Applied Energy 114 (2014) 790–797

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Applied Energy

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Comparative evaluation of biomass production and bioenergygeneration potential of Chlorella spp. through anaerobic digestion

0306-2619/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.apenergy.2013.08.021

⇑ Corresponding author. Tel.: +91 11 26591158; fax: +91 11 26591121.E-mail addresses: sanjukec@gmail.com (S.K. Prajapati), anushree_malik@yahoo.

com (A. Malik), vkvijay14@hotmail.com (V.K. Vijay).

Sanjeev Kumar Prajapati, Anushree Malik ⇑, Virendra Kumar VijayCentre for Rural Development and Technology, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India

h i g h l i g h t s

� Higher biomass production potential of Chlorella pyrenoidosa among tested species.� Empirical formulae and theoretical COD estimation for algal biomass.� Determination of maximum TMP and SMP of three species of Chlorella.� Biogas production potential in range of 0.340–0.464 m3 kg�1 VS added.� Highest biogas production from C. pyrenoidosa biomass.

a r t i c l e i n f o

Article history:Received 4 January 2013Received in revised form 24 May 2013Accepted 7 August 2013Available online 29 August 2013

Keywords:ChlorellaBMPSMPAnaerobic digestionBiogasDigestibility

a b s t r a c t

Microalgae Chlorella spp. are being considered of great research interest for biofuel application. The cur-rent study was focused on the comparative exploration of biogas production potential of three Chlorellaspp. namely C. minutissima, C. vulgaris and C. pyrenoidosa. Among the tested algae C. pyrenoidosa wasfound to be the best in both biomass production potential and biogas generation. After 12 days of culti-vation, biomass productivity was found to be 0.90 ± 0.04, 0.98 ± 0.11 and 0.92 ± 0.01 g L�1, respectively,for C. minutissima, C. pyrenoidosa and C. vulgaris. The corresponding estimated annual areal yields were27.37, 27.98 and 29.20 tons dry biomass ha�1 y�1, respectively. The elemental and biochemical composi-tion of the algal biomass was also determined and the theoretical/stoichiometric methane potential (TMPand SMP) of respective algal biomass was estimated. The estimated TMP and SMP values ranged from0.563 to 0.592 and 0.598 to 0.699 m3 kg�1 VS, respectively. C. pyrenoidosa was found to have the highestTMP and SMP. Moreover, biogas production potential was also determined through BMP protocols. Rel-atively higher biogas yield of 0.464 ± 0.066 m3 biogas kg�1 VS added with 57% (v/v) CH4 content wasobtained for C. pyrenoidosa biomass during 30 day digestion. Moreover, the digestate analyses showedthat all parameters (pH, alkalinity, VFA and NH3–N concentration) were in the stable range. In-contrastwith the good biogas potential, the digestibility of the Chlorella biomass was around 50%. Current findingsrevealed that there is need of extensive comparative analysis in order to find out the interspecific varia-tions of the algae with respect to biofuel production.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Microalgae have been subjected to extensive investigations forbiofuel production of liquid (e.g., bioethanol and biodiesel) andgaseous fuels such as biogas and bio-hydrogen [1]. The majoradvantage of using microalgae as biofuel feedstock include theirability to grow and replicate at faster rates, possibility of cultiva-tion on non-arable lands as well as the less water uptake and landrequirement compared to terrestrial biofuel crops [2–5]. Microal-

gae also have ability to grow in range of industrial and domesticwastewaters with simultaneous phycoremediation and biomassproduction [6]. Moreover, ability of microalgae to uptake and fixCO2 from waste gas streams such as flue gases indicates the possi-bility of integration of algal biofuel production with CO2 sequestra-tion [7]. Reports on microalgal treatment of high strengthwastewaters including livestock waters, biogas plant slurry andagro-industrial wastewaters, etc., are also available in the litera-ture [8,9]. The phycoremediation potential of microalgae can alsobe utilized for the treatment of high strength wastewater or greywaters in rural areas [10] to make wastewater suitable for agricul-tural application with simultaneous production of substantial bio-mass for bioenergy production.

S.K. Prajapati et al. / Applied Energy 114 (2014) 790–797 791

Among the various microalgae, Chlorella sp. has been given verymuch attention in biofuel research. Chlorella vulgaris, owing to itshigh lipid content has been explored extensively for bio-diesel pro-duction [11]. C. vulgaris has also been widely reported for its appli-cation in phycoremediation of wastewater and flue gases [12].Moreover, recently the biogas (286 mL CH4 g�1 VS) and bio-hydro-gen (10.8 mL H2 g�1 VS) production potential of C. vulgaris biomasshave also been reported [13]. Similarly, C. minutissima has beenused in phycoremediation coupled biomass [14] and biofuel pro-duction [15]. C. pyrenoidosa has also been tested for phycoremedi-ation of soybean processing wastewater [16]. However, the reportson application of C. pyrenoidosa in biofuel application are almostnonexistent.

