Biomass and Bioenergy 127 (2019) 105266

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Biomass and Bioenergy

journal homepage: www.elsevier.com/locate/biombioe

Research paper

Optimization of biomethane production from anaerobic Co-digestion of T

microalgae and septic tank sludge
Dingnan Lua,∗, Xiao Liub, Onur G. Apulc, Lin Zhangd, David K. Ryane, Xiaoqi Zhang (Jackie)a
a
Department of Civil and Environmental Engineering, University of Massachusetts Lowell, One University Ave, Lowell, MA, 01854, USA
b
Department of Biological Sciences, University of Massachusetts Lowell, One University Ave, Lowell, MA, 01854, USA
c
Department of Civil and Environmental Engineering, University of Massachusetts Lowell, One University Av, Lowell, MA, 01854, USA
d
Department of Physical and Environmental Science, Texas A&M University-Corpus Christi, 6300 Ocean Drive, Corpus Christi, TX, 78412, USA
e
Chemistry Department, University of Massachusetts Lowell, 265 South Riverside St., Lowell, MA, 01854, USA

A R T I C LE I N FO A B S T R A C T

Keywords: The objective of this study was to analyze the effects of the total organic concertation and the proportion of
Anaerobic co-digestion microalgae to optimize biomethane production during anaerobic co-digestion of microalgae and septic tank
Biomethane production sludge. To achieve this goal, a statistical tool, central composite design, was applied to design the co-digestion
Microalgal biomass experiments. Using multiple variance analysis, the experimental data were analyzed, and the results were
Septic tank sludge
presented by the response surface methodology, which illustrated that peak methane production could be
Volatile fatty acids
Ammonia inhibition
achieved when the total volatile solids concentration was in the range of 16–20 g/L and the proportion of
microalgal biomass ranged from 26 to 47%. An inhibitory effect was observed from volatile fatty acids accu-
mulation when the total volatile solids concentration was supplied as high as 35 g/L. Organic nitrogen miner-
alization took place in all groups, ranging from 23 to 62%. There was no direct evidence showing that produced
ammonium ion nitrogen or the free ammonia nitrogen had inhibitive effects on methanogenic activity.

1. Introduction Several researchers have used different waste materials, such as

To effectively extract energy from microalgal biomass, many dif-
ferent conversion methods have been studied, such as anaerobic di-
gestion, ethanol fermentation, lipid extraction, and anaerobic microbial
fuel cell [1]. Among these methods, anaerobic digestion of microalgae
is one of the most effective methods with relatively higher maturity
[2–4]. However, challenges due to the intrinsic properties of microalgae
must be solved before they become a mainstream feedstock for reusable
energy production. The main adverse intrinsic property associated with
the anaerobic digestion of microalgae is the high proportion of proteins
which can result in low C/N ratios in a digester [4–6]. The typical C/N
ratio varies from 4.2:1 to 7.8:1 for microalgal species, which have been
utilized for anaerobic digestion [7]. Microalgal feedstocks with low C/N
ratios (less than 20:1) could result in high TAN release in anaerobic
digesters and potentially inhibit the methanogenic bacterial activity [6]
To overcome this limitation, anaerobic co-digestion may be applied.
This strategy is to co-digest the microalgal biomass with other carbon-
rich waste, hence balancing the C/N ratio and decreasing ammonia
release.
waste paper, sewage sludge, and activated sludge, to perform anaerobic
co-digestion to increase the C/N ratio, aiming to balance the carbon and
nitrogen requirements for the bacterial community and maintain a fa-
vorable ammonia concentration in a digester [6,8,9]. Samson and
LeDuy [10] performed anaerobic co-digestion which combined sewage
sludge with Spirulina maxima and found a 2-fold increase in the biogas
production. Thorin et al. [11] undertook a semi-continuous co-diges-
tion of microalgae and sewage sludge under thermophilic conditions.
They reported that increased methane production can be sufficient for
covering the required heat for heating the substrates. González-Fer-
nández et al. [12] investigated the addition of microalgal biomass to pig
manure and observed that methane production was taking place to a
higher extent in samples with a higher proportion of microalgae. Park
and Li [13] studied anaerobic co-digestion of Nannochloropsis salina and
lipid-rich waste fats, oil, and grease, and observed much higher me-
thane production. They also found the anaerobic co-digestion could
bear high organic loading due to a more balanced C/N ratio with less
potential ammonia inhibition and digester imbalance. Wang et al. [9]
reported better methane production in the co-digestion of waste

∗
Corresponding author.
E-mail addresses: Dingnan_lu@student.uml.edu (D. Lu), xiao_liu@student.uml.edu (X. Liu), onur_apul@uml.edu (O.G. Apul), lin.zhang@tamucc.edu (L. Zhang),
David_Ryan@uml.edu (D.K. Ryan), Jackie_zhang@uml.edu (X. Zhang).

https://doi.org/10.1016/j.biombioe.2019.105266
Received 18 September 2018; Received in revised form 23 May 2019; Accepted 5 June 2019
0961-9534/ © 2019 Elsevier Ltd. All rights reserved.
D. Lu, et al. Biomass and Bioenergy 127 (2019) 105266

