Biomass and Bioenergy 122 (2019) 156–164

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Biomass and Bioenergy

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

Research paper

Anaerobic co-digestions of agro-industrial waste blends using mixture T

design
Md Anisur Rahmana,b,c,∗, Chayan Kumer Sahaa, Alastair James Wardc, Henrik Bjarne Møllerc,
Md Monjurul Alama
a
Department of Farm Power and Machinery, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh
b
Bangladesh Sugarcrop Research Institute, Ishurdi, 6620, Pabna, Bangladesh
c
Aarhus University, Department of Engineering, Blichers Allé 20, DK-8830, Tjele, Denmark

ARTICLE INFO ABSTRACT

Keywords: Batch assays were performed under a stressed condition during the anaerobic digestion process to assess the
Agro-industrial waste effect of component proportions on the quantity and pattern of biochemical methane yield of two sets of mix-
Anaerobic Co-digestions tures, consisting of poultry droppings (PD) with sugarcane bagasse (SB) and press mud (PM) for set A and PD
Mixture design with sugar beet roots and tops (SBRT) and PM for set B. Augmented simplex centroid design was used to design
Methane yield
the mixture composition for the anaerobic co digestion. The reactor performances were assessed under identical
conditions using cumulative methane yield (CMY) and percentage volatile solids destruction (PVSD) as the
criteria. The maximum volume of methane were obtained 48.5 NL CH4 kg−1 VS at mixture proportion of 66.67%
of PM, 16.665% of PD and 16.665% of SB for set A and 44 NL CH4 kg−1 VS at mixture proportion of 50% of PM
and 50% of PD for set B. Synergistic effect were observed by adding a greater proportion of PM for both the sets
of A and B. Antagonistic effect were obtained by adding a greater proportion of SB for set A and a higher
proportion of SBRT for set B. Response surface methods and modeling were used to find out optimum mixture
combinations for maximizing the methane production. From the optimization plots, the use of PD having the
antagonistic effect was found for both the sets of A and set B.

1. Introduction
Agro-industrial wastes cover a wide spectrum of byproducts derived
from crops, livestock and agro-based industries. The poultry industry is
one of the largest and fastest growing in the agro-food sector worldwide
[1]. The growth of the poultry industry has been accompanied by a
corresponding increase in poultry droppings (PD), including litter,
blood, carcasses, feathers, eggshells and wastewater; inadequate man-
agement of these wastes can result in pollution of soil and water with
nutrients, pathogenic micro-organisms and heavy metals [2]. Agro-en-
vironmental legislation and targets for increased use of renewable en-
ergy (along with specific incentives for energy from wastes) have led to
the implementation of waste-to-energy processes in the poultry sector,
such as anaerobic digestion to produce biogas.
From the 19th century, the main reason for growing sugarcane in
different countries in the world is for production of sugar as the main
product. Due to energy crises, scientists and researchers have realized
the value of sugarcane and its by-products and co-products. The sugar
industry produces a number of by-products during the process of sugar
production including bagasse (SB), pressed mud (PM) or filter cake, ash,
mill effluent, and trash. The sugar production process releases pressed
mud (PM) and sugarcane bagasse (SB) by-products at a rate of 3–4%
and 25–30% of the fresh sugar cane input, respectively. In general, SB is
directly burned to produce steam and electricity as part of a co-gen-
eration scheme for the sugar mills. The residual SB is stockpiled as an
industrial waste with low economic value. PM is mainly treated as
waste material or occasionally used as a low quality fertilizer. Most of
these wastes contain biodegradable matter. This could potentially be a
resource using anaerobic technology for biogas production [3–5].
Among the several techniques for disposal of these wastes, anae-
robic co-digestion could be an effective and least-cost treatment method
for the following reasons: (i) AD of organic wastes is preferred over the
other options because of lower capital investment, operation and
maintenance costs, etc. [6]; (ii) the production of high-value renewable
energy in the form of methane-rich biogas [7]; (iii) balancing the C:N
ratio of the co-substrates to the feedstock as well as balancing macro
and micronutrients, pH, inhibitors/toxic compounds and dry matter
content [8]; (iv) nuisance odors would be reduced and (v) the content

∗
Corresponding author. Department of Farm Power and Machinery, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh.
E-mail address: anis@bsri.gov.bd (M.A. Rahman).

