Waste Management xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Waste Management
journal homepage: www.elsevier.com/locate/wasman

Evaluation of biogas production potential of kitchen waste in the

presence of spices
Nidhi Sahu, Abhinav Sharma, Priyanka Mishra, B Chandrashekhar, Ganesh Sharma, Atya Kapley,
R.A. Pandey ⇑
CSIR-National Environmental Engineering Research Institute [CSIR-NEERI], Nehru Marg, Nagpur 440020, India

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

Article history: Anaerobic digestion (AD) of kitchen waste (KW) for biogas production is a major challenge to all over the
Received 13 October 2016 world due to significant compositional variations in KW, such as different types and quantities of spices
Revised 24 July 2017 used for preparing food. Spices may affect the AD process owing to their antimicrobial activity. In this
Accepted 25 August 2017
paper, the effect of spices (garlic, red chili, cinnamon, coriander, clove, turmeric, cardamom, black pepper)
Available online xxxx
on AD of KW has been investigated. Batch experiments were carried out to determine the maximum bio-
gas production potential, methane production rate and lag phase for biogas production. Analysis of the
Keywords:
results revealed different magnitude of inhibition of the AD process of KW in the presence of different
Kitchen waste
Spices
spices. Cinnamon, cardamom and clove resulted >85%, black pepper resulted 75%, while coriander, chili,
Biomethanation turmeric and garlic resulted 55–70% reduction in cumulative biogas yield. Elemental analysis showed
Gompertz kinetic model high concentration of heavy metals in the spices, which along with other bioactive components of the
Antimicrobial effects spices could be responsible for the inhibitory effect of the spices on biomethanation. Microbial examina-
tion of the digestate also showed a decrease in population of fermentative and methanogenic bacteria in
the presence of spices.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction production technology for its proper treatment and management.

Globally, approximately 1.3 billion tons of kitchen waste (KW)
is generated per year (FAO, 2011). The amount of generated KW
is increasing continuously and due to the environmental impacts
of its improper treatment, management of KW has become an
environmental and social concern. Anaerobic digestion (AD) is a
very promising approach for efficient management of organic
waste such as KW (Iqbal et al., 2014; Xiao et al., 2015). AD is the
process by which organic matter is broken down to produce biogas
and biofertilizer. AD has the advantage of biogas production, lead-
ing to efficient resource recovery along with contribution to con-
servation of non-renewable energy sources.
KW is composed of organic substances like carbohydrate, pro-
tein, cellulose and fats (Fang et al., 2012) which are rapidly biode-
graded, making it an attractive feedstock for running an anaerobic
digester. However, AD plants face difficulties in obtaining fairly
clean feedstock which results in technical problems. Moreover, sig-
nificant compositional variations in KW found in different parts of
the world poses a challenge to the development of a robust biogas
⇑ Corresponding author.
E-mail address: rapandey108@gmail.com (R.A. Pandey).
In India where a wide range of spices are used to prepare food,
presence of high quantities of spices in the KW is inevitable. Spices
and herbs such as red chili, coriander, garlic, turmeric, black pep-
per, cardamom, cinnamon, and clove are commonly used in large
quantities to enhance the flavor, color and aroma of the food.
Spices are known for their antimicrobial and antioxidant prop-
erties (Lai and Roy, 2004; Asimi et al., 2013). Therefore, the pres-
ence of spices in KW might affect the AD process. It has been
reported that spices owe their antimicrobial properties due to
the presence of specific biochemical components (including euge-
nol in clove, thymol in thyme and oregano, carvacrol in oregano,
vanillin in vanilla, allicin in garlic, cinnamic aldehyde in cinnamon
and allyl isothiocyanate in mustard) (Škrinjar and Nemet, 2009).
Some of these compounds act as broad spectrum antimicrobial
agents and others show high antibacterial activity against either
Gram positive or Gram negative bacteria (Ceylan and Fung, 2004).
Spices also contain metal salts which may affect the microbial
population of digester due to their cytotoxicity. However, other
components present in spices such as vitamins and minerals may
stimulate growth and/or biochemical activity (Ceylan and Fung,
2004). There is little information available on the effect of spices
on AD process for biogas production. Hence, the present investiga-
tion was carried out to evaluate the composition of selected Indian

http://dx.doi.org/10.1016/j.wasman.2017.08.045
0956-053X/Ó 2017 Elsevier Ltd. All rights reserved.

