Environmental Challenges 5 (2021) 100262

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Environmental Challenges
journal homepage: www.elsevier.com/locate/envc

Application of Biochar on methane production through organic solid waste

and ammonia inhibition
Apourv Pant a,∗, J.P.N. Rai b
a
Research Scholar, Department of Environmental Sciences, Govind Ballabh Pant University of Agriculture and Technology, GBPUAT Pantnagar, Pantnagar,
Uttarakhand 263145, India
b
Department of Environmental Sciences, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, Uttarakhand 263145, India

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

Keywords: The present study discusses the role of biochar in enhancing methane (biogas) production from organic solid waste
Biochar amendments (OSW) employing a co-culture of Pseudomonas aeruginosa and Methanosarcina mazei. The high porosity, alkalinity,
Gompertz model and high ion-exchange capacity of biochar make it an efficient support material for microbial cell growth and
Methane enhancement
proliferation. Here the effect of different doses of biochar on biogas production parameters, i.e., cumulative
Ammonia inhibition
methane production, maximum methane production rate, and lag phases, are studied. The synergistic effect of
Anaerobic digestion
biochar for its supplemental methane production via ammonia mitigation potential is also studied. The results
illustrate a maximum methane yield of 109±0.42 mlCH4 gCarboinitial with a maximum of 54.83±0.20% COD
removal was achieved at 12.5 g/L biochar concentration. Also, there is a significant improvement in the lag phase
from 13.2 ± 0.3 h at control (without biochar) to 8.8±.15 h at the same 12.5 g/L effective biochar concentration.
The effect of ammonia addition revealed low methane production rates, which were subsequently reduced with
the biochar amendment that conversely increased the methane production rates in each experimental batch. Thus
the result showed that biochar addition could significantly affect methane production rates, ammonia inhibition
potential and also showed increased volatile fatty acid generation.

1. Introduction found its application in daily household uses in most rural and urban
towns. The primary method is the biogas plants (Gobar-gas plants) sub-
Energy is the essential requirement for all types of activities derived sidized by India’s government under the National Biogas and Manure
from the burning of fossil fuels or other non-renewable energy sources. Management Programme (NBMMP), 2014.
These conventional energy sources cause much disturbance to the envi- At present, 50–60% of methane is generated from anaerobic diges-
ronment by releasing vast amounts of toxic gasses and chemicals into the tion processes only. Nowadays, several methane production concepts
ecosystem. These pollutants in converse cause global-scale phenomenon and technologies are used. The concept of the generation of methane
such as global warming, air and soil pollution, acid rain, and biodiver- from more potential methanogenic bacteria is gaining interest in re-
sity loss. Therefore, there is a need for a cheap and eco-friendly alterna- cent years (Enzmann et al., 2018). A considerable number of likely mi-
tive energy source. Biogas is a promising fuel that gives a high calorific crobial species have been isolated in recent years for their industrial-
value of 55 KJ/g compared to traditional fuels; liquefied petroleum gas, scale methane production applications, such as Methanosarcina bark-
kerosene, wood, charcoal. Biogas is an environmentally friendly alterna- eri, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and
tive that consists of 55–70% CH4 and 30–50% CO2 , with a trace amount Methanobacterium wolfei, Methanobrevibacter ruminantium, Methanoflo-
of other impurities and H2 S. Conventionally biogas enhancing methods rens stordalenmirensis, etc. (Karrasch et al., 1990; Borner et al., 1991;
include various pre-treatments (thermal, chemical, and biological) or Schmitz et al., 1992). This study uses a co-culture of facultative anaerobe
solvent absorption, pressure/ temperature adsorption, and membrane Pseudomonas aeruginosa and Methanosarcina mazei. P. aeruginosa con-
separation (Bauer et al., 2013). Recently some newer technologies were sumes the leftover oxygen present in the reaction mixture and helps
used to increase process efficiency and production, such as metal and or- create a perfect anaerobic environment for Methanosarcina maezi. This
ganic materials-based adsorption structure and pressure swing adsorp- co-culture technique helps increase methane production by maintain-
tion (Chaemchuen et al., 2013; Shen et al., 2015a). Biogas is often a ing a strict anaerobic microenvironment inside the reaction bottles
readily available and commercialized form of energy in India, which (Yeung et al., 2017; Pant and Rai, 2018). Methanosarcina spp. are a

∗
Corresponding author.
E-mail address: [email protected] (A. Pant).