There have been various attempts on anaerobic digestion of al-gal biomass. For instant, biogas production potential in the range of287–587 mL g�1 VS have been reported for various algal biomassincluding Chlorella kessleri, Scendesmus obliquus andChlamydo-monas reinhardti [17]. Recently, Zamalloa et al. [18] have reportedthe biomethane potential of around 0.36 and 0.24 L CH4 g�1 VSadded for P. tricornutum and S. obliquus biomass, respectively.Moreover, Ehimen et al. [19] have worked towards the optimiza-tion of anaerobic digestion of Chlorella biomass residue resultingfrom bio-diesel production process. Ras et al. [20] have also re-ported the methane production of 147 (during 16 days HRT) and240 mL g�1VSS (during 28 days HRT) from anaerobic digestion ofC. vulgaris biomass under semi-continuous mode. However, despitethe good theoretical biomethane potential (0.8 L g�1 VS) reportedby Sialve et al. [21], there is no previous experimental attempton exploring the biogas production potential of C. pyrenoidosaand C. minutissima.

Apart from the biogas production potential, determination ofCOD of biomass or organics solids by experimental methods is of-ten considered very tedious and prone to produce erroneous re-sults due to incomplete oxidation of solids or biomass residues[22,23]. Alternatively, estimation of theoretical COD has been re-ported to be a better approach [22]. Estimation of theoretical spe-cific methane potential also provides quick and easy insight intothe bioenergy generation potential of any organic substrate includ-ing algal biomass. Demonstration of such theoretical estimationhas systematically been done by Sialve et al. [21] using biochemi-cal composition of algal biomass. However, this may result in thewrong estimation as in spite of similar composition, the biochem-ical profiling among the different algal strains may vary signifi-cantly. Therefore, empirical formulae based estimation ofmethane potential may be appropriate alternative to the biochem-ical estimation approach as demonstrated elsewhere [22].

The current study was focused on the determination and com-parison of bioenergy potential of three commonly used Chlorellastrains through anaerobic digestion in order to identify the bestsuitable algal substrate for biogas production. In addition, two dif-ferent methods for estimation of theoretical COD and methane po-tential were also tested for their consistency, reliability andsuitability for further applications.

2. Materials and methods

2.1. Algae culture and growth medium

Three species of Chlorella namely C. vulgaris,C. minutissima andC. pyrenoidosa were used in the present study. Pure cultures of al-gae were procured from algal culture collection of VivekanandaInstitute of Algal Technology (VIAT), Chennai (India); Centre forConservation and Utilization of Blue Green Algae, IARI New Delhi(India) and National Collection of Industrial Microorganisms(NCIM), NCL Pune (India), respectively. BG11 broth (HIMEDIA,

M1541-500) was used as standard growth medium. After receiv-ing, the aliquots of each alga was transferred to separate sterileBG11 agar slants (2% agar) and broth. The cultures were than main-tained in a plant growth chamber (Daihan Labtech, LGC-5101) un-der cool fluorescent light (�2500 Lux) at 25 ± 1 �C with 12:12 hlight:dark cycle.

2.2. Biomass production and harvesting

Biomass production potential was estimated in 250 mL flaskwith 50 mL working volume under controlled conditions as re-ported in our previous study [10]. In order to get sufficient biomassfor biochemical analysis and anaerobic digestion studies, algaewere cultivated (under non-axenic conditions) in fabricated photo-bioreactor (PBR) with 20 L working volume. Tap water medium[10] having 12.3 mg L�1 nitrogen (as NaNO3), 1.1 mg L�1 phospho-rous (as KH2PO4) and sodium carbonate (20 mg L�1) was used asgrowth medium. The initial pH of the growth medium was ad-justed at 7.0. Inoculum (10% v/v) was taken from the algal culture(optical density �2.0 at 680) maintained in plant growth chamber(Section 2.1). After inoculation, the culture bottles were incubatedunder natural atmospheric conditions (direct sun light and tem-perature �30–42 �C) during day time and under illumination of�1000 lux using cool fluorescent light during night. In order to pre-vent the settling of algal biomass, mixing was provided by bub-bling air (0.5–1.0 L min�1) through aquarium pump. Afterincubation for 12–14 days, the air bubbling was stopped and bot-tles were kept overnight for harvesting of algal biomass by auto(gravity) settling [24].

2.3. Biomass composition analyses

After harvesting, algal biomass were dried overnight at 65–70 �C and grinded in a mortar pestle to make fine powder for ele-mental and biochemical analyses. The elemental analysis (CNH)was done using CHN analyzer. Volatile solids (VS) and ash contentof biomass was determined through standard methods [25].