Nomenclature R2 coefficients of variance

RSM response surface methodology
AIN ammonium ion nitrogen TAN total ammonia nitrogen
BBM Bold Basal medium TCOD total chemical oxygen demand
CCD Central composite design TKN total kjeldahl nitrogen
C/N ratio carbon to nitrogen ratio TOC total organic carbon
FAN free ammonia nitrogen VS volatile solids
pKa dissociation constant VFAS volatile fatty acids

activated sludge with Chlorella sp. They observed that with the addition
of activated sludge, not only the net biogas yields improved but the
biogas production rate was accelerated significantly. All the examples
listed above demonstrate advantages in methane production when
performing anaerobic co-digestion of microalgae with a carbon-rich
waste co-substrate.
The feasibility of using septic tank sludge as a co-substrate to digest
with microalgae has been proven [14]. The exceptional performance of
septic tank sludge can be attributed to its high concentration of organic
carbon (∼10,000 mg/L) and the presence of important hydrolytic en-
zymes and bacteria [15]. Particularly, the addition of septic tank sludge
to microalgae can result in a more favorable C/N ratio (20:1-30:1) to
balance the high protein content of microalgal biomass. Besides, the
introduced enzymatic activities from septic tank sludge can sig-
nificantly accelerate hydrolysis of microalgal biomass, which resulted
in a higher glucose release rate (0.14 mg/L/min) in the early anaerobic
digestion period [14]. Many anaerobic bacteria, such as Clostridium
acetobutylicum, Clostridium cellulolyticum, and Acetivibrio cellulolyticus,
have been proven to actively hydrolyze cellulose. And these re-
presentative anaerobic bacteria can be easily found in soil, sewage, and Fig. 1. Microscopic image of the harvested Chlorella sp. (1000× magnifica-
animal feces [16] which are the potential components of the septic tank tion).
sludge. It is reasonable to believe that the cellulolytic activities from the
septic tank sludge is fundamentally triggered by the presence of these 2. Methods
anaerobic bacteria. Furthermore, methanogens and various bacterial
species involved in the anaerobic degradation of organic matter, in- 2.1. Raw materials
cluding hydrolytic, fermentative, acidogenic, acetogenic, homo-
acetogenic and sulfate-reducing bacteria should also be present [17]. The microalgae strain was isolated from the primary clarifiers at the
Although the co-digestion of microalgae and septic tank sludge has Lowell Regional Wastewater Utility (LRWU) (Lowell, MA). The ob-
been proven to outperform the biogas production from the digestion of tained green spherical-shaped microalgae, identified as Chlorella sp.
microalgae alone, there is scarce biochemical information available (see Fig. 1), was then cultivated using the modified BBM in the study.
regarding the co-digestion of microalgae and septic tank sludge. Spe- The detailed procedures of microalgae isolation, identification, and
cifically, it is not clear how the organic loading and the split between cultivation were described in our previous works [14,19]. The micro-
the co-substrates will affect methane production when conducting the algal stock was prepared by centrifugation at 3000 rpm for 10 min
anaerobic co-digestion of microalgae and septic tank sludge. followed by washing with distilled water. The centrifugation-washing
The main objective of this study was to investigate the effects of two was repeated three times to ensure the microalgae culture was free from
variables, namely the VS concentration (g/L) and the percentage of the chemical residues of modified BBM. The prepared microalgae stock
microalgal biomass, on the anaerobic co-digestion of microalgae and was kept suspended in tap water and stored in sealed beakers at 4 °C.
septic tank sludge. Aiming to study both parameters simultaneously, Septic tank sludge was obtained directly from a hauling truck at the
CCD followed by RSM was performed. CCD is a useful statistical tool to LRWU and transported immediately to the environmental laboratory at
arrange experiment groups when multiple factors were studied in a the University of Massachusetts Lowell. The septic tank sludge was
limited number of runs, and RSM can help visualize the statistical data collected from residential septic tanks near the city of Lowell, MA.
that was generated by the experimental arrangement from CCD [18]. The microalgae stock and septic tank sludge were characterized by
There are no commonly agreed methods to design and conduct anae- measuring pH, VS, TCOD, TKN, and TOC (see Table 1). The con-
robic co-digestion studies, which adds difficulty to compare different centration ratio of TOC and TKN were used to calculate the initial C/N
co-digestion studies. Therefore, this study can be served as an example ratio.
to help researchers design other anaerobic co-digestion studies, which
might involve two or more co-substrates. Moreover, the concentrations
2.2. Mesophilic anaerobic experiment
of VFAs and ammonia were monitored to help understand the me-
chanisms during the anaerobic co-digestion of microalgae and septic
The selected variables for the study were the substrate concentra-
tank sludge.
tion measured in terms of VS (g/L) and the proportion of microalgal
biomass added in terms of percentage of volume (%). The initial sub-
strate concentration range was arranged in accordance with Ward et al.
[7], who reported a wide range of initial VS concentrations from dif-
ferent studies performed anaerobic digestion of Chlorella sp. On the
other hand, the selected range for proportions of microalgal biomass

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D. Lu, et al. Biomass and Bioenergy 127 (2019) 105266