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2 N. Sahu et al. / Waste Management xxx (2017) xxx–xxx
spices, specifically metal concentration, and the effect of the spices
on AD of KW. Further, the effect of spices on the microbial flora
present in the biogas digester was determined. The experimental
data was further explained by applying Gompertz model to derive
kinetic parameters.
2. Materials and methods
Biomethanation potential (BMP) of KW was studied by a series
of batch experiments using serum bottles. The experiments pri-
marily involved measurement of methane (CH4) and carbon diox-
ide (CO2) in the biogas produced during the anaerobic digestion
of KW in the presence of various spices. Some microbiological tech-
niques were also involved in order to determine the population of
facultative anaerobes (fermentative bacteria) and anaerobic (ace-
togens/methanogens) bacteria in the digestate.
2.1. Chemicals, reagents and media composition
All the reagents and chemicals were purchased from Merck,
India and used as per the standard protocols without further purifi-
cation. The spices-red chili (Capsicum annuam), coriander (Corian-
drum sativum), garlic (Allium sativum), clove (Syzygium
aromaticum), black pepper (Piper nigrum), cardamom (Elettaria car-
damomum), turmeric (Curcuma longa) and cinnamon (Cinnamo-
mum verum) were purchased from the local market. Luria –
Bertani agar (LBA) medium and anaerobic agar (AA) medium were
purchased from Himedia laboratory Ltd.
2.1.1. Seed inoculum
The anaerobic sludge from a running biogas plant was collected
and used as a source of methanogenic bacteria for the experiments.
The sludge was characterized for pH, total solid (TS) and volatile
solid (VS). Before inoculation, the sludge was activated metaboli-
cally by adding anaerobic growth activation medium (medium
composition per liter: NH4HCO3, 3.77 g; K2HPO4, 0.125 g; NaHCO3,
2 g; CuSO4
5H2O, 0.005 g; MgCl2
6H2O, 0.1 g; MnSO4
4H2O,
0.015 g; FeSO4
7H2O, 0.025 g; CoCl2
6H2O, 0.125 g; C12H22O11,
11.1 g). 50 mL of activation medium was added to 1 L of sludge
and incubated overnight at 37 °C with shaking at 120 rpm to avoid
settling of biomass.
2.1.2. Kitchen waste
Basic ingredients of Indian food such as cooked rice, wheat
flour, pulses (legume seeds such as lentils) and potato were
selected as the components of the KW. These components were
selected after determining the common components used daily
for preparing food on the basis of an inventory of the community
kitchen. All these components were taken in equal VS basis and
ground to make uniform in composition. The composition of KW
was fixed in all the experiments and prepared freshly before con-
ducting the experiments. The prepared KW was characterized for
TS, VS and moisture count (MC).
2.2. BMP procedure
The BMP test was performed to measure the maximum biogas
production potential of KW with and without spices. 10 gm of
KW was mixed with 940 mL of activated sludge and 50 mL of pre-
viously prepared BMP medium as described by Angelidaki et al.
(2009) for nutritional supplements. 100 ml of the above digestion
mixture was transferred to 125 mL serum vials. Based on the max-
imum quantity of spices used in Indian food recipes, different con-
centrations of the selected spices (10–200 mg/L) were added to
serum vials to study their effects on biomethanation. Sludge with
Please cite this article in press as: Sahu, N., et al. Evaluation of biogas production potential of kitchen waste in the presence of spices. Waste Management
(2017), http://dx.doi.org/10.1016/j.wasman.2017.08.045
KW only (SKW) was used as positive control and, sludge without
KW and spice was used as negative control. In order to check the
effect of only metal ions on CH4 production, the digestion mixture
was supplemented with 2 mg/L of metal ions using CoCl2, NiCl2,
PbCl2 and ZnCl2 as the sources.
Anaerobic condition inside the vials was maintained by flushing
pure nitrogen gas through the medium. Serum vials were sealed
tightly with silicon stoppers and incubated at 37 °C in an orbital
shaker at 200 rpm. The experiment was performed in triplicate.
Pressure of biogas generated in the head space of serum vials
was measured using digital pressure meter (HND-P, Kobold) and
analyzed for CH4 and CO2 percentage by using a gas chro-
matograph equipped with a thermal conductivity detector (GC-
TCD) and Porapak Q column (Agilent) daily. Liquid samples were
withdrawn before and after the completion of experiment and
enumerated for bacterial colony forming units (CFUs). The average
values from the triplicate experiments were noted down and used
for interpretation.
2.3. Metal composition analysis of spices
Samples for metals analysis were prepared by acid digestion as
described (APHA, 2008). Nitric acid was used for the digestion.
After digestion, the samples were filtered using 0.22 lm filter
paper and analyzed for metals using inductively coupled plasma
resonance mass spectrometer (ICP-MS, Agilent).
2.4. Enumeration of microbial flora during digestion process
In order to determine the effect of spices on the bacterial pop-
ulation, CFU count of facultative anaerobes (fermentative bacteria)
and anaerobic (acetogens/methanogens) bacteria was performed
in AA and LBA media, respectively. For this, the sludge obtained
from the digester was spread on LBA and AA media (spread plate
method) and incubated for 48 h at 37 °C. After incubation micro-
bial count was done using a digital colony counter.
2.5. Kinetic analysis
The kinetics of biogas production during AD of SKW with and
without spices was evaluated by fitting the modified Gompertz
kinetic model equation to the experimental data (Lo et al., 2010).
The model gives an estimate of the maximum biogas production
potential, rate of biogas production and the lag phase. The modi-
fied Gompertz equation is written as (Nopharatana et al., 2007)
Eq. (1):
  