https://doi.org/10.1016/j.envc.2021.100262
Received 4 March 2021; Received in revised form 30 August 2021; Accepted 31 August 2021
2667-0100/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
A. Pant and J.P.N. Rai Environmental Challenges 5 (2021) 100262

diverse group that shows high growth rates (doubling time 1.1– 1.2 2. Material and methods
days) and high pH (0.8–10) tolerance, compared to other methanogens
(Conklin et al., 2006; Liu et al., 2011; Shin et al., 2011). Methanosarcina 2.1. Feedstock
can tolerate a high concentration of ammonium up to 7000 mg TAN L−1
(total ammonia nitrogen) and can operate in low pH conditions such as The organic waste was collected from the agriculture farm of Pant-
pH 5.0 (Smith, 1966; Calli et al., 2005; Schnurer and Nordberg, 2008). nagar University. It was composed of cow dung waste mixed with lo-
Methanosarcina sp. has the advantage of utilizing both acetoclastic and cal household biological waste such as peels of vegetables, fruits, rice,
the hydrogenotrophic methanogenesis pathway due to their specific tol- bread, and paper. The organic compost is prepared by grinding and mix-
erance towards inhibitors such as fluoroacetate and methyl fluoride ing the waste in smaller-sized particles (2 mm) and then further filtered
(Thauer et al., 2008; Liu et al., 2011). Thus, Methanosarcina sp. is the and dewatered. The feedstock was collected in plastic zip lock bags and
better choice for anaerobic methane production studies. It helps achieve stored in the refrigerator at −4 °C to avoid biological degradation. The
stable growth, higher organic loading rates, and high ammonia toler- characteristics of OSW are mentioned in Table 1 (See supporting file).
ance at low retention times (> 4 days). On the other hand, there are
several studies on the use of Pseudomonas aeruginosa in methanogenic 2.2. Biochar
experiments. In an experiment by Potivichayanon et al. (2011) on bak-
ery waste, Pseudomonas cells increased the methane production from Biochar was procured online from Greenfield Eco Solutions Com-
24.90 to 44.33%; also glycerol, which is essential for anaerobic di- pany, India. Biochar is manufactured through pyrolysis of woody
gestion, showed an increase from 12.83 to 48.10%. Pseudomonas also biomass, i.e., heating the biomass to 400–5000 C in a low oxygen en-
helps in methane formation by forming biosurfactants that help in bet- vironment. Composition of biochar was surface area: 124 m2 g− 1; con-
ter degradation of fatty molecules in the anaerobic digester (Jadav et al., ductivity <1500 μS cm−1 , particle size: 5.4–20.6 mm 83.15% carbon,
2017). 8.23% oxygen, 4.21% hydrogen, 0.39% nitrogen, and 0.44% sulfur (dry
Anaerobic digestion is an oxygen deficit biological process that re- weight basis).
quires the wet phase conditions to operate and produces valuable fuel
gasses such as methane and hydrogen. Anaerobic digestion, in recent 2.3. Experimental design conditions
years, provided a promising and effective approach for organic waste re-
duction, generation of important fuel gasses, and excellent bio-manure The experiment was conducted in the ecotechnology lab at the de-
for crops (Mata-Alvarez et al., 2000; Ariunbaatar et al., 2014). Several partment of environmental sciences, GBPUAT, Pantnagar. The lab exper-
reactions in the anaerobic digestion process include acidogenesis, ace- iments were conducted using 500 ml Duran reagent bottles fitted with
togenesis, hydrolysis, and methanogenesis (Weiland, 2010). Hydrolysis rubber screw caps, which were used as anaerobic fermenters; further,
of organic solid wastes being the rate-limiting step in an anaerobic di- the bottles were connected to the mass-spectrometer with the help of
gestion process (Choi et al., 2006; Rittmann et al., 2008). There is also rubber pipes to analyze gas samples. The gas flow was controlled using
a problem of inhibition while dealing with high N-containing wastes by a second-hand quadrupole (fitted with a pump with a turbo and a rotary)
excess ammonia (Ward et al., 2008). There are specific methods reported to detect low methane levels (ppm) with excellent results. Each bottle
such as ammonia stripping; struvite precipitation, and addition of zeo- was filled with 300 ml of organic solid waste (25 gm/l OSW); pure nitro-
lites that can reduce or scavenge the ammonia ions from the medium gen gas was passed through each bottle for 5 min, sealed with a rubber
(Borja et al., 1993; Ho and Ho, 2012; Calli et al., 2005; Rajagopal et al., cap, and covered with aluminum foil. All glassware and media were au-
2013), but they are all inorganic materials that add towards increased toclaved at 125 °C and 15 psi pressure for about 35 min to avoid any type
COD in the waste effluent. Therefore, there is a need to increase or- of contamination. Then each bottle was inoculated with 50 ml of inocula
ganic solubility and accelerate the biodegradation rate by providing consisting of 25 ml of Pseudomonas aeruginosa and 25 ml Methanosarcina
more surface area for microbial action, thus reducing the sludge load mazei (102 CFU/ml) using a sterile loop. P. aeruginosa creates a perfect
(Khalid et al., 2011). Biochar amendment gives a suitable eco-friendly anaerobic environment for Methanosarcina mazei by consuming the ex-
option in anaerobic digestion processes. Biochar has gained popularity cess oxygen in the reaction mixture. Thus co-culture technique favors
in recent years due to its multidisciplinary application in the agriculture, the increased methane production by maintaining the strict anaerobic
environment, and energy sector (Chen et al., 2019) and proposed im- microenvironment inside the bottles. Different biochar concentrations
pacts on soil carbon and fertility (Ameloot et al., 2013). Biochar provides were supplemented to each bottle with the help of a sterile spatula,
excellent support material for the growth of diverse microorganisms be- as mentioned in Table 1. Before being used in batch experiments, the
cause of its structure and high organic content. Biochar thus increases samples were ground and sieved to a size fraction of 1.8–2 mm for uni-
the soil water retention property, conductivity, porosity, and nutrient form surface area. The pH of the biochar samples was maintained at
retention property (Glaser et al., 2002; Lehmann and Rondon, 2006; 8.50 ± 0.14 (mixed with deionized water at 1:10, w/v). All were re-
Kookana, 2010). Biochar is an excellent additive in the anaerobic di- peated in triplicates with control for each experiment prepared as blank
gestion process because it provides biofilm formation and mitigates am- in Duran reagent bottles, without the addition of biochar, besides the
monia and acid inhibition (Torri and Fabbri, 2014). A 6.7% dose of inocula. The bottles were then incubated at 35–40 °C in the Remi CS-
hydro-char, a type of biochar produced from hydrothermal carboniza- 2014 incubator, and pH was maintained at an optimum range of 6.8–
tion, prevents mild ammonia inhibition and increased methane yield 7.8, using 1 N sodium hydroxide and 1 N hydrogen chloride solution.
up to 32.0% (Mumme et al., 2014). Biochar also helps in reducing the In this experimental set-up, in one batch, only ammonia was added. In
methanogenic lag phase by 30.3% and increases methane production another set-up, batches of biochar were supplemented in increasing con-
by 86.6% (Luo et al., 2015). Similarly, Inthapanya et al. (2012) found centration and ammonia, as depicted in Table 2. The amount of methane
that 1% pyrolytic rick husk increases methane production by 31.0%, production was regularly monitored every 24 h. The effect of different
and further addition did not increase gas production, thus showing the biochar concentrations on ammonia mitigation is also estimated.
concentration-dependent effect of biochar.
This study investigates the role of different concentrations of biochar 2.4. Analytical methods
on cumulative methane yield and its ammonia mitigation potential. This
includes the co-culture of Pseudomonas aeruginosa and Methanosarcina Total Kjeldahl nitrogen (TKN), Total solids (TS), Chemical oxygen
mazei on organic solid waste as a substrate in an anaerobic digestion demand (COD), Volatile solids (VS), Lipids, and Ammonia were assessed
system. by standard methods (APHA 2005). Phenol-sulfuric corrosive strategy