Table 1 production, measured as the volume of methane produced per gram of

Initial characterizations of microalgae stock and septic tank sludge. VS added. The factors, Xi, were coded as xi according to Eq. (2):
Microalgae stock Septic tank sludge Xi − Xi∗
xi = ; where i = 1,2,3, …, k
TCOD (g/L) 20.6 ± 1.0 23.7 ± 1.3 ΔXi (2)
VS (g/L) 15.1 ± 1.1 15.3 ± 0.7
where xi is the dimensionless coded value of a factor, Xi is the actual
TOC (g/L) 13.5 ± 1.9 14.5 ± 2.3
TKN (mg/L) 2300 ± 210 400 ± 56 value of a factor for the ith test, Xi∗ is the actual value of a factor at the
NH3–N (mg/L) 99 ± 50 366 ± 37 central point and ΔXi is the actual step change of a factor [18]. CCD in
Organic-N (mg/L)a 2201 ± 194 34 ± 3 this study played a role as the experimental guideline during the pro-
C/N ratiob 5.9 ± 0.3 36.2 ± 2.4
cedure of feedstocks preparation and anaerobic co-digestion (see
pH 7.61 6.59
Fig. 2), and the actual factor values and their coded values are listed in
a
Calcualted by TKN - NH3–N. Table 2.
b
Calculated by TOC/TKN; triplicate measurements were conducted; Data
shown as mean ± SD. 2.4. RSM and statistical analyses

was 0–100% for the co-digestion experiment. In order to prepare a
desirable VS concentration and microalgae proportion in a digester, the
raw feedstocks of the microalgae slurry and the septic tank sludge had
gone through three sequential processes: 1. Prepare both raw feedstocks
into the same VS concentration (i.e., 15 g/L) by centrifugation; 2. Dis-
card supernatant (or fill additional distilled water) in proportion to a VS
difference between a centrifuged raw feedstocks and a desirable level;
3. Shake centrifuge tubes to resuspend settled sludge and pour prepared
microalgae slurry and septic tank sludge into a digester based on vo-
lume ratio to meet a request for specific microalgae proportion. As an
example, take the digesters consisting 30 g/L of VS and 85% of mi-
croalgae. The raw feedstocks were first centrifuged to obtain the VS
concentration of 15 g/L. Then the clean supernatant was carefully dis-
carded from centrifuge tubes by half of the total volume to reach the
desirable VS concentration (i.e., 30 g/L). Lastly, 85 mL of microalgae
slurry and 15 mL of septic tank sludge (both had been shaken to re-
suspend) were added into the serum bottles to have these digesters
consisting 30 g/L of VS and 85% of microalgae.
The anaerobic co-digestion was carried out in a series of 200 mL
serum bottles with a liquid mixture volume of 110 mL and a headspace
of 90 mL for the gas. The serum bottles were sealed by rubber stoppers
and secured with aluminum foil to keep them airtight. After the set-up
of each reactor, the headspace was flushed with nitrogen for 15 min to
remove residual oxygen in the headspace. Anaerobic digestion was
performed by placing the serum bottles in a reciprocal shaker bath
(VWR™ model 1227) and run at 120 oscillations per minute at 35 °C for
30 days. The volume of produced biogas was calculated by measuring
the pressure in the bottle's headspace. The experimental design is
shown in Table 2. All the reactors were carried out in triplicate.
Anaerobic inoculum consisted of digested sludge from a mesophilic
anaerobic reactor (New Brunswick Scientific Co., Inc.) fed with artifi-
cial wastewater [20] under semi-continuous operation. Prior to the co-
digestion experiment, the inoculum (VS at 15 g/L) had been degassed at
35 °C for one week. Observation indicated no significant methane (0.06
mL/gVS) being produced after the one-week incubation.
RSM was performed for the result optimization of the CCD analysis.
RSM is a collection of statistical and mathematical techniques which
can visualize a response influenced by several variables [21]. This
method uses a second-order polynomial to describe the response, see
Eq. (3):
Y = β0 + β1 X1 + β2 X2 + β11 X12 + β22 X22 + β12 X1 X2 + E (3)
where Y represents the predicted response, β0 , β1, β2 , β11, β22 , and β12 are
the regression coefficients. X1 and X2 are the factors' coded values. E is
the standard error.
The CV was calculated as the ratio of the standard error of the es-
timate to the mean value of the observed response. The R2 was calcu-
lated to evaluate the proportion of data variability which is predicted
by the second-order polynomial model. The p-value of each regression
coefficient was calculated to validate the model. Excel v.2016 and
STATISTICA v.10 were used to perform the multiple regression and
graphical analyses.
2.5. Ammonia (NH3) and ammonium (NH4+) speciation analysis
In order to quantify the proportions of molecular ammonia and
ammonium ion, pKa for ammonium ion at specific was calculated using
the following equation [22]:
2729.92
pK a = 0.09018 +
T + 273.15 (4)
o
where T is the temperature of culture solution ( C). The ammonia re-
moval rate was determined using the pKa calculated by Eq. (4), and the
proportional relation between FAN (mg/L) and TAN (mg/L) can be
expressed as the following:
FAN 1
=
TAN 1 + 10 (pK a− pH ) (5)
where pKa is the negative logarithm of the dissociation constant of
8.95 at 35 °C (calculated by Eq. (4)), and pH is the negative logarithm of