Rm
P ¼ A exp  exp ðk  tÞ þ 1 ð1Þ
A
where, P = the cumulative methane produced at time (t) (mL/gVS),
A = maximum biogas production potential (mL/gVS), Rm = rate of
biogas production (mL biogas/gVS/Day), k = lag phase of biogas pro-
duction (d), and t = the time taken for cumulative biogas production
(P) calculated (d). The equation was solved using solver function in
Microsoft excel by non-linear regression.
3. Results
The characteristics of inoculum and KW are compiled in Table 1.
Batch digestion experiments were carried out to study the biogas
production potential of KW and to elucidate the effect of spices
on digestion in terms of daily biogas production, cumulative biogas
and CH4 yield as described in this section.
 N. Sahu et al. / Waste Management xxx (2017) xxx–xxx 3

Table 1 sludge alone (negative control) are shown in Fig. 1(a). The cumula-
Characteristics of kitchen waste (KW) and sludge. tive biogas production using SKW during this study was found
Composition of Food TS (g/L) VS (g/L) 340 mL/gVS which was much higher than the activated anaerobic
Rice 81.6 ± 2.51 73.5 ± 2.26 sludge alone i.e. 119 mL/gVS (Fig. 1(a)). As a result, total biogas
Wheat Flour 90.3 ± 1.53 88.5 ± 1.49 yield obtained with activated anaerobic sludge was 65% lower as
Pulse 87.3 ± 1.5 81.8 ± 1.88 compared to SKW.
Potato 55.6 ± 3.51 51.2 ± 3.23
Sludge 6.2 ± 0.55 4.42 ± 0.39
3.1.2. Production of biogas from SKW in the presence of spices
Table 2 summarizes the cumulative biogas produced from SKW
in the presence of spices, showing varied effects at different con-
3.1. Effect of spices on biogas production and methane percentage centrations of spices. In case of coriander, garlic, turmeric, chili
and black pepper highest biogas yield was obtained when the spice
3.1.1. Production of biogas from SKW concentration was between 30 and 50 mg/L. The biogas yield was
The experimental results of daily and cumulative biogas pro- found to decrease at spices concentration above 50 mg/L. The pres-
duction from SKW (positive control) and activated anaerobic ence of coriander, chili, turmeric, garlic and black pepper in the

Fig. 1. Experimental daily biogas production (primary axis) and cumulative biogas production (secondary axis) from (a) Sludge alone, Sludge + KW (SKW, positive control)
and in the presence of spices (b) chili and (c) cinnamon in SKW.

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Table 2
The experiment biogas production potential (mL/g VS) of Sludge + kitchen waste (SKW) in the presence of spices.

Experimental cumulative biogas production (mL/gVS) Average cumulative Biogas % Decrease in biogas production
production(mL/gVS) as compared to SKW
10 mg/L 30 mg/L 50 mg/L 100 mg/L 200 mg/L#
Chili 100 102 157 109 93 117 66
Turmeric 159 137 109 92 78 124 63
Coriander 120 151 213 109 1 148 56
Clove 24 29 31 54 16 35 90
Cardamom 18 24 31 86 90 40 88
Cinnamon 32 45 35 24 6 34 90
Black peeper 78 89 90 85 81 86 75
Garlic 61 124 138 102 101 106 69
Sludge 119 65
SKW 340
#
Values are omitted for average cumulative biogas production calculation.

Fig. 2. Experimental daily biogas production (primary axis) and cumulative biogas production (secondary axis) from SKW in the presence of (a) coriander, (b) garlic and (c)
turmeric.

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SKW resulted in decreased biogas production by 56–75% (Table 2).
Highest inhibition of biogas yield was observed in the presence of
cardamom, clove and cinnamon (>85% lesser biogas produced).
AD of SKW with spices showed similar trends for the biogas
yields to that shown without spices (Figs. 1–3). Among all the
spices used, coriander showed least inhibition to the daily biogas
yield (60 mL biogas/gVS/Day) during the first 4 days of experiment
after which it decreased drastically (Fig. 2(a)). In the presence of
chili and garlic the daily biogas yield reached as high as 20 and
30 mL/gVS/Day, respectively (Figs. 1(b) and 2(b)). The cumulative
biogas yield was also highest in the presence of these spices
(>100 mL/gVS). Regarding all the spices, a high biogas production
rate was observed for a period of 8 days amounting 62–96% of total
biogas produced during which most of the cumulative biogas yield
was obtained. The peak value of daily biogas yield typically
occurred before the 4th day of digestion and was negligible after
the 15th day.
3.1.3. Methane and carbon dioxide content in the biogas produced
from SKW in the presence of spices
The average daily CH4 yield in terms of percentage during the
experiments with standard deviation (shown as error bars) is
showed in Fig. 4(a). As the total biogas production decreased, %
CH4 also decreased in the presence of spices such as cardamom,
cinnamon, black pepper and clove. Therefore, the effect of these
spices on CH4 and biogas production was more profound as com-
pared to others. Turmeric, coriander and chili supported higher
biogas formation, as well as higher CH4 content (35–45%) as

