Applied Energy 88 (2011) 3454–3463

Contents lists available at ScienceDirect

Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Anaerobic digestion of microalgae residues resulting from the biodiesel

production 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 Zealand
b
Department of Food Science, University of Otago, 276 Leith Walk, Dunedin 9016, New Zealand
c
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: The recovery of methane from post transesterified microalgae residues has the potential to improve the
Received 11 August 2010 renewability of the ‘microalgae biomass to biodiesel’ conversion process as well as reduce its cost and
Received in revised form 4 October 2010 environmental impact. This paper deals with the anaerobic digestion of microalgae biomass residues
Accepted 9 October 2010
(post transesterification) using semi-continuously fed reactors. The influence of substrate loading con-
Available online 19 November 2010
centrations and hydraulic retention times on the specific methane yield of the anaerobically digested
microalgae residues was investigated. The co-digestion of the microalgae residues with glycerol as well
Keywords:
as the influence of temperature was also examined. It was found that the hydraulic retention period was
Microalgae residues
Anaerobic digestion
the most significant variable affecting methane production from the residues, with periods (>5 days) cor-
Glycerol responding to higher energy recovery. The methane yield was also improved by a reduction in the sub-
Co-digestion strate loading rates, with an optimum substrate carbon to nitrogen ratio of 12.44 seen to be required for
Methane the digestion process.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction highlighted factors which could potentially limit energy recovery

The use of photosynthetic micro-organisms (microalgae) for
biodiesel production has been discussed extensively in the litera-
ture [1–8]. The biodiesel production process also results in the
co-production of glycerol (C3H5(OH)3) as well as microalgae resi-
dues. With the proposed commercial production of biodiesel from
microalgae, 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 livestock
feeds [9]. Otherwise, the residues would be considered as process
wastes, representing a cost liability for its disposal and treatment.
Chisti [5] discussed the recovery of energy from the microalgae
residues after biodiesel production, highlighting its potential to
meet most of the energy demands of the preceding processes.
Chisti [5] theoretically estimated that an average heating value of
9360 MJ/metric t of microalgae residues was recoverable as meth-
ane (CH4). The anaerobic digestion of the microalgae residues was
further examined by Sialve et al. [10] to improve the energetics of
the microalgae biodiesel production process. That study [10]
⇑ Correspondence. Tel.: +64 3479 7812; fax: +64 3479 0964.
E-mail address: zhifa@physics.otago.ac.nz (Z.F. Sun).
from 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] using
post transesterified residues of Chlorella mono-cultures. That study
investigated the batch anaerobic digestion of Chlorella residues
subjected to two preceding treatments, with average CH4 yields
of 222–267.5 mL/g total solids of the microalgae residue digested
obtained [11]. Furthermore, co-digesting the microalgae residues
with the glycerol co-product in quantities equivalent to those pro-
duced was observed to increase the CH4 yields by 5–8% compared
to 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 be
encountered using this method. Due to the design and time frame
of the batch tests, valuable information on the substrate digestibil-
ity and process efficiency when the anaerobic digesters are fed
continuously, 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