Total carbohydrate was determined through phenol–sulfuricacid method [10,26]. Briefly, the powdered biomass (100 mg)was hydrolyzed with 2.5 N HCl (5 mL) in boiling water bath for3 h, cooled at room temperature and neutralized with solid sodiumcarbonate. After neutralization, an aliquot of 0.1 mL was pipetteout in a clean test tube and diluted to 1 mL. After dilution, 1 mLphenol solution and 5 mL 96% sulfuric acid were added, well mixedand cooled to 25–30 �C in a water bath. The color intensity of thesamples was measured at 490 nm and the total carbohydrateswere than calculated using standard calibration curve. Protein con-tent was calculated by multiplying the total nitrogen (obtainedthrough CHN analyzer) by 6.25 [27].

Total lipid was extracted using chloroform–methanol mixture(1:1 v/v) mixed with the sample in the proportion of 1:1 and thanestimated through modified Bligh and Dryer’s method [10]. Forefficient extraction of lipids, algal cell wall was disrupted throughheat treatment at 100–110 �C up to 5 min (with heat pulses of 30 sto avoid sample loss by over boiling) in a microwave oven [28].

2.4. Estimation of methane production potential

After biochemical and elemental composition analysis, themaximum possible methane potential of selected algal biomasswas estimated through two different methods. In the first method,the elemental composition of the algal biomass was utilized. Basedon the elemental composition, empirical formula of algal biomasswas developed and the maximum possible stoichiometric methanepotential (SMP) was calculated through the equation given bySymons and Buswell [29] adopted from Sialve et al. [21].

792 S.K. Prajapati et al. / Applied Energy 114 (2014) 790–797

CaHbOcNd þ4a� b� 2c þ 3d

4

� �H2O

! 4aþ b� 2C � 3d8

� �CH4 þ

4a� bþ 2c þ 3d8

� �CO2

þ dNH3 ð1Þ

The SMP was than calculated using Eq. (2) given as (2)

SMP ¼ 18

4aþ b� 2c � 3d12aþ bþ 16c þ 14d

� �Vm ð2Þ

where Vm is the molar volume of methane at STP.In the second approach, it was assumed that the algal lipid, pro-

tein and carbohydrate chemical formulae were C57H104O6, C6

H13.1O1N0.6 and (C6H10O5)n, respectively [21], and the specificmethane yield for these three organic components of algal biomasswere used for methane production potential estimation. The calcu-lation for theoretical methane potential (TMP) was done throughthe following equation.

TMP ¼ 1100

A� CL þ B� CP þ C � CCð Þ ð3Þ

where A is the specific methane yield of lipid (1.014 L CH4 g�1 VS); Bthe specific methane yield of protein (0.851 CH4 g�1 VS); C the spe-cific methane yield of carbohydrates (0.415 CH4 g�1 VS) and CL, CP,CC are the % (on TS basis) of lipid, protein and carbohydrates, respec-tively, in algal biomass.

2.5. Determination of biogas production potential through BMP test

The biogas production potential of selected algal biomass wasdetermined through biochemical methane potential (BMP) testprotocol [30]. Briefly, experiments were conducted in 500 mLcapacity BOD bottles (Borosil) with hermetically sealed stoppersand controlled gas opening valves. The working volume was keptup to 300 mL and remaining 200 mL was left for gas storage. Theinoculum was aseptically and anaerobically transferred to experi-mental bottles from an actively running cattle dung based lab scalebiogas plant. Specific methanogenic activity (SMA) of the inoculumestimated using 1 g L�1 acetate solution was found to be around82.12 L CH4 kg�1 VSS d�1 (0.2102 g COD CH4 g�1 VSS d�1). The ini-tial substrate (biomass) concentration of 5 g VS L�1 and substrateto inoculum ratio of 3.0 (on VS basis) was used for the experiments.Distilled water was used to make-up the working volume up to300 mL whenever needed. Bottle containing only seed culture,was used as the control. After inoculation, the prepared bottleswere kept under stationary conditions at 36 ± 1 �C for incubationand the volume of biogas produced was measured after every24 h for 30 days. Biogas production from the control bottle wasalso studied simultaneously. The net biogas production from algalbiomass was determined using the following equation

Bnet ¼ Bx � B0 ð4Þ

where Bnet is the net gas yield from algal biomass (m3 kg�1 VSadded); Bx the gas yield in experimental bottles (m3 kg�1 VS added)and B0 the gas yield in control bottles (m3 kg�1 VS added).

2.6. Analytical methods

2.6.1. Algal growth measurementAlgal growth was measured in terms of culture density (OD680),

chlorophyll (Chl-a) content and the dry cell weight. Well mixed ali-quot (3–5 mL) of the growing culture was withdrawn from the cul-tivation unit and the optical density (OD) was measured at 680 nmusing UV/Vis spectrophotometer (Lamda 35, PerkinElmer). The bio-mass (g dry biomass L�1) was determined gravimetrically and

chlorophyll was extracted and estimated using modified methanolextraction method [10].

2.6.2. COD estimation of algal biomassFor estimation of theoretical COD (CODth) the stoichiometric

equation for complete oxidation of algal biomass was used. Itwas assumed that the nitrogen (N) remains in the reduced form(i.e., NH3) during the oxidation. The CODth reported as mg-O2/g-VS of algal biomass (CaHbOcNd) was estimated using the formulaadopted from Angelidaki and Sanders [31].