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A. Pant and J.P.N. Rai Environmental Challenges 5 (2021) 100262

Table 1
Batch design conditions for determining the experimental effect of biochar on methane yield.

Batches 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
OSW(g/L) 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25 25
Biochar concentration(g/L) 0 2.5 3.5 5 7.5 10 12.5 15 17.5 20 22.5 25 27.5 30 32.5 35

Table 2
Experimental design conditions for quantifying the effect of biochar on methane pro-
duction profile and ammonia inhibition.

Batch No. OSW (g/L) Biochar conc. (g/L) Ammonia conc. (g/L)

Ammonia with Nil biochar concentration

1 20 Nil 0.5
2 20 Nil 1
3 20 Nil 1.5
4 20 Nil 2
5 20 Nil 2.5
6 20 Nil 3
7 20 Nil 3.5
8 20 Nil 4
9 20 Nil 4.5
10 20 Nil 5
11 20 Nil 5.5
12 20 Nil 6
13 20 Nil 6.5
14 20 Nil 7
15 20 Nil 7.5
16 20 Nil 8
Ammonia with 12.5 g/L Biochar concentration
1 20 12.5 0.5
2 20 12.5 1
3 20 12.5 1.5
4 20 12.5 2
5 20 12.5 2.5
6 20 12.5 3
7 20 12.5 3.5
8 20 12.5 4
9 20 12.5 4.5
10 20 12.5 5
11 20 12.5 5.5
12 20 12.5 6
13 20 12.5 6.5
14 20 12.5 7
15 20 12.5 7.5
16 20 12.5 8