2.3. CCD and statistical analyses Table 2

Arrangement of CCD for the two factors used in this study.
The central composite design is a second order factorial design
Category Reactor no. Coded values Actual values
employed when multiple variables are studied and the number of runs
for a full factorial design is too large to be performed [18]. This fac- Initial VS Microalgae Initial Microalgae (%)
torial design can consist of a 2k factorial nucleus and 2*k axial points, VS (g/L)
where k is the number of factors evaluated (in this study k was 2). All
Central point R0 0 0 20 50
the evaluated levels of the two factors were codified from −1 to +1, Factorial R1 −1 −1 10 15
and the central point was codified as the point of (0, 0). The axial points nucleus R2 −1 1 10 85
and their distance to the central point (α) are calculated as Eq. (1): R3 1 −1 30 15
R4 1 1 30 85
α = 2k/4 (1) Axial points R5 0 −1.4142 20 0
R6 0 1.4142 20 100
The α value in this study was 1.414, calculated by using Eq. (1),
R7 −1.4142 0 5 50
when the k value was equal to 2. R8 1.4142 0 35 50
The selected response for this CCD analysis was the methane

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D. Lu, et al. Biomass and Bioenergy 127 (2019) 105266

Fig. 2. Schematic diagram of the experimental procedures.

the hydrogen ion activity on the selected day. Therefore, Eq. (5) can be specifically, both the first-order coefficients (β1 and β2) of x1 (i.e., VS
rewritten by inputting the calculated pKa value at 35 °C, then the final concentration) and x2 (i.e., microalgal proportion) and the second-
equations for FAN and AIN can be expressed as the following: order coefficients (β11 and β22) of x12 and x 22 . The β1 and β2 had close
negative values, which means that both x1 and x2 had negative linear
FAN 1
= correlations with Y. In practical terms, with a fixed microalgae pro-
TAN 1 + 10 (8.95 − pH ) (6)
portion then an increase in VS loading will lead to a decrease in biogas
AIN 10 (8.95 − pH ) production. Similarly, with a static supply of VS loading, biogas pro-
=
TAN 1 + 10 (8.95 − pH ) (7) duction will decrease when increasing in microalgae proportion. The
second-order coefficients β11 and β12 also had negative values, indicated
From above Eq. (6) and Eq. (7), it can be found when a digester is
the parabolas open downwards in their respective two-dimensional
running at a constant temperature, the ammonia molecular and am-
surface. Since this quadratic function inputs, x1 and x2, were an ordered
monia ion speciation totally depends on the pH.
pair of real numbers and a function's curve is the collection of all or-
dered triples (x1, x2 , f (x1, x2) ) , the resulted function was a three-di-
2.6. Analytical techniques
mensional surface with its mouth opening downwards. In other words,
the vertex had the highest methane production on the three-dimen-
VS was measured in accordance with the Standard Methods [23].
sional plot. The magnitude of a second-order coefficient directly de-
The TCOD and TAN were carried out by using a Hach DR/2000 spec-
termines the shape of a parabola. In other words, it is able to indicate
trophotometer (Hach Inc., Loveland, CO, USA). The TKN assay was
the sensitivity of methane production affected by both variables, which
done by using Hach DR/3900 spectrophotometer (Hach Inc., Loveland,
will be discussed in the next two paragraphs. Lastly, the coefficient of
CO, USA) coupled with a DRB 200 block digester (Hach Inc., Loveland,
x1x2 (β12) had the p-value greater than 0.05, which implied the inter-
CO, USA). The TOC measurement was performed by using an Ionics
action effect between the two selected variables were relatively insig-
model 1555B carbon analyzer (GE Analytical Instruments, Boulder, CO,
nificant to the result of methane production.
USA). The biogas pressure in the bottle's headspace was measured every
For the graphical interpretation of the interactions, the use of a
other day with a digital pressure meter Cole Parmer 500PSI (Chicago,
three-dimensional plot of the regression model is highly useful [25]. In
IL), and the biogas composition (CH4 and CO2) was evaluated si-
this study, the two variables x1 and x2 and the result of y (i.e., methane
multaneously by using a gas chromatograph: SRI Model 8610 equipped
production) were used as the axes of the surface response plot (see
with a flame ionization detector (FID) for CH4 and CO2. The con-
Fig. 3A). The predicted values for the response of methane production
centration of VFAs, including formate, acetate, propionate, and buty-
are listed in Table 3. The three-dimensional shape of the response
rate, was determined by using a Dionex ion chromatography (IC)
surface was a hump, indicated a peak methane production had occurred
system equipped with an ED50 electrochemical detector.
inside the designed CCD boundary. A contour plot, made by projecting
the three-dimensional plot on the plane of x1−x2, was used to depict
3. Results and discussion
the range of the correlated x1 and x2 values, in which the resulted
methane production was higher than a specific value, such as, 180, 210,
3.1. Statistical and graphical analyses
240, 270, 300, and 330 mL/gVSfed (see Fig. 3B). Therefore, the most