0306-2619/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apenergy.2010.10.020
 E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463
Nomenclature
BV/A ratio of butyric + valeric acid to acetic acid concentra-
tion (mg butyric and acetic acid L1/mg acetic acid L1)
C/N carbon to nitrogen ratio
d days
HHV higher heating value (MJ/m3; MJ/kg)
HRT hydraulic retention time (d)
MR % microalgae residues per volatile solids digested sub-
strate
P/A propionic to acetic acid ratio (mg propionic acid L1/mg
acetic acid L1)
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-continuously
fed stirred anaerobic reactor. The paper aims to answer the follow-
ing questions:
(i) What influence would various combinations of substrate
loading concentrations and retention periods have on the
anaerobic 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 during
the digestion process?
2. Materials and methods
2.1. Digestion substrates and Inoculum
2.1.1. Post transesterified microalgae residues and glycerol
The Chlorella source, as well as methods used for its cultivation,
harvesting and drying was same as described in [11]. The dried
Chlorella biomass was subjected to an acid catalysed in situ transe-
sterification process and the residues were obtained via filtration
as described in [6]. The microalgae residues were pooled and deep
frozen at 24 °C and later thawed in the quantities required for the
anaerobic digestion tests.
Glycerol solution (85% mass purity, Merck AaG) was used as the
co-digestate. This was chosen to represent the crude glycerol likely
to be available industrially as was presented in [11].
2.1.2. Inoculum
The inoculum source, type, as well as its adaptation for the
digestion of the high protein microalgae residues was same as
the 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 carried
out using standard methods [12]. The determined VS of the micro-
algae residues, inoculum and glycerol were 0.946, 0.570 and
1.000 g/g TS respectively.
2.2. Anaerobic digesters
To determine the influence of the investigated variables (except
temperature) on the anaerobic digestion process and CH4 forma-
tion, experiments were conducted using 2 L Erlenmeyer flasks as
3455
RSM response surface methodology
SC substrate concentration, kg volatile solids substrate/m3
digester
TS 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 minute
STP standard temperature and pressure
CCD central composite design
reactors with a working volume of 1.5 L. Continuous stirring of
the digester was provided by Teflon covered magnetic stirring bars
operated at 300 rpm. The anaerobic reactors were sealed air tight
with silicon stoppers designed with three glass tubes to facilitate
the removal of effluent, addition of fresh substrate and collection
of the produced gas. The daily withdrawal of the reactor effluents,
as well as the addition of residue feedstocks, was carried out using
syringes. The process temperature was maintained by immersing
the 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 Pyrex
glass with stainless steel casings. The total reactor volume was
5 L with a working volume of 4 L. To ensure a uniform temperature
and 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 hermetically
sealed surrounding water jacket with the heated water supplied by
a thermostatic water bath. Effluent removal and loading of the feed
daily into the digester was accomplished by the use of a syringe
system. For the continuous measurement of the process a pH and
Ò
temperature 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 the
head of the reactors. The specific CH4 production of the digested
materials 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. The
eudiometers in the duplicate reactors contained saturated sodium
chloride (NaCl) solution to minimise the solubility of the biogas
acidic gases. For all measurements, the CH4 and biogas volumes
were corrected for standard temperature and pressure (STP).
2.3. Experimental design
2.3.1. Effect of varying retention times, substrate concentrations and
microalgae residue fraction of digestion feedstock
To evaluate the influence of the hydraulic retention times
(HRT), substrate concentration (SC), and substrate C/N ratios on
the CH4 yield, an empirical fitting technique, the response surface
methodology (RSM) [14] was used. The RSM used for the investiga-
tion of the above factors in this study was the central composite
design (CCD) method. This involved the analysis of the effects of
retention 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. The
experiments were carried out in duplicates with the anaerobic
digestion 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 residues
with glycerol.
2.3.2. Temperature
