Accepted Manuscript

Impact of Metal Nanoparticles on Biogas Production from Poultry Litter

Amro Hassanein, Stephanie Lansing, Rohan Tikekar

PII: S0960-8524(18)31709-7
DOI: https://doi.org/10.1016/j.biortech.2018.12.048
Reference: BITE 20805

To appear in: Bioresource Technology

Received Date: 8 November 2018

Revised Date: 14 December 2018
Accepted Date: 15 December 2018

Please cite this article as: Hassanein, A., Lansing, S., Tikekar, R., Impact of Metal Nanoparticles on Biogas
Production from Poultry Litter, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.12.048

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Impact of Metal Nanoparticles on Biogas Production from Poultry Litter

Amro Hassaneina,b, Stephanie Lansinga,*, Rohan Tikekarc

a
Department of Environmental Science and Technology, University of Maryland, College

Park, MD 20742, USA.

b
Faculty of Environmental Agricultural Sciences, Arish University, North Sinai, Egypt
c
Department of Nutrition and Food Science, University of Maryland, College Park, MD

20742, USA.
*
Corresponding author Tel.: +1 301 405 1197; fax: +1 301 314 9023. E-mail address:

slansing@umd.edu

Abstract

The effects of metal nanoparticle (NP) addition during anaerobic digestion (AD) of

poultry litter was tested using two sequential experiments: Exp. A) four NPs (Fe, Ni, Co, and

Fe3O4) at three concentrations; and Exp. B) NP combinations (Fe, Ni, and Co) at four

concentrations. Scanning electronic microscopy (SEM) and elemental analysis were used to

confirm NP inclusion after dispersion (before AD) and track nanoparticles post-AD, and new

technique for NP extraction post-AD was developed. Before AD, NPs ranged from 30.0 -

80.9 nm for Fe, Ni, and Co, and 94.3 – 400 nm for Fe3O4. Methane production increased with

NPs addition compared to poultry litter-only, with the highest increases observed with NPs

concentrations (in mg/L) of 12 Ni (38.4% increase), 5.4 Co (29.7% increase), 100 Fe (29.1%

increase), and 15 Fe3O4 (27.5% increase). Nanoparticle mixtures greatly decreased H 2S

production. The SEM post-AD detected Fe, Ni, and Fe3O4 at concentrations  100mg/L.

Keywords: Anaerobic digestion, Nanotechnology, Methane, Hydrogen Sulfide

1
1.0 Introduction

Anaerobic digestion (AD) reduces organic pollution while creating renewable energy in

the form of methane (CH4)-enriched biogas, however, AD is a relatively slow microbial-

based process that is dependent on temperature, pH, hydraulic retention time (HRT), and

C:N ratio (Feng et al., 2014). In the United States, the poultry industry generates over 9

million tons of poultry litter per year from broiler operations (Perera and Bandara, 2010).

Poultry litter consists of the animal waste, feed residue, and the bedding material, which

contains wood chips. Although AD has been used extensively for treating dairy manure,

food waste, and swine waste, it remains underutilized in the poultry industry due to the

complex nature of poultry litter and high ammonium (NH4) concentrations (Gungor-Demirci

and Demirer, 2004). Recent research to improve the AD of poultry litter includes multi-stage

digestion (Rao et al., 2011), reducing NH4 concentrations (Abouelenien et al., 2010), co-

digestion with other animal wastes (Gungor-Demirci and Demirer, 2004), and enhancement

of hydrolysis through thermochemical pre-treatment and bioaugmentation (Costa et al.,

2012). In addition, hydrogen sulfide (H2S) concentrations in poultry litter-derived biogas are

often high, ranging from 5,000–30,000 ppm (Arif et al., 2018; Schieder et al., 2003). The

H2S must be removed to avoid corrosion of storage tanks, compressors, and engines, and to

reduce health and safety concerns due to its high toxicity (Hu et al., 2018).

The use of additives in AD could significantly improve performance (Yadvika et al.,

2004). Trace metals are microbial nutrients that play a significant role in AD performance

(Demirel and Scherer, 2011). Specifically, the biochemical processing in methanogens has

been shown to be reliant on Zn, Fe, Ni, Mo, and Co for enzyme synthesis (Thauer et al.,

2008). Trace metal additions have been shown to increase degradation rates and increase CH4

production in pure and mixed cultures (Abdelsalam et al., 2016; Gustavsson et al., 2013).

2
However, high concentrations of trace metals can lead to toxicity and microbial inhibition.

Previous AD studies have shown that Fe additions, including zero valent iron (ZVI), in dairy

manure and poultry litter AD increased CH4 production, decreased organic pollutants in the

effluent, and shortened the HRT in the reactor (Demirel and Scherer, 2011; Feng et al.,

2010).

Nanotechnology is the engineering and art of manipulating matter at the nanoscale (1-100

nm) and considered to be one of the most important advancements in science and technology

of the last few decades (Lucia and Rojas, 2009). Nanoparticles (NP), compared to atomic or

bulky counterparts, have shown superior physical and chemical properties due to their

mesoscopic effect, small object effect, quantum size effect, and surface effect. Recently,

Fe3O4 magnetic nanoparticles (MNPs) have been investigated due to their super

paramagnetism, high coercivity, non-toxicity, and biocompatibility. Mu et al. (2011a) found

that adding three metal oxide nanoparticles (nano-TiO2, nano-Al2O3, and nano-SiO2) to AD

did not inhibit CH4 generation, while nano-ZnO was inhibitory at doses > 6 mg/gTSS.

