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Human waste anaerobic digestion as a promising low-carbon strategy: Operating

performance, microbial dynamics and environmental footprint

Na Duan, Duojiao Zhang, Benyamin Khoshnevisan, Panagiotis G. Kougias, Laura

Treu, Zhidan Liu, Cong Lin, Hongbin Liu, Yuanhui Zhang, Irini Angelidaki

PII: S0959-6526(20)30461-3
DOI: https://doi.org/10.1016/j.jclepro.2020.120414
Reference: JCLP 120414

To appear in: Journal of Cleaner Production

Received Date: 15 November 2019

Revised Date: 15 January 2020
Accepted Date: 3 February 2020

Please cite this article as: Duan N, Zhang D, Khoshnevisan B, Kougias PG, Treu L, Liu Z, Lin C, Liu H,
Zhang Y, Angelidaki I, Human waste anaerobic digestion as a promising low-carbon strategy: Operating
performance, microbial dynamics and environmental footprint, Journal of Cleaner Production (2020), doi:
https://doi.org/10.1016/j.jclepro.2020.120414.

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CRediT author statement

Na Duan： ：Conceptualization, Writing original draft, Editing; Duojiao Zhang:

Investigation; Benyamin Khoshnevisan: Software, Writing original draft; Panagiotis
G. Kougias: Reviewing and Editing; Laura Treu: Software, Editing; Zhidan Liu:
Editing; Cong Lin: Reviewing and Editing; Hongbin Liu: Reviewing and Editing;
Yuanhui Zhang: Reviewing and Editing; Irini Angelidaki: Reviewing and Editing;
Human waste anaerobic digestion as a promising low-carbon strategy:

operating performance, microbial dynamics and environmental

footprint

Na Duan 1, 2,*, Duojiao Zhang1, Benyamin Khoshnevisan3, Panagiotis G. Kougias4,

Laura Treu4, Zhidan Liu1, Cong Lin 1, Hongbin Liu3,*, Yuanhui Zhang1, Irini

Angelidaki4

1
College of Water Resources and Civil Engineering, China Agricultural University,

Beijing, 100083, China

2
Jiangsu Key Laboratory of Anaerobic Biotechnology (Jiangnan University), Wuxi

214122, P. R. China

3
Key Laboratory of Nonpoint Source Pollution Control, Ministry of Agriculture/

Institute of Agricultural Resources and Regional Planning, Chinese Academy of

Agricultural Sciences, Beijing, 100081, China;

4
Department of Environmental Engineering, Technical University of Denmark, Kgs.

Lyngby, DK-2800, Denmark

*Corresponding author

E-mail address: duanna@cau.edu.cn (N. Duan); Tel.: +86-10-6273-7329 (N. Duan)

E-mail address: liuhongbin@caas.cn (H.B. Liu); Tel.:+86-10-8210-8763 (H.B. Liu)

1
Abstract

Human waste (HW) poses environmental and public health risk, thereby its sustainable

management is becoming a serious growing challenge. Anaerobic digestion (AD) has

long been introduced as an environmental-friendly and sustainable waste management

technology with bio-methane as final product. Energy production via AD of HW would

significantly contribute to low-carbon energy production systems and circular

bio-economy. In this study, optimal conditions, inhibiting factors, and microbial

community changes in continuously fed biogas reactors during anaerobic digestion of

HW at elevated influent feedstock concentration (IFC) were investigated. The highest

methane yield (327±21 mL g VS-1) was obtained at IFC of 3% TS. Increased IFC

deteriorated the process and affected the microbiome dynamicity. Specifically, methane

production was reduced by 50% with a concomitant increment of ammonia, Na+, and

K+ concentration. Two archaeal species (Methanosaeta and WSA2) were dominating the

microbial community at stable period. Two uncharacterized microbial groups (WWE1

and WSA2) were present and a potential syntrophic interaction between these two

members was hypothesized to play a crucial role in achieving a well-performing process.