The 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 HRT
of 10 and 15 d and with substrate C/N ratios of 8.53 and 12.44.
The studied temperatures were attained after a gradual increase
of the digester temperatures by 2.5 ± 0.5 °C/week after three
hydraulic cycles starting with a temperature of 25 °C. The investi-
gations of the influence of temperature in this study were limited
to the mesophilic temperature range primarily since the inoculum
used was obtained from mesophilic digesters. Furthermore, this
temperature level was considered to require less energy and there-
fore operational cost inputs.
2.4. Analytical procedures for monitoring the anaerobic digester
performance
The analysis of the microalgae residues anaerobic digestion was
mainly restricted to the relationships of the variables to CH4 pro-
duction in this study. However, to further assess the performance
of the digester, indicators such as the digestate total volatile fatty
acids (C1–C5) concentrations and total alkalinity were also
highlighted.
2.4.1. VFA concentration determination
The volatile fatty acids (acetic, propionic, butyric and valeric
acids) concentrations (VFA) were determined using an Agilent
6850 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 and
a flame ionisation detector (FID). A DB-FFAP column (30 m length,
0.25 mm ID and 0.25 lm thickness) was used for the separation
with 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 of
10 °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 alkalinity
The total alkalinity expressed as mg CaCO3/L digestate, was
measured as in [15] by titration to pH 4.5 with 0.05 M sulphuric
acid (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 determining
NH3–N concentrations in the range of 0.5–1000 mg NH3–N/L di-
rectly from the digestate. The measurements were carried out with
the Orion 951201 ammonia electrode (Thermo-scientific instru-
ments), with higher concentrations determined following dilution.
2.4.4. Extent of VS destruction
The extent of substrate VS destruction via the anaerobic diges-
tion process was determined as in [16]. This method utilises the
molar concentrations of the CH4 and CO2 of the produced biogas.
This estimation is based on the assumption that all of the carbon
in 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 yield
from the microalgae residues, the MATLAB computing package
(Version R2009a, The MathWorks Inc., Massachusetts, USA) was
used for the regression analysis of the experimental data. An
empirical relationship between the experimental parameters and
the observed response (specific CH4 yield) was proposed. Eq. (1)
shows the second order polynomial fit equation obtained for the
CH4 yield (Y) subject to the independent variables x1 (HRT), x2
(SC), and x3 (C/N) investigated in this study.
Y ¼ 0:3693 þ 0:0765x1  0:0014x2 þ 0:0017x3
 8:2277e5 x1 x2  5:8955e6 x2 x3 þ 8:3766e5 x1 x3
 0:0027x21 þ 2:2381e5 x22  5:3557e4 x23 ð1Þ
where Y, x1, x2 and x3 are the CH4 yield, HRT, SC and C/N ratio
respectively.
The significance levels of the regression coefficients in Eq.1 are
shown in Table 1. The statistical student t-values were calculated
and used to determine the significance of each coefficient as put
forward in [14]. The significance level (%) of the respective vari-
ables was adjudged by comparing the obtained t-values with the
tcritical value, as seen in Table 1. Higher values than the tcritical value
were 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 used
to adequately predict the CH4 yields from the variables within the
experimental 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 the
specific 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 selected
for 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 digestion
period of 12–14 d. The possibility of reducing the digestion time
requirement, by manipulating the process variables (i.e. SC and
C/N), was one of the aims of this study. This was since this could
correspond to a potential reduction in the process operational costs
due to reduced digester size requirements.
For all investigated levels of substrate C/N, it was observed that
an increase in the digestion time with a corresponding reduction in
the 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 from
the microalgae residues. Using a HRT of 15 d, a substrate loading
concentration 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 the
Chlorella residues alone for the digestion process. The observed
CH4 yields were similar to those shown in [11] for post transeste-
rified microalgae residues.
Increasing the substrate C/N ratio (via glycerol addition) was
found to positively influence CH4 production. This may be
 E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463 3457