Abdelsalam et al. (2016) found that adding NPs (Co, Ni, Fe, and Fe3O4) to AD of livestock

urine and feces increased the CH4 yield by 2.0, 2.2, 1.7 and 2.2 times, respectively, compared

to the control, with the nano-Ni addition yielding the highest CH4 production. No prior

studies have evaluated NP additions to poultry litter AD and tracked the NP degradation in

the AD environment. In addition, while Fe NPs were shown to reduce H 2S in biogas and

significantly increase CH4 production in certain cases (Li et al., 2007; Wang et al., 2016),

prior studies have not determined the most effective concentration of NPs in poultry litter to

remove H2S effectively or the effect of NP mixtures on both H2S and CH4 concentrations in

the produced biogas.

The objectives of this study were: 1) to determine the effects of four types of NPs (Fe, Ni,

Co and Fe3O4) at various concentrations on CH4 and H2S production during the AD of

3
poultry litter, 2) to determine the effects of mixtures of NPs (Fe, Ni, and Co) at various

concentrations on CH4 and H2S production, and 3) to verify NP inclusion prior to AD, track

the degradation of NPs post-AD, and verify an appropriate method to collect the metal NPs

after AD. The results of this study can be used to better understand the effect of

nanotechnology in increasing biogas production and utilization and how to track NP size and

distribution in AD digestate.

2.0 Materials and Methods

2.1 Experimental design

The study consisted of two sequential experiments (Figure 1) using biochemical

methane potential (BMP) tests. The first experiment (Exp. A) quantified the effect of NP

addition (Fe, Ni, Co, and Fe3O4) on AD of poultry litter using three NP concentrations in

triplicate reactors, and the second experiment (Exp. B) quantified the effect of using

nanoparticles mixtures (NPM) of Fe, Ni and Co through testing of four different NPM

concentrations in triplicate reactors, with comparisons to triplicate poultry litter-only reactors

in both experiments. Tables 1 and 2 detail the quantity of poultry litter, inoculum, and

specific concentrations (in mg/L and mg/g VS) of NP additions for Exp. A and B. In Exp. A,

all triplicate reactors, including the poultry litter-only reactors, contained inoculum (185.9 g),

poultry litter (4.11 g), and 0.1 mM sodium dodecylbenzene sulfonate (SDS) solution (10 mL)

used for nanoparticle dispersion. In one set of triplicate poultry litter-only reactors, DI water

(10 ml) was added in place of SDS to test the effect of the solution on CH 4 production. In

Exp. B, all triplicate reactors contained inoculum (185.9 g), poultry litter (4.11 g), and 0.1

mM SDS solution (32.7 mL), with one triplicate set of poultry litter-only reactors containing

only DI water (32.7 mL) in place of the SDS solution.

4
2.2 Substrate and inoculum utilized

Poultry litter was collected from a poultry house in Pocomoke City, MD, USA and

stored at < -4°C for one week. Total solids (TS) and volatile solids (VS) of the poultry litter

and inoculum were measured according to Standard Methods (APHA, 2005). A substrate to

inoculum ratio of 1:1 (by VS) was used for each reactor. The inoculum was collected from a

digester located at the Beltsville Agricultural Research Center (BARC) in Beltsville, MD,

USA. While the source of the poultry litter and inoculum for Exp. A and B were the same,

the TS and VS concentrations were slightly different. For Exp. A, the poultry litter had an

average TS and VS concentration of 80.8 ± 0.028% and 65.2 ± 0.22%, respectively, and the

inoculum had a TS and VS concentration of 2.19 ± 0.003% and 1.45 ± 0.003%, respectively,

with a pH of 7.70 ± 0.051. For Exp. B, the poultry litter had an average TS and VS

concentration of 77.2 ± 0.445% and 61.6 ± 0.851%, respectively, and the inoculum had a TS

and VS concentration of 2.19 ± 0.025% and 1.39 ± 0.005%, respectively, with a pH of 7.72 ±

0.024. There were significant differences between the poultry litter utilized for % TS (p-value

<0.001) and % VS (p-value = 0.005) based on wet weight, however, there was not a

significant difference in VS dry weight (p-value = 0.237) in Exp. A and B indicating

differences in moisture content but not necessarily in organic content.

The total Fe, Ni, and Co were determined in the poultry litter using USEPA 3051

method (USEPA, 1994). The poultry litter used in Exp. A had Fe, Ni, and Co concentrations

of 173, 11.4, and 0.48 mg/kg, respectively. The poultry litter used in Exp. B had Fe, Ni, and

Co concentrations of 126, 13.4, and 0.48 mg/kg, respectively.

2.3 Biochemical Methane Potential (BMP) Test

The BMP tests were conducted at the University of Maryland’s Department of

Environmental Science and Technology’s (ENST) Water Quality Laboratory. The BMP test

5
is a laboratory batch study used to characterize CH 4 production potential developed by Owen

et al. (1979). Substrate and inoculum were added into 500 mL serum bottles, purged with N 2

gas to establish anaerobic conditions, capped, and incubated at 35ºC in an environmental

chamber. Biogas, CH4, and H2S concentrations were monitored at regular intervals for 69

days for Exp. A, and 79 days for Exp. B. Experiments were conducted until biogas

production had largely ceased, with the daily biogas production < 1% of the total biogas

production for most treatments. Triplicate controls containing only inoculum were incubated

to allow subtraction of biogas not attributed to the manure substrate (Table 2).

