Applied Energy 88 (2011) 3454–3463

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Applied Energy

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Anaerobic digestion of microalgae residues resulting from the biodieselproduction process

E.A. Ehimen a, Z.F. Sun a,⇑, C.G. Carrington a, E.J. Birch b, J.J. Eaton-Rye c

a Department of Physics, University of Otago, 730 Cumberland Street, Dunedin 9016, New Zealandb Department of Food Science, University of Otago, 276 Leith Walk, Dunedin 9016, New Zealandc Biochemistry Department, University of Otago, 710 Cumberland Street, Dunedin 9016, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 August 2010Received in revised form 4 October 2010Accepted 9 October 2010Available online 19 November 2010

Keywords:Microalgae residuesAnaerobic digestionGlycerolCo-digestionMethane

0306-2619/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.apenergy.2010.10.020

⇑ Correspondence. Tel.: +64 3479 7812; fax: +64 34E-mail address: zhifa@physics.otago.ac.nz (Z.F. Sun

The recovery of methane from post transesterified microalgae residues has the potential to improve therenewability of the ‘microalgae biomass to biodiesel’ conversion process as well as reduce its cost andenvironmental impact. This paper deals with the anaerobic digestion of microalgae biomass residues(post transesterification) using semi-continuously fed reactors. The influence of substrate loading con-centrations and hydraulic retention times on the specific methane yield of the anaerobically digestedmicroalgae residues was investigated. The co-digestion of the microalgae residues with glycerol as wellas the influence of temperature was also examined. It was found that the hydraulic retention period wasthe most significant variable affecting methane production from the residues, with periods (>5 days) cor-responding to higher energy recovery. The methane yield was also improved by a reduction in the sub-strate loading rates, with an optimum substrate carbon to nitrogen ratio of 12.44 seen to be required forthe digestion process.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

The use of photosynthetic micro-organisms (microalgae) forbiodiesel production has been discussed extensively in the litera-ture [1–8]. The biodiesel production process also results in theco-production of glycerol (C3H5(OH)3) as well as microalgae resi-dues. With the proposed commercial production of biodiesel frommicroalgae, a major question arises: ‘‘What to do with the microal-gae residues obtained after the transesterification process?’’

The use of the protein rich microalgae residues has been dis-cussed to be potentially used as a nutrient additive in livestockfeeds [9]. Otherwise, the residues would be considered as processwastes, representing a cost liability for its disposal and treatment.

Chisti [5] discussed the recovery of energy from the microalgaeresidues after biodiesel production, highlighting its potential tomeet most of the energy demands of the preceding processes.Chisti [5] theoretically estimated that an average heating value of9360 MJ/metric t of microalgae residues was recoverable as meth-ane (CH4). The anaerobic digestion of the microalgae residues wasfurther examined by Sialve et al. [10] to improve the energetics ofthe microalgae biodiesel production process. That study [10]

ll rights reserved.

79 0964.).

highlighted factors which could potentially limit energy recoveryfrom this feedstock using the anaerobic conversion route.

Despite the theoretical work conducted by previous authors[5,10], there have been few experimental investigations on CH4

production using post transesterified microalgae biomass residues.The only demonstration found was that described in [11] usingpost transesterified residues of Chlorella mono-cultures. That studyinvestigated the batch anaerobic digestion of Chlorella residuessubjected to two preceding treatments, with average CH4 yieldsof 222–267.5 mL/g total solids of the microalgae residue digestedobtained [11]. Furthermore, co-digesting the microalgae residueswith the glycerol co-product in quantities equivalent to those pro-duced was observed to increase the CH4 yields by 5–8% comparedto the digestion of the residues alone [11].

The batch fermentation set-up used in [11] provided useful pre-liminary empirical data on the practical CH4 yields from microal-gae residues, however, certain experimental limitations can beencountered using this method. Due to the design and time frameof the batch tests, valuable information on the substrate digestibil-ity and process efficiency when the anaerobic digesters are fedcontinuously, cannot normally be acquired. A continuous experi-mental set-up suited to investigate useful process parameters,their interactions and influence on the efficiency of the CH4 pro-duction process, is thus required.