CODth ¼1000

44aþ b� 2c

12aþ bþ 16c

� �M ð5Þ

where M is the molecular weight of oxygen (=32 g/mole).

2.6.3. Biogas volume measurement and composition analysisThe volume of biogas produced was measured after every 24 h

using acidic water displacement (pH � 2.0) as soon as the bottleswere taken out from the incubator. Biogas yield from the algal bio-mass was then calculated and reported in m3 biogas g�1 VS added.Biogas composition was analyzed using a gas chromatograph(Agilent 7890A) equipped with stainless steel column packed withPorapack-Q 80/100 mesh (Supelco) and thermal conductivitydetector (TCD). The carrier gas used was Argon at a flow rate of30 mL min�1 at 75 psi pressure and 60 �C. Temperature of ovenand detector were 50 and 250 �C, respectively.

2.6.4. Digestate analysisAfter completion of experiments, the digestate from the bottles

was withdrawn and processed according to Shanmugam and Hor-an [22] for the analysis of total alkalinity (T.Alk), total volatile fattyacids (TVFA) and ammoniacal nitrogen (NH3–N).

T.Alk was determined using potentiometric titration. The pH ofsample was brought to 4.5 by adding 0.16 N H2SO4 and T.Alk wascalculated as follow:

T:Alkðmg CaCO3 L�1Þ ¼ A� N � 50;000B

ð6Þ

where A is the mL of standard acid used; N the normality of stan-dard acid used and B is the mL of the sample.

The TVFA content of digestate (as equivalent mg L�1 of aceticacid) were estimated through spectrophotometric method as re-ported in previous study [10]. The NH3–N was estimated usingHach high range ammonia TNT reagent and DR/890 colorimeter(range: 0–50 mg L�1) utilizing salicylate method.

The volatile solid reduction (VSr) resulting from the BMP testwas estimated for comparing the digestibility of different algal bio-mass. VS content of the digestate was estimated and VSr was calcu-lated using following equation:

VSrð%Þ ¼VSf � VSw

VSf

� �� 100 ð7Þ

where VSf and VSw are volatile solid concentration (g L�1) in the feedand digestate, respectively.

2.7. Statistical analysis

All the experiments were performed in triplicate unless other-wise stated. The standard deviation and means was analyzed forsignificance using biostatistics software SPSS 17.0 through oneway ANOVA. Duncan multiple range test was used to comparethe significance of differences among tested algae at P values of<0.05. Results are reported as either mean ± SD or error bars.

Table 1Biomass (biochemical and elemental) composition of selected algae (represented asmean ± SD, for n P 3).

Parameters C. minutissima C. pyrenoidosa C. vulgaris

Volatile solids (% of TS) 73.55 ± 0.38 72.35 ± 0.53 60.83 ± 1.18Lipid (% of TS) 16.32 ± 0.57 13.65 ± 0.75 20.35 ± 1.07Protein (% of TS) 43.78 ± 0.51 40.92 ± 0.33 36.48 ± 0.48Carbohydrate (% of TS) 14.59 ± 0.43 25.30 ± 0.99 15.39 ± 2.34Carbon (% of TS) 41.54 ± 2.13 45.90 ± 0.47 42.87 ± 0.22Hydrogen (% of TS) 5.85 ± 0.12 5.95 ± 0.03 6.37 ± 0.06Nitrogen (% of TS) 7.02 ± 0.08 5.55 ± 0.05 5.84 ± 0.08Oxygen (% of TS) 19.24 ± 0.42 14.95 ± 0.62 15.71 ± 0.75Ash content (% of TS) 26.45 ± 0.382 27.65 ± 0.53 39.17 ± 1.18C/N ratio 5.89 ± 0.12 8.27 ± 0.03 7.35 ± 0.09

S.K. Prajapati et al. / Applied Energy 114 (2014) 790–797 793

3. Results and discussion

3.1. Biomass potential of selected algae

The biomass productivity (on 12th day of cultivation) wasfound to be 0.90 ± 0.04, 0.98 ± 0.11 and 0.92 ± 0.01 g L�1, respec-tively, for C. minutissima, C. pyrenoidosa and C. vulgaris. The corre-sponding Chl-a concentration (lg mL�1) and the culture OD680

was 9.34 ± 0.13, 14.30 ± 0.59, 13.22 ± 0.10 and 2.46 ± 0.27,3.74 ± 0.14, 2.85 ± 0.18, respectively. Hence C. pyrenoidosa possessslightly higher biomass production potential as reflected fromdry weight, Chl-a as well as OD680 measurements (Fig. 1). More-over, the growth (in terms of Chl-a) of the tested Chlorella spp.was significantly higher than the values reported for C. vulgaris[32]. Similarly, the dry cell weights obtained were almost threetimes of values previously reported for C. vulgaris FACHB-31 inBG11 medium [33]. Hence, from the present study it is proved thatthe tap water medium can be used as a better alternate of high costBG11 medium. Moreover, the obtained productivities were rela-tively lower than the values reported for algal consortium at ele-vated CO2 (6%) level [32]. Hence, it is possible that the biomassproduction can further be enhanced through cultivating selectedalgae at elevated CO2 levels.