The produced biogas volume was determined using a graduated, gas-tight, wet-tipped 50

mL glass syringe inserted through a three-way valve connected to the septum and

equilibrated to atmospheric pressure. The produced biogas was analyzed for CH 4, CO2 and

H2S content by injecting 0.10 mL sample, using a luer-lock, gas tight syringe, into an Agilent

HP 7890A gas chromatograph (Agilent Technologies, Santa Clara, CA, USA), equipped with

a thermal conductivity detector (TCD) using the following parameters: (1) injection

temperature of 250 ºC; (2) detector temperature of 250 ºC; (3) oven temperature of 60 ºC; and

(3) a carrier gas flow rate of 8.6 mL He/min.

2.4 Nanoparticles preparations

Four types of NPs (Fe, Ni, Co, Fe3O4) were used in Exp. A and three types were used in

Exp. B (Fe, Ni, and Co). NPs were purchased as powder (US Research Nanomaterials, Inc.

Texas, USA) and transferred into a dark glass bottle. The NP transfer process was conducted

inside a glove box using N2 as inert gas to avoid oxidation or heating. Sodium

dodecylbenzene sulfonate (SDS) at 0.1mM was used as a dispersing reagent to ensure NPs

dispersion prior to use, as SDS has been shown to not significantly effect CH4 production

(Wang et al., 2016; Mu et al. 2011b). Two blank tests were performed to verify this: 1) a SDS

6
concentration of 0.1 mM without NPs inclusion, and 2) no SDS and no NPs (Tables 1 and 2).

To increase NP dispersion prior to AD, a probe ultrasonication (Misonix S-4000, US) was

used for 25 min at 40 KHz, within an ice bath, to decrease the process temperature (Wang et

al., 2016) (Figure 1).

2.5 Scanning electronic microscopy (SEM)

A scanning electronic microscopy (SEM) with energy-dispersive X-ray spectroscopy

(EDXA) function was used to confirm the NPs after dispersion but before AD, and to track

the NPs after AD (TESCAN XEIA FEG SEM, Czech Republic).

Prior to SEM, the NPs were present at low concentrations within a mixed, high solids,

heterogeneous manure and inoculum mixture and needed to be effectively collected and

prepared. No previous study had detailed methods to collect the NPs inside of an AD system.

In order to effectively collect the NPs, three methods were tested: 1) liquid drops (no solids)

were collected from four reactors after AD of the following NPs (in mg/L) (100 Fe, 12 Ni,

5.4 Co, and 100 Fe3O4), dried inside a closed incubator (35 °C) for approximately 20

minutes, and then coated with carbon before SEM and EDXA scans; 2) liquid drops and

solids particulates were collected from the four reactors, as described above; and 3) liquid

and solid samples were collected from inside the reactors using a magnetic stir bar covered

with plastic parafilm. This was achieved by placing the post-AD reactor on a magnetic stirrer

(medium speed; laminar flow) for 20 minutes. During the stirring process, the NPs were

attracted to the bottom of the reactor due to the magnetic nature of the stir plate on which the

reactor was placed. Once the magnetic stir bar covered with plastic parafilm was inserted into

the reactor, it forced the NPs from the bottom of the reactor onto the inserted stir bar, which

had a higher magnetic force. After collection, the plastic parafilm was carefully removed, and

a section of the collected material attached to the plastic parafilm was transferred to SEM

7
sample plate, with air drying of the sample on the plate (Figure 1). For all methods, carbon

coating was used in the SEM scans, instead of gold NP coating, to avoid adding additional

NPs prior to the scan.

2.6 Statistical analyses

ANOVA and Tukey-Kramer analyses were performed on the cumulative CH4 production

values of the triplicate vessels to analyze the effect of adding the NPs to AD. The statistical

analyses were conducted using SPSS with an alpha value of 0.05. Values are given as

averages with standard error (SE).

3.0 Results and Discussion:

3.1 Exp. A: Effect of NP addition (Fe, Ni, Co, and Fe3O4) on AD of poultry litter

In Exp. A., nanoparticle (NPs) inclusion significantly increased CH4 production by 23.8 –

38.4% compared to poultry litter-only AD for all four NPs included at each of the three

concentrations tested (p-value = 0.008; Figure 2). The highest increases in CH4 production,

compared to the poultry litter-only reactor, were observed with the following concentrations

of NPs (in mg/L): 12 Ni (38.4% increase; p-value < 0.001), 5.4 Co (29.7% increase; p-

value = 0.001), 100 Fe (29.1% increase; p-value =0.002), and 15 Fe3O4 (25.6% increase; p-

value = 0.003 (Figure 2; Table 3). The poultry litter-only reactors produced an average of 266

mL CH4/g VS. The poultry litter-only AD with 0.1 mM SDS (NP dispersion solution) was

not significantly different than poultry litter-only reactors with DI water added (p-value =

0.999), which was consistent with previous research by Wang et al. (2016) and showed that

the SDS inclusion was an appropriate NP dispersion solution choice, as it did not affect CH 4

production.