https://doi.org/10.1016/j.biombioe.2019.01.036
Received 19 April 2018; Received in revised form 8 November 2018; Accepted 23 January 2019
Available online 01 February 2019
0961-9534/ © 2019 Elsevier Ltd. All rights reserved.
M.A. Rahman et al.
of pathogens in the digested effluent would be reduced as well as better
management of waste disposal [9]. Co-digestion not only results in high
biogas yields but also improves the process stability as different types of
wastes are being mixed [10]. The solids content, nutritional balance,
biogas yield and process stability can be improved by co-digesting PD
with other organic substances such as sewage waste, cow dung, water
hyacinth, vegetable processing wastes, food wastes, lignocellulosic crop
residues (wheat straw, maize stalk, meadow grass) etc. [11–16]. From
the literature, it was observed that during batch experiments, the ratios
of the mixture components were selected randomly. In the case of some
continuous experiments, the substrates were fed and mixed based on
the operating parameters such as hydraulic retention time and organic
loading rate [17]. On the contrary, the mixture composition largely
influences the biogas yield, reactor stability, solids destruction effi-
ciency, etc. [18].
Mixture experiments are commonly encountered in industrial pro-
duct formulations (e.g., food processing, chemical formulations, textile
fibers, pharmaceutical drugs) but the application in many fields of agro-
industrial research is limited [19]. It has many advantages, such as
improved process yields, reduced variability and closer conformance to
nominal or target requirements, reduced development time and re-
duced overall costs [20].
The mixture design is a class of the response surface experiments in
which the independent variables are the proportions of the components
under investigation [21]. In a mixture design, the total amount of
material is held constant because the response depends on the relative
proportions of the components (ingredients) in the mixture and not on
the amount of the mixture [22]. The mixture design studies aim not
only to develop better or innovative formulations providing optimal
requests, but also to create general conceptions about responses and
interactions between independent factors [23]. A more general goal of
this studyis to use mixture designs to determine which mixture or single
substrateis most desirable, productive, or effective.
In a normal start-up of a batch digester, a certain amount of in-
oculum should be added together with the substrate to provide the
required microorganisms to start reactions. Low amount of inoculum
refers to a small number of microbes that will put pressure on the
process [24]. A critical review of literature reveals that there was no
comprehensive study available to understand the effect of only the
proportions of the components on the biodegradation process under
stressed condition of AD process from agro-industrial wastes of PD, PM,
SB and SBRT. Therefore, an attempt has been made in the present in-
vestigation to (i) study only the effect of substrate composition on
methane production and generation patterns as well as the synergistic
and antagonistic effects of composition under a stressed condition of the
AD process of the mixture and (ii) to optimize the response variable
using mixture design.
2. Materials and methods
2.1. Substrates and inoculums for co-digestion
PD was collected from the poultry farm of Bangladesh Agricultural
University, Mymemsingh. SB and PM were collected from North Bengal
Sugar Mills of Natore district and SBRT were collected from the ex-
perimental fields of Bangladesh Sugarcrops Research Institute, located
in Ishurdi upazila under Pabna district. SBRT was chopped and natu-
rally dried to 6% moisture content. After collecting, SB and SBRT were
kept at 4 °C and, PD and PM were stored at −18 °C. After this SB and
SBRT were dried in an oven (Memmert, Germany) at 100 °C for 48 h.
The dried SB and SBRT were ground (500 μm) using a milling machine
(Wanma 4874, India). The ground materials were preserved in air tight
polyethylene packets for further use. Appropriate amounts of de-mi-
neralized water were added to the dried SB and SBRT and raw sub-
strates PD and PM to bring the feedstock to the design total solids (TS)
concentration (11%) by using the equation no. (1). In this study TS
157
Biomass and Bioenergy 122 (2019) 156–164
concentration was designed at ≈11%, because it was found to be the
optimum TS concentrations for PD, PM and SB in our previous study
[25].
kg TSraw
= 1
kg TSreq (1)
Where TSreq and TSraw are the required percentage of TS concentration
and TS concentration of the raw feedstock, respectively. All the pre-
pared substrates were stored in a refrigerator at 4 °C until character-
ization. At the beginning of the experiment, anaerobically digested
material (cow dung) from a preceding continuously-fed experiment
operated at mesophilic temperature was used as inoculum for this
study. The inoculum was pre-incubated for 21 days at 35 °C tempera-
ture in order to deplete the residual biodegradable organic material
(degasification). The inoculum had 6.87% TS, 5.58% volatile solids
(VS), 1.32% ash content, 1.0 gL−1 total ammoniacal nitrogen (TAN),
2.95 mgL−1 total volatile fatty acids (VFA) and a pH of 7.95.
2.2. Analytical methods
All the feedstocks selected for the digestion were analyzed for their
physical and chemical properties. TS, VS, pH and TAN, were analyzed
by standard methods [26]. To determine the TS in the substrates,
samples were dried at 105 °C for 24 h and weighed before and after this
period. To determine the VS in the samples, the oven dried crucibles
were kept in a muffle furnace at 550 °C for 5.5 h. The crucibles were
removed from the furnace and cooled in air until most of the heat had
dissipated. The sample was then weighed for calculation of VS. Total
nitrogen (TN) was measured by Kjeldahl method and total organic
carbon (TOC) was measured by wet oxidation/Walkley-Black method
[27]. The total volatile fatty acids (TVFA) and total alkalinity (TA) of
the solutions were measured by titration technique using 848 Titrino
plus, Metrohm, Switzerland [28]. These parameters were analyzed for
the feedstock mixtures used in the batch reactors before and after di-
gestion. The biogas generated was analyzed with a BIOGAS 5000,
analyzer (Geotech, UK). All the measurements were performed in du-
plicate and the averages were taken for further interpretation. All the
chemicals used for the analysis were of analytical grade.
2.3. Experimental setup
Experiments were performed in air-tight glass reactors of volume
500 ml with butyl rubbers stoppers. All the experiments were con-
ducted in duplicates at 35 °C and the average was taken for inter-
pretation. Total volume of the substrate in each reactor was maintained
at 300 ml. Calculated amount of inoculum (10% v/v) as described by
Yavini et al. and Budiyono et al. [29,30] and substrate were added into
the bottles separately with each feedstock composition as treatment. In
each bottle approximately 170 ml headspace was left for biogas col-
lection. In order to maintain anaerobic conditions, headspaces of the
bottles were flushed with nitrogen gas for 2 min and the bottles were
closed with air tight butyl rubber stoppers. The bottles were static
throughout, except manual mixing during gas measurements. The vo-
lume of biogas generated in the batch reactor was measured using a
glass syringe, (Switzerland).
2.4. Mixture design and statistical analysis
To assess the effect of co-digestion on methane production as re-
sponse variable under 3 component factors, augmented simplex-cen-
troid design (ASCD) was used [21]. The factors represent the fraction of
each feedstock in the mixture, which ranges from 0 to 1 without con-
straints on the design space. If ‘q’ represents the number of ingredients
in the system under study and ‘xi’ represents the proportion of ‘i’ th
constituent in the mixture, then
M.A. Rahman et al.
q
xi = x1 + x2 + …+ x q = 1.0; x i > 0; i = 1, 2, 3, …q
i=1
The augmented simplex centroid design (ASCD) allows to fit the
special cubic model or special quartic model. The ASCD has more
power for detecting lack of fit than does the {3, 3} simplex centroid
design [17]. Data were fitted to a special quartic polynomial model
applying the least squares regression to estimate the unknown coeffi-
cients in Eq. (3):
q q q conditions (0 °C and 1.013 bar) by using equation (4):
Special quartic : Y= i xi + ij x i x j + iijk x i x j xk
i=1 i<j i<j< k
q q
2 2
+ ijjk x i x j xk +
i< j<k i<j <k
where Y represents the output or response variable of the process. i
represents the expected response to the pure blend x i = 1 and x j = 0
when j ≠ i. The portion i = 1 i x i is called the linear blendingportion.
q
When there is curvature arising from nonlinear blendingbetween
component pairs, the parameters ijk (regression co-efficient) represent
either synergistic or antagonistic blending.
Fig. 1 shows the {3, 2} simplex centroid design augmented with the
axial points. An augmented simplex centroid design, consisting of
(23−1) runs in addition to three additional (7 + 3 = 10) runs where
the blends consist of all the three mixture components was used. The
vertices of the triangle (1, 2, 3) correspond to the three single propor-
tion. The midpoints of the three sides of the triangle (4, 5, 6) consist of
the binary combinations and the central point (centroid-7) and the
three augmented points (8, 9, 10) ascribed to the ternary combinations.
Two sets, A (PD, SB and PM) and B (PD, SBRT and PM), each
Fig. 1. Augmented simplex centroid design plan used for experiments.
Biomass and Bioenergy 122 (2019) 156–164
consisting of ten mixes of the three wastes were prepared by using
(2) ASCD. The waste compositions tested are shown in Table A (supple-
mentary data file). All the batch reactors were maintained at 35 °C as
well as same environmental conditions during digestion (90 days).
In the general mixture problem, the measured responses are as-
sumed to depend only on the proportions of the ingredients present in
the mixture. Cumulative methane yield (CMY) and percentage VS di-
gestibility (PVSD) were measured as response variables.
The resulting cumulative biogas yields were normalized to standard
2
Vm × Ts × Pm
Vs =
Tm × Ps (4)
ijkk x i x j xk where, Vs is the volume of measured gas at STP (mL), Vm is the volume
of measured gas at ambient condition (mL), Tm is the ambient tem-
(3) perature (0K), Pm is the ambient pressure (atm), Ts is the standard
temperature (0 °C or 273 K) and Ps is the standard pressure (1 atm).
2.5. Response analysis
The performance efficiency of the reactor and conversion efficiency
of feedstock was estimated from the response variables CMY and PVSD.
The CMY was obtained by summing up the values of methane generated
at regular intervals. At the end of the experimental period, the value
was calculated for all the mixtures taken for the study. PVSD is a very
important parameter which indicates the conversion efficiency of or-
ganic matter into biogas. PVSD was calculated using the equation no.
(5):
VSinitial VSfinal
% VS digestibility =
VSinitial (5)
158
M.A. Rahman et al. Biomass and Bioenergy 122 (2019) 156–164