From the growth data, the estimated volumetric productivitieswere found to be 0.075, 0.08 and 0.076 g L�1 d�1, respectively, forC. minutissima, C. pyrenoidosa and C. vulgaris. The productivities ob-tained were similar to the reported values for Chlorella sp.227 cul-tivated in partially treated municipal wastewater [34]. However,the observed values were relatively lower than those reported forChlorella sp. cultivated in nutrient supplemented tap water underflues gas supply [35]. Moreover, the biomass production potentialwas estimated to be 27.37, 29.20 and 27.98 tons dry biomass ha�1 -y�1 (assuming 10 cm water depth), respectively, for C. minutissima,C. pyrenoidosa and C. vulgaris. The estimated biomass potential oftested algal strains was in line with the values for algal consortiumcultivated in raceway pond and vertical tank reactors (19–22 tons ha�1 y�1) reported previously [36].

3.2. Biochemical and elemental composition

The VS content, biochemical (lipid, protein, carbohydrates) andelemental (C, H, N, O) composition of algal biomass from differentChlorella species are given in Table 1. Among the tested algae, C.minutissima and C. pyrenoidosa have significantly higher VS thanthat of C. vulgaris biomass. Despite lesser VS content(60.83 ± 1.18% of TS), C. vulgaris was found to be rich in lipids(20.35 ± 1.07% of TS) when compared with others species. C. minu-tissima and C. pyrenoidosa biomass were relatively rich in proteins

Fig. 1. Comparison of biomass production potential (in terms of Chl-a, dry weightand culture optical density at 680 nm) of the tested strains of Chlorella.

(43.78 ± 0.51% of TS) and carbohydrates (25.30 ± 0.99% of TS),respectively. The biochemical composition of tested algae was inline with values reported previously [37]. However, the lipid con-tent of C. pyrenoidosa was higher than values (2% of TS) reportedby Singh et al. [37] while it was significantly lower than the lipidcontent of C. pyrenoidosa (37.00 ± 9.34% of TS) reported by Suet al. [16]. Further, the higher carbohydrate content of C. pyrenoid-osa biomass obtained in the present study revealed that it can alsobe utilized as good source for bioethanol production. Moreover, thedigestibility of C. pyrenoidosa was expected to be relatively betterover other tested algae due to high content of easily digestible car-bohydrate and protein fractions. On the other hand, the digestibil-ity of C. vulgaris and C. minutissima was expected to be relativelylower due to poor digestibility of substantial amount of lipid pres-ent in their biomass.

Similar to the biochemical composition, the elemental composi-tion and C/N ratios of algal biomass were also significantly differentfrom each other (Table 1). The C/N ratio ranged from 5.89 ± 0.12 (C.minutissima) to 8.27 ± 0.03 (C. pyrenoidosa). The Biochemical andelemental composition observed here indicates that different spe-cies of Chlorella have different biomass composition and hence thebiogas/methane yield should also be different.

3.3. Empirical formulae, theoretical COD and methane productionpotential

From the elemental composition (Table 1), the empirical formu-lae of the algal biomass were developed and used for the computa-tion of CODth and expected SMP. The preliminary and empiricalformulae as well as the comparisons of SMP with the TMP calcu-lated from the biochemical composition of the algae are shownin the Table 2. As expected from the biomass composition, signifi-cant differences in the TMP as well SMP were observed among thetested algae. The calculated values of CODth, SMP and TMP for C.pyrenoidosa (2366.98 mg O2 g�1 VS, 0.699 and 0.592 L CH4 g�1 VS,respectively) were relatively higher than that for the other testedalgae. The high CODth and SMP value of C. pyrenoidosa may beattributed to the higher carbon content in its biomass comparedto other algae. Moreover, the COD to VS ratios (g O2 g�1 VS) werein the range of 2.00–2.37, which were significantly higher thanthe values reported (based on experimental COD) by Zamalloaet al. [18] for algal biomass of Scenedesmus obliuuus (1.3:1) andPhaeodatylum treicornutum (1.4:1). The observed difference also re-flected the possibility of underestimation of biomass COD throughexperimental determination. The application of such theoreticalapproach has also been successfully applied for estimation ofmethane potential and COD of various solid waste includingleather fleshing, SRB sludge and municipal solid waste [22].

The SMP values obtained in the present study were relativelyhigher than the calculated TMP. The observed difference in theestimated TMP and SMP could be explained from the fact that that

Table 2Comparison of empirical formula, CODth, TMP and SMP of the tested algal biomass.