Table 1
Significance levels of regression coefficients.

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

Intercept 0.3693 0.0249 2.0451 97.79
x1 (HRT) 0.0765 0.0038 2.2529 98.64
x2 (SC) 0.0014 0.0007 0.2254 58.89
x3 (C/N ratio) 0.0017 0.0022 0.8276 79.48
x1x2 8.2277e5 4.1280e5 0.2257 58.90
x1x3 8.3766e5 8.2228e5 0.1153 54.58
x2x3 5.8955e6 2.0470e5 0.0326 51.30
x21 0.0027 0.0002 2.2348 98.58
x22 2.2381e6 1.1054e5 0.0503 51.99
x23 5.3557e4 6.7256e5 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 and
a process temperature of 35 °C.

attributable to the improved digestibility of the substrates, since
the 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/N
ratio 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 levels
of 5, 10, 20, 30, 40 and 50 kg VS/m3 respectively. Within the
boundaries of this experimental study, no improvement in the
CH4 recovery was achieved with C/N ratios >12.44 for all loading
concentrations and anaerobic digestion times, as shown in Fig. 1.
From Fig. 1 it was observed that with a digestion time 5 d the
reactor exhibited process inhibition (CH4 yield of <0.05 m3/kg VS),
or even complete digester failure (zero CH4 production), for all the
loading concentrations investigated. The inhibited CH4 yields
observed with a HRT of 5 d can be attributed to the increased
washout 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 would
correspondingly 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, at
different substrate C/N ratios. The highest CH4 yields were ob-
tained using the least SC (i.e. 5 kg VS/m3digester). The observed
reduction in the CH4 production with increases in the SC appear
to be due to the organic overloading of the digester, resulting in
the reduction or inhibition of the degradative capacity of the
bacteria [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 in
Section 3.5 with the investigation on the influence of the digester
VFAs 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 study
was limited to the mesophilic temperature range. Apart from the
fact that the inoculum used for this study had been adapted for
mesophilic digestion, this temperature range was selected to
investigate the anaerobic digestion of the microalgae residues to
potentially 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, 35
and 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 HRT
used 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 of
temperature variations on the digestion of two substrate C/N ratios
i.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 the
digester from 25 to 35 °C. An increase of 53.65% and 60.64% was
demonstrated when the digestion temperature was increased from
25 to 35 °C for both C/N levels investigated. A further increase in
temperature to 40 °C was, however, shown to have a minimal
influence on the CH4 production for both samples.
The obtained results may however not fully characterise the
influence of temperature on the anaerobic digestion of the residue
substrates. This was due to the fact that the fermentative microbial
mass used was initially adapted to a temperature level similar to
the ‘optimum’ level before the temperature level was lowered to
25 °C and subsequent increases applied. The adaptation period
could therefore favour the selection of specific bacterial masses
that thrive at this temperature.
3.4. Influence of C/N ratios on biogas methane content and anaerobic
digestion efficiency
Fig. 2 shows the CH4 content in the biogas (v/v) obtained using a
HRT 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 experimental
boundaries, a reduction in the CH4 fraction of the biogas (v/v)
was observed with an increase in the C/N ratio of the co-digested
biomass substrate for all the loading concentrations. The reduction
Table 2
Influence of varying mesophilic temperatures on methane yield.
Substrate C/N ratios
12.44
Temperature (°C)
25 30
Specific CH4 yield (m3 CH4/kg VS) 0.192 0.208
% CH4 in biogas (v/v) 62.0 61.7
Total alkalinity (mg CaCO3/L) 12,850 13,150
VFA (mg acetate/L) 6005 5111
pH 7.05 7.09
in the biogas CH4 fraction was attributed to the relative ease with
which the glycerol fraction was digested. Increases in the substrate
C/N ratio (via increases of the glycerol fraction) correspond to an
increase in the substrate oxygen content i.e. from 36.22% (C/N ratio
of 5.40) to 48.94% oxygen content (for 24.17 C/N samples), and
hence an increase in the CO2 fraction (v/v) resulting from the
methanogenic process.
The anaerobic digestion efficiency was estimated on the basis of
the substrate VS destruction and the observed molar concentration
of CH4 and CO2. An increase in the substrate loading concentration
coupled with a reduction of the HRT was observed to result in a
reduction in the anaerobic digestion efficiency. For example, with
the HRT fixed at 15 d, Fig. 3 shows the variation of the digestion
efficiency with the studied C/N ratios and loading rates. The diges-
tion efficiency was observed to improve by 37.1% on increasing the
C/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 an
indicator of the effectiveness of the anaerobic reactors has been
suggested [19–23]. The different VFAs levels of the digestate could
therefore 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, the
specific CH4 yield (m3 CH4/kg VS digested) was used to monitor
the 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 a
substrate of at least 50% indicates the digester is ‘‘healthy’’ [20].
The specific CH4 yield of 0.20 m3 CH4/kg VS, which corresponds
to a 50% VS reduction was considered as the indicator of a healthy
digester in this study, with lesser yields signalling impending di-
gester failure.
Irrespective of the retention times, loading concentration and
substrate 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 in
the activity of the acidogenic phase bacteria, coupled with a likely
inhibition of the methane forming bacteria and/or a slower rate of
the acid intermediates consumption by the methanogenic
process.
The high of VFA concentrations (>5000 mg total VFA/L), indica-
tive of instability in the anaerobic digester, was observed for all the
reactors with SC > 40 kg/m3, regardless of the substrate C/N ratios
and HRT used. Furthermore, a reduction in the digestion retention
times significantly increased the VFA accumulation, indicating a
comparably faster acid formation process in relation to the CH4
forming phase. This suggests that the methanogenic process could
be the rate limiting step for the anaerobic digestion of Chlorella
residues.
8.53
Temperature (°C)
35 40 25 30 35 40
0.295 0.265 0.188 0.227 0.302 0.308
65.3 63.1 64.5 68.3 67.9 69.2
14,820 14,550 12,580 13,200 16,340 16,200
1533 2023 6850 4086 1135 989
7.17 7.15 7.08 7.11 7.12 7.16
 E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463 3459