Co-digesting the microalgae residues with the transesterifica-tion glycerol by-product has the potential to improve the carbon

Nomenclature

BV/A ratio of butyric + valeric acid to acetic acid concentra-tion (mg butyric and acetic acid L�1/mg acetic acid L�1)

C/N carbon to nitrogen ratiod daysHHV higher heating value (MJ/m3; MJ/kg)HRT hydraulic retention time (d)MR % microalgae residues per volatile solids digested sub-

strateP/A propionic to acetic acid ratio (mg propionic acid L�1/mg

acetic acid L�1)

RSM response surface methodologySC substrate concentration, kg volatile solids substrate/m3

digesterTS total solids (g)VFA volatile fatty acids concentration (mg acetate/L)VS volatile solids (g/g total solids)t metric ton (1000 kg)rpm revolutions per minuteSTP standard temperature and pressureCCD central composite design

E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463 3455

to nitrogen (C/N) ratio of the digestion feedstock enhancing CH4

production [10]. This was further investigated in this study.This study evaluates CH4 recovery from microalgae residues fol-

lowing biodiesel production with the use of a semi-continuouslyfed stirred anaerobic reactor. The paper aims to answer the follow-ing questions:

(i) What influence would various combinations of substrateloading concentrations and retention periods have on theanaerobic digestion process and CH4 yield?

(ii) What substrate C/N ratios would lead to optimum CH4

yield?(iii) What effect would temperature increases in the mesophilic

operational range (25–40 �C) have on the extent of the mic-roalgae residue digestion?

(iv) How is the reactor stability and performance affected duringthe digestion process?

2. Materials and methods

2.1. Digestion substrates and Inoculum

2.1.1. Post transesterified microalgae residues and glycerolThe Chlorella source, as well as methods used for its cultivation,

harvesting and drying was same as described in [11]. The driedChlorella biomass was subjected to an acid catalysed in situ transe-sterification process and the residues were obtained via filtrationas described in [6]. The microalgae residues were pooled and deepfrozen at �24 �C and later thawed in the quantities required for theanaerobic digestion tests.

Glycerol solution (85% mass purity, Merck AaG) was used as theco-digestate. This was chosen to represent the crude glycerol likelyto be available industrially as was presented in [11].

2.1.2. InoculumThe inoculum source, type, as well as its adaptation for the

digestion of the high protein microalgae residues was same asthe methods described in [11].

Prior to their use for the anaerobic digestion trials, the sub-strates and inoculum were characterised on the basis of their vol-atile solids (VS) expressed as g/g total solids (TS). This was carriedout using standard methods [12]. The determined VS of the micro-algae residues, inoculum and glycerol were 0.946, 0.570 and1.000 g/g TS respectively.

2.2. Anaerobic digesters

To determine the influence of the investigated variables (excepttemperature) on the anaerobic digestion process and CH4 forma-tion, experiments were conducted using 2 L Erlenmeyer flasks as

reactors with a working volume of 1.5 L. Continuous stirring ofthe digester was provided by Teflon covered magnetic stirring barsoperated at 300 rpm. The anaerobic reactors were sealed air tightwith silicon stoppers designed with three glass tubes to facilitatethe removal of effluent, addition of fresh substrate and collectionof the produced gas. The daily withdrawal of the reactor effluents,as well as the addition of residue feedstocks, was carried out usingsyringes. The process temperature was maintained by immersingthe reactors in temperature controlled water baths.

To investigate the influence of temperature (mesophilic tem-perature range) on the digestion process, a laboratory scale contin-uously stirred tank reactor (CSTR) was constructed using Pyrexglass with stainless steel casings. The total reactor volume was5 L with a working volume of 4 L. To ensure a uniform temperatureand proper mixing of the digester, the process stirring was accom-plished using a mechanical stirrer operated at a speed of 310 rpm.The temperature of the digester was regulated using a hermeticallysealed surrounding water jacket with the heated water supplied bya thermostatic water bath. Effluent removal and loading of the feeddaily into the digester was accomplished by the use of a syringesystem. For the continuous measurement of the process a pH andtemperature probe (Mettler Toledo Inlab

�Expert Pro) was inserted

in the digester.Collection and measurement of the gas produced by the diges-

ter was carried out using eudiometer units (ISO/DIS 14853 (1999)),as described in [13], connected to the gas collection tubes at thehead of the reactors. The specific CH4 production of the digestedmaterials was determined by the use of a 5% molar sodium hydrox-ide (NaOH) solution in the eudiometers. To determine the percent-age of CH4 in the biogas (v/v), duplicate reactors were used. Theeudiometers in the duplicate reactors contained saturated sodiumchloride (NaCl) solution to minimise the solubility of the biogasacidic gases. For all measurements, the CH4 and biogas volumeswere corrected for standard temperature and pressure (STP).