8
 The trace metals, added as NPs in this study, were likely utilized as micronutrients by

microbial biomass in the AD reactors. Trace metals have been shown to increase degradation

rates and CH4 production (Abdelsalam et al., 2016; Demirel and Scherer, 2011; Feng et al.,

2010; Gustavsson et al., 2013), with Fe, Co, and Ni shown to specifically stimulate

methanogenic growth (Krongthamchat et al., 2006). A recent study by Molaey et al. (2018a)

suggests that poultry manure may be deficient in some trace metals, such as Ni and Co and

that supplementation can aid long-term digester stability. Molaey et al. (2018b) stated that the

presence of sufficient amounts of Ni was important for the growth of ammonia-resistant

hydrogenotrophic methanogens and Co was important for the syntrophic acetate oxidation

pathway. However, not all previous studies have found that NPs inclusion increased CH 4

production (Su et al., 2013). Yang et al. (2013) found that adding Fe NPs (1.1 mM of Fe NPs

at 55nm) and non-nano Fe (1.1 mM Fe at <212 µm) to waste sludge AD resulted in a

decrease in CH4 production by 20 and 70%, respectively, compared to the control. Su et al.

(2013) found that using 20 nm Fe at a high concentration (1000 mg/L) increased CH4 by

40.4% from waste activated sludge. Yet, our study, found a 23.7 – 29.1% increase in CH4

production in concentration ranges of 15 – 100 mg/L with Fe NPs and Fe3O4 NPs. The largest

CH4 increases with Fe were 29.1% (100 mg/L Fe NPs) and 27.5% (15 mg/L Fe3O4 NPs).

In our study, the Ni NP addition produced the highest quantity of CH 4 of all treatments

flowed by the Co treatment (Table 3). Previous studies have reported increases in CH4

production from maize silage and manure of 30 and 100% with 10 µm and <20 nm Ni added,

respectively (Abdelsalam et al., 2017; Pobeheim et al., 2010), while Feng et al. (2010)

reported that various concentrations of Co NPs (0.06 to 6 mg/L) increased the CH 4

production by 7 - 15%. Abdelsalam et al. (2016) reported that using Ni NPs (>20nm) with

dosage of 0.5 – 2 mg/L for digesting dairy manure for 50 days increased CH4 production by

55 – 101%. Ni has been shown to be one of most important trace elements used by bacteria

9
during AD (Abdelsalam et al., 2017; Yirong et al., 2013). Gustavsson et al. (2013) observed

that Ni inclusion increased AD stability and increased CH4 yield. Altas (2009) indicated that

Ni doses of 0.5 – 16 mg/L increased cumulative CH4 production, which was in agreement

with our results that confirmed that Ni NPs with concentration 12 mg/L increased cumulative

CH4 production.

Previous research has shown that using magnetic NPs can increase CH4 production due to

the ability of specific NPs to improve biological activity during long-term exposure, as the

metal ions released from NPs play an important role in microbial communities involved in

biological treatment processes (Ni et al. 2013; Wang et al., 2016). Direct interspecies electron

transfer via metal NPs may play an important role in facilitating the methanogenesis process

during the AD (Wang et al., 2016). It has been reported that the reduced electron carriers

could regenerate into H2 or formate and then serve as an electron donor for CH4 production

by combining with CO2 (Summers et al. 2010; Cruz et al. 2014). Additionally, Ganzoury et

al. (2015) stated that the increased surface area from using NPs had a positive effect on AD.

The results showed that using NPs can not only increase CH4 production, but can also

decrease the number of days required to reach peak CH4 production (Figure 3). The poultry

litter-only reactors produced 266 mL CH4/g VS over 69 days of digestion, while the Ni

treatment (12 mg/L) produced 261 mL CH4/g VS during the first 10 days of digestion, with a

total of 336 mL CH4/g VS (Figure 3). Figure 3 shows that 95.1% of the CH4 from the Ni

treatment (12 mg/L) was produced during the first 29 days of the experiment, and therefore,

illustrated that 29 days could be a suitable HRT for future work. There was not a significant

difference in H2S concentrations in all treatments in Exp. A (Table 3; p-values all > 0.05).

3.2 Exp. B: Effect of nanoparticles mixtures (NPM) of Fe, Ni and Co in AD of poultry litter

10
 In Exp. B, compared to poultry litter-only reactors (305 mL CH4/g VS), there was a

significant increase (8.6%) in CH4 production for the ‘mid-low NPM’ concentration (334 mL

CH4/g VS; p-value = 0.010), which contained a mixture of 400 mg/L Fe, 48 mg/L Ni, and

21.6 mg/L Co NPs added, and a 7.8% increase in CH4 production at the ‘mid-high NPM’

concentration (331 mL CH4/g VS; p-value = 0.018), which contained 400 mg/L Fe, 24 mg/L

Ni and 10.8 mg/L Co NPs added. There was not a significant difference between the ‘low

NPM’ concentration (321 mL CH4/g VS; p-value = 0.056) and the ‘high NPM’ concentration

(307 mL CH4/g VS; p-value = 0.999) compared to the poultry litter-only reactors. Gonzalez-

Estrella et al. (2013) reported that using high concentrations of Fe NPs could decrease the

overall biogas and CH4 production, up to 91%, which could have occurred in our high-range

treatment, with 1,000 mg/L Fe, 120 mg/L Ni, and 54 mg/L Co NPs added, with no effect in

CH4 production observed in the ‘low NPM’ concentration tested (100 mg/L Fe, 12 mg/L Ni,

and 5.4 mg/L Co NPs).