Fig. 3. Experimental daily biogas production (primary axis) and cumulative biogas production (secondary axis) from SKW in the presence of (a) black pepper, (b) cardamom
and (c) clove.

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Fig. 4. Effect of spices on (a) methane (CH4) and (b) carbon dioxide (CO2) contents (in vol%) in produced biogas. The error bars represent standard deviation.

compared to other spices (11–25%). Presence of garlic favored
higher biogas formation (105 mL/gVS), however, the CH4 percent-
age in the presence of garlic was lower (about 25%). Fig. 4(b) shows
the daily % CO2 in the biogas generated during the digestion. It was
observed that % CO2 remained between 30 and 40% in most of the
cases, even in the presence of black pepper, cinnamon, cardamom
and clove in which the cumulative biogas volume as well as % CH4
is drastically reduced. In those cases where both % CO2 and % CH4
are not severely affected, the biogas volume is also higher. The %
CH4 is much lower than % CO2 in the presence of some spices,
which indicates inhibition of methanogenesis. The cumulative
CH4 and CO2 produced in the experiments is also showed in
Fig. S1, supplementary information. The volume of CH4 produced
in the presence of spices was lesser than in the blank SKW. In some
cases (clove, cinnamon, cardamom, black pepper), the cumulative
CO2 volume was also reduced, therefore, leading to very low cumu-
lative biogas yield. Inhibition of hydrogenotrophic methanogenesis
(hence % CH4) may lead to accumulation of % CO2 along with rise in
hydrogen (H2), ammonia (NH3) and hydrogen sulphide (H2S) con-
centration in the biogas which might make up the remaining vol-
ume of the gas.
Analysis of variance (ANOVA) for % CH4 produced in the pres-
ence of spices was conducted using the spice concentration and
set of experiments (triplicate) as the sources of variation. Differ-
ence between % CH4 production obtained at various concentrations
of spices was found significant (p < 0.05) (Table S1, Supplementary
information). The results also demonstrated no significant differ-
ence in the % CH4 production between the triplicate set of experi-
ments at any spice concentration (p > 0.05) except in case of
cardamom (p < 0.05), which indicated the reproducibility of
results.
3.2. Kinetic parameters
The kinetic parameters of biogas production were estimated by
the Gompertz modified model using the experimental data and are
presented in the Table 3. Among all the spices used in the study
turmeric, coriander and garlic showed higher cumulative biogas
(P value) than other spices. Clove and cinnamon showed low P val-
ues while cardamom and black pepper showed moderate P values
(Table 3). Upon increasing spice concentration, the value of P ini-
tially increased in most of the cases and after attaining a maximum
value it decreased with further increase in the spice concentration.
Exceptions were turmeric and cardamom. Increase in the turmeric
concentration caused decrease in the P value, while cardamom was
the only spice which showed an increase in P value, even as the
concentration of the spice was increased. Black pepper did not

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Table 3
Estimated kinetic parameters for biogas production using modified Gompertz model.

Spice name Concentration (mg/L) Biogas yield potential (mL/gVS) Biogas production rate (mL/gVS/Day) Lag phase (day) R2
Chili 10 96 0.94 0.56 0.988
30 97 0.77 0.50 0.972
50 150 0.66 0.35 0.967
100 108 0.96 0.53 0.995
200 88 0.72 0.41 0.968
Turmeric 10 157 0.77 0.34 0.980
30 135 0.88 0.48 0.990
50 107 0.95 0.56 0.994
100 91 1.03 0.45 0.997
200 77 1.00 0.63 0.995
Coriander 10 119 0.98 0.42 0.994
30 142 0.82 0.53 0.966
50 198 1.05 0.58 0.976
100 106 0.98 0.52 0.992
200 1 0.68 0.09 0.999
Clove 10 24 1.40 0.41 0.991
30 29 1.33 0.53 0.996
50 30 1.30 0.37 0.988
100 53 1.43 0.54 0.996
200 16 1.66 0.49 0.991
Cardamum 10 18 1.43 0.41 0.990
30 24 2.46 0.74 0.992
50 30 1.25 0.43 0.995
100 84 1.21 0.44 0.995
200 88 1.05 0.32 0.992
Cinnamon 10 32 1.41 0.51 0.997
30 44 1.51 0.45 0.996
50 35 1.59 0.53 0.998
100 24 1.61 0.42 0.997
200 6 0.66 0.40 0.828
Black Pepper 10 77 1.15 0.46 0.997
30 88 1.17 0.46 0.998
50 89 1.22 0.44 0.999
100 85 0.94 0.39 0.999
200 81 0.78 0.41 0.998
Garlic 10 59 0.97 0.48 0.994
30 119 1.10 0.38 0.991
50 135 1.26 0.58 0.993
100 102 1.31 0.63 0.997
200 100 1.19 0.47 0.999
Mix 10 51 1.54 0.61 0.999
30 46 1.57 0.58 0.998
50 42 1.52 0.56 0.995
100 22 1.53 0.48 0.992
200 18 2.16 0.52 0.996
Blank 350 1.69 0.11 0.998
Sludge 111 0.70 0.40 0.966