2.3. Experimental design

2.3.1. Effect of varying retention times, substrate concentrations andmicroalgae residue fraction of digestion feedstock

To evaluate the influence of the hydraulic retention times(HRT), substrate concentration (SC), and substrate C/N ratios onthe CH4 yield, an empirical fitting technique, the response surfacemethodology (RSM) [14] was used. The RSM used for the investiga-tion of the above factors in this study was the central compositedesign (CCD) method. This involved the analysis of the effects ofretention times (5 < HRT < 15 d), substrate concentration(5 < SC < 40 kg VS/m3) and biomass carbon to nitrogen (5 < C/N < 25) on the anaerobic digestion of microalgae residues. Theexperiments were carried out in duplicates with the anaerobicdigestion temperature maintained at a fixed temperature of

3456 E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463

35 ± 0.5 �C throughout the reaction. The different substrate C/N ra-tios were obtained by the co-digestion of the microalgae residueswith glycerol.

2.3.2. TemperatureThe second set of experiments investigated the influence of

mesophilic digestion temperatures (25 �C < T < 40 �C) on CH4 pro-duction. The 5 L laboratory digester previously described in Sec-tion 2.2 were operated using a SC of 5 kg VS/m3 only, with a HRTof 10 and 15 d and with substrate C/N ratios of 8.53 and 12.44.The studied temperatures were attained after a gradual increaseof the digester temperatures by 2.5 ± 0.5 �C/week after threehydraulic cycles starting with a temperature of 25 �C. The investi-gations of the influence of temperature in this study were limitedto the mesophilic temperature range primarily since the inoculumused was obtained from mesophilic digesters. Furthermore, thistemperature level was considered to require less energy and there-fore operational cost inputs.

2.4. Analytical procedures for monitoring the anaerobic digesterperformance

The analysis of the microalgae residues anaerobic digestion wasmainly restricted to the relationships of the variables to CH4 pro-duction in this study. However, to further assess the performanceof the digester, indicators such as the digestate total volatile fattyacids (C1–C5) concentrations and total alkalinity were alsohighlighted.

2.4.1. VFA concentration determinationThe volatile fatty acids (acetic, propionic, butyric and valeric

acids) concentrations (VFA) were determined using an Agilent6850 series II gas chromatograph (GC) system. Samples of the di-gester effluents were prepared for GC analysis according to [12]and analysed by the GC equipped with a 7683A autosampler anda flame ionisation detector (FID). A DB-FFAP column (30 m length,0.25 mm ID and 0.25 lm thickness) was used for the separationwith helium as the carrier gas. The injector and detector tempera-tures were at 250 �C and 300 �C respectively, and the oven temper-ature was gradually increased from 100 �C to 250 �C at the rate of10 �C/min. A standard mixture containing 1 g/L of acetic, propionic,butyric and valeric acids, obtained from Merck KGaA chemicals,was used to calibrate the chromatograph.

2.4.2. Total alkalinityThe total alkalinity expressed as mg CaCO3/L digestate, was

measured as in [15] by titration to pH 4.5 with 0.05 M sulphuricacid (H2SO4).

2.4.3. Ammonia nitrogen (NH3–N)The ammonia nitrogen was measured using the standard test

method B of ASTM D1426-08 due to its accuracy in determiningNH3–N concentrations in the range of 0.5–1000 mg NH3–N/L di-rectly from the digestate. The measurements were carried out withthe Orion 951201 ammonia electrode (Thermo-scientific instru-ments), with higher concentrations determined following dilution.

2.4.4. Extent of VS destructionThe extent of substrate VS destruction via the anaerobic diges-

tion process was determined as in [16]. This method utilises themolar concentrations of the CH4 and CO2 of the produced biogas.This estimation is based on the assumption that all of the carbonin the destroyed feedstock VS is converted to CH4 and CO2 follow-ing the anaerobic digestion process.

3. Results and discussions

3.1. Analysis of the methane yields obtained varying HRT, SC and MR%at 35�C

To investigate the influence of the variables on the CH4 yieldfrom the microalgae residues, the MATLAB computing package(Version R2009a, The MathWorks Inc., Massachusetts, USA) wasused for the regression analysis of the experimental data. Anempirical relationship between the experimental parameters andthe observed response (specific CH4 yield) was proposed. Eq. (1)shows the second order polynomial fit equation obtained for theCH4 yield (Y) subject to the independent variables x1 (HRT), x2

(SC), and x3 (C/N) investigated in this study.

Y ¼ �0:3693þ 0:0765x1 � 0:0014x2 þ 0:0017x3

� 8:2277e�5x1x2 � 5:8955e�6x2x3 þ 8:3766e�5x1x3

� 0:0027x21 þ 2:2381e�5x2

2 � 5:3557e�4x23 ð1Þ

where Y, x1, x2 and x3 are the CH4 yield, HRT, SC and C/N ratiorespectively.