69

5 Kg VS/m3
68 10 kg VS/ m3
20 kg VS/ m3
30 kg VS/ m3
67 40 kg VS/ m3
% CH 4 in biogas (v/v) 50 kg VS/ m3

66

65

64

63

62
0 5 10 15 20 25 30
Substrate C/N ratio
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.

70

60
Anaerobic digestion efficiency (%)

50

40

30
5 Kg VS/m3
10 kg VS/ m3
20 20 kg VS/ m3
30 kg VS/ m3
40 kg VS/ m3
10 50 kg VS/ m3

0
0 5 10 15 20 25 30
Substrate C/N ratio
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 of
35 °C.

The chromatographic analysis of the effluent VFAs revealed a
variation in the presence of acetic, propionic, butyric and valeric
acids at different digestion conditions and specific CH4 yields. For
all levels of C/N ratios and with SC rates of 5 kg VS/m3 using a
HRT of 10 and 15 d, the predominant digestate VFAs were acetic
and propionic acids. Similar results were obtained with a SC of
10 and 20 kg VS/m3 with a HRT of 15 d. This was indicative of an
active degradation of the biomass component macromolecules
[22]. However at higher loading concentrations, an increase in
the 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) obtained
at the onset of digestion inhibition. The accumulation of the buty-
ric and valeric acids appears to exhibit inhibitory effects on the
methanogenic process. This can be seen in the reduced specific
CH4 yields, with an almost complete digester failure observed with
concentrations levels >6500 mg butyric and valeric acids/L as seen
in Fig. 5.
Foree and McCarty [24] demonstrated that the digestion tem-
perature and retention time had an influence on the individual
VFA components available during the anaerobic digestion process
of untreated microalgae. However, this study did not investigate
3460 E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463

0.35

Methane yield (m 3CH4 /kg VS digested sample )

0.3

0.25

0.2

0.15

0.1

0.05

0
0 5000 10000 15000 20000 25000 30000
Total VFA (mg VFA/L)
Fig. 4. Observed relationship between total VFA concentration and specific methane yields for anaerobic digestion runs at 35 °C.

0.35

0.3
Methane yield (m 3 CH 4 /kg VS sample)

0.25

0.2

0.15

0.1

0.05

0
0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
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.