Table 1 approximately 9–30 [33]. According to the literature, all the substrates
Characterization of mixtures before digestion for sets A and B. (PD, PM and SBRT) in this study were within the optimum range except
Reactor no PD:SB:PM C/N ratios TS (%) VS % (db) pH SB. pH of single substrate PD before digestion was close to neutral
(7.04) but reactors other than PD (A1) single and/or mixtures of sub-
A1 100:0:0 10.39 10.49 72.23 6.97 strates (A2-A10 and B2eB10)were slightly acidic (Table 1).
A2 0:100:0 41.78 10.21 95.70 6.48
A3 0:0:100 9.59 10.96 51.73 6.73
A4 50:50:0 26.08 10.35 83.97 6.89 3.2. Methane yield
A5 0:50:50 25.68 10.59 73.72 6.67
A6 50:0:50 9.99 10.72 61.98 6.91
The average specific methane yields were 31 ± 1.4, 5 ± 0.1,
A7 33.33:33.33:33.33 20.58 10.46 73.22 6.92
A8 66.67:16.665:16.665 22.58 10.43 72.73 6.90 42 ± 6.5, 11 ± 0.3, 38 ± 0.2, 44 ± 0.12, 26 ± 2.7,24 ± 2.2,
A9 16.665:66.67:16.665 34.23 10.34 84.46 6.84 23 ± 4.0 and 47 ± 0.8 (set A, 1–10 respectively) and 31 ± 1.4,
A10 16.665:16.665:66.67 23.72 10.61 62.48 6.80 1.71 ± 0.1, 42 ± 6.5, 4.34 ± 0.13, 5.3 ± 1.2, 44 ± 0.92,
11 ± 1.4, 12 ± 0.4, 2.29 ± 0.15 and 20 ± 3.27 NL CH4 kg−1 VS(set
Reactor no PD:SBRT:PM C/N ratios TS% VS% (db) pH
B, 1–10 respectively). The produced specific methane yields of sub-
B1 100:0:0 10.39 10.49 72.23 6.97 strates from mono-digestion and/or co-digestions of substrates as per
B2 0:100:0 25.59 10.40 88.10 6.43 designed treatments were low. Among the reasons, low inoculum/
B3 0:0:100 9.59 10.96 51.73 6.73 substrate ratio (ISR) (0.1) was considered to be the main cause. Our
B4 50:50:0 17.99 10.44 80.17 6.83
previous research results demonstrated that specific methane yields of
B5 0:50:50 17.59 10.68 69.92 6.67
B6 50:0:50 9.99 10.72 61.98 6.91 same single substrates were 254 ± 19 for PD, 121 ± 4.1 for PM,
B7 33.33:33.33:33.33 15.19 10.52 70.69 6.90 205 ± 5.0 for SB and 23 ± 1.1 NL CH4 kg−1 VS for SBRT, respectively
B8 66.67:16.665:16.665 18.58 10.47 71.46 6.92 where ISR was 1.0 [25]. Previous studies also validated the results of
B9 16.665:66.67:16.665 23.54 10.46 79.39 6.70
the present study; Hashimoto [34] demonstrated that the ultimate
B10 16.665:16.665:66.67 18.32 10.64 61.21 6.90
methane yield of milled straw drastically decreased from 323 to 18 NL
CH4 kg−1 VS when ISR decreased from 2.18 to 0.19 on a VS basis and
3. Results and discussion 0.9 to 0.10as v/v basis. Moset et al. [35] revealed the ultimate methane
yield of maize straw decreased from 465.5 to 12.1 NL CH4 kg−1 VS for
3.1. Characterizations of substrates ISR decreased from 2.0 to 0.25 (VS basis). Córdoba et al. [36] also
stated that when a low amount of inoculum was used, the adaptation
The TS% of single and/or mixtures of substrate(s) of all reactors time of microorganisms resulted in a much higher than expected de-
were kept between 10 and 11% for this study [25]. The VS (% of DM) laying of methane production and extending the time needed to achieve
among the single substrates was highest for SB-A2 (95.50%) followed by adequate performance of the process. Low ISR in a digester and the
SBRT-B2 (88.10%), PD-A1 (72.23) and PM-A3 (51.73), respectively. The corresponding low number of methanogens leading to accumulation of
total VS% for the rest of the reactors (A4-A10 and B4eB10) was as VS VFA produced during the acidogenesis step has been reported by Liu
proportions of single substrate accordingly. The C:N ratios of PD, PM, et al. (2009) [24]. The average total VFA were found in the range from
SB and SBRT were calculated as 10.49 (A1), 9.59(A3), 41.78(A2) and 5.0 to 10.16 gL−1 from all the reactors of set A and set B after digestion
25.59 (B2). Also, the C:N ratios for rest of the reactors (mixtures of (Table 3). Ahring et al. [37]showed their research results that the in-
substrates) were calculated as C:N proportions of single substrates of dividual VFA did not inhibit the overall biogas process at concentra-
the mixture. The C:N ratio of substrate in AD greatly influences biogas tions up to 3.35 gL−1. A slight decrease in methane production rate was
production [31,32]. Siddiqui et al. (2011) showed that the metabolic observed when VFA was present at a concentration of 7.0 gL−1, but did
activity of methanogens was optimized by a C:N ratio range of not reduce the methanogenic activity at this concentration. They also
examined that total VFA of 13.5 gL−1 decreased the methanogenic