produce considerable change on biogas production potential upon
increasing the spice concentration.
Analysis of Rm values indicated that the change in spice concen-
tration did not considerably affect the rate of biogas production,
except in few cases such as turmeric and black pepper which
increased or decreased the Rm values as the spice concentration
was increased. Overall, clove, cardamom, cinnamon, and garlic pro-
vided higher Rm values (>1.16 respectively) as compared to other
spices. In most of the cases, the Rm values were lesser than that
obtained in SKW (1.69 mL/gVS/Day) proving that addition of spices
reduced the biogas production. Rm value was found highest in case
of cardamom (2.46 mL/gVS/Day) at 30 mg/L concentration. The
values of lag-phase of biogas production, k (the time required for
biogas production), were found minimum in case of SKW
(0.11 day) which increased in the presence of spices to 0.43–
0.55 days. Higher value of k in presence of spices showed the spices
affected the microbial growth.
3.3. Metal analysis
The metal composition of spices is presented in the Table 4. The
concentration of metals in the spices is higher than the reported in
previous studies carried out in other parts of the world (Umar and
Salihu, 2014) especially Pb (1000–1300 mg/kg), Zn (2500–
3200 mg/kg) and Mn (300–7600 mg/kg). Cd concentration is the
lowest and more or less equal in all the spices analyzed. Ni concen-
tration in chili and coriander was found to be 792 mg/kg and
77 mg/kg, while Co concentration in chili and coriander was found
to be 132 mg/kg and 136 mg/kg, respectively.
4. Discussion
The TS and VS contents of KW as shown in Table 1 indicated
that KW was rich in organic carbon. which was accountable for