the influences of varying process temperatures. The results re- posed to be used as an indicator of the digester performance.
ported are confined to experimental runs conducted at 35 °C. Impending digester failure appears to be predictable with BV/A ra-
The ratio of the propionic to acetic acid concentrations (P/A) has tios > 1.2 (Fig. 6b).
been discussed the literature as a good indicator of anaerobic di-
gester performance [20,25,26]. Hill and Feinberg [20] demon- 3.5.2. Process alkalinity
strated that a P/A ratio of >1.4, and acetate levels of >800 mg/L, Coupling the process alkalinity with the observed total VFA con-
could signal impending digester failure. In this study, the observed centrations was another criterion used for the assessment of the
variation of the P/A ratio with specific CH4 yields (Fig. 6a), suggests performance of the anaerobic digestion process in this study. Using
that the digester performance was not adequately predicted by the the process VFA to alkalinity ratio, three critical values were con-
P/A ratio. Poor digester performance was observed at P/A ratios as sidered by Callaghan et al. [15] for monitoring the CH4 production
low as 0.6, whereas reasonable performance was obtained for P/A process. These are:
ratios as high as 1.2 as seen in Fig 6a.
Based on these results, and taking into account that increasing <0.4 (would ensure digester stability),
butyric and valeric acid concentrations led to process inhibition, 0.4–0.8 (some instability may occur),
the ratio of butyric and valeric acid to acetic acid (BV/A) was pro- P0.8 (significant instability would be encountered).
 E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463 3461

(a) 0.35

0.3

Methane yield (m3 CH 4 /kg VS)

0.25

0.2

0.15

0.1

0.05

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4
P/A ratio
(b) 0.35

0.3
Methane yield (m3 CH4 /kg VS)

0.25

0.2

0.15

0.1

0.05

0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
BV/A ratio
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.

18000 4.000

16000
Total alkalinity (mg CaCO3 /L)

3.500
14000
VFA/Alkalinity ratio

3.000
12000
2.500
10000
2.000
8000
1.500
6000
1.000
4000

2000 0.500

0 0.000
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
Specific methane yield (m 3 CH4 /kg VS substrate)

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- in the process pH to <6.5, which was shown to result in an inhibi-
scribed above appears to adequately characterise the Chlorella res- tion or complete disruption of the CH4 production, as indicated by
idues digestion. The maximum and minimum CH4 yields (0.295 the low specific CH4 yields in those systems as shown in Fig. 9.
and 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-
3.5.4. Process ammonia nitrogen (NH3–N)
bility indicated by a specific CH4 yield of 0.2 m3 CH4/kg VS was
The high protein (60.19%) and corresponding nitrogen (9.39%)
seen to have a VFA/alkalinity ratio of 0.435.
content of the post transesterified Chlorella residues [11] used in
the digestion process could potentially result in the by-production
3.5.3. Process pH of toxic ammonia concentrations.
As highlighted in Fig. 8, a stable pH range (6.6–7.32) was ob- Within the experimental boundaries, it was observed that with
served for most of the experimental runs, thus indicating a high an increase in HRT and the SC, an increase in the digestate total
buffering capacity of the digestion process. The buffer system ammonia nitrogen was observed. This was attributed to more
was, however, shown not to adequately sustain high VFA accumu- complete digestion of the microalgae residue proteins with time,
lation, as encountered with increases in the SC as well as a reduc- with an accompanying accumulation of ammonia in the liquid
tion of the HRT, i.e. for all SC at HRT of 5 d (Fig. 8). This led to a drop phase.

7.4

7.2 5 d HRT-C/N 5.40

10 d HRT-C/N 5.40
15 d HRT-C/N 5.40
7 5 d HRT-C/N 6.56
15 d HRT-C/N 6.56
5 d HRT-C/N 8.53
Process pH

6.8 10 d HRT-C/N 8.53

15 d HRT-C/N 8.53
5 d HRT-C/N 12.44
6.6
10 d HRT-C/N 12.44
15 d HRT-C/N 12.44

6.4 5 d HRT-C/N 24.17

10 d HRT-C/N 24.17
15 d HRT-C/N 24.17
6.2 10 d HRT-C/N 6.56

6
0 10 20 30 40 50 60
Loading concentration (kg VS sample/m 3 )
Fig. 8. Observed process pH with substrate concentrations for different HRT and substrate C/N ratios for anaerobic digestion runs at 35 °C.