Table 2
Characterization of mixtures after digestion for sets A and B.
Reactor no PD:SB:PM TS (%) VS % (wb) VS % (db) pH VFA (gL−1) NH4eN (mgL−1) Alkalinity (mgL−1)

A1 100:0:0 5.97 3.98 66.64 7.46 7.00 900 90

A2 0:100:0 7.50 7.03 93.76 6.50 7.97 1100 69
A3 0:0:100 8.07 3.50 43.37 6.84 7.84 900 53
A4 50:50:0 6.59 5.32 80.76 7.18 8.81 700 10
A5 0:50:50 7.03 4.93 70.09 6.29 9.76 800 40
A6 50:0:50 6.16 3.38 54.84 7.02 6.84 850 58
A7 33.33:33.33:33.33 6.50 4.32 66.41 6.92 8.75 650 98
A8 66.67:16.67:16.67 6.20 4.09 65.95 7.06 6.25 800 151
A9 16.67:66.67:16.67 7.24 5.82 80.33 6.59 8.76 800 47
A10 16.67:16.67:66.67 8.32 3.68 44.23 6.96 6.96 700 108

Reactor no PD:SBRT:PM TS% VS% (wb) VS% (db) pH VFA (gL−1) NH4eN (gL−1) Alkalinity (mgL−1)

B1 100:0:0 5.97 3.98 66.64 7.46 7.00 900 90

B2 0:100:0 4.69 4.11 87.58 5.47 10.16 700 28
B3 0:0:100 8.07 3.50 43.37 6.84 7.84 900 53
B4 50:50:0 7.32 6.16 84.13 6.95 9.19 750 26
B5 0:50:50 7.59 4.91 64.66 6.15 9.01 750 8
B6 50:0:50 6.16 3.38 54.84 7.01 6.07 700 128
B7 33.33:33.33:33.33 7.25 4.68 64.51 6.87 8.88 750 6
B8 66.67:16.67:16.67 5.41 3.57 65.87 7.04 8.49 700 17
B9 16.67:66.67:16.67 7.78 6.55 84.29 5.50 9.14 850 54
B10 16.67:16.67:66.67 7.65 3.59 46.92 6.85 6.04 650 57