The NP mixtures (Fe, Ni, and Co) significantly reduced H2S production for all four

NPMs concentrations tested compared to poultry litter-only digestion (Figure 4). The results

showed that the most effective treatments to reduce H2S production was the ‘high NPM’ (0

mL H2S/g VS), as the inoculum-only reactors had higher H2S concentrations than the ‘high

NPM’ reactors. The H2S concentration did increase with decreasing NPM concentrations,

with 15.71 and 49.4 mL H2S/g VS in the ‘mid-High NPM’ and ‘Low NPM’ reactors, with

average H2S concentrations of 2,302 and 7,217 ppm, respectively (p-values < 0.001 and

0.002, respectively; Table 3; Figure 4). All NPM reactors had lower H2S concentrations than

the poultry litter-only reactors (56.1 mL H2S/g VS and an average concentration of 8,241 H2S

ppm).

A strong linear correlation (r2 = 0.997) was shown between H2S reduction and the four

NPMs concentrations tested (Figure 4c), with H2S reductions of 100, 71.9, 40.9 and 11.9%

11
for the four NPM concentrations tested (from ‘high’ to ‘low NPM’ concentrations) compared

to the poultry litter-only reactors. This strong correlation indicates that H 2S reductions were

linear with increasing concentrations of the Fe, Ni, Co mixture, with H2S reduction likely

through precipitation of the H2S through FeS and/or NiS formation (Díaz et al., 2015).

Li et al. (2007) showed that Fe2+ released from nanoscale zero-valent Fe could combine

with S2− during the AD process inhibiting H2S production, even with a relatively low dose

(0.5mg Fe NPs/L of wastewater). Additionally, precipitated sulfide has a relatively low

solubility in water, and therefore, sulfide can be adsorbed onto the iron particle via formation

of surface complexes (Li et al., 2007). The increase in surface area when using likely has a

positive effect on removing H2S.

It should be noted that the lowest concentration tested in Exp. B (‘low NPM’) was equal

to the highest concentrations of the individual NPs tested in Exp. A. There was not a

significant reduction in H2S in Exp. A, with individual additions of Fe and Ni NPs. There was

likely a synergistic effect with the NP mixtures in that both FeS and NiS formation occurred

at the ‘low NPM’ concentration tested in Exp. B, resulting in 11.9% reduction with NP

mixtures.

3.3 Tracking nanoparticles after digestion:

The SEM with EDXA function was used to confirm the NPs purity, size, and morphology

after dispersion (before AD) and after the 69-day AD period of Exp. A and 79-day AD period

of Exp. B. The SEM indicated that prior to AD, the Fe, Ni, Co, and Fe3O4 sizes in Exp. A

were 57.2 - 80.9 nm, 58.3 - 79.7 nm, 30.0 - 50.8 nm, and 94 - 3,400 nm, respectively.

Detecting nanoparticles after AD using a magnetic stirring bar covered with plastic

parafilm was shown to be the only successful method to collect the metal NPs after both

experiments when the NP treatments were >100 mg/L NP. The first two methods tested

12
(without utilization of parafilm and magnetic stirring) did not detect any NPs using SEM.

After Exp. A, the Fe and Fe3O4 NPs were detected with the high concentration addition (100

mg/L NP). After Exp. B, the Fe NPs were detected in all samples, and the size was confirmed

for the ‘high NPM’ concentration (1,000 mg/L Fe NP). The Ni NPs were only detected in the

‘high NPM’ concentration (Ni 120 mg/L). The Co NPs were not detected in any treatments

due to the low concentrations in the collected samples. The SEM showed bacteria cell uptake

of the Fe3O4 NPs within the cell structure, while the EDXA confirmed the presence of Fe.

Mu et al. (2011a) reported that NPs uptake may stimulate methanogens by increasing their

metabolic intermediates and key enzyme activities involved in the hydrolysis, acidification,

and methanogenesis stages of AD.

Confirming the size and presence of the NP additions after AD is important, as previous

results have suggested that the size of the NP has an effect on CH4 production. Abdulsalam et

al. (2017) reported a 39.3% increase in CH4 production with cattle manure diluted 1:1 with

DI water and digested with 20 mg/L Fe NPs (9 nm size). While other previous studies have

reported decreases (20, 15, 20, and 70%) in CH4 production during AD when using larger Fe

NPs (55nm, 46-60nm, 74µm, and <212µm in size, respectively) (Gonzalez-Estrella et al.,

2013; Su et al., 2013; Yang et al., 2013). Jiang et al. (2008) found that NP size strongly

affected the binding and activation of membrane receptors and subsequent protein

expression. In our study, cumulative CH4 production from Fe, Ni, and Co NPs treatments

were higher than Fe3O4 treatment (Figure 2), which may have been influenced by the

confirmed NPs size within the AD. The NP size before AD ranged from 30.0 - 80.9 nm for

Fe, Ni, and Co NPs, while the Fe3O4 NP size ranged from 94.3 - 400 nm. It appears that the

effect of NPs used in AD depends not only on the size of NPs but also the concentration, the

NP type, NP mixtures, the substrate being digested, and hydraulic retention time (Abdelsalam

13
et al., 2017; Su et al., 2013; Pobeheim et al., 2010, Abdelsalam et al., 2016; Casals et al.,

2014).