The significance levels of the regression coefficients in Eq.1 areshown in Table 1. The statistical student t-values were calculatedand used to determine the significance of each coefficient as putforward in [14]. The significance level (%) of the respective vari-ables was adjudged by comparing the obtained t-values with thetcritical value, as seen in Table 1. Higher values than the tcritical valuewere considered to be more significant. It was observed that x1

(HRT) and x21 had the most significant influence on the CH4 yield.

The R2 value of 0.979 obtained meant the fit model could be usedto adequately predict the CH4 yields from the variables within theexperimental boundaries

Using Eq. 1, the anaerobic digestion process was then graphi-cally represented using response surface and contour plots show-ing the individual and cumulative effects of the variables on thespecific CH4 yield.

3.2. Influence of variable interactions on methane yield

Fig. 1 shows the response surface plots of the influence of vary-ing C/N ratios (5.4–24.17) and HRT (5–15 d) on the specific CH4

yield (m3 CH4/kg VS) with loading concentrations of 5–50 kg VS/m3 and an operating temperature of 35 �C.

The maximum retention time investigated, 15 d, was selectedfor this study based on the findings of preliminary anaerobic diges-tion studies in [11] using batch reactors. That study showed that�98% of the optimum CH4 yields were obtained after a digestionperiod of 12–14 d. The possibility of reducing the digestion timerequirement, by manipulating the process variables (i.e. SC andC/N), was one of the aims of this study. This was since this couldcorrespond to a potential reduction in the process operational costsdue to reduced digester size requirements.

For all investigated levels of substrate C/N, it was observed thatan increase in the digestion time with a corresponding reduction inthe loading concentrations of the substrate, led to increased CH4

yields. The trends however showed that digestion times of 11–15 d were still required to achieve maximum CH4 production fromthe microalgae residues. Using a HRT of 15 d, a substrate loadingconcentration and C/N ratio of 5 kg VS/m3 and 5.40 respectively,a practical specific CH4 yield of 0.245 (±0.015) m3 CH4/kg VS sub-strate was obtained. This C/N ratio corresponds to the use of theChlorella residues alone for the digestion process. The observedCH4 yields were similar to those shown in [11] for post transeste-rified microalgae residues.

Increasing the substrate C/N ratio (via glycerol addition) wasfound to positively influence CH4 production. This may be

Table 1Significance levels of regression coefficients.

Variable Regression coefficient Standard deviation t-Value Significance level (%)

Intercept �0.3693 0.0249 2.0451 97.79x1 (HRT) 0.0765 0.0038 2.2529 98.64x2 (SC) �0.0014 0.0007 0.2254 58.89x3 (C/N ratio) 0.0017 0.0022 0.8276 79.48x1x2 �8.2277e�5 4.1280e�5 0.2257 58.90x1x3 8.3766e�5 8.2228e�5 0.1153 54.58x2x3 �5.8955e�6 2.0470e�5 0.0326 51.30x2

1�0.0027 0.0002 2.2348 98.58

x22

2.2381e�6 1.1054e�5 0.0503 51.99

x23

�5.3557e�4 6.7256e�5 1.0652 85.49

*Degrees of freedom = 77, tcritical-value = 1.9911, Root squared value (R2) = 0.9790, root mean square error (RMSE) = 0.0171.

Fig. 1. Response surface plots showing the influence of substrate C/N ratios and HRT on the methane yield (m3 CH4/kg VS) with loading concentrations of 5–50 kg VS/m3 anda process temperature of 35 �C.

E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463 3457

attributable to the improved digestibility of the substrates, sincethe liquid glycerol fraction would be more accessible to the fer-mentative bacterial mass [17].

With a retention time of 15 d, an increase in the substrate C/Nratio from 5.4 to 12.44 was observed to improve the specific CH4

yields by 20.0%, 29.8%, 30.0%, 53.0%, 48.0% and 61.0% at SC levelsof 5, 10, 20, 30, 40 and 50 kg VS/m3 respectively. Within theboundaries of this experimental study, no improvement in theCH4 recovery was achieved with C/N ratios >12.44 for all loadingconcentrations and anaerobic digestion times, as shown in Fig. 1.

From Fig. 1 it was observed that with a digestion time 5 d thereactor exhibited process inhibition (CH4 yield of <0.05 m3/kg VS),or even complete digester failure (zero CH4 production), for all theloading concentrations investigated. The inhibited CH4 yields

observed with a HRT of 5 d can be attributed to the increasedwashout the unreacted substrates as well as active micro-organ-isms (especially slow-growing bacteria) from the anaerobic diges-ter [18]. Hence, the Chlorella residues digestion wouldcorrespondingly improve with increases in the HRT due to the en-hanced exposure of the substrate to the active digester bacteria.