and colorimetric technique were used for quantifying carbohydrates and 𝜆= Lag phase duration (h)
protein. Further, the samples were subjected to 0.45 𝜇m Millipore filter e = 2.7182
paper which removes particulate COD and helps in calculating soluble Inhibition coefficient calculated for specific inhibitor as follows (Eq.
chemical oxygen demand (SCOD). After that, SCOD was evaluated by (3)):
the standard method given in APHA (2005). The following formula (Eq.
𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓 𝑓 𝑖𝑐𝑖𝑒𝑛𝑡
(1)) is used for calculating particulate COD:
𝑀𝑒𝑡ℎ𝑎𝑛𝑒 𝑦𝑖𝑒𝑙𝑑 𝑓 𝑟𝑜𝑚 𝑂𝑆𝑊 𝑤𝑖𝑡ℎ 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟
𝐶 𝑂𝐷𝑝𝑎𝑟𝑡𝑖𝑐𝑢𝑙𝑎𝑡𝑒 = 𝐶 𝑂𝐷𝑇 𝑜𝑡𝑎𝑙 − 𝐶 𝑂𝐷𝑠𝑜𝑙𝑢𝑏𝑙𝑒 = 100 − ∗ 100 (3)
(1) 𝑀𝑒𝑡ℎ𝑎𝑛𝑒 𝑦𝑖𝑒𝑙𝑑 𝑓 𝑟𝑜𝑚 𝑂𝑆𝑊 𝑤𝑖𝑡ℎ𝑜𝑢𝑡 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑜𝑟

2.5. Methane and carbon dioxide (CH4 and CO2 ) analysis 2.6. Statistical analysis

Gas chromatography (GC-2014) Shimadzu is used for methane and The results were analyzed with Minitab® 17.1.0 statistical analysis
carbon dioxide analysis. The column is equipped with a 1.2 m X 3-mm software using a one-way ANOVA statistical test.
diameter capillary column (Porapak Q) and a thermal conductivity de-
tector (TCD). Operating conditions temperature for injector, column, 3. Results and discussion
and detector were 120 °C, 100 °C, and 150 °C, respectively. Same GC
conditions were also used for analyzing volatile fatty acids using a flame 3.1. Effect of biochar amendments on methane production profile
ionization detector (FID).
For evaluating methane potential and maximum methane production Modified Gompertz model equation (Eq. (2)) was correlated with
rate, a modified Gompertz equation was used (Eq. (2)): cumulative methane production data to obtain kinetic parameters using
{ [ ]} regression analysis. There is a significant impact on methane production
𝑅𝑚 𝑋𝑒
𝐺𝑚 = 𝑃𝑥 exp − exp (𝜆 − 𝑡) + 1 (2) rate with biochar amendment. An enhancement in cumulative methane
𝑃 production from 218±2.0 ml to 944.96±4.9 ml is observed when in-
Where Gm = Methane production (ml) at particular reaction time.’ creasing the biochar concentration from nil to 15 g/L (Table 3). And
Px = Total Methane production potential also, there was subsequent volumetric methane production from 0.72
Rm = Rate of maximum methane production (ml/h) LCH4 /Lsubstrate to 3.18 LCH4 /L substrate .

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A. Pant and J.P.N. Rai Environmental Challenges 5 (2021) 100262

Fig. 1. Effect of different concentrations of biochar on methane yield (mlCH4 /gCarboinitial ) (Note: The P-value is < 0.001 at 95% significance, which shows
results are highly significant (see supporting file).

Fig. 2. Biochar supplementation effect on volatile fatty acid profile (Note: The P-value is < 0.001 at 95% significance, which shows results are highly significant (see
supporting file).

Fig. 3. Ammonia supplementation affects methane yield (Note: The P-value is < 0.001 at 95% significance, which shows that results are highly significant (see supporting
file).

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A. Pant and J.P.N. Rai Environmental Challenges 5 (2021) 100262