159
M.A. Rahman et al. Biomass and Bioenergy 122 (2019) 156–164

Table 3 YCMY (A) = .31. 3 X1 + 5.32 X2 + .42. 3 X3 .26. 7X1 X2 + .7. 3 X1 X3 + .83. 3 X2 X3
Results of response values for sets A and B.
.62. 2 x12 X2 X3 14 X1 x22 X3 + 274 X1 X2 X32
Run/Reactor Independentvariables Dependentvariables
Set A
PD SB PM CMY(NL CH4 kg−1 VS) PVSD YPVSD (A) = .47. 4 X1 + .29. 4 X2 + .38. 7X3 .26. 2 X1 X3 + .61. 8 X2 X3
.473. 2 x12 X2 X3 .662. 2 X1 x22 X3 + .237. 7X1 X2 X32
A1 100 0 0 31 47.47
A2 0 100 0 5 28.48
A3 0 0 100 42 38.81 YCMY (B ) = 30.63 X1 + 1.34 X2 + .41. 6 X3 .49. 5 X1 X3 + .126. 3 X1 X3
A4 50 50 0.0 11 38.83
A5 0.0 50 50 38 36.65
+ .873. 2 X2 X3 593 x12 X2 X3 + 1119 X1 X22 X3 2279 X1 X2 X32
A6 50 0.0 50 44 49.17
A7 33.33 33.33 33.33 26 43.63 YCMY (B ) = .47. 5 X1 + .56. 0 X2 + .38. 9 X3 .97. 9 X1 X2 + .24. 4 X1 X3
A8 66.67 16.66 16.66 24 46.14
A9 16.66 66.67 16.66 23 33.47 .49. 9 X2 X3 + .121. 3 x12 X2 X3 + 299 X1 X22 X3 .87. 7X1 X2 X32
A10 16.66 16.66 66.67 47 44.51
Regression coefficients for both the responses are shown in Table C
Run/Reactor Independentvariables Dependent variables, (supplementary data file). ANOVA was also performed for both the
Set B
models. The values of R2, a measurement of fitness of the regression
PD SBRT PM CMY (NL CH4 kg−1 VS) PVSD
equations are referenced in Table C. These results reveal that the ex-
B1 100 0 0 31 47.47 perimental data is in good agreement with the predicted values. The
B2 0 100 0 1.71 56.01 parameter's T value is used to determine the significance of the re-
B3 0 0 100 42 38.81
gression coefficients and P value is defined as the smallest level of
B4 50 50 0.0 4.34 27.17
B5 0.0 50 50 5.3 34.84
significance leading to rejection of the null hypothesis [42].
B6 50 0.0 50 44 49.17
B7 33.33 33.33 33.33 11 37.55 3.3.1. Regression analysis for set A
B8 66.67 16.66 16.66 12 52.50
The co-efficient of all the linear terms was positive which indicates
B9 16.66 66.67 16.66 2.29 22.13
B10 16.66 16.66 66.67 20 45.26 all the coefficients were having a synergetic effect on the responses
where the effect of PM was maximum for CMY and PD for PVSD.
However, the quadratic terms of the model reflects synergetic and an-
activity. Xu et al. (2014) investigated that during the AD of kitchen tagonistic effects from the coefficients. The coefficient of quadratic
waste in batch reactors, the initial inhibitory concentration of acetate terms β13 and β23 were having synergetic effect but were insignificant
was between 1.5 and 2.5 gL−1 and methanogenic activity was inhibited (T value = 0.28 and 3.18; P = 0.826 and 0.194, respectively) and the
completely at total VFA concentration of 5.8–6.9 gL−1 [38]. other quadratic terms β12 was found to be antagonistic yet insignificant
Angelidakiet al. (1993) opined that it is not feasible to define an ab- because of its T (negative values) and P values (−1.07 and 0.48) re-
solute VFA level indicating the state of the process. Different anaerobic spectively for the response CMY. From the quartic term, β1123 was
systems have their own normal levels of VFA, determined by the found to be an insignificant antagonistic effect on the response
composition of the substrates digested or by the operating conditions (T = −1.16 and P = 0.452) for CMY. The other two quartic terms for
[39]. Veeken et al. [40] also stated that inhibition regimes were always CMY, β1223 and β1233 were found to be insignificant antagonistic and
accompanied by not only high VFA concentrations, but also sometimes synergetic effects (T = 0.01 and 0.52; P = 0.995 and 0.696, respec-
low pHs (5.0–5.5) because the hydrolysis rate of biowaste depended on tively) depicted in Table D (supplementary data file). However, all the
the pH value not only on VFA. However, reactors B2 and B9 showed pH quadratic terms for CMY could be ignored from the model. The results
value within 5.0–5.5 might also have contributed to low methane of the analysis of variance (Table D) indicate no significant interaction
production (Table 2). In accordance with these literature, the findings effects showing that the 3 components are most effective when used
of this study revealed that the methanogenic activity may have de- alone. In case of PVSD, the quadratic term β13was found to be negative
creased under the stressed condition of the AD process and it showed with negative T value and hence ignored, whereas terms β12 and β23
the reduced rate of methane production due to VFA accumulation. showed a synergetic effect. Between these two terms β23 showed sig-
nificant synergetic effect because values T = 15.04 and P = 0.042. This
3.3. Model fitting and regression analysis means that PVSD was higher for the mixes, combination of SB and PM.
However, all the quartic terms were found to be insignificant. After
The response data as the dependent variables based on independent eliminating the insignificant terms from the model, it could be re-
variables was obtained from the experiments and is depicted in Table 3. written as the following:
All the independent and response variables were fitted to linear,
YCMY (A) = 31.32x1 + 5.32x2+ 42.32x3
quadratic, special cubic, full cubic and special quartic models. The re-
sidual errors were calculated for each model to check the goodness of Y PVSD(A) = 47.4x1 + 28.4x2 + 38.7x3 + 61.8x2x3
the fit. Model summary statistics are given in Table B (supplementary
data file). Standard error of regression, S was used as a measure of
model fit in regression and analysis of variance (ANOVA) [41]. For a 3.3.2. Regression analysis for set B
given study, the better the equation predicts the response, the lower the The results were similar to set A for both the response variables
value of S. Another parameter which was considered to evaluate the (CMY and PVSD) in case of linear terms in the model that indicate no
model was the R2 (coefficient of determination) value, as the value significant interaction effects showing that the 3 components are most
reflects its relationship with one or more predictor variables. The best effective when used alone. None of the quadratic and quartic terms
model was selected using the criteria having low standard error for were found to be significant and hence can be ignored for CMY and
regression and high coefficient of determination. After applying the PVSD. Therefore, after eliminating the insignificant terms from the
criteria, full cubic and special quartic models were found to be the best model, it could be rewritten as the following:
suited models for both sets A and B. The polynomial model selected was YCMV(B) = 30.63x1 + 56x2+ 38.9x3
special quartic for both sets A and B for both the responses and is given
below: Y PVSD(B) = 47.5x1 + 56.0x2 + 38.9x3

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M.A. Rahman et al. Biomass and Bioenergy 122 (2019) 156–164

Fig. 2. Residual plot for CMY of set A.

Fig. 3. Mixture contour plots for CMY and PVSD of sets A and B.