Fig. 1 also shows a decline in the specific CH4 yields with an in-crease in the substrate loading rates for all the digestion times, atdifferent substrate C/N ratios. The highest CH4 yields were ob-tained using the least SC (i.e. 5 kg VS/m3digester). The observedreduction in the CH4 production with increases in the SC appearto be due to the organic overloading of the digester, resulting inthe reduction or inhibition of the degradative capacity of thebacteria [18]. With reduced retention times, as seen in Fig. 1, an

3458 E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463

increase in the SC could therefore result in imbalances in the bac-terial population, consequently leading to volatile acids accumula-tion and digester failure. This assumption was further addressed inSection 3.5 with the investigation on the influence of the digesterVFAs on the specific CH4 yield.

3.3. Influence of varying process temperatures on methane yield

Of the three digestion temperature levels: psychrophilic(<25 �C), mesophilic (25–40 �C) and thermophilic (40–55 �C) usu-ally considered in anaerobic process investigations, this studywas limited to the mesophilic temperature range. Apart from thefact that the inoculum used for this study had been adapted formesophilic digestion, this temperature range was selected toinvestigate the anaerobic digestion of the microalgae residues topotentially minimise the digester heating demands.

Table 2 shows a summary of the results obtained for the anaer-obic digestion of post transesterified Chlorella biomass at 25, 30, 35and 40 �C at an organic loading concentration of 5 kg VS/m3 diges-ter and a hydraulic retention time of 15 d.

The loading concentration of 5 kg VS/m3 digester and 15 d HRTused in this part of the investigation was selected because the spe-cific CH4 yields were found to be highest at these levels (Sec-tion 3.2). To further examine any trends, the influence oftemperature variations on the digestion of two substrate C/N ratiosi.e. 8.53 and 12.44 were investigated.

A significant increase in the specific CH4 yield of the continu-ously co-digested glycerol-microalgae biomass residues was ob-served with increases in the mesophilic temperatures of thedigester from 25 to 35 �C. An increase of 53.65% and 60.64% wasdemonstrated when the digestion temperature was increased from25 to 35 �C for both C/N levels investigated. A further increase intemperature to 40 �C was, however, shown to have a minimalinfluence on the CH4 production for both samples.

The obtained results may however not fully characterise theinfluence of temperature on the anaerobic digestion of the residuesubstrates. This was due to the fact that the fermentative microbialmass used was initially adapted to a temperature level similar tothe ‘optimum’ level before the temperature level was lowered to25 �C and subsequent increases applied. The adaptation periodcould therefore favour the selection of specific bacterial massesthat thrive at this temperature.

3.4. Influence of C/N ratios on biogas methane content and anaerobicdigestion efficiency

Fig. 2 shows the CH4 content in the biogas (v/v) obtained using aHRT of 15 d and process temperature of 35 �C, with varying sub-strate C/N ratios of 5.4–24.17 and loading concentrations of 5–50 kg VS/m3 digester volume. Within the study experimentalboundaries, a reduction in the CH4 fraction of the biogas (v/v)was observed with an increase in the C/N ratio of the co-digestedbiomass substrate for all the loading concentrations. The reduction

Table 2Influence of varying mesophilic temperatures on methane yield.

Substrate C/N ratios

12.44Temperature (�C)

25 30 35

Specific CH4 yield (m3 CH4/kg VS) 0.192 0.208 0.295% CH4 in biogas (v/v) 62.0 61.7 65.3Total alkalinity (mg CaCO3/L) 12,850 13,150 14,820VFA (mg acetate/L) 6005 5111 1533pH 7.05 7.09 7.17

in the biogas CH4 fraction was attributed to the relative ease withwhich the glycerol fraction was digested. Increases in the substrateC/N ratio (via increases of the glycerol fraction) correspond to anincrease in the substrate oxygen content i.e. from 36.22% (C/N ratioof 5.40) to 48.94% oxygen content (for 24.17 C/N samples), andhence an increase in the CO2 fraction (v/v) resulting from themethanogenic process.

The anaerobic digestion efficiency was estimated on the basis ofthe substrate VS destruction and the observed molar concentrationof CH4 and CO2. An increase in the substrate loading concentrationcoupled with a reduction of the HRT was observed to result in areduction in the anaerobic digestion efficiency. For example, withthe HRT fixed at 15 d, Fig. 3 shows the variation of the digestionefficiency with the studied C/N ratios and loading rates. The diges-tion efficiency was observed to improve by 37.1% on increasing theC/N ratio of the residues from 5.4 to 24.17.