4.0 Conclusions

This study demonstrated the impact of metal NP addition in poultry litter AD. The results

suggest that higher concentrations of NP mixtures (in mg/L) (> 100 Fe, 12 Ni, and 5.4 Co)

are more effective at reducing H2S than increasing CH4 production, while lower NP

concentrations increased CH4 production and reduced the days required to rich the maximum

biogas production. Detection of NP presence and size at concentrations  100 mg/L within a

heterogeneous, high solids mixture after digestion was successfully shown. In the future,

these NP detection methods for AD digestate can be used to evaluate the environmental risk

of field application when using NPs in AD.

Acknowledgments

This research was supported by the Maryland Agricultural Experiment Station. A special

thank you to the University of Maryland Waste to Energy laboratory. Any opinions, findings,

conclusions, or recommendations expressed in this publication are those of the author(s) and

do not necessarily reflect the view of the Maryland Agricultural Experiment Station.

References

1. Abdelsalam, E., Samer, M., Attia, Y.A., Abdel-Hadi, M.A., Hassan, H.E., Badr, Y., 2016.

Comparison of nanoparticles effects on biogas and methane production from

anaerobic digestion of cattle dung slurry. Renew Energ, 87, 592-598.

2. Abdelsalam, E., Samer, M., Attia, Y.A., Abdel-Hadi, M.A., Hassan, H.E., Badr, Y., 2017.

Influence of zero valent iron nanoparticles and magnetic iron oxide nanoparticles on

14
 biogas and methane production from anaerobic digestion of manure. Energ, 120, 842-

853.

3. Abouelenien, F., Fujiwara, W., Namba, Y., Kosseva, M., Nishio, N., Nakashimada, Y.,

2010. Improved methane fermentation of chicken manure via ammonia removal by

biogas recycle. Bioresour Technol, 101(16), 6368-73.

4. APHA, 2005. Standard methods for the examination of water and wastewater. American

Public Health Association (APHA): Washington, DC, USA.

5. Altaş, L., 2009. Inhibitory effect of heavy metals on methane-producing anaerobic granular

sludge. J. Hazard. Mater., 162(2-3), 1551-1556.

6. Arif, S., Liaquat, R., Adil, M., 2018. Applications of materials as additives in anaerobic

digestion technology. Renew Sust Energ Rev, 97, 354-366.

7. Casals, E., Barrena, R., García, A., González, E., Delgado, L., Busquets‐ Fité, M., Font,

X., Arbiol, J., Glatzel, P., Kvashnina, K., 2014. Programmed iron oxide nanoparticles

disintegration in anaerobic digesters boosts biogas production. Small, 10(14), 2801-

2808.

8. Costa, J.C., Barbosa, S.G., Alves, M.M., Sousa, D.Z., 2012. Thermochemical pre- and

biological co-treatments to improve hydrolysis and methane production from poultry

litter. Bioresour Technol, 111, 141-147.

9. Cruz Viggi, C., Rossetti, S., Fazi, S., Paiano, P., Majone, M. and Aulenta, F., 2014.

Magnetite particles triggering a faster and more robust syntrophic pathway of

methanogenic propionate degradation. Environ. Sci. Technol., 48(13), pp.7536-7543.

10. Demirel, B., Scherer, P., 2011. Trace element requirements of agricultural biogas

digesters during biological conversion of renewable biomass to methane. Biomass

Bioenergy , 35(3), 992-998.

15
11. Díaz, I., Ramos, I., Fdz-Polanco, M., 2015. Economic analysis of microaerobic removal

of h2s from biogas in full-scale sludge digesters. Bioresour Technol, 192, 280-286.

12. Feng, X.M., Karlsson, A., Svensson, B.H., Bertilsson, S., 2010. Impact of trace element

addition on biogas production from food industrial waste–linking process to microbial

communities. FEMS Microbiol. Ecol., 74(1), 226-240.

13. Feng, Y., Zhang, Y., Quan, X., Chen, S., 2014. Enhanced anaerobic digestion of waste

activated sludge digestion by the addition of zero valent iron. Water Res, 52, 242-50.

14. Ganzoury, M.A. and Allam, N.K., 2015. Impact of nanotechnology on biogas production:

a mini-review. Renew Sust Energ Rev, 50, pp.1392-1404.

15. Gonzalez-Estrella, J., Sierra-Alvarez, R., Field, J.A., 2013. Toxicity assessment of

inorganic nanoparticles to acetoclastic and hydrogenotrophic methanogenic activity in

anaerobic granular sludge. J. Hazard. Mater., 260, 278-285.

16. Gungor-Demirci, G., Demirer, G.N., 2004. Effect of initial cod concentration, nutrient

addition, temperature and microbial acclimation on anaerobic treatability of broiler

and cattle manure. Bioresour Technol, 93(2), 109-17.

17. Gustavsson, J., Shakeri Yekta, S., Sundberg, C., Karlsson, A., Ejlertsson, J., Skyllberg,

U., Svensson, B.H., 2013. Bioavailability of cobalt and nickel during anaerobic

digestion of sulfur-rich stillage for biogas formation. Appl. Energ, 112, 473-477.

18. Hu, X., Chi, Q., Wang, D., Chi, X., Teng, X., Li, S., 2018. Hydrogen sulfide inhalation-

induced immune damage is involved in oxidative stress, inflammation, apoptosis and

the th1/th2 imbalance in broiler bursa of fabricius. Ecotoxicol. Environ. Saf., 164,

201-209.