Algae Preliminary formula Empirical formula CODth (mg O2 g�1 VS) SMP (L CH4 g�1 VS) TMP (L CH4 g�1 VS)

C. minutissima C3.442H5.803N0.501O1.203 C7H12NO2 2069.71 0.598 0.563C. pyrenoidosa C3.821H5.899N0.468O0.872 C8H13NO2 2366.98 0.699 0.592C. vulgaris C3.569H6.324N0.417O0.982 C9H15NO2 2298.60 0.689 0.566

794 S.K. Prajapati et al. / Applied Energy 114 (2014) 790–797

the specific methane yields taken from Sialve et al. [21] for calcu-lation of TMP are particularly for fixed empirical formula of lipids,proteins and carbohydrate. However, different algal biomass mayhave different kind of lipids, proteins and carbohydrates with dif-ferent empirical formulae. The SMP estimation was done directlyfrom the empirical formulae developed from elemental composi-tion. Hence, although the biochemical composition based determi-nation of TMP gave good estimation, the empirical formula basedSMP seems to be a better approach for maximum methane poten-tial estimation.

3.4. Biogas potential of algal biomass

The biogas production potential of algal biomass was measuredunder controlled temperature (36 ± 1 �C) conditions for 30 days.Daily and cumulative biogas production profile is shown in Fig. 2aand b, respectively. The biogas measured (in m3 biogas kg�1 VSadded) ranged from 0.34 ± 0.114 for C. minutissima to 0.464 ±0.066 for C. pyrenoidosa with C. vulgaris being at 0.369 ± 0.067. Themaximum methane content was 48.86 ± 0.74, 57.05 ± 0.89 and53.02 ± 0.46% (v/v of biogas), respectively, for C. minutissima,C. pyrenoidosa and C. vulgaris biomass. The values observed aresimilar to those reported in literature (0.287–0.587 m3 kg�1 VSadded) for other algal biomass including green algae C. reinhardtii,

Fig. 2. Variation of (a) daily biogas evolution and (b) cumulative biogas frombiomass of different strains of Chlorella with elapsed time (control refers to biogasproduction from the bottles containing inoculum only).

Dunaliella salina andScenedesmus obliquus, C. kessleri, euglenoidspeciesEuglena gracilis and prokaryotic cyanobacterium Arthrospiraplatensis [17]. Moreover, similar biogas yields (0.401–0.487 m3

kg�1 VS added) were observed in case of Chroococcus sp. biomassduring our previous study [10].

The biogas production started without any lag period in allexperimental bottles except control (Fig. 2a). This confirmed thegood activity of the microbial flora in the inoculum as well as therapid digestibility of some cells (may be due to the cell wall disrup-tion of some algal cells during harvesting/transferring stage). Thebiogas production reached to its maxima on 4th (C. vulgaris), 6th(C. minutissima) and on 11th day (C. pyrenoidosa) depending uponthe species tested. For first few days of experiments, biogas pro-duction from the bottles containing biomass of C. vulgaris and C.minutissima was higher and subsequently started decreasing afterreaching their respective maxima. However, biogas productionwas very less from the bottle containing C. pyrenoidosa biomassduring first 4 days, remained almost constant during 4–8 daysand started increasing dramatically from day 9th onward untilreached maxima. After 11th day, the biogas production starteddecreasing constantly. The different performance of three speciesduring the initial phase could arise out of variations in their cellu-lar and cell wall constitution, which needs to be investigated fur-ther. Moreover, the observed (Fig. 2a) daily fluctuations (rise andfalls) could be attributed to the heterogeneity of system resultinginto the poor interaction of the anaerobic microflora with thesubstrate under unmixed and stationery conditions. Similar non-uniform biogas evolution pattern was also observed during ourprevious study with Chroococcus spp. biomass [10]. From Fig. 2b,it was observed that the cumulative biogas production profile ofC. minutissima and C. vulgaris were closer to each other but signif-icantly different from C. pyrenoidosa. From the cumulative biogasproduction, the rates of biogas production (Rg) were determinedfor every 5 day interval as well as overall 30 day (Fig. 3). The pat-tern of Rg obtained was similar to the daily biogas production pro-files of respective algal biomass. The maximum Rg values (mLbiogas g�1 added VS d�1) of 15.80 ± 0.57 (0–5 d), 22.06 ± 1.16(11–15 d) and 18.68 ± 0.54 (0–5 d) were obtained for biomass ofC. minutissima,C. pyrenoidosa and C. vulgaris, respectively. Similarly,the overall Rg for C. pyrenoidosa (17.35 ± 0.28) was significantly

Fig. 3. Comparison of biogas production rates (Rg) for every 5 day interval and theoverall biogas production rate from different algal biomass.

S.K. Prajapati et al. / Applied Energy 114 (2014) 790–797 795

higher than that of C. minutissima (11.02 ± 0.43) and C. vulgaris(12.06 ± 0.28).