AD process treating HW showed promising results for valorization of HW to clean

energy-biomethane both in environmental and economic aspects. Specifically, 1 t of

HW VS could obtain a greenhouse gases (GHG) mitigation of -54 to -272 kg CO2,eq via

the AD process. The LCA results demonstrated that such a bioenergy system would also

bring about environmental savings in ecosystem quality and resource damage categories.

2
Although ammonia inhibition at elevated IFC found as a potential inhibitory factor, it

can be easily overcome using co-digestion strategies or nitrogen recovery at upstream.

Key words: human waste; anaerobic digestion; methane; microbial community;

influent feedstock concentration; environmental footprint

1. Introduction

Human wastes (HW) are a growing environmental concern, as it is estimated that

approximately 127 kg of feces are generated per person each year (Asl and Hosseini,

2000), while the total amount is around 7.6 hundred million tons. HW may constitute a

public health risk, especially in regions with inefficient centralized treatment facilities

or inadequate decentralized technologies, like some developing countries or countryside.

Therefore, having considered the environmental threat and potential health risks, urgent

need for adequate treatment and subsequence reuse of HW is felt.

HW is rich in high strength organic matter and nutrients (Singh et al., 2017), thus,

it has been considered as a renewable feedstock for different purposes (Lu et al., 2017).

For example, HW can be utilized to produce biofuels like methane, bioethanol,

biodiesel through various processes, including pyrolysis, anaerobic digestion (AD),

hydrothermal liquefaction and others (Gomaa and Abed, 2017; Lu et al., 2017).

AD is widely applied for treating various fecal wastes, since it can offer a good

alternative to minimize environmental impacts of excrement storage and simultaneously

produce methane. Previous studies found that HW can be also a good substrate for

biogas production with comparable performance under lab conditions (Snell, 1943;

3
Singh et al., 1993; Meher et al., 1994; Park et al., 2001; Lalander et al., 2018). Although

a great number of studies have already focused on AD of HW, limited information is

available regarding the process stability and its inhibition factors during AD treatment

of HW. Colon et al. (2015) investigated the effect of high ammonia concentration on

biogas production using an artificial waste with similar properties of human excreta,

indicating that ammonia was a crucial parameter during the AD process of HW. Zhang

et al. (2017) also verified the important role of ammonia in the AD process treating HW.

The rapid development of sequencing technologies (i.e. next-generation sequencing,

16S rRNA amplicon sequencing) could help better understand the underlying

mechanisms of AD by analyzing the microbial communities in biogas reactors, as well

as the relationship between microbiomes and process parameters. A recent survey,

comparing the microbial community composition before and after anaerobic digestion

of HW in a batch experiment, showed that Methanomicrobia and Cloacimonetes were

the predominant archaea and bacteria, respectively (Gomaa and Abed, 2017). Notably,

besides ammonia, what may affect the process stability with the increased feeding

concentration in a continuous AD system has not been specifically addressed. In

addition, the microbial dynamicity, the correlation of operational taxonomic unit (OTU),

and process performance should be further investigated.

The sanitation of 7.6 hundred million tonnes human wastes, produced annually, is a

major concern worldwide due to the fact that the collection and treatment systems, even

in developed countries, are too costly, too complex, and use too much energy (Colón et

4
al., 2015; Lalander et al., 2013). The currently available waste water treatment plants

have been developed based on aeration, nitrification, and denitrification systems to

decrease COD and remove nitrogen from waste waters. AD of HW under optimal

conditions would lead to considerable biogas production. The produced biogas can be

either combusted in combined heat and power production (CHP) plants or upgraded to

biomethane; thus would substitute for fossil energies and contribute to sustainable

development (Shirzad et al., 2019; Khoshnevisan et al., 2018). Life cycle assessment

(LCA) is a stablished tool that evaluates a range of environmental impacts across the

lifetime of a product or process (Khoshnevisan et al., 2020). LCA aims to

comprehensively assess the impacts of a product by looking at all resulting effects on

the natural world, including human health, ecosystem