3.5. Monitoring the digester performance

3.5.1. Volatile fatty acids (VFA)Owing to the important role that volatile fatty acids (VFAs) play

as intermediates in the CH4 metabolic chain, the use of VFAs as anindicator of the effectiveness of the anaerobic reactors has beensuggested [19–23]. The different VFAs levels of the digestate couldtherefore aid in predicting the digester performance and help iden-tify underlying process problems, such as overloading.

With CH4 production of primary importance in this study, thespecific CH4 yield (m3 CH4/kg VS digested) was used to monitorthe digester performance. The criterion used to determine the suc-cess or impending failure of the digester was based on the assump-tion that a volatile solids reduction via the anaerobic digestion of asubstrate of at least 50% indicates the digester is ‘‘healthy’’ [20].The specific CH4 yield of 0.20 m3 CH4/kg VS, which correspondsto a 50% VS reduction was considered as the indicator of a healthydigester in this study, with lesser yields signalling impending di-gester failure.

Irrespective of the retention times, loading concentration andsubstrate C/N ratio levels, it was found that the total VFA values(in mg VFA/L) increased with a decrease in the specific CH4 yield(Fig. 4). The observed digester VFA may be due to an increase inthe activity of the acidogenic phase bacteria, coupled with a likelyinhibition of the methane forming bacteria and/or a slower rate ofthe acid intermediates consumption by the methanogenicprocess.

The high of VFA concentrations (>5000 mg total VFA/L), indica-tive of instability in the anaerobic digester, was observed for all thereactors with SC > 40 kg/m3, regardless of the substrate C/N ratiosand HRT used. Furthermore, a reduction in the digestion retentiontimes significantly increased the VFA accumulation, indicating acomparably faster acid formation process in relation to the CH4

forming phase. This suggests that the methanogenic process couldbe the rate limiting step for the anaerobic digestion of Chlorellaresidues.

8.53Temperature (�C)

40 25 30 35 40

0.265 0.188 0.227 0.302 0.30863.1 64.5 68.3 67.9 69.214,550 12,580 13,200 16,340 16,2002023 6850 4086 1135 9897.15 7.08 7.11 7.12 7.16

62

63

64

65

66

67

68

69

0 5 10 15 20 25 30

% C

H i

n bi

ogas

(v/v

)4

Substrate C/N ratio

5 Kg VS/m3

10 kg VS/ m3

20 kg VS/ m3

30 kg VS/ m3

40 kg VS/ m3

50 kg VS/ m3

Fig. 2. Influence of substrate C/N ratio on percentage methane in the biogas (v/v) produced from the microalgae residues at a HRT of 15 d and process temperature of 35 �C.

0

10

20

30

40

50

60

70

0 5 10 15 20 25 30

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(%)

Substrate C/N ratio

5 Kg VS/m3

10 kg VS/ m3

20 kg VS/ m3

30 kg VS/ m3

40 kg VS/ m3

50 kg VS/ m3

Fig. 3. Influence of substrate C/N ratio on the anaerobic digestion efficiency of co-digested microalgae residues with a retention period of 15 d and process temperature of35 �C.

E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463 3459

The chromatographic analysis of the effluent VFAs revealed avariation in the presence of acetic, propionic, butyric and valericacids at different digestion conditions and specific CH4 yields. Forall levels of C/N ratios and with SC rates of 5 kg VS/m3 using aHRT of 10 and 15 d, the predominant digestate VFAs were aceticand propionic acids. Similar results were obtained with a SC of10 and 20 kg VS/m3 with a HRT of 15 d. This was indicative of anactive degradation of the biomass component macromolecules[22]. However at higher loading concentrations, an increase inthe butyric and valeric acids concentrations was observed(Fig. 5). The butyric and valeric acid rose from 0–2% of the total

VFA (w/w) in high CH4 producing digesters to 10% (w/w) obtainedat the onset of digestion inhibition. The accumulation of the buty-ric and valeric acids appears to exhibit inhibitory effects on themethanogenic process. This can be seen in the reduced specificCH4 yields, with an almost complete digester failure observed withconcentrations levels >6500 mg butyric and valeric acids/L as seenin Fig. 5.

Foree and McCarty [24] demonstrated that the digestion tem-perature and retention time had an influence on the individualVFA components available during the anaerobic digestion processof untreated microalgae. However, this study did not investigate

0

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0 5000 10000 15000 20000 25000 30000

Met

hane

yie

ld (m

CH

/kg

VS

dige

sted

sam

ple

)3

4

Total VFA (mg VFA/L)

Fig. 4. Observed relationship between total VFA concentration and specific methane yields for anaerobic digestion runs at 35 �C.