3.4. Interpretation of residual graphs
The residuals from a fitted model are the differences between the
responses observed at each combination of values of the explanatory
variables and the corresponding prediction of the response computed
using the regression function. These residual plots can be used to assess
the quality of the regression. Therefore, if the residuals appear to be-
have randomly, it suggests that the model fits the data well. A normal
probability plot of the residuals can be used to check whether the
variance is normally distributed as well. If the resulting plot is

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M.A. Rahman et al. Biomass and Bioenergy 122 (2019) 156–164

Fig. 4. Mixture 3D surface plots for CMY and PVSD of sets A and B.

Fig. 5. Overlaid contour plot for CMY and PVSD of sets A and B.

approximately linear, we proceed assuming that the error terms are
normally distributed. The histogram of the residual can be used to
check whether the variance is normally distributed. A symmetric bell-
shaped histogram which is evenly distributed around zero indicates that
the normality assumption is likely to be true. If the histogram indicates
that random error is not normally distributed, it suggests that the
model's underlying assumptions may have been violated. The residual
plot for set A is shown in Fig. 2 for CMY. The results can also be shown
with the help of histograms. A histogram of the residuals shows the
distribution of the residuals for all observations. Fig. 2 also plots the
residuals versus the fitted values (predicted response). It was observed
from Fig. 2 that the experimental points were reasonably aligned sug-
gesting the normal distribution and the residuals were found to be
scattered randomly about zero, which means that the error terms are
normally distributed.