Table 3 capability easily increases its biomass while digesting different complex
Cumulative methane production (P), maximum methane produc- carbon sources (Xu et al., 2015). A further decrease in methane produc-
tion rate (Rmax ), and lag phase (𝜆) through different concentra- tion was observed when biochar concentration exceeded the 12.5 g/L
tions of biochars.
limits. Viggi et al. (2017) reported a gradual decrease to nil lag phases in
Batches Modified Gompertz Kinetics all biochar-amended bottles before the onset of VFAs degradation com-
P(ml) Rmax (ml/h) 𝜆 (h) R2
pared to the unamended control bottles where the lag phase of almost
10days recorded (Fig. 1).
1(Control) 218±2.0 18.6 ± 1.1 13.2 ± 0.3 0.996
This Figure depicts the effects of various concentrations of biochar
2 517.26±4.1 43.76±0.3 11.53±0.3 0.994
3 610±2.2 51.86±1.4 10.83±0.1 0.998
on methane yield (mlCH4 /gCarboinitial ) based on initial carbohydrate.
4 712.10±1.5 63.86±1.4 10.63±0.2 0.992 From the figure, it was concluded that at 12.5 g/L biochar concen-
5 821.8 ± 1.6 72.13±0.3 9.9 ± 0.1 0.989 tration, a maximum methane yield was achieved, i.e., 23.45 ± 2.1
6 911.93±1.6 80±0.9 9.16±0.1 0.996 mlCH4 /gCarboinitial in control to 109.66 ± 2.5 mlCH4 /gCarboinitial .
7 944.96±4.9 86.23±1.6 8.83±0.1 0.997
However, Mumme et al. (2014), have found no effect on methane yield
8 913.76±1.6 81.73±0.4 9.2 ± 0.3 0.997
9 882.3 ± 1.9 78.16±0.9 9.73±0.2 0.998 even at 8.3 g/L pyrochar concentration. This behavior is attributed to
10 856.83±4.1 75.26±1.1 10.16±0.2 0.998 complex biochar–microbe interactions and the function of continuously
11 815.16±2.7 72.16±0.2 10.3 ± 0.1 0.999 fed anaerobic digesters that need to be studied. The biological methane
12 780.56±1.9 69.03±0.2 10.72±0.1 0.996
content of 46.4 ± 0.7 to 78.0 ± 0.5% is reported in Table 4. Further,
13 739.23±1.2 65.46±0.8 10.83±0.1 0.994
14 703.5 ± 0.8 61.06±0.2 11.06±0.2 0.997
the system reports the only CO2 , with no hydrogen detected in sam-
15 672.13±1.9 57.16±0.4 11.27±0.1 0.994 ples. Therefore, it may be concluded that biochar supplementation helps
16 629.13±3.0 54.16±0.7 11.53±0.20 0.998 in enhanced methane production by supplementing anaerobic bacterial
growth (Viggi et al., 2017; Cai et al., 2016).
Note: The P-value is < 0.001 at 95% significance, which shows
that results are highly significant (see supporting file). Further,
all the R2 values above are close to 1, which explains the vari- 3.2. Effect of biochar amendment on direct cod removal
ability of methane production rate can easily be predicted and
recorded using the modified Gompertz equations. In anaerobic processes, the organic waste is readily metabo-
lized, leading to increase COD removal and methane production rate
(Hutnan et al., 2013). Table 4 showed that the biochar supplementation
Similar observations were reported by Sunyoto et al. (2016). In- increases the COD removal rate in each experiment significantly than
creasing the biochar concentration above 33 g L−1 inhibits the further the control. This depicts the role of biochar amendment in improving
methane yield; the positive effect of biochar is directly related to the microbial activities while increasing the biochar concentration from nil
biochar’s electron-donating capacity (EDC) (Viggi et al., 2017). Signifi- to 12.5 g/L also increases the COD removal efficiency from 32.7 ± 0.3%
cantly few scientists reported the full-scale study of the effect of biochar to 54.8 ± 0.2%. However, after increasing the biochar concentration
on methane yield in an anaerobic system like Meyer-Kohlstock et al. above 12.5 g/L, the COD removal efficiency started decreasing, with a
(2016) observed the increasing trend of cumulative methane produc- minimum of 41.2 ± 1.0% biochar concentration of 35 g/L.
tion from 5 to 10% while increasing the biochar concentration from Biochar application helps in reducing NH3 emission and subse-
5% (dry weight of organic waste) to 10%. Biochar composition plays quently increases COD removal, corresponding to high methane pro-
an important role; for example, in its in-situ experiments, Linville et al. duction (Maurer et al., 2017). Different fractions of OSW such as lipid,
(2017) observed that fine shell biochar increases the methane yield by carbohydrate, and protein were estimated related to the COD removal.
77.5%–98.1% CH4 compared to coarse shell biochar of 78.9% CH4 . In contrast, carbohydrates contribute the maximum towards methane
Similarly, an on-field experiment conducted by Wang et al. (2019) ob- production, followed by lipids and proteins, respectively (Table 4). The
served a 0.5–37.5% increase in CH4 when N-fertilizer is amended with percentage contribution of carbohydrates, proteins and lipids towards
biochar. The fine quality or surface area of biochar is crucial for op- COD removal was observed as 69.1 ± 0.2% to 81.1 ± 0.3%, 4.6 ± 0.1 to
timum methanogenic activity; the high surface area of biochar results 14.2 ± 0.7% and 8.9 ± 0.2 to 28.1 ± 0.2%, respectively. Nielfa et al.
in higher methane activity (>90%) by enhancing the methane yield, (2015) and Labatut et al. (2011) have compared the COD and BMP
bio methanation rate constant, and maximum methane production rate method for determining the methane yield with lower error; he found
by up to 7.0%, 8.1%, and 27.6%, respectively (Shen et al., 2015b). that COD methods showed well for co-digestion while BMP showed
Torri and Fabbri (2014) studied the biochar effect on APL (Aqueous good results in terms of complex substrate dairy manure or corn silage.
pyrolysis liquid), increasing the theoretical methane yield by 65±5%. Nielfa et al. (2015) observed that lipids contribute more towards bio-
The higher surface area of biochar helps in biofilm formation that gas production (1m3 per kg of volatile solids) than proteins and car-
accommodates a wide range of microorganisms, thus enhancing micro- bohydrates. Effective carbohydrates, lipid, and protein (40:40:20) are
bial activity and shortening the lag phase, and boosting the exponential important for methane production as they can balance the acidification
phase, i.e., Enhance methane production (Cooney et al., 2016). Biochar and methanation in the system (Johnson and Johnson, 1995; Xue et al.,
supports microbial metabolism and growth by acting as a good elec- 2019). The carbon fraction of carbohydrates are easily digested com-
tron exchanger (APHA 2015). Similarly, in Gompertz, kinetic param- pared to the lignin–associated cellulosic components that result in in-
eters, i.e., cumulative methane potential (p), lag phase (l), and max- creased methane production and more effective substrate utilization
imum methane production rate (Rmax ), we’re dependent on effective (Mulat et al., 2018).
biochar concentration (Table 3). Here maximum methane production
rate (Rmax) increased from 18.6 ± 1.1 ml/ h at control (0 g/L) to 3.3. Biochar amendment effects on volatile fatty acid generation rate
86.23 ± 1.6 ml/h at 12.5 g/L concentration of biochar. Subsequent
biochar amendment decreases the Lag phase from 13.2 ± 0.3 h at control Volatile fatty acids generation is directly correlated with the
to 8.83 ± 0.1 h at 12.5 g/L concentration of biochar. Similar results were methane production rate in an anaerobic digestion process.
reported by Sunyoto et al. (2016), which showed a decrease in lag phase Therefore, quantifying its concentration and distribution is in-
by 41–45% and an increased maximum production rate by 23.0– 41.6% dicative of methane production and monitoring. An enhanced
and CH4 production potential by 1.9–9.6% of CH4 . The Syntrophic asso- concentration of volatile fatty acids reported in all the batched
ciation of Pseudomonas aeruginosa and Methanosarcina mazei increases with biochar supplementation, propionate, acetate, and butyrate,
the cumulative methane yield, while Methanosarcina’s high metabolic was detected in all the batches (Fig. 2).