0

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Butyric + valeric acid concentrations (mg/L)

Fig. 5. Variation in the digester butyric and valeric acids concentrations with specific methane yields for the anaerobic digestion runs at 35 �C.

3460 E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463

the influences of varying process temperatures. The results re-ported are confined to experimental runs conducted at 35 �C.

The ratio of the propionic to acetic acid concentrations (P/A) hasbeen discussed the literature as a good indicator of anaerobic di-gester performance [20,25,26]. Hill and Feinberg [20] demon-strated that a P/A ratio of >1.4, and acetate levels of >800 mg/L,could signal impending digester failure. In this study, the observedvariation of the P/A ratio with specific CH4 yields (Fig. 6a), suggeststhat the digester performance was not adequately predicted by theP/A ratio. Poor digester performance was observed at P/A ratios aslow as 0.6, whereas reasonable performance was obtained for P/Aratios as high as 1.2 as seen in Fig 6a.

Based on these results, and taking into account that increasingbutyric and valeric acid concentrations led to process inhibition,the ratio of butyric and valeric acid to acetic acid (BV/A) was pro-

posed to be used as an indicator of the digester performance.Impending digester failure appears to be predictable with BV/A ra-tios > 1.2 (Fig. 6b).

3.5.2. Process alkalinityCoupling the process alkalinity with the observed total VFA con-

centrations was another criterion used for the assessment of theperformance of the anaerobic digestion process in this study. Usingthe process VFA to alkalinity ratio, three critical values were con-sidered by Callaghan et al. [15] for monitoring the CH4 productionprocess. These are:

<0.4 (would ensure digester stability),0.4–0.8 (some instability may occur),P0.8 (significant instability would be encountered).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 0.2 0.4 0.6 0.8 1 1.2 1.4

Met

hane

yie

ld (m

CH

/kg

VS)

34

P/A ratio

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

Met

hane

yie

ld (

m C

H /

kg V

S)3

4

BV/A ratio

(a)

(b)

Fig. 6. (a and b) The relationship between the observed specific methane yield to the P/A and BV/A ratios for anaerobic digestion runs at 35 �C.

E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463 3461

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

Specific methane yield (m CH /kg VS substrate)34

Tot

al a

lkal

init

y (m

g C

aCO

/L

)3

0.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

4.000

VF

A/A

lkal

init

y ra

tio

Fig. 7. Relationship between observed total alkalinity levels and total VFA/alkalinity ratios with specific methane yields for anaerobic digestion runs at 35 �C (e = alkalinity,mg CaCO3/L; � = VFA/alkalinity ratio).

3462 E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463

It was observed from Fig. 7, that the VFA/alkalinity ratio as de-scribed above appears to adequately characterise the Chlorella res-idues digestion. The maximum and minimum CH4 yields (0.295and 0.002 m3 CH4/kg VS) were obtained at a total VFA/alkalinity ra-tio of 0.103 and 4.059 respectively. The onset of the reactor insta-bility indicated by a specific CH4 yield of 0.2 m3 CH4/kg VS wasseen to have a VFA/alkalinity ratio of 0.435.

3.5.3. Process pHAs highlighted in Fig. 8, a stable pH range (6.6–7.32) was ob-

served for most of the experimental runs, thus indicating a highbuffering capacity of the digestion process. The buffer systemwas, however, shown not to adequately sustain high VFA accumu-lation, as encountered with increases in the SC as well as a reduc-tion of the HRT, i.e. for all SC at HRT of 5 d (Fig. 8). This led to a drop

6

6.2

6.4

6.6

6.8

7

7.2

7.4

0 10 20 30

Pro

cess

pH

Loading concentration (kg

Fig. 8. Observed process pH with substrate concentrations for different

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

6 6.2 6.4 6.6

Met

hane

yie

ld (m

CH

/kg

VS

sam

ple)

3

4

Meas

Fig. 9. Relationship between measured digester pH and spec

in the process pH to <6.5, which was shown to result in an inhibi-tion or complete disruption of the CH4 production, as indicated bythe low specific CH4 yields in those systems as shown in Fig. 9.

3.5.4. Process ammonia nitrogen (NH3–N)The high protein (60.19%) and corresponding nitrogen (9.39%)

content of the post transesterified Chlorella residues [11] used inthe digestion process could potentially result in the by-productionof toxic ammonia concentrations.