0.35
Methane yield (m CH4 /kg VS sample)

0.3

0.25

0.2
3

0.15

0.1

0.05

0
6 6.2 6.4 6.6 6.8 7 7.2 7.4
Measured pH
Fig. 9. Relationship between measured digester pH and specific methane yields for anaerobic digestion runs at 35 °C.
 E.A. Ehimen et al. / Applied Energy 88 (2011) 3454–3463
With an increase in the substrate C/N ratio, a reduction in the
ammonia 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 the
digestion bacteria where its toxicity will be expressed [27] was
anticipated in this study. It was expected that the acetoclastic
methanogenic bacteria would be the most sensitive to free ammo-
nia as reported by Angelidaki and Ahring [27]. However, the
observed ammonia nitrogen concentrations in this study (880–
4300 mg NH3–N/L digestate, for all investigated levels) appear
not to directly affect the process stability. For example, a nitrogen
ammonia 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 of
ammonia inhibition. The high VFA concentration in this study
could have promoted a slightly acidic digester environment, which
would largely aid the protonation of the free NH3 to the ionized
ammonium species NHþ 4 , by enhancing the shift of the chemical
equilibrium in Eq. (2) to the right.
NH3 þ Hþ $ NHþ4 ð2Þ
Hence, at the recorded pH levels, most of the ammonia nitrogen
in this study may be below the toxic concentration levels. McCarty
[28] determined that ammonia inhibition in the anaerobic digester
occurs with concentrations of 1.5–3.0 g N/L at a pH level of over
7.4.
In addition, the digester acclimatisation period, as well as the
inoculum and digestion temperature used in this study, might have
helped reduce the toxic effects of NH3. The ammonia nitrogen con-
centrations in this study was observed to fall within the broad
inhibiting concentration range of 1.7–14 g N/L discussed in [27]
when different process conditions which might influence the
ammonia inhibition are considered.
4. Conclusion
The results obtained in this paper have shown that it is feasible
to subject the microalgae residues, post transesterification, to the
anaerobic digestion process with the CH4 yields mainly influenced
by digestion times. The results obtained in this experimental study
were based on the use of post transesterified residues after the
in situ transesterification method using the freshwater microalgae
specie (Chlorella sp.).
Increases in the hydraulic retention period have been shown to
significantly affect the digestion process with periods longer than
5 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 anaerobic
digestion of microalgae residues has the potential to improve the
overall energy recovered as CH4. Increasing the substrate C/N ratio
to 12.44 by co-digesting the microalgae residues with glycerol was
observed to increase CH4 production by >50%, compared with the
CH4 production when the residues were digested alone.
The substrate loading concentration was also found to be as an
important parameter with lower digester loading rates favouring
higher 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 microalgae
biodiesel production.
3463
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 Novis
of Landcare Research for providing the Chlorella culture used in this
study and Mr. Muthasim Fahmy for his help with the process
modelling.
References
[1] Hill AM, Feinberg DA. Fuel from microalgae lipid products. SERI/SP-231-2348.
Solar Energy Research Institute, Golden, Colorado. US; 1984.
[2] Nagle N, Lemke P. Production of methyl ester fuels from microalgae. Appl
Biochem Biotechnol 1990;24(25):355–61.
[3] Miao X, Wu Q. Biodiesel production from heterotrophic microalgal oil.
Bioresour Technol 2006;97:841–6.
[4] Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25:294–306.
[5] Chisti Y. Biodiesel from microalgae beats bioethanol. Trends Biotechnol