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A. Pant and J.P.N. Rai Environmental Challenges 5 (2021) 100262

Table 4
Biochar supplemented effect on methane production profile and COD removal.

Batches CH4 (%) CO2 (%) H2 (%) COD removal (%) Carbohydrate removal (%) Protein removal (%) Lipid removal (%)

1 46.4 ± 0.7 54.4 ± 0.4 Nil 32.7 ± 0.3 77.4 ± 0.4 4.6 ± 0.1 20.5 ± 0.1
2 53.7 ± 0.2 47.7 ± 0.4 Nil 39.7 ± 0.4 76.1 ± 0.1 4.6 ± 0.2 21.4 ± 0.2
3 58.8 ± 0.2 42.9 ± 0.7 Nil 43.9 ± 0.3 73.6 ± 0.4 6.9 ± 0.2 22.8 ± 0.3
4 68.5 ± 0.6 31.5 ± 0.4 Nil 48.9 ± 0.1 78.8 ± 0.2 6.2 ± 0.6 17.5 ± 0.1
5 74.9 ± 0.4 28.1 ± 0.2 Nil 52.8 ± 0.4 69.1 ± 0.2 5.0 ± 0.2 28.1 ± 0.2
6 76.2 ± 0.3 27.1 ± 0.3 Nil 53.6 ± 0.2 74.9 ± 0.1 8.9 ± 0.3 19.1 ± 0.2
7 78.0 ± 0.5 26.7 ± 0.3 Nil 54.8 ± 0.2 79.1 ± 0.2 13.4 ± 0.3 14.5 ± 0.8
8 76.5 ± 0.4 25.4 ± 0.2 Nil 50.3 ± 0.6 76.4 ± 0.3 9.4 ± 0.4 20.7 ± 0.5
9 77.5 ± 0.3 26.4 ± 0.2 Nil 49.4 ± 0.3 81.1 ± 0.3 10.9 ± 0.2 15.1 ± 0.2
10 76.1 ± 0.2 27.4 ± 0.1 Nil 49±0.1 73.7 ± 0.6 10.3 ± 0.1 22.4 ± 0.3
11 74.9 ± 0.1 29.1 ± 0.2 Nil 47.2 ± 0.3 71.0 ± 0.3 11.4 ± 0.2 24.5 ± 0.4
12 71.4 ± 0.2 31.4 ± 0.4 Nil 45.2 ± 0.4 77.8 ± 0.8 9.4 ± 0.3 19.9 ± 0.1
13 67.9 ± 0.3 35.4 ± 0.4 Nil 43.2 ± 0.2 73.4 ± 0.2 8.3 ± 0.2 14.7 ± 0.3
14 62.9 ± 0.1 39.2 ± 0.5 Nil 43.5 ± 0.2 79.6 ± 0.8 10±0.3 12.8 ± 0.2
15 59.3 ± 0.4 42.4 ± 0.4 Nil 41.1 ± 0.2 80.9 ± 0.7 14.2 ± 0.7 8.9 ± 0.2
16 57.3 ± 0.5 45.6 ± 0.3 Nil 40.0 ± 0.1 80.3 ± 0.5 13.8 ± 0.1 12.2 ± 0.3

Note: Note: The P-value is < 0.001 at 95% significance, which shows results are highly significant (see supporting file).