Within the experimental boundaries, it was observed that withan increase in HRT and the SC, an increase in the digestate totalammonia nitrogen was observed. This was attributed to morecomplete digestion of the microalgae residue proteins with time,with an accompanying accumulation of ammonia in the liquidphase.

40 50 60

VS sample/m )3

5 d HRT-C/N 5.40

10 d HRT-C/N 5.40

15 d HRT-C/N 5.40

5 d HRT-C/N 6.56

15 d HRT-C/N 6.56

5 d HRT-C/N 8.53

10 d HRT-C/N 8.53

15 d HRT-C/N 8.53

5 d HRT-C/N 12.44

10 d HRT-C/N 12.44

15 d HRT-C/N 12.44

5 d HRT-C/N 24.17

10 d HRT-C/N 24.17

15 d HRT-C/N 24.17

10 d HRT-C/N 6.56

HRT and substrate C/N ratios for anaerobic digestion runs at 35 �C.

6.8 7 7.2 7.4

ured pH

ific methane yields for anaerobic digestion runs at 35 �C.

E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463 3463

With an increase in the substrate C/N ratio, a reduction in theammonia nitrogen levels was obtained compared with the corre-sponding SC and HRT of samples with lower C/N ratios.

Inhibition of the anaerobic digestion process, due to passive dif-fusion of unionized free ammonia (NH3) across the cell wall of thedigestion bacteria where its toxicity will be expressed [27] wasanticipated in this study. It was expected that the acetoclasticmethanogenic bacteria would be the most sensitive to free ammo-nia as reported by Angelidaki and Ahring [27]. However, theobserved ammonia nitrogen concentrations in this study (880–4300 mg NH3–N/L digestate, for all investigated levels) appearnot to directly affect the process stability. For example, a nitrogenammonia concentration of 3500 mg/L was obtained for the 6.56 C/N ratio sample digested with a HRT of 15 d and SC of 5 kg VS/m3

which had a specific CH4 yield of 0.245 m3 CH4/kg VS.The influence of the process pH on the ammonia species propor-

tion in the digester may have played a role in reducing the risk ofammonia inhibition. The high VFA concentration in this studycould have promoted a slightly acidic digester environment, whichwould largely aid the protonation of the free NH3 to the ionizedammonium species NHþ4 , by enhancing the shift of the chemicalequilibrium in Eq. (2) to the right.

NH3 þHþ $ NHþ4 ð2Þ

Hence, at the recorded pH levels, most of the ammonia nitrogenin this study may be below the toxic concentration levels. McCarty[28] determined that ammonia inhibition in the anaerobic digesteroccurs with concentrations of 1.5–3.0 g N/L at a pH level of over7.4.

In addition, the digester acclimatisation period, as well as theinoculum and digestion temperature used in this study, might havehelped reduce the toxic effects of NH3. The ammonia nitrogen con-centrations in this study was observed to fall within the broadinhibiting concentration range of 1.7–14 g N/L discussed in [27]when different process conditions which might influence theammonia inhibition are considered.

4. Conclusion

The results obtained in this paper have shown that it is feasibleto subject the microalgae residues, post transesterification, to theanaerobic digestion process with the CH4 yields mainly influencedby digestion times. The results obtained in this experimental studywere based on the use of post transesterified residues after thein situ transesterification method using the freshwater microalgaespecie (Chlorella sp.).

Increases in the hydraulic retention period have been shown tosignificantly affect the digestion process with periods longer than5 d required for efficient anaerobic conversion of the biomass res-idues to CH4.

When available, the integration of glycerol from biodiesel pro-duction, or from other conventional oil feedstocks, in the anaerobicdigestion of microalgae residues has the potential to improve theoverall energy recovered as CH4. Increasing the substrate C/N ratioto 12.44 by co-digesting the microalgae residues with glycerol wasobserved to increase CH4 production by >50%, compared with theCH4 production when the residues were digested alone.

The substrate loading concentration was also found to be as animportant parameter with lower digester loading rates favouringhigher specific CH4 yields.

Methane recovery from post transesterified microalgae resi-dues, coupled with optimising the biomass and lipid productivities[29] and the use of novel drying technologies for microalgae [30]could potentially improve the commercial viability of microalgaebiodiesel production.

Acknowledgements

The authors are most grateful to Jackie Shand and the postgrad-uate students of the Photosystem II lab, Biochemistry Dept.,University of Otago, New Zealand for the use of their facilities,instruction and assistance. We also wish also thank Dr. Phil Novisof Landcare Research for providing the Chlorella culture used in thisstudy and Mr. Muthasim Fahmy for his help with the processmodelling.

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