The results of methane production (mL/gVSfed) are presented in

Table 3. The following second-order polynomial equation, Eq. (4), is the Table 3
Coded values and the response of methane production for the anaerobic co-
regression model obtained between the coded values and methane
digestion of microalgae and septic tank sludge.
production:
Reactor no. Variables Results
YCH 4 = 327.3 − 21.3x1 − 22.6x2 − 73.1x12 − 28.3x 22 − 1.9x1 x2 (8)
Initial VS Microalgae (%) Actual YSM Predicted YSM (mL
The results of the statistical analysis are shown in Table 4. This (g/L) (mL CH4/ CH4/VSfed)
model was statistically significant as proved by the F-test, since the F VSfed)
value of 1.04 was much less than 3.44, the Fcritical value at the 0.05
alpha level. Furthermore, the R2 was 0.96, meaning only 4% of the R0 20 50 327.3 (37.5) 327.1
R1 10 15 267.5 (31.9) 267.6
conditions can't be explained by this regression model. The smaller R2 10 85 239.7 (24.1) 226.5
adjusted R2 value of 0.89 was mainly due to the limited number of R3 30 15 233.4 (36.7) 228.7
regressors, however, it was still a satisfactory fit of the quadratic model R4 30 85 198.0 (16.4) 180.0
to the experimental data. The CV value of 5.14% was much less than R5 20 0 302.4 (41.3) 302.0
R6 20 100 220.1 (29.8) 238.5
10% indicating this model is reasonably reproducible [24].
R7 5 50 205.8 (12.5) 211.3
The specific coefficients of Eq. (4) and the corresponding p-values R8 35 50 138.7 (24.6) 151.0
are listed in Table 4. From the five calculated coefficients,
β1, β2 , β11, β22 , and β12 , four of them had p-values less than 0.05, Data are presented as mean of triplicate. SD is shown in brackets.

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D. Lu, et al. Biomass and Bioenergy 127 (2019) 105266

Table 4
Statistical analysis of the evaluated factors (Initial VS and microalgae %) re-
garding the response of methane production.
Analysis of variance (ANOVA)

Variable Value

R2 0.96
Adj. R2 0.89
F 1.04
Fcritical 3.44
Coef. of variance (%) 5.14

Coefficient probability
Coefficient p-value*

β1 −21.3 < 0.001

β2 −22.6 0.048
β11 −73.1 0.041
β22 −28.4 0.007
β12 −1.9 0.851

∗
p-value < 0.05 is statistically significant at 95% level of confidence.

Fig. 4. (A) Response profile for methane production when X2 = 0 (50% of

microalgae); (B) Response profile for methane production when X1 = 0 (20 mg/
L of VS concentration) (Gray areas highlight the span of the methane produc-
tion responding to the change of X1 and X2 coded values).

stretching from −0.35 to 0.00 in the x1 direction. Based on this range of

Fig. 3. (A) Response surface plot for methane production, (B) Projection of the
peak methane production area on the two-dimensional surface.
internal ellipse, where the predicated methane production should be
greater than 330 mL/gVSfed, had its major axis stretching from ap-
proximately −0.7 to −0.1 in the x2 direction and the minor axis
coded value, the real range of VS concentration and microalgal pro-
portion could be calculated by using Eq. (2). The calculated results
showed a peak methane production can be achieved when the VS
concentration is between 16 and 20 g/L and the microalgae proportion
is between 26 and 47%. Among all the experimental groups, group R0
had the closest methane production to the theoretical peak production
of 333 mL/gVSfed.
As shown in Fig. 3A, the three-dimensional plot had different de-
grees of curvature in the x1−y and x2−y surfaces. The curvature of a
response surface depends on the coefficient of the squared term in a
quadratic polynomial equation [26]. From the response surface plot, it
can be observed that the degree of curvature, determined by x12 , was
higher than the x 22 , revealing that the varying VS concentration had a
greater effect on the response surface than the factor of microalgae
proportion. By comparing the absolute value of β11 (73.1) with β22
(28.4), the fitted model again revealed a higher sensitivity of methane
production affected by the VS concentration.
In Fig. 4A, when the microalgal proportion was at a constant level of
50%, the span of the methane production is from 195 to 329 mL/gVSfed
on its left-hand side, and from 131 to 329 mL/gVSfed on its right-hand
side. This behavior could be related to insufficient VS loading (left) and
the inhibition of VFAs due to organic overloading (right), as discussed
in the next section. A similar tendency can be found in Fig. 4B, but the
methane production response was much less sensitive when the VS
concentration was constant at 20 g/L. However, it can be found that,
when digesting septic tank sludge alone (left-hand side), an anaerobic
digester is still able to achieve a relatively higher methane production
of 303 mL/gVSfed, compared to the digestion of microalgae alone which
could only reach 230 mL/gVSfed (right-hand side). This tendency
clearly demonstrated, without conducting any pretreatment, that the
septic tank sludge showed a better potential for methane production
than the microalgal biomass, which was highly consistent with the re-
sult reported by Lu and Zhang [14].