Experimental batches recorded a lower propionate concentration fol- Table 5

lowed by higher butyrate and acetate when a lower amount of biochar, Combined effects of ammonia concentrations and biochar addition on cu-
i.e., < 12.5 g/L, was applied. But as the concentration of biochar in- mulative methane production, maximum methane production rate, and
lag phase.
creased (> 12.5 g/L), a subsequent increase in propionate production
with a relative reduction in butyrate and acetate concentration was ob- Experimental Batches Gompertz kinetics model
served. This indicates that the butyrate metabolic pathway is favored P (ml) Rmax (ml/h) 𝜆 (h) R2
at lower biochar concentrations, while organic overloading (higher
Ammonia addition + Nil biochar
biochar concentration) shifts towards the propionate pathway. A prob-
1 226.5 ± 0.8 20.0 ± 0.3 13.1 ± 0.2 0.998
able reason for this type of metabolic shift is due to cis-unsaturated 2 248.4 ± 0.6 22.2 ± 0.3 12.5 ± 0.4 0.999
fatty acids type inhibition that inhibits the methane production while 3 281.8 ± 1.1 26.0 ± 0.3 11.8 ± 0.2 0.996
increasing the propionic acid production when we increase the biochar 4 268.1 ± 0.2 24.5 ± 0.4 12.0 ± 0.2 0.997
concentration >12.5 g/L (Demeyer and Henderickx, 1967; Pereira et al., 5 249.5 ± 0.8 23.5 ± 0.2 12.3 ± 0.3 0.995
6 238±0.4 22.3 ± 0.3 12.4 ± 0.3 0.998
2004). Awasthi et al. (2018) also found that an 8–10% biochar amend-
7 220±0.7 21.2 ± 0.3 12.6 ± 0.2 0.994
ment increased methane production by reducing the VFAs profiles. Con- 8 210.5 ± 0.7 20.4 ± 0.3 12.8 ± 0.3 0.999
sequently, a higher dosage of biochar (> 8%) provides higher buffer ca- 9 199.4 ± 0.7 19.6 ± 0.7 13.1 ± 0.2 0.998
pacity, increased porosity, and optimum moisture level of OSW that re- 10 188.5 ± 0.5 18.4 ± 0.2 13.2 ± 0.4 0.994
11 175.7 ± 0.7 17.6 ± 0.3 13.5 ± 0.2 0.989
sults in optimized pH and temperature profile throughout the anaerobic
12 163.4 ± 0.5 16.5 ± 0.3 13.7 ± 0.2 0.996
treatment duration that conversely enhance the rate of composting and 13 143.6 ± 0.5 15.5 ± 0.3 13.8 ± 0.1 0.997
its end quality products, i.e., methane (Awasthi et al., 2017). Further, 14 130.1 ± 1.2 14.4 ± 0.3 14.3 ± 0.3 0.995
increasing the contact time/incubation period up to four weeks, there 15 110.3 ± 0.6 13.5 ± 0.4 15.0 ± 0.2 0.998
is a subsequent reduction in VFAs profiling (> 95%) with an increase in 16 102.4 ± 0.7 12.4 ± 0.3 15.5 ± 0.5 0.999
Ammonia addition + biochar
methane yield (Amani et al., 2011).
1 889.3 ± 0.8 77.0 ± 0.4 10.1 ± 0.2 0.998
2 909.6 ± 0.7 77.4 ± 0.3 10.6 ± 0.1 0.999
3.4. Biochar amended effect on ammonia inhibition rate4 3 917.8 ± 0.9 77.9 ± 0.3 9.8 ± 0.4 0.996
4 887.6 ± 0.5 80.6 ± 0.2 10.2 ± 0.2 0.986
5 865.5 ± 0.5 78.6 ± 0.4 10.7 ± 0.2 0.997
Increased ammonia production directly inhibits the methane pro-
6 830.2 ± 0.7 75.6 ± 0.4 11.0 ± 0.1 0.998
duction rate. Table 5 depicts the enhancement effect on cumulative 7 808.2 ± 0.6 73.7 ± 0.5 11.3 ± 0.3 0.995
methane production and maximum methane production rate and lag 8 776.1 ± 1.0 69.6 ± 0.4 11.7 ± 0.1 0.998
phase parameters by suitable ammonia addition. A 1.889 g/L ammo- 9 766.1 ± 0.4 68.4 ± 0.1 12.1 ± 0.2 0.994
nia concentration observed a slight increase in methane production 10 725.4 ± 0.8 64.7 ± 0.4 12.3 ± 0.1 0.998
11 701.0 ± 1.0 62.3 ± 0.7 13.1 ± 0.3 0.989
and methane yield (Fig. 3). Subsequent higher methane production of
12 667.1 ± 1.0 59.8 ± 0.2 13.3 ± 0.3 0.996
281.8 ± 1.1 ml was obtained at 1.889 g/L ammonia concentration com- 13 634.5 ± 0.9 53.6 ± 0.3 13.5 ± 0.3 0.994
pared to 226.5 ± 0.8 ml methane at control. Similarly, there is an in- 14 603.6 ± 1.5 49.9 ± 0.1 13.8 ± 0.2 0.997
creased methane yield from 25.6 mlCH4 /gCarboinitial in control to 34.6 15 577.7 ± 1.5 46.3 ± 0.4 14.2 ± 0.2 0.998
16 546.5 ± 0.6 44.0 ± 0.2 14.6 ± 0.1 0.997
mlCH4 /gCarboinitial at 1.889 g/L ammonia concentration (Fig. 3). At the
same time, a fluctuation of maximum methane production rate and lag Note: Note: The P-value is < 0.001 at 95% significance, which shows re-
phase is observed at a lower concentration of ammonia (< 1.889 g/L) sults are highly significant (see supporting file).
(Table 5). With the subsequent increase in ammonia concentration,
there is an apparent cut decrease in methane production (Table 5) and
methane yield (Fig. 3). Biochar causes the available ammonia to get ab- crobial growth at lower concentrations, followed by a slight increase in
sorbed and reduces its inhibition property by converting free ammonia methane production at a lower concentration. However, a higher ammo-
to bioavailable nitrogen (Tu et al., 2020). Taghizadeh-Toosi et al. (2012) nia concentration interrupts anaerobic digestion by restricting microbial
in an N15 tracer study, observed that the biochar amendment increases growth and its metabolic activities. A comprehensive review by many
the ammonia bio-availability to plants. In a similar study, Mumme et al. scientists also suggests that ammonia inhibition is an important param-
(2014) has reported a mild ammonia inhibition via physical adsorption eter when it comes to anaerobic digestion (AD) processes and methano-
by pyrochar as 2.1 g TAN kg−1 . This shows that ammonia supports mi- genesis (Rajagopal et al., 2013; Yenigün and Demirel, 2013). NH3 /NH4