19. Jiang, W., Kim, B.Y., Rutka, J.T., Chan, W.C., 2008. Nanoparticle-mediated cellular

response is size-dependent. Nat. Nanotechnology, 3(3), 145.

16
20. Krongthamchat, K., Riffat, R., Dararat, S., 2006. Effect of trace metals on halophilic and

mixed cultures in anaerobic treatment. Int. J. Environ. Sci. Technol. , 3(2), 103-112.

21. Li, X.-q., Brown, D.G., Zhang, W.-x., 2007. Stabilization of biosolids with nanoscale

zero-valent iron (nzvi). J. Nanopart. Res., 9(2), 233-243.

22. Lucia, L.A. and Rojas, O. eds., 2009. The nanoscience and technology of renewable

biomaterials. John Wiley & Sons.

23. Molaey, R., Bayrakdar, A. and Çalli, B., 2018a. Long-term influence of trace element

deficiency on anaerobic mono-digestion of chicken manure. Journal of environmental

management, 223, pp.743-748.

24. Molaey, R., Bayrakdar, A., Sürmeli, R.Ö. and Çalli, B., 2018b. Anaerobic digestion of

chicken manure: Mitigating process inhibition at high ammonia concentrations by

selenium supplementation. Biomass and Bioenergy, 108, pp.439-446.

25. Mu, H., Chen, Y., Xiao, N., 2011a. Effects of metal oxide nanoparticles (tio2, al2o3, sio2

and zno) on waste activated sludge anaerobic digestion. Bioresour Technol, 102(22),

10305-11.

26. Mu, H. and Chen, Y., 2011b. Long-term effect of ZnO nanoparticles on waste activated

sludge anaerobic digestion. Water research, 45(17), pp.5612-5620.

27. Ni, S. Q., Ni, J., Yang, N., & Wang, J. (2013). Effect of magnetic nanoparticles on the

performance of activated sludge treatment system. Bioresour Technol, 143, 555-561.

28. Owen, W.F., Stuckey, D.C., Healy, J.B., Young, L.Y., McCarty, P.L., 1979. Bioassay for

monitoring biochemical methane potential and anaerobic toxicity. Water Res., 13(6),

485-492.

29. Perera, P., Bandara, W., 2010. Potential of using poultry litter as a feedstock for energy

production.

17
30. Pobeheim, H., Munk, B., Johansson, J., Guebitz, G.M., 2010. Influence of trace elements

on methane formation from a synthetic model substrate for maize silage. Bioresource

Technology, 101(2), 836-839.

31. Rao, A.G., Prakash, S.S., Joseph, J., Reddy, A.R., Sarma, P.N., 2011. Multi stage high

rate biomethanation of poultry litter with self mixed anaerobic digester. Bioresour

Technol, 102(2), 729-35.

32. Schieder, D., Quicker, P., Schneider, R., Winter, H., Prechtl, S., Faulstich, M., 2003.

Microbiological removal of hydrogen sulfide from biogas by means of a separate

biofilter system: Experience with technical operation. Water Sci. Technol. , 48(4),

209-212.

33. Su, L., Shi, X., Guo, G., Zhao, A., Zhao, Y., 2013. Stabilization of sewage sludge in the

presence of nanoscale zero-valent iron (nzvi): Abatement of odor and improvement of

biogas production. Journal of Material Cycles and Waste Management, 15(4), 461-

468.

34. Summers, Z.M., Fogarty, H.E., Leang, C., Franks, A.E., Malvankar, N.S. and Lovley,

D.R., 2010. Direct exchange of electrons within aggregates of an evolved syntrophic

coculture of anaerobic bacteria. Science, 330(6009), pp.1413-1415.

35. Thauer, R.K., Kaster, A.K., Seedorf, H., Buckel, W., Hedderich, R., 2008. Methanogenic

archaea: Ecologically relevant differences in energy conservation. Nat Rev Microbiol,

6(8), 579-91.

36. USEPA, 1994. Method 3051, microwave assisted acid digestion of sediments, sludges,

soils and oils. In: Test Methods for Evaluating Solid Waste, Physical/Chemical

Methods, third ed. U.S.

18
37. Wang, T., Zhang, D., Dai, L., Chen, Y., Dai, X., 2016. Effects of metal nanoparticles on

methane production from waste-activated sludge and microorganism community shift

in anaerobic granular sludge. Sci. Rep., 6, 25857.

38. Yadvika, Santosh, Sreekrishnan, T.R., Kohli, S., Rana, V., 2004. Enhancement of biogas

production from solid substrates using different techniques--a review. Bioresour

Technol, 95(1), 1-10.

39. Yang, Y., Guo, J., Hu, Z., 2013. Impact of nano zero valent iron (nzvi) on methanogenic

activity and population dynamics in anaerobic digestion. Water Res., 47(17), 6790-

6800.

40. Yirong, C., Banks, C.J. and Heaven, S., 2013. Comparison of mesophilic and

thermophilic anaerobic digestion of food waste. AD13 Recovering (bio) Resources

for the World.

19
Figure Captions

Fig. 1: The methods used in Exp. A and Exp. B to add nanoparticles (NP) to the triplicate

reactors, quantify CH4 production during digestion, extract the NPs from the reactors, and

verify NP presence before and after digestion.