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Table 4
Metal concentration (mg/kg) in the selected spices.
Element Clove Garlic Cinnamon
Cd 71 80 74
Co 0 92 96
Cu 323 513 362
Mn 7524 348 2330
Mo 69 42 46
Ni 100 36 247
Pb 1092 1262 1082
Zn 2581 3050 2975
the higher biodegradability and suitability of KW for biogas pro-
duction. The biogas production potential of SKW and activated
anaerobic sludge is presented in the Fig. 1(a). Fig. 1(a) indicated
that the extent of biogas production per gVS of SKW (340 mL/
gVS) is higher than that of acclimatized anaerobic biomass
(119 mL/gVS). The results were obtained in the same range as
per the literature reports (Kubaska et al., 2010; Qiao et al., 2011;
Li et al., 2011). Biogas production potential of KW in the presence
of spices was found lesser than that of SKW which shows the inhi-
bitory effect of spices on the biogas production process. This may
be attributed to presence of antimicrobial property of the some
of the spices used in the Indian curry preparation (Asimi et al.,
2013). On the basis of the average cumulative biogas production
obtained for each spice concentration, it may be suggested that
spice concentration of 30–50 mg/L is least inhibitory. Lower biogas
and CH4 production by the ruminant rumen in the presence of
spices has also been reported by many researchers (Patra 2012;
García-González, 2008; Chaudhry and Mohammad, 2012).These
studies have suggested that the reduction in CH4 and biogas forma-
tion occurs in the presence of spices and the magnitude of inhibi-
tion depends on the chemical composition of spice or herb (i.e.
phenolic compounds, essential oils, etc.) and the type of food and
microorganism.
Essential oil of spices has been reported for their strong antimi-
crobial activities (Bag and Chattopadhyay, 2015). Concentration of
essential oil in spices is shown in Table 5. The amount of essential
oil in the spices is in the order: coriander < garlic < cinna-
mon < chili < black pepper < turmeric < cardamom < clove. Among
the spices used, coriander, chili and garlic supported higher biogas
production which may be attributed to the low essential oil con-
centration in the spices. Lesser the concentration of essential oil,
lesser is the inhibitory effect of the spice on biogas production,
as observed from the experiments. Apart from bioactive compo-
nents, coriander also contains vitamins and minerals which may
further support microbial growth. This could explain why upon
increasing the concentration of coriander (up to 50 mg/L) increased
the biogas production (213 mL/gVS) found in the present study;
nevertheless, higher concentration of coriander also reduced the
biogas production potential. Inhibition of CH4 formation in the
presence of high concentration of coriander has also been reported
earlier in rumens (Jahani-Azizabadi et al., 2014). Garlic is very rich
in sulfur containing constituents mainly c-glutamyl cysteines
(0.9%) and S-alkenyl cysteine sulfoxides, such as alliin (1.8%)
(Amagase et al., 2001). Allicin is known for high antimicrobial
action which could be the reason for inhibition of biogas produc-
tion at higher concentration of garlic. Patra and Yu (2012) showed
that several essential oils such as garlic oil can significantly
decrease CH4 production (by 42% at 0.25 g/L dosage), NH3 produc-
tion, and the abundance and diversity of archaea with increasing
doses and exert adverse effects on ruminal feed digestion and fer-
mentation. From this information, it is speculated that the spices
containing high concentration of essential oils may inhibit the
CH4 yield in anaerobic digesters fed with KW. Benchaar et al.
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Chili Turmeric Cardamom Black pepper Coriander
74 82 74 78 80
132 96 84 60 136
416 282 411 687 456
440 582 1698 1503 728
14 17 20 11 9
792 43 53 105 77
1153 1268 1040 1145 1162
2954 2963 2971 2861 3138
(2008) has reported that essential oils inhibit the fermentative bac-
teria resulting in lower methane production in rumens.
It has been found that heavy metals play important roles in bio-
chemical reactions. Many heavy metals have been overwhelmingly
reported to be inhibitory and toxic to bacteria depending on their
concentrations. Spices are also known for their high metal content
and the toxicity of these metals towards fermentative and metha-
nogenic population which may be responsible for inhibition of bio-
gas production. In our investigation, the maximum metal dosing to
the digestion mixture was approximately 1–2 mg/L due to the
addition of spices. Therefore, another set of experiments was con-
ducted by dosing metals (Cd, Ni, Pb and Zn) in the form of their
salts and the effect on CH4 yield was checked. Fig. 6 depicts the
trends of daily % CH4 yield in the presence of 2 mg/L of metals ions
viz. Cd, Ni, Pb and Zn. Compared to blank SKW, significant differ-
ences in % CH4 was observed in case of Cd, Pb and Zn (p < 0.05)
while the difference was not found to be significant in case of Ni.
This proves that these metals inhibited CH4 generation. The recom-
mended concentration of heavy metals such as Co, Mo, Ni and Pb is
less than 1 mg/L above which the AD is inhibited (Weiland, 2006).
The analysis of metals in the spices (Table 4) showed that chili
and coriander contain significantly high concentration of Co, while
chili contains high concentration of Ni. Ni and Co both are known
to enhance acetoclastic methanogenesis during AD of food waste
(Gustavsson et al., 2013), therefore, higher biogas production in
the presence of chili and coriander can be attributed to high Co
and Ni content. Wellinger et al. (2013), has estimated the mini-
mum threshold concentration of various metals such as Na, Cu,
Cd, Zn, Ni and Pb on biomethanation process and reported that
Zn, Ni, Cu and Cd start inhibiting the biomethanation process at
less than 20 mg/L concentration. Pb is reported for inhibition of
acetate production while Zn increases acetate production during
initial hours after which acetate production is inhibited (Mudhoo
and Kumar, 2013). The toxic effect of heavy metals is attributed
to the disruption of enzyme structure and function by binding of
the metal ions with thiol and other functional groups on the pro-
tein molecules, or by replacing natural prosthetic group metals of
the enzymes responsible for methanogenesis (Yang et al., 2011).
At higher concentration of spices, excess of metal ions may also
cause precipitation of other essential metals leading to the reduc-
tion in the bioavailability of such metal ions, and hence, inhibition
of biomethanation (Osuna et al., 2003). The metals along with
essential oils present in the spices might be the most possible rea-
son for inhibition of methane yield in our experiments on AD of
KW.
Spices have been reported to specifically inhibit the activity of
methanogens and hence, CH4 formation (Kamra et al., 2015).
Patra et al. (2006), reported the extracts of garlic and clove inhibit
in vitro CH4 production. Therefore, though biogas production in the
presence of garlic was higher as compared to other spices but the
CH4 yield was much lower in our study. Garlic and cinnamalde-
hyde alter AD process in favour of propionate and lowering CH4
yield (Blanch et al., 2016). Batch experiments using rumen
 N. Sahu et al. / Waste Management xxx (2017) xxx–xxx 9