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D. Lu, et al. Biomass and Bioenergy 127 (2019) 105266

3.2. Methane production and VFAs concentration group R7 due to lower initial organic loading.

As Table 3 shows, the methane production of 327.3 ± 37.5 mL/

gVSfed was observed from group R0 (VS20, ALG50%), which had the 3.3. Fate of organic nitrogen
initial organic concentration of VS 20 g/L and was supplied with 50%
microalgae and 50% septic tank sludge. This value was similar to the To study organic nitrogen mineralization during the anaerobic co-
results obtained by González-Fernández et al. [12] who achieved an digestion of microalgae and septic tank sludge, the concentrations of
average methane production of 314.6 mL/gVSfed by the co-digestion of organic nitrogen and TAN were analyzed at the beginning and end of
algae and swine manure, and Wang et al. [9] who accomplished the the experiment (see Fig. 6).
highest methane production of 295.8 mL/gVSfed by the co-digestion of The organic nitrogen reduction was observed in all the experimental
50% microalgae and 50% waste activated sludge (measured by VS) for groups (Fig. 6A). The degrees of organic nitrogen reduction ranged
45 days. from 43 to 62% for all the reactors mixed with microalgae (i.e. R0, R1,
In Fig. 5A, it clearly showed that group R0 (VS20, ALG50%) and R2, R3, R4, R6, R7, and R8). Among the above groups, R6 (VS20,
group R5 (VS20, ALG0%) had the highest methane production. Based ALG100%) had the lowest organic nitrogen reduction efficiency of 43%.
on their overlap in standard deviation, the differences on the methane This result is most likely due to the strong cell wall resistance of mi-
production between R0 and R5 were not significant. Similar results croalgae which reduced the efficiency of microalgae decomposition by
were found by Lu and Zhang [14] who reported that septic tank sludge
itself had a strong capability to produce methane. As expected, R6
(VS20, ALG100%) had the lowest methane production (both actual and
predicted, see Table 3) among these three conditions. According to
Takeda [27] and Takeda [28], typical microalgae, like Chlorella vulgaris
and Scenedesmus obliquus, possess hemi-cellulosic cell walls which have
a high resistance to microbial degradation. Therefore, the primary ob-
stacle of anaerobic digestion of microalgae alone is the resistance to the
breakdown of the cell wall. Another potential reason for the higher
methane production in group R0 is that it had a more favorable C/N
ratio of 21:1. According to Sialve et al. [5], when the C/N ratio is below
20 there is an imbalance between the carbon and nitrogen requirements
for the anaerobic microbial community, which can reduce the perfor-
mance of anaerobic digestion.
For the three groups (R0, R5, and R6) started with 20gVS/L, a peak
concentration of VFAs was detected in each group during the digestion
period (see Fig. 5B). Comparing the peak VFAs concentrations in these
three groups, group R0 had the highest peak concentration of 32.42 mM
on day 8, followed by 15.5 mM on day 6 in group R5, and then 12.5 mM
on day 16 in group R6. Examining the VFAs profile obtained from group
R5, it appears that the easily digestible organic matter from the septic
tank sludge was hydrolyzed and converted to VFAs quicker than the
other two groups. Due to the strong cell wall's resistance, the peak VFAs
concentration occurred later in group R6 than that in groups R0 and R5.
The VFAs profiles of groups R6(VS20, ALG100%) and R0(VS20,
ALG50%) clearly demonstrated the decomposition of the recalcitrant
microalgae cell wall was significantly enhanced by the addition of
septic tank sludge. The result illustrates that septic tank sludge can
enhance the digestibility of pure microalgal biomass.
On the other hand, when comparing the VFAs concentrations in the
groups with the same microalgae composition (50%), but different VS
concentrations (VS20, VS5, and VS35), group R8(VS35, ALG50%) fed
with the highest initial VS concentration showed an over-accumulation
of VFAs and the lowest methane production of 138.7 mL/gVSfed (see
Fig. 5C and D). Unlike the other groups, the VFAs in group R8 stayed at
a level of approximately 35.0 mM after reaching a peak concentration
of 54.1 mM at day 10. Siegert and Banks [29] evaluated the inhibitory
effects of high VFAs concentration during mesophilic anaerobic diges-
tion of cellulose and glucose. It was reported that VFAs could inhibit the
cellulolytic activity at concentrations above 27.0 mM and the metha-
nogenic activity at concentrations above 54.0 mM. The above two cri-
tical concentrations were both reached in groups R0 at day 6 (28.4 mM)
and R8 (54.1 mM) at day 10. Since the VFAs concentration in group R0
slightly exceeded the first reported threshold only for a short period, it
didn't affect the group's final methane production. It appears the me-
thanogenic activity in group R8 (VS35, ALG50%) was inhibited as in- Fig. 5. (A) Accumulated methane production and (B) VFA concentrations
dicated by the poor methane production and high VFAs accumulation during the experiment for groups R0(VS20 ALG50%), R5(VS20 ALG0%), and
in the system. Group R7 (VS5, ALG50%) had similar methane produc- R6(VS20 ALG100%); (C) Accumulated methane production and (D) VFA con-
tion to group R0 until day 10, but there is very little VFAs accumulation centrations during the experiment for groups R0(VS20 ALG50%), R7(VS5
suggesting very rapid hydrolysis and conversion to methane occurred in ALG50%), and R8(VS35 ALG50%). Data are expressed as mean ± SD, n = 3.