3.5. Characteristics of digestate and process stability

After the completion of experiment, the pH of the digestatefrom all the bottles was in the neutral range (7.1–7.9). The T.Alkin case of C. minutissima,C. pyrenoidosa and C. vulgaris was2304.00 ± 87.42, 2288.89 ± 177.02 and 1962.39 ± 78.38 mg equiva-lent of CaCO3 L�1, respectively. The corresponding TVFA concentra-tion were 225.33 ± 6.83, 428.67 ± 9.31, 447.00 ± 20.49 mg L�1,respectively. The NH3–N concentration in digestate was 165.00 ±2.24 (C. minutissima), 185.00 ± 4.47 (C. pyrenoidosa) and 203.33 ±5.16 mg L�1 (C. vulgaris). Although the T.Alk and NH3-N of C. minu-tissima,C. pyrenoidosa digestate was relatively closer, there was sig-nificant difference in the TVFA concentration. Moreover, T.Alk incase of C. vulgaris was lowest but the NH3-N and TVFA concentra-tions were significantly higher compared to other tested algae. Allparameters of the digestate were in the stable range of AD processreported elsewhere [27,38].

Inspite of having the higher nitrogen content compared to otherChlorella species, the NH3–N concentration of C. minutissima dige-state was lowest. This could be attributed to the low digestibilityof the C. minutissima biomass. The observed digestibility of testedalgae (in terms of VSr%) was significantly higher for C. pyrenoidosabiomass (51.17 ± 2.08%) than that of C. minutissima and C. vulgarisbiomass (39.00 ± 3.61 and 45.50 ± 2.29%, respectively). Moreover,there were some intact algal cells in the digestate when examinedunder the microscope (Fig. 4) which also confirmed the poordigestibility of algal biomass. The low digestibility of C. minutissimamay be either due to the low C/N ratio (Table 1) or high resistanceof the algal cell wall. Microalgae are known to have very complexand poorly understood cell walls. Moreover, intraspecies variationin cell walls as well as variations in a single strain of Chlorellagrown under different conditions have also been reported [39].The cell wall of C. pyrenoidosa has been reported mainly (>80%)composed of cellulose [40] and while the cell walls of C. vulgarisare known to have rigid wall components embedded within a moreplastic matrix and protected by a durable polymer of unknowncomposition [39]. Similarly, the cell wall of C. minutissima may alsohave more protective cell wall which could be difficult to digest bymicrobes during anaerobic digestion.

Among the Chlorella spp., C. vulgaris, has been extensively ex-plored as feed stock for bio-diesel, bio-ethanol and biogas produc-tion [13,41,42] with some attention on C. minutissima as biodieselfeedstock [15]. Moreover, few reports on C. pyrenoidosa in waste-water treatment and biomass production also exist [16,43]).

Fig. 4. Phase contrast microphotographs (100�) of resulted digestate showing intact cehighlighted with the arrow).

However, C. pyrenoidosa has not been given much importance inbiofuel applications. Moreover, to the best of our knowledge noprevious report on biogas production from C. pyrenoidosa is avail-able in the literature except theoretical methane potential reportedby Sialve et al. [21]. But as reflected from the current observa-tions,C. pyrenoidosa was found to be the best suitable algal biomassfor anaerobic digestion among the tested algae. The suitability of C.pyrenoidosa biomass for anaerobic digestion could be due to eitheror combination of the following factors: (i) high SMP value, (ii)more favorable C/N ratio and (iii) relatively good digestibility com-pared to other tested biomass. Although it was the best among thetested algae, the digestibility of C. pyrenoidosa was around 50%.

Recently, there have been some attempts in order to enhancethe digestibility of algal biomass by pretreatment methods suchas mechanical, thermal and biological treatments [44–46]. How-ever, the cost and energy inputs of such pretreatments are alsoconsiderable. Moreover, optimization of C/N ratio by appropriateco-digestion strategies is another possible route to enhance the al-gal biogas production by enhancing activity of anaerobic micro-flora. For instance, Yen and Brune [47] achieved a significantincrease in methane production (1.17 mL L�1 d�1 vs. 0.57 mL L�1

d�1) from waste paper and algal biomass blend (1:1) comparedto anaerobic digestion of pure algal biomass. Similarly, Zhonget al. [27] have reported 61.69% increase in methane production(325 mL g�1 VS compared with 201 mL g�1 VS of algae digestionalone) from co-digestion of algal biomass with corn straw at 20/1C/N ratio.

Hence, it can be concluded that there is a huge scope of enhanc-ing the biogas production from the C. pyrenoidosa biomass. How-ever, an economically viable pretreatment method for algalbiomass is yet to be identified. Also, the process optimization forco-digestion of algae with carbon rich waste is of great interestand importance for development of bioenergy generation processform algal biogas through anaerobic digestion.