6
A. Pant and J.P.N. Rai Environmental Challenges 5 (2021) 100262

Fig. 4. Combined effects of ammonia and biochar supplementation on methane yield (Note: The P-value is < 0.001 at 95% significance, which shows that results are
highly significant (see supporting file).

ratio disturbs the pH change by absorbing protons (H+ ) and subse- 13.2 ± 0.3 h to 8.83±0.1 h in control and 12.5 g/L biochar experimental
quently causes inhibition of specific enzyme reactions and increases the batches.
cell system’s maintenance energy (Muller et al., 2004; Wittmann et al., Further, statistical analysis of the results using the one-way ANOVA
1995). test indicates the model’s suitability. Results with p-values come out to
Results depicted in Table 5 indicate lower ammonia toxicity when be less than 0.001 at a 95% level of significance. Thus the effective con-
using biochar. It also shows increased methane production and yields centration of biochar for all the Gompertz parameters found out to be
experimental batches supplemented with biochar and ammonia com- 12.5 g/L, above which there is the onset of inhibition. It can also be con-
pared to batches without ammonia. Experimental batches 1, 2, and cluded that biochar proves to treat the ammonia inhibition that directly
3 with biochar showed increased cumulative methane production to affects methane production effectively. As a biochar supplement of a
889.3 ± 0.8, 909.6 ± 0.7, and 917.8 ± 0.9 ml with ammonia com- concentration up to 12.5 g/L, there is a significant increase in ammonia
pared with experimental batches only ammonium concentration of inhibition and methane production.
0.889 g/L without biochar. This supports the observation that lower
ammonia concentration is suitable for microbial growth, i.e., good Declaration of Competing Interest
methane yield and vice versa. Further, increasing the ammonia con-
centration > 1.889 g/L and constant biochar concentration of 12.5 g/L, The authors declare that they have no known competing financial
there is a decline of cumulative methane production compared with the interests or personal relationships that could have appeared to influence
batched having biochar supplemented with ammonia. The ammonia ad- the work reported in this paper.
dition on methane yields indifferent experimental batches supplemented
with/without biochar addition is shown in Figs. 3 and 4, respectively. Acknowledgement
Thus results indicate the effectiveness of biochar in effective ammonia
mitigation and increasing the methane yield. The author would like to acknowledge the financial and instrumental
Similarly, several researchers concluded that ammonia is one of support provided by the Department of Environmental Sciences, Ecotox-
the inhibitory substances in anaerobic processes (Luz et al., 2018; icology lab, College of Basic Sciences and Humanities, Govind Ballabh
Mumme et al., 2014), where biochar is a promising solution for provid- Pant University of Agriculture and Technology Pantnagar, Uttarakhand,
ing reaction surface area for anaerobic growth and mitigating the am- India.
monia inhibition, thereby increasing the methane yield (Li et al., 2019;
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