6
D. Lu, et al.
Fig. 6. (A) Concentration of organic nitrogen and (B) total ammonia nitrogen
(mg/L) at the beginning (white column) and end (gray column) of the 30-day
co-digestion period with initial (triangle) and final (square) pH for R0(VS20
ALG50%), R1(VS10 ALG15%), R2(VS10 ALG85%), R3(VS30 ALG15%),
R4(VS30 ALG85%), R5(VS20 ALG0%), R6(VS20 ALG100%), R7(VS5 ALG50%),
and R8(VS35 ALG50%).
anaerobic bacteria. A similar tendency was found by González-Fer-
nández et al. [12] who observed a rate of nitrogen mineralization in
anaerobic digestion was significantly reduced with the addition of
algae. In group R0 (VS20, ALG50%), we observed the highest organic
nitrogen reduction of 62%. Together with the higher methane produced
in group R0, the results demonstrated higher protein mineralization
from the microalgal biomass.
On the other hand, the group with the septic tank sludge alone i.e.,
R5 (VS20, ALG0%) had only 23% reduction in organic nitrogen (initial
organic nitrogen was 109 ± 20 mg/L). Comparing the lowest initial
organic nitrogen in R5 with R6 (VS20, ALG100%) (2569 ± 147 mg/L),
it can conclude that the majority portion of organic nitrogen in this
study was contributed from microalgal biomass. A calculated reduction
rate of 42% was obtained by averaging the initial and final organic
nitrogen concentrations in groups R5 and R6 (see Fig. 7). As expected,
the experimental group R0 (VS20, ALG50%) with an organic nitrogen
reduction of 62%, surpassing the mathematically calculated reduction
of 42%, again demonstrating the benefits of using the septic tank sludge
to enhance the microalgae decomposition.
3.4. Interactions between temperature, pH, and inorganic nitrogen
speciation
AIN and FAN are the two principal forms of inorganic ammonia
nitrogen in aqueous solution, and FAN has been suggested to be the
major cause of inhibition since it is freely membrane-permeable
[30,31]. The hydrophobic FAN molecules may diffuse passively into
cells, causing a proton imbalance and/or potassium deficiency [32–34].
To determine the proportions of AIN and FAN in the digester, NH3 and
NH4+ speciation analysis was done by using Eq. (4), Eq. (6) and Eq. (7).
From the above equations, it is worth noting that temperature and pH
are the two fundamental environmental factors that govern the spe-
ciation process of NH3 and NH4+ [35]. To suppress the formation of
FAN and reduce the impact of FAN inhibition, interactions between
temperature, pH, and FAN speciation were estimated and compared
mathematically.
The temperature of this anaerobic digestion study was pre-
determined to be 35 °C. Comparing to thermophilic conditions, this
mesophilic temperature might result in a relatively lower kinetic
7
Biomass and Bioenergy 127 (2019) 105266
advantage. Kim et al. [36] studied the effects of different temperatures
on anaerobic digestion of food waste and found the first order kinetic
constant was higher at 50 °C (0.61 s−1) than at 35 °C (0.35 s−1).
However, thermophilic digestion temperature can cause a shift of am-
monia speciation and favor the formation of FAN. For example, using
the pKa values of 8.95 at 35 °C and 8.41 at 55 °C as calculated by Eq. (4)
and plugging these values into Eq. (6), we will get a much higher FAN
concentration at 55 °C (three-to four-fold higher than that at 35 °C),
which can significantly increase the risk of ammonia inhibition. Simi-
larly, Angelidaki and Ahring [37] particularly studied the effects of
different temperatures and ammonia loads and observed a poor process
performance at high temperature and high ammonia conditions. Based
on the final TAN concentrations (see Fig. 6B) and the mesophilic op-
eration temperature, it was found that AIN was the dominant species in
all the experimental groups. Specifically, group R8 had the highest final
TAN concentration of 2326 mg/L, a final pH of 6.15, and FAN and AIN
concentration ratio of 1:512, which could only result in a small amount
of FAN concentration of about 4.5 mg/L theoretically at 35 °C.
As mentioned in Section 2.5, at a constant temperature, the con-
centration of FAN highly depends on the pH. An increase in pH would
result in a higher FAN/AIN ratio due to the shift in the speciation of
NH3 and NH4+, thereby increasing the inhibition on methanogenesis.
And an inhibited methanogenesis would result in an accumulation of
VFAs in a digester and further affect the pH value. For example, in
group R8 its pH was likely pulled down by an over-accumulation of
VFAs (see Fig. 5D) and thereby constraining its FAN concentration at a
relatively lower level. This interesting interaction among FAN, pH, and
VFAs was also observed in other studies. Chen et al. [31] used the term
“inhibited steady state” to describe this situation where the process is
running stably but with low methane production. Angelidaki and
Ahring [38] undertook anaerobic digestion of livestock waste and ob-
served stable digestion of cattle manure at an extremely high TAN
concentration of 6000 mg/L, but with low methane production and a
high VFAs concentration.
3.5. Practical implications and future work
Several practical implications can be drawn based on the observed
results. 1: An anaerobic co-digestion study can be successfully designed
by CCD, and then its results can be simply visualized via RSM. 2:
Microalgae are not suitable for anaerobic single-substrate digestion due
to their high nitrogen content and cell wall recalcitrance, but with the
addition of septic tank sludge both issues can be remedied. 3: An
