et al., 2003); the release of SCODthrough the disruption of the cell walls of the microorganisms; andimproved fat emulsification (Palmowski et al., 2006).

Apart from the solubilization study, a biodegradability studywas performed to determine whether the increase in SCOD also

Elapsed time (h)

0 100 200 300 400 500

Cu

mu

lative m

eth

an

e (m

l g

-V

S-1

)

0

100

200

300

400

5 J ml-1

10 J ml-1

20 J ml-1

50 J ml-1

100 J ml-1

200 J ml-1

•

Fig. 5. Effect of specific energy input on microalgae disintegration.


0

0

20

50 100 150 200

40

60

80

100

En

ha

nc

ed

g

as

p

ro

du

ctio

n (%

)

Energy (J ml-1

)

Algae (This study)

Sludge (Pérez-Elvira, 2009)

Fig. 6. Comparison of enhanced gas production with algae and sludge.

K.Y. Park et al. / International Biodeterioration & Biodegradation xxx (2013) 1e5 5

yielded a subsequent increase in biogas production. To compare thedigestion progression of the different samples, the determinedbiogas volume was calculated by regression analysis using theGompertz equation. Cumulative methane production showed thatsonication at 5e20 J ml�1 did not affect methane production(Fig. 5). The cumulative methane production of disintegratedC. vulgaris indicated a strong relationship with the ultrasound dose.Lower doses of ultrasound (below 50 J ml�1) did not improvemethane generation. At doses higher than 50 J ml�1, gas productionwas proportional to the specific energy dose. Ultrasonic disinte-gration techniques have turned out to be efficient means forimproving methane conversion yields. Fig. 6 shows the enhancedgas production after sonication and a comparison with the gasproduction in sludge by using the data from a previous report(Pérez-Elvira et al., 2009). The sonication of microalgae was moreeffective than sludge sonication at higher doses of energy. Volatilesolids reduction VSR data were also obtained during the digestion.For the disintegrated samples, the VSR was increased according tothe energy input and degradation reached up to 60.7% at an energydose of 200 J ml�1. These results indicate that the hydrolysis ofmicroalgal cells is the rate-limiting step in the anaerobic digestionof microalgal biomass.

4. Conclusions

This study investigated the integration of algae cultivation,disintegration, and biogas production during anaerobic digestion ofwastewater sludge. Nitrogen and phosphorus uptake were ach-ieved when C. vulgaris was grown in swine wastewater. Anaerobicco-digestion studies showed that the addition of C. vulgaris biomassto sludge improved VS reduction compared to the digestion ofsludge alone. However, the digestion of algae led to poor biogasgeneration. Ultrasonic treatment improved the solubility of thesubstrate. Disintegration reached up to 70% at 200 J ml�1 of energy.Ultrasound at doses of 50 J ml�1 did not lead to an increase inbiodegradability. For specific energy higher than 50 J ml�1, thehigher the energy that was applied, the higher was the increase inbiogas production.

Acknowledgments

This subject was supported by the Korea Ministry of the Envi-ronment (MOE) as an "Eco-Innovation Project"(Project No.:E212-40005-0035-0). This work was financially supported by the KoreaMOE as the “Waste to energy recycling human resource develop-ment Project” (YL-WE-12-001).


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anaerobic digestion of microalgal biomass with ultrasonic disintegration

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