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M.A. Rahman et al.
Fig. 6. Optimization plots for CMY and PVSD of sets A and B.
3.5. Interpretation of contour plots and 3D surface plots
Using Minitab software, contour plots and 3D surface plots were
generated to find the optimum proportion of the mixes for both sets A
and B. Contour and surface plots are useful for establishing desirable
response values and mixture blends. A contour plot provides a two-
dimensional view where all points that have the same response are
connected to produce contour lines of constant responses. A surface plot
provides a three-dimensional view that may provide a clearer picture of
the response surface. Figs. 3 and 4 show the contour and 3D surface
plots of CMY and PVSD for both the sets A and B. It was observed that
both sets A and B gave similar results for the contour and surface plots.
From the contour plots of CMY, the zones of maximum response vari-
ables were located towards the side of the triangle having PD and PM as
the vertices. In case of PVSD for sets A and B (Fig. 3), maximum re-
sponse was observed with co-digestion of PD and PM. This indicates
that to certain extent, these waste proportions may be added to improve
CMY and having simultaneous effect of solids reduction. The PM con-
tent was found to be significant proportion in the mixture to maximize
the response variable. The addition of PD and SBRT showed antag-
onistic effect on the response variables for set A and set B respectively.
This can be verified clearly by observing the mixture surface plots
(Fig. 4). The white zones of the overlaid contour plots (Fig. 5)indicate
the feasible region that satisfies both the criteria of optimum response
variables.
3.6. Interpretation of mixture proportion optimization
Optimization of mixture proportions is used to identify the combi-
nation of input variable settings that jointly optimize a single response
or a set of responses. It is useful in determining the optimum operating
conditions that will maximize the response. Optimal solution was ob-
tained usingMinitab-18 and a plot was drawn accordingly. The optimal
solutions obtained for both sets A and B are shown in Fig. 6. The op-
timum mixture ratios obtained from the analysis were 33.33% of SB and
66.67% of PM for set A and 48.48% of SBRT and 51.52% of PM for set
B. The respective maximum predicted CMY and PVSD was found to be
48.5 NLkg−1 VS and 49.05% for set A and 43.85 NLkg−1 VS and
49.07% for set B, respectively. The composite desirability of the re-
spective mixtures was found to be 1.00 and 0.89 for sets A and B re-
spectively, which is a significant factor needed to be taken into account
while considering the optimum composition. The composite desirability
nearing to one indicates positive effect on maximizing the response
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Biomass and Bioenergy 122 (2019) 156–164
variables. From the optimization plots, the use of PD in major propor-
tion having the antagonistic effect was observed for the responses both
sets of A and B. However, the proportions of PD can be increased to a
certain extent without compromising on the response variables for both
sets of A and set B.
3.7. Model validation
From the experimental data, it was observed that the maximum
values for CMY was found to be 47NLkg−1 VS and 44 NLkg−1 VS (for
mixture no. A10 and B6) and maximum values for PVSD was found
49.17%and 56.01% (for mixture no. A6 and B2). In order to validate the
model, experiments were conducted with the optimum compositions
obtained. The predicted optimum results were found to be 48.5NLkg−1
VS of CMY and 49.05% of PVSD at composition of 33.33% SB and
66.67% PM for set A and 38.80 NLkg−1 VS of CMY and 55.44% of PVSD
at composition of 49.49% PD and 50.51% PM for set B. The results
obtained from the experiment were found to be comparable with the
prediction. The small variation in the results can be attributed to
change in the composition of substrates used for the validation of ex-
periments.
4. Conclusions
Batch experiments for co-digestions of PD, SB and PM (set A) and
PD, SBRT and PM (set B) were performed under stressed condition to
evaluate only the effect of proportions of components in the mixtures.
In a co-digestion experiment, the proportion of each component in
mixture plays a significant role. Mixture experiment is appropriate to
use when one wishes to determine if synergism exists in mixing com-
ponents which increases productivity or desirability of a product. A
more general goal of a researcher might be to use mixture designs to
determine which mixture or mono substrate is most desirable, pro-
ductive, or effective. From the results of predicted optimization, it was
found that the mixtures containing SB in minor proportion and PM in
major proportion presented the highest CMY for set A and addition of
PD and PM in almost equal portion in set B showed the highest CMY.
Additions of PD and SBRT in major proportion in the mixtures of SB and
PD for the sets A and B were found to be antagonistic, respectively as it
acidifies rapidly and resulting in dropping the pH significantly. The low
pH in mixture media will also inhibit the methanogens which are the
key organisms to convert the acids into methane. The batch mode ex-
periment is the limitation of this study, because a full scale digester
M.A. Rahman et al.
plant is used for industrial purposes, which usually operates in a con-
tinuous mode. Despite the difficulty of comparing batch and con-
tinuous, the batch can show results that are relative to each other or
could be said that good batch performance can suggest good CSTR
performance. Likewise, the source of inoculum can have an effect on
results, but direct comparisons of the results qualitatively within the
same batch experiment is possible. Further investigation could be made
by continuous mode for better performance and the acquisition of
quantitative data.
Acknowledgment
The authors would like to acknowledge the International Finance
Corporation (IFC) and DANIDA, Bangladesh for providing the scholar-
ship under the “Green Energy Knowledge Hub Project (No-600237)” to
allow this research to be undertaken.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biombioe.2019.01.036.
References
[1] N.S. Bolan, A.A. Szogi, T. Chuasavathi, B. Seshadri, M.J. Rothrock,
P. Panneerselvam, World Poultry Sci. J. 66 (4) (2010) 673–698.
[2] P. Gerber, C. Opio, H. Steinfeld, Poultry production and the environment – a review,
FAO, 2007.
[3] R.L. Yadav, S. Solomon, Potential of Developing Sugarcane By-product Based
Industries in India, Indian Institute of Sugarcane Research, P.O. Dilkusha, Rae
Bareli Road, Lucknow-226 002, India, 2006, pp. 104–111.
[4] S. Meunchang, S. Panichsakpatana, R.W. Weaver, Co-composting of filter cake and
bagasse; by-products from a sugar mill, Bioresour. Technol. 96 (2005) 437–442
http://doi.org/10.1016/j.biortech.2004.05.024.
[5] P. Horecky, M. Saska, Thermophilic Anaerobic digestion of filter cake from su-
garcane factory, Audubon Sugar institute, Louisiana State University Agricultural
Centre, 2004.
[6] S. Peter, D.M. Katherine, I.M. Roderick, R. Lutgarde, Anaerobic codigestion ofmu-
nicipal solid waste and biosolids under various mixing conditions, I.
Digesterperformance , Water Res. 35 (2001) 1804–1816.
[7] K. Singh, K. Lee, J. Worley, L.M. Risse, K.C. Das, Anaerobic digestion of poultry
litter: a review, Appl. Eng. Agric. 26 (4) (2010) 677–688.
[8] H. Hartmann, B.K. Ahring, Anaerobic digestion of the organic fraction of municipal