anaerobic digester is relatively resistant to acidification due to the ad-
dition of microalgal biomass, however, organic over-loading can result
in an inhibited steady state to take place and cause low methane pro-
duction.
Fig. 7. Concentration of organic nitrogen (mg/L) at the beginning (whit
column) and end (gray column) of the experiment, and reduction (%) for
R0(VS20 ALG50%), R5(VS20 ALG0%), calculated average (R5 and R6), and
R6(VS20 ALG100%).
D. Lu, et al.
Anaerobic co-digestion is a pragmatic method to resolve many dif-
ficulties in a single-substrate digestion process. However, with the in-
creased use of anaerobic co-digestion, the complexity of the process
also increases. Particularly, how to select suitable co-substrates that
have complementary characteristics and design an optimized process
are still a challenging task. The CCD combined with RSM used in this
study demonstrated that it will allow researchers to determine the
suitability of selected co-substrates and their optimal combination ratio.
Microalgae, typically rich in proteins and consequently have a high
nitrogen level, are not a suitable substrate for single-substrate digestion.
In addition, the recalcitrant cellulosic cell wall [39] further increases
the difficulty of anaerobic digestion. This study showed that the septic
tank sludge with a high total organic carbon concentration can balance
the low C/N ratio from the microalgae. Furthermore, the highly accli-
mated hydrolytic bacteria coming from the septic tank sludge can ef-
fectively assist in breaking the microalgae cell wall. Based on the C/N
ratios of the microalgae and the septic tank sludge found in this study
(6:1 and 36:1), we recommend an optimal co-digestion process when
the VS ratio of the microalgae and septic tank sludge is between 1:3 to
1:2.
Usually, the digester will go “sour” if the system pH drops below 6.8
due to an overaccumulation of VFAs [29]. However, with the addition
of microalgae, the co-digestion process is less likely to turn “sour” due
to the release of TAN. From our observation, the co-digestion process,
especially the methanogenesis, would suffer from a “mild” inhibition by
the VFAs accumulation when the organic loading was above 35 gVS/L
in group R8 (VS35, ALG50%). Similarly, the RSM results showed the
highest methane production happened when the VS concentration was
in the range of 16–20 g/L (see Fig. 3B). Therefore, it is necessary to set
an upper limit to the organic loading to maintain the methanogenic
activity and high methane production.
Unlike the sewage sludge, the detailed composition information of
the septic tank sludge is scarce. Robidoux et al. [40] evaluated different
toxic substances in the septic tank sludge received at a municipal
wastewater treatment plant and suggested that copper and lead are the
most concerned substances because of their high possibility of a pre-
sence in the septic tank sludge. We believe it is important to analyze
these heavy metals in the future study since they could have adverse
effects on the anaerobic digestion. Especially copper which is highly
toxic to the VFAs-degrading acetogens [41]. As mentioned above, due
to the concern for the potential inhibition from ammonia, this anae-
robic co-digestion study was performed at a relatively low temperature.
However, it would be of interest to find out the most suitable co-di-
gestion temperature, which may be higher than what was used in the
current study, to further enhance the efficiency of the anaerobic di-
gestion.
4. Conclusions
The anaerobic co-digestion of microalgae and septic tank sludge was
studied by factorial design of experiments. Two selected factors in-
cluding organic concentrations and microalgal proportions were proved
to have significant effects on methane production. The results of the
statistical analysis, such as F-test (1.04), correlation coefficient (0.96),
and coefficients of variance (5.14%), all demonstrated that methane
production had a strong dependence on the two studied factors. The
surface response plot showed the peak methane production, greater
than 300 mL/gVSfed, could be achieved when the total volatile solids
concentration was in the range of 16–20 g/L and the proportion of
microalgal biomass ranged from 26 to 47%. The inhibitive effect of the
volatile fatty acids was observed when the co-digestion started with 35
gVS/L, and no ammonia inhibition was observed in this study.
Informed consent
No conflicts, informed consent, human or animal rights applicable.
8
Biomass and Bioenergy 127 (2019) 105266
Author contributions
DL, XZ, LZ, and DR Conception and design; DL and XL Collection
and assembly of data; DL, XZ, and OA Analysis and interpretation of
data; DL, XZ, OA, LZ, and DR Drafting of the article. All authors Final
approval of the article.
Author agreement to authorship
The authors confirm that the manuscript has been read and ap-
proved by all named authors and there are no other persons who have
satisfied the criteria for authorship but are not listed. The authors fur-
ther confirm that the order of authors listed in the manuscript has been
approved.
Acknowledgement
The authors would like to acknowledge the financial support from
the University of Massachusetts Lowell (UML). The authors thank the
staff at the Lowell Regional Wastewater Utility for assistance in ob-
taining septic tank sludge and clarifier supernatant.
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