2008;26:126–31.
[6] Ehimen EA, Sun ZF, Carrington CG. Variables affecting the in-situ
transesterification of microalgae lipids. Fuel 2010;89:677–84.
[7] Gao C, Zhai Y, Ding Y, Wu Q. Application of sweet sorghum for biodiesel
production by heterotrophic Chlorella protothecoides. Appl Energy
2010;87:756–61.
[8] Huang GH, Chen F, Wei D, Zhang XW, Chen G. Biodiesel production by
microalgal biotechnology. Appl Energy 2010;87:38–46.
[9] Dubinsky Z, Aaronson S, Berner T. Some economic considerations in the mass
culture of microalgae. In: Shelef G, Soeder CJ, editors. Algae biomass.
Amsterdam: Elsevier/North-Holland biomedical press; 1980. p. 819–32.
[10] Sialve B, Bernet N, Bernard O. Anaerobic digestion of microalgae as a necessary
step to make microalgal biodiesel sustainable. Biotechnol Adv
2009;27:409–16.
[11] Ehimen EA, Connaughton S, Sun Z, Carrington CG. Energy recovery from lipid
extracted transesterified and glycerol co-digested microalgae biomass. GCB
Bioenergy 2009;1:371–81.
[12] APHA. Standard methods for the examination of water and wastewater. 21st
ed. Washington DC, US: American Public Health Association; 2005.
[13] Guwy AJ. Equipment used for testing anaerobic biodegradability and activity.
Rev Environ Sci Biotechnol 2004;3:131–9.
[14] Box GEP, Hunter JS, Hunter WG. Statistics for experimenters. Design,
innovation and discovery, 2nd ed. New Jersey. US: Wiley Interscience; 2005.
[15] Callaghan FJ, Wase DAJ, Thayanithy K, Forster CF. Continuous co-digestion of
cattle slurry with fruit and vegetable wastes and chicken manure. Biomass
Bioenergy 2002;22:71–7.
[16] Varel VH, Isaac HR, Bryant MP. Thermophilic methane production from cattle
waste. Appl Environ Microbiol 1977;33:298–307.
[17] Siles López JA, MdlA MartinSantos, Chica Pérez AF, Martín Martín A. Anaerobic
digestion of glycerol derived from biodiesel manufacturing. Bioresour Technol
2009;100:5609–16.
[18] Khanal SK. Anaerobic biotechnology for bioenergy production: principles and
applications. Iowa, US: Wiley-Blackwell; 2008.
[19] Mendez-Acosta HO, Palacios-Ruiz B, Alcaraz-Gonzalez V, Steyer JP, Gonzalez-
Alvarez V, Latrille E. Robust control of volatile fatty acids in anaerobic
digestion processes. Ind Eng Chem Res 2008;47:7715–20.
[20] Hill DT, Cobb SA, Bolte JP. Using volatile fatty acid relationships to predict
anaerobic digester failure. Trans ASAE 1987;30:496–501.
[21] Nielsen HB, Uellendahl H, Ahring BK. Regulation and optimisation of the
biogas process: Propionate as a key parameter. Biomass Bioenergy
2007;31:820–30.
[22] Samson R, LeDuy A. Detailed study of anaerobic digestion of Spirulina maxima
algal biomass. Biotechnol Bioenergy 1986;28:1014–23.
[23] Switzenbaum MS, Giraldo-Gomez E, Hickey RF. Monitoring of the anaerobic
methane fermentation process. Enzyme Microbiol Technol 1990;12:722–30.
[24] Foree EG, McCarty PL. Anaerobic decomposition of algae. Environ Sci Technol
1970;4:842–9.
[25] Nordstedt RA, Thomas MV. Start-up characteristics of anaerobic fixed bed
reactors. Trans ASAE 1985;28:1242–7.
[26] Bolte JP, Hill DT, Woods TH. Anaerobic digestion of screened waste liquids in
suspended particle-attached growth reactors. Trans ASAE 1986;29:543–9.
[27] Angelidaki I, Ahring BK. Anaerobic thermophilic digestion of manure at
different ammonia loads. Appl. Microbiol Biotechnol 1993;38:560–4.
[28] McCarty PL. Anaerobic waste treatment fundamentals III. Toxic materials and
their control. Public Works 1964;95:91–4.
[29] Hu Q, Sommerfeld M, Jarvis E, Ghiradi M, Posewitz M, Siebert M, et al.
Microalgal triacylglycerols as feedstock for biofuel production: perspectives
and advances. Plant J 2009;54:621–39.
[30] Catton W, Carrington CG, Sun ZF. Performance assessment of contact heat
pump drying. Int J Energy Res; 2010. doi: 10.1002/er.1704.