solid waste: Influence of co-digestion with manure, Water Res. 39 (2005)
1543–1552 https://doi.org/10.1016/j.watres.2005.02.001.
[9] N.J. Horan, L. Fletcher, S.M. Betmal, S.A. Wilks, C.W. Keevil, Die-off of enteric
bacterial pathogens during mesophilic anaerobic digestion, Water Res. 38 (2004)
1113–1120 https://doi.org/10.1016/j.watres.2003.12.004.
[10] J. Mata-Alvarez, S. Mace, P. Llabres, Anaerobic digestion of organic solid wastes,an
overview of research achievements and perspectives, Bioresour. Technol. 74 (2000)
3–16 https://doi.org/10.1016/S0960-8524(00)00023-7.
[11] M.R. Sebola, H.B. Tesfagiorgis2, E. Muzenda, Methane production from anaerobic
co-digestion of cow dung, chicken manure, pig manure and sewage waste,
Proceedings of the world congress on engineering-WCE, July 1 - 3, London, U.K.
vol. I, 2015.
[12] E.C. Chukwuma, I.C.E. Umeghalu1, L.C. Orakwe1, E.E. Bassey, J.N. Chukwuma,
Determination of optimum mixing ratio of cow dung and poultry droppings in
biogas production under tropical condition, Afr. J. Agric. Res. 8 (18) (2013)
1940–1948 https://doi.org/10.5897/AJAR12.1781.
[13] J.H. Patil, M.L.A. Raj, Bhagyashree, V. Dinesh, Rohith, Kinetic modeling of anae-
robic co-digestion of water hyacinth and poultry litter, Res. J. Chem. Sci. 3 (6)
(2013) 69–73.
[14] B. Molinuevo, M.C. Garcia, M. C. Leon, M. Acitores, Anaerobic co-digestion of an-
imal wastes (poultry litter and pigmanure) with vegetable processing wastes,
Agricultural technological Institute of Castilla and Leon, Finca Zamadueñas,
Valladolid, Castilla and Leon, Spain, www.Ramiran.Uvlf.Sk/Doc08/Ramiran…/
Molinuevo.Pdf.
[15] T.R.T. Yusof, H. Che Man, Nor Aini A. Rahman, H.S. Hafid, Optimization of me-
thane gas production from co-digestion of food waste and poultry manure using
artificial neural network and response surface methodology, J. Agric. Sci. 6 (7)
(2014) 27–37 https://doi.org/10.5539/jas.v6n7p27.
[16] M.A. Rahman, H.B. Moller, C.K. Saha, M.M. Alam, R. Wahid, L. Feng, Optimal ratio
164
Biomass and Bioenergy 122 (2019) 156–164
for anaerobic co-digestion of poultry droppings andlignocellulosic-rich substrates
for enhanced biogas production, Energy Sustain. Develop. 39 (2017) 59–66 https://
doi.org/10.1016/j.esd.2017.04.004.
[17] M. Krupp, J. Schubert, R. Widmann, Feasibility study for co-digestion of sewage
sludge with OFMSW on two wastewater treatment plants in Germany, Waste
Manag. 25 (2005) 393–399.
[18] P.V. Rao, S.S. Baral, Experimental design of mixture for the anaerobic co-digestion
of sewage sludge, Chem. Eng. J. 172 (2011) 977–986 https://doi.org/10.1016/j.cej.
2011.07.010.
[19] K. Bondari, Mixture experiments and their applications in agricultural research,
Paper 209-30, Experimental Statistics, Coastal Plain Station, University of Georgia,
Tifton, GA 31793-0748.
[20] R.S. Prakasham, T. Sathish, P. Brahmaiah, R.C.H. Subba, R.R. Sreenivas, P.J. Hobbs,
Biohydrogen production from renewable agri-waste blend: optimization usingmixer
design, Int. J. Hydrogen Energy 34 (2009) 6143–6148 https://doi.org/10.1016/j.
ijhydene.2009.06.016.
[21] J. Goupy, L. Creighton, Introduction aux plans d’expériences, 3e edition, Dunod,
Paris, 2006 ISBN 2 10 049744 8.
[22] J.A. Cornell, Experiments with Mixtures: Designs, Models and the Analysis
ofMixture Data, third ed., Wiley, 2002.
[23] E.C.R. Maia, D. Borsato, I. Moreira, K.R. Spacino, P.R.P. Rodrigues, A.L. Gallina,
Study of the biodiesel B100 oxidative stability in mixture withantioxidants, Fuel
Process. Technol. 92 (2011) 1750–1755 https://doi.org/10.1016/j.fuproc.2011.04.
028.
[24] G. Liu, R. Zhang, H.M. El-Mashad, R. Dong, Effect of feed to inoculum ratioson
biogas yields of food and green wastes, Bioresour. Technol. 100 (2009) 5103–5108
https://doi.org/10.1016/j.biortech.2009.03.081.
[25] M.A. Rahman, C.K. Saha, L. Feng, H.B. Moller, M.M. Alam, Anaerobic Digestion of
Agro-Industrial Wastes in Bangladesh: Influence of Total Solid Concentration,
(unpublished)under review to Journal of Energy and Environment.
[26] APHA-(American Public Health Association), Standard methods for the examina-
tion of water and wastewater, 21th ed, APHA, Washington, 2005.
[27] G.N. Bartlett, B. Craze, M.J. Stone, R. Crouch (Eds.), Guidelines for Analytical
Laboratory Safety, Department of Conservation & Land Management, Sydney, 1994.
[28] G.K. Anderson, G. Yang, Determination of bicarbonate and total volatile acid con-
centration in anaerobic digesters using a simple titration, Water Environ. Res. 64
(1992) 53–59 https://doi.org/10.2175/WER.64.1.8.
[29] T.D. Yavini, A.I. Chia, A. John, Evaluation of the effect of total solids concentration
on biogas yields of agricultural wastes, Int. Res. J. Environ. Sci. 3 (2) (2014) 70–75.
[30] Budiyono, I. Syaichurrozi, S. Sumardiono, Effect of total solid content to biogas
production rate from vinasse, IJE Trans. B: Appl. 27 (2) (2014) 177–184 http://doi.
org.10.5829/idosi.ije.2014.27.02b.02.
[31] X. Wang, G. Yang, Y. Feng, G. Ren, X. Han, Optimizing feeding composition and
carbonnitrogen ratios for improved methane yield during anaerobic co-digestion of
dairy, chicken manure and wheat straw, Bioresour. Technol. 120 (2012) 78–83
https://doi.org/10.1016/j.biortech.2012.06.058.
[32] M. Kayhanian, Ammonia inhibition in high-solids biogasification: an overview and
practical solutions, Environ. Technol. 20 (1999) 355–365.
[33] Z. Siddiqui, N. Horan, K. Anaman, Optimization of C:N ratio for co-digested pro-
cessed industrial food waste and sewage sludge using the BMP test, Int. J. Chem.
React. Eng. 9 (2011) S4.
[34] A.G. Hashimoto, Effect of inoculum/substrate ratio on methane yield and produc-
tion rate from straw, Biol. Wastes 28 (4) (1989) 247–255 https://doi.org/10.1016/
0269-7483(89)90108-0.
[35] V. Moset, N. Al-zohairi, H.B. Møller, The impact of inoculum source, inoculum to
substrate ratio and sample preservation on methane potential from different sub-
strates, Biomass Bioenergy 83 (2015) 474–482 https://doi.org/10.1016/j.
biombioe.2015.10.018.
[36] V. Córdoba, M. Fernández, E. Santalla, The effect of substrate/inoculum ratio on the
kinetics of methane production in swine wastewater anaerobic digestion, Environ,
Sci Pollut Res (2017) 1–10 http://doi.org/10.1007/s11356-017-0039-6.
[37] B.K. Ahring, M. Sandberg, I. Angelidaki, Volatile fatty acids as indicators of process
imbalance in anaerobic digestors, Appl. Microbiol. Biotechnol. 43 (1995) 559–565.
[38] Z. Xu, M. Zhao, H. Miao, Z. Huang, S. Gao, W. Ruan, In situ volatile fatty acids
influence biogas generation from kitchen wastes by anaerobic digestion, Bioresour.
Technol. 163 (2014) 186–192 https://doi.org/10.1016/j.biortech.2014.04.037.
[39] I. Angelidaki, L. Ellegaard, B.K. Ahring, A mathematical model for dynamic simu-
lation of anaerobic digestion of complex substrates, focusing on ammonia inhibi-
tion, Biotechnol. Bioeng. 42 (1993) 159–166.
[40] A. Veeken, S. Kalyuzhnyi, H. Scharff, B. Hamelers, Effect of pH and VFA on hy-
drolysis of organic solid waste, J. Environ. Eng. 126 (12) (2000) 1076–1081.
[41] N. Abdullah, N.L. Chin, Simplex-centroid mixture formulation for optimised com-
posting of kitchen waste, Bioresour. Technol. 101 (2010) 8205–8210 http://doi.
org/10.1016/j.biortech.2010.05.068.
[42] T. Mahe, O. Prakash, S.H. Hasan, Enzymatic detection of As(III) in aqueoussolution
using alginate immobilized pumpkin urease: optimization of processvariables by
response surface methodology, Bioresour. Technol. 100 (2009) 4462–446 http://
doi.org/10.1016/j.biortech.2009.04.009.