methanogenic archaea have revealed that garlic oil constituents
such as diallyl sulphide, allyl mercaptan and allicin at 300 mg/l
concentration reduce CH4 production by 74% and 69%, respectively
which might have been caused due to due to the direct inhibition
the methanogen as reported by Busquet et al. (2005). Higher inhi-
bition in the presence of clove and cinnamon may be attributed to
higher heavy metal concentration and also to the presence of sig-
nificant amount of eugenol (Table 5) which is well reported for
its strong antimicrobial activity (Ali et al., 2005; Michiels et al.,
2007). In addition, bioactive compounds of chili show negligible
antimicrobial effects (Lim, 2016), hence, are showing lesser inhibi-
tory effect on biogas production as compared to most of the spices.
Gompertz model fits well with the experimental data as evident
from the coefficient of determination (R2) values, ranging from
0.83 to 0.99 (Table 3). Large variations in the value of P was
obtained among all the spices and were found to be lesser than

Table 5
Phenolic contents (TPC), Essential oil (EO) concentration with minimal inhibitory concentration (MIC) and major bioactive compounds present in the spices used in this study.

Spice name Common TPC (mg GAE/g Approximate EO MIC of EO (lL/ Major Antimicrobial References
Name DW) content% mL) Component
Capsicum annuam Chili 4.135 <0.5 capsacin Shaha et al. (2013)
Syzygiumaromaticum Clove 144 16–18 0.4–2.5 Eugenol Shan et al. (2007) and Burt (2004)
Cinnamomumverum Cinnamon 63 0.5–2.0 0.125 Cinnamaldehyde, Eugenol Shan et al. (2007) and Mith et al.
(2014)
Coriandrumsativum coriander 2.2769 0.1–0.36 230 linalool Souri et al. (2008) and Ebrahimi
et al. (2010)
Curcuma longa turmeric 0.679 4.0–5.0 >0.2 Curcumin Maizura et al. (2011) and Burt, 2004
Allium sativum Garlic 0.18 0.3–0.5 3000 Allicin Bozin et al. (2008) and Ross et al.
(2001)
Piper nigrum Black 3 2.0–4.5 4.2 Piperidine Shan et al. (2007)
pepper
Elettariacardamomum Cardamom 0.8419 4.0–10 4.2 Limonene Souri et al. (2008)

Fig. 5. CFU count of digestate in AA and LBA media represented as Heat map. Green color represents the value for too numerous to count, yellow color represents the value
from 1000 to 2000 (CFU count) and red color represents the value from 100 to1000 (CFU count). (*AA = Anaerobic Agar, **LBA = Luria Bertani Agar).

Please cite this article in press as: Sahu, N., et al. Evaluation of biogas production potential of kitchen waste in the presence of spices. Waste Management
(2017), http://dx.doi.org/10.1016/j.wasman.2017.08.045
10 N. Sahu et al. / Waste Management xxx (2017) xxx–xxx
Fig. 6. Trends of % methane produced after addition of 2 mg/L metal ions (Co, Cd, Pb
and Zn) to the kitchen waste digestion mixture.
SKW (350 mL/g VS). Similar values of P for KW have been reported
in the literature (Li et al., 2011). This observation also supports the
inhibitory effect of the spices on biomethanation as discussed ear-
lier. Presence of bioactive components might be a reason behind a
longer lag phase for bacterial growth and biomethanation in the
presence of spices. The heat-map shown in Fig. 5 depicts the inten-
sity of CFU count for various concentrations of the spices in the two
media. CFU count was used to determine the population of aceto-
genic and methanogenic bacteria in the presence of different con-
centration of the spices, which could directly indicate if the spices
affected their growth and population during digestion. At lower
concentration of spices (<50 mg/L) the CFU count was very high
in both the media suggesting that both hydrolytic and methano-
genic bacteria grew well in this condition without any resistance.
Upon increasing the concentration of spices, the CFU counts in both
media decreased but the CFU count of LBA bacteria remained lower
than that of AA bacteria in most of the cases, which indicated that
the spices are more effective against the hydrolytic bacteria than
the methanogenic bacteria. These observations further confirm
that the spices show inhibitory effect on bacterial growth and AD
(Leite et al., 2005). The bioactive components present in the spices
inhibit the hydrolysis process by inhibiting growth rate of hydroly-
tic bacteria which in turn reduces the rate of VFA production (Rai
and Chikindas, 2011), necessary for optimum methanogenesis,
ultimately decreasing biogas production.
Stoichiometric mass balance for AD yields CH4 and CO2 in the
ratio of 50:50 for carbohydrate, and protein digestion and 63:37
for fat digestion by assuming complete degradation of substrate
(Ruggeri and Tommasi, 2015) as shown in Eqs. (2)–(4).
Carbohydrate C6 H12 O6 ¼ 3CH4 þ 3CO2