Fig. 2: Cumulative CH4 production in Exp. A testing three concentrations of nanoparticle

(NP) additions to poultry litter manure digesters and comparing to poultry litter-only

digestion. The NP additions were Fe (in A), Ni (in B), Co (in C), and Fe3O4 (in D). Error bars

represent standard error of triplicate reactors.

Fig. 3: Average cumulative CH4 production from the nanoparticle (NP) concentration that

had the highest CH4 values for each NP addition tested in Exp. A, and the poultry litter-only

reactors. Methane production was divided into time intervals of 4 - 7 days for Days 1 – 29,

with the last interval representing Days 30 - 69.

Fig. 4: Hydrogen sulfide (H2S) concentration shown in (A) for the nanoparticles mixtures

(NPM) at the ‘low NPM,’ ‘mid-low NPM.’ ‘mid-high NPM,’ and ‘high NPM’ concentrations

tested and poultry litter-only reactors in Exp. B, with cumulative H2S production shown in

(B). Error bars represent standard error of triplicate reactors. The H 2S concentration and

percent reduction compared to the poultry litter-only reactors are shown in (C), with linear

regression of the reduction values over the NPM concentration gradient.

20
Tables

Table 1. Inoculum (185.9 g), poultry litter (4.11 g), and 0.1 mM sodium dodecylbenzene

sulfonate (SDS) solution (10 mL) for nanoparticle dispersion were added to each triplicate

reactor based on a 1:1 ratio (by VS), with the concentration of nanoparticles (NP) added for

each treatment in Exp. A shown.

Treatment
Fe Ni Co Fe3O4
15 mg/L 3 mg/L 1.4 mg/L 15 mg/L
Low
(1.68 mg/g VS) (0.34 mg/g VS) (0.15 mg/g VS) (1.68 mg/g VS)
50 mg/L 6 mg/L 2.7 mg/L 50 mg/L
NPs Mid
(5.58 mg/g VS) (0.67 mg/g VS) (0.30 mg/g VS) (5.58 mg/g VS)
100 mg/L 12 mg/L 5.4 mg/L 100 mg/L
High
(11.17 mg/g VS) (1.34 mg/g VS) (0.60 mg/g VS) (11.17 mg/g VS)

21
Table 2. Inoculum (261.4.9 g), poultry litter (5.92 g), and sodium dodecylbenzene sulfonate

(SDS) solution (32.7 mL) for nanoparticle dispersion were added to each triplicate reactor

based on a 1:1 ratio (by VS), with the concentration of the nanoparticles mixtures (NPM)

added for each treatment in Exp. B shown.

Treatment Fe Ni Co
100 mg/L 12 mg/L 5.4 mg/L
Low NPM
(8.22 mg/g VS) (0.99 mg/g VS) (0.44 mg/g VS)
200 mg/L 24 mg/L 10.8 mg/L
Mid-Low NPM
(16.44 mg/g VS) (1.97 mg/g VS) (0.89 mg/g VS)
400 mg/L 48 mg/L 21.6 mg/L
Mid-High NPM
(42.88 mg/g VS) (3.95 mg/g VS) (1.78 mg/g VS)
1,000 mg/L 120 mg/L 54 mg/L
High NPM
(82.19 mg/g VS) (9.86 mg/g VS) (4.44 mg/g VS)

22
Table 3: Average CH4 (% and cumulative production ± SE) values and H2S cumulative

production, with the percent reduction in H2S for each nanoparticle (NP) treatment compared

to the poultry litter-only reactors for Exp. A and Exp. B.

Cumulative Cumulative
CH4
Treatment CH4 H2 S
(% )
(mL /g VS) (mL /g VS)
Low 56.5  0.2 329  7 74.4  12.2
Fe NPs Mid 56.5  0.4 331  16 66.7  9.7
High 57.3  0.5 343  10 71.2  13.8
Low 57.2  0.7 338  12 67.3  3.8
Ni NPs Mid 57.4  0.3 336  8 65.1  6.3
High 57.6  0.3 368  21 73.1  9.9
Exp. A Low 56.6  0.2 342  1 65.3  11.0
Co NPs Mid 56.7  0.2 334  6 62.3  11.2
High 56.6  0.5 345  8 61.8  3.1
Low 56.7  0.4 339  9 60.9  15.1
Fe3O4 Mid 56.4  0.5 335  6 52.3  22.3
High 55.6  0.2 334  12 60.4  15.7
Poultry litter 54. 6  0.4 265  11 66.0  13.3
NPM Low 51.1  0.79 321  4 49.4  0.4
NPM Mid-Low 50.8  0.36 334  2 33.1  1.4
Exp. B NPM Mid-High 52.8  1.26 331  5 15.7  0.6
NPM High 51.4  1.93 308  15 0.00  0.0*
Poultry litter 49.1  1.29 305  5 56.1  0.5
*
The CH4 and H2S production from the inoculum was subtracted from all treatments,
resulting in zero values when the treatment values were less than the control.

23
Fig. 1

24
Fig. 2

25
Fig. 3

26
Fig. 4

27
Highlights
 Metal nanoparticles (NPs) were added to anaerobic digestion (AD) of poultry litter

 Adding Ni NPs (12 mg/L) increased CH4 production by 38.4% (368 mL CH4/g VS)

 Fe, Co and Fe3O4 NPs added individually also increased CH4 with no effect on H2S

 Higher concentrations of NP mixtures greatly reduced H2S with no effect on CH4

 A method for extraction of NPs from digestate for SEM detection was developed

28