3.6. Energy generation potential of selected algal biomass

As concluded above, biomass of C. pyrenoidosa was best suitablefor biogas production. Moreover, C. pyrenoidosa, has the highestbiomass production potential (29.20 tons dry biomass ha�1 y�1)among the tested algae with approximately 72% (on TS basis) vol-atile fraction. Hence, based on the lab scale observations, the esti-mated annual biogas production potential of C. pyrenoidosa isaround 13,549 m3 biogas ha�1 y�1 (�7729.59 m3 CH4 ha�1 y�1).Considering the energy content of CH4 (�37.78 MJ m�3) [10], C.pyrenoidosa has the biomethane energy yield of 292.02 GJ ha�1 y�1,which is equivalent to the renewable power generation of

lls of (a) C. minutissima, (b) C. pyrenoidosa and (c) C. vulgaris (intact algal cells are

796 S.K. Prajapati et al. / Applied Energy 114 (2014) 790–797

�81117.32 kW h ha�1 y�1. The estimated renewable power yieldwas significantly higher than the yields reported for algal biomasscultivated in wastewater [36]. However, relatively higher annualpower generation potential has been reported for algal biomassof Chroococcus sp. [10].

3.7. Scale-up feasibility of the algal biogas process

The lab scale experimentation has proved that algal biomasscan be good substrate for bioenergy generation by anaerobic diges-tion. However, the limitations in the process scale up cannot be ne-glected and have to be overcome in order to make algal biogas afeasible renewable fuel on sustainable basis. The major issues forthe large scale production of algal biomass has been extensivelyexplored and analyzed by Pate et al. [48]. These include high de-mand for cultivation land, water, high cost of nutrients (N, P andtrace nutrients) and the carbon (CO2) source [48]. However, algaecan be grown on non-arable land using wastewater as nutrientsource [6,10,34,48]. Moreover, the carbon requirements of largescale algae cultivation systems can be meet through supply thewaste CO2 from fossil-fired power plants, cement plants, fermenta-tion industries, and others [7,9,48,49]. Hence, the large scale algalcultivation for biogas production seems to be possible with re-duced capital inputs using wastewater and waste CO2 streams.Moreover, it has been reported that elevated CO2 levels as wellas the native bacterial community of wastewater have synergisticeffect on the algal growth resulting in enhanced biomass produc-tion [4,6,10,48,50]. Hence, it is possible that the higher biomassand bioenergy yields may obtained with the selected algae whencultivated in wastewater under elevated CO2 levels. Feasibilityanalysis of algal bioenergy generation in integration with waste-water treatment has been recently published [10].

Apart from the troubles in large scale cultivation, the lowdigestibility of the algal biomass may become a major hurdle inthe scale-up of AD process. However, the digestibility of algal bio-mass may be enhanced using various pretreatment methods as dis-cussed earlier. On the other hand, all the process parameters of ADviz., pH, NH3–N, TVFA, etc., were in the sable range during the labscale experiments. However, in the real engineering application atlarge scale, ammonia accumulation can occur due to low C/N ratioof algal biomass. The ammonia accumulation may further affectthe performance by inhabiting the growth of anaerobic microbialflora and thus resulting in process failure. The problem of ammoniaaccumulation at large scale may be handled by balancing the C/Nratio of feed using suitable carbon rich co-substrate.

4. Conclusions

The present study evidenced that microalgae Chlorella spp. havegreat potential for bioenergy generation by anaerobic digestion. Asignificant variation in the biochemical composition, theoreticaland experimental biogas yields of three species of Chlorella was ob-served. From the findings, it could also be concluded that relativelyless explored C. pyrenoidosa performed better in terms of biomassas well as biogas production potential. Moreover, the digestibilityof C. pyrenoidosa was relatively lower during first few days of theexperiments but started increasing dramatically from 5th day on-ward. Also, the overall digestibility of C. pyrenoidosa was highest.Although being good substrate for anaerobic digestion there waspresence of some intact cells in the digestate. Hence there is needfor pretreatment of algal biomass in order to enhance the digest-ibility and hence the biogas production. Also the co-digestion withcarbon rich waste may be applied to algal C/N ratio optimum foranaerobic digestion.

Overall, C. pyrenoidosa was found best suited among the testedChlorella spp. for biomass production and subsequent biogas gener-ation through anaerobic digestion. However, further attempts onoptimization of co-digestion and pretreatment methods are needto make bioenergy generation from algal biomass as viable optionto overcome the future energy scenario. Moreover, coupling of al-gal bioenergy generation with wastewater treatment may makethe process more feasible at large scale in real engineeringapplications.

Acknowledgements

The present research work was financially supported by theMinistry of New and Renewable Energy, Govt. of India. Authorswould like to thank Prof. (Dr.) V. Sivasubramanian, Director, VIATChennai (India) for providing the pure culture of C. vulgaris as gift.The technical assistance provided by Mr. Pushpender Kumar andMs. Poonam Choudhary (JRF, IIT Delhi) and Mr. Sabal Sing andMr. Vinod Kumar (Lab Assistants, IIT Delhi) throughout the workand Mr. Amit (Biogas Lab., IIT Delhi) in GC-analysis of biogas, is alsoacknowledged. Thanks are due to the reviewers for their valuablesuggestions and text enhancement.

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