Proteins C12 H25 O7 N3 S þ 5H2 O
¼ 6CH4 þ 3NH3 þ H2 S þ 6CO2









Fats C12 H24 O6 þ 3H2 O ¼ 7:5CH4 þ 4:5CO2






The KW used for this study is represented by the formula
C117.12H253.49O85.49N5.6S, determined by analysis of elemental com-
position of the KW (Sahu et al., 2016). Therefore, according to Bus-
well equation, CH4 and CO2 production using the KW after
complete digestion under ideal conditions is represented by the
following equation:
C117:12 H253:49 O85:49 N5:6 S þ 15:71H2 O
¼ 66:52CH4 þ 50:6CO2 þ 5:6NH3 þ H2 S



The molar composition of the biogas according to Buswell equa-
tion would be 56.49% CH4, 38.17% CO2, 4.53% NH3 and 0.81% H2S. In
the present study, SKW yielded higher % of CH4 than CO2 (Fig. 4).
But in the presence of spices, the volume of biogas (Figs. 1–3),
Please cite this article in press as: Sahu, N., et al. Evaluation of biogas production potential of kitchen waste in the presence of spices. Waste Management
(2017), http://dx.doi.org/10.1016/j.wasman.2017.08.045
CO2 and CH4 (Fig. S1, supplementary information) and their per-
centage in the biogas (Fig. 4) are decreased indicating inhibition
of methanogenesis. In some cases of present study CH4 and CO2%
was <30% and >50%, respectively. Lower CH4 production may be
due to sensitivity of methanogens towards the spices. Inhibition
of hydrogenotrophic methanogenesis would lead to accumulation
of CO2 and H2 along with organic acids (Abbassi-Guendouz et al.,
2012). Further, inhibition of hydrogenotrophic methanogenesis
(and CH4%) may cause other metabolic pathway to redirect the
process for maintaining energy flow of the system by using nitrate,
sulphate or other available electron acceptors which results in the
increase in CO2% along with rise in H2, NH3, H2S concentration in
the biogas. This might make up the remaining volume of the gas
and acting a sink for the H2 present in the substrate. However,
these gases were not analyzed in our experiments and so it is dif-
ficult to establish the mass balance of H2 and other elements. The
shift in CO2/CH4 ratio and non-fulfillment of elemental mass bal-
ance between the substrate and products represented by Eqs.
(2)–(4) could be due to incomplete microbial degradation of sub-
strate, inhibition of methanogenesis in presence of ingredients
having antimicrobial property (spices, heavy metals and essential
oil) and by product accumulation (VFA, ammonia, etc.) (Patra and
Yu (2012)). Moreover, substrate carbon used for the biomass syn-
thesis is not accounted for the mass balance and the dead biomass
present in the anaerobic sludge could be used for biogas produc-
tion. Other factors such as quality of anaerobic biomass & its
microbial population, substrate quality and other environmental
conditions also affect the biogas and CH4 yield.
The presence of high concentration of spices in KW would make
biogas production in a community level anaerobic digester very
difficult. Hence, it is important to adopt some measures to mitigate
the inhibitory effect of spices on biogas production by AD. Co-
digestion of food and kitchen waste with other substrates has also
been reported earlier (Yong et al., 2015; Ratanatamskul and
Manpetch, 2016). Co-digestion of food waste with raw vegetable
cuttings or other wastes such as municipal waste can dilute and
reduce the effect of spices on methanogenesis. In future, investiga-
tion may be done to check the feasibility of co-digestion of such
KW with other suitable organic wastes.
5. Conclusion
Spices can significantly decrease methane production, and the
abundance and diversity of bacteria with their increasing doses
in KW. Each of the spices used in the investigation showed differ-
ð2Þ ent effects on biomethanation of KW, which is attributed to the
composition of metals and bioactive compounds in the spices. Fur-
ther research work can be carried out to improve the performance
ð3Þ of anaerobic digesters fed with KW containing high concentration
of spices. The work described in the present study forms a basis for
looking at active ingredients of various spices for determining pos-
ð4Þ
sible interference of these spices with biogas production using KW.
The findings may also be used for determining the composition and
loading rate of KW for efficient digester performance.
Acknowledgement
The author (Nidhi Sahu) is thankful to Council of Scientific and
Industrial Research (CSIR), New Delhi under CSIR award No. 31/16
(120)/2014-EMR- I for providing financial assistance. Authors are
ð5Þ
also thankful to the Director, CSIR-NEERI, for his invaluable help
in extending all necessary facilities at the institute. Authors would
like to acknowledge Knowledge Resource Center (KRC), CSIR-
NEERI, Nagpur for providing literature resources, IT services and
KRC number (KRCn2016nSEPnEBGDn2) to the manuscript. Authors
 N. Sahu et al. / Waste Management xxx (2017) xxx–xxx
are also show gratitude to Mr. G.S. Kanade and Mr V.M. Shinde for
their assistance in instrumental analysis.
Conflict of interest
There is no conflict of interest.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.wasman.2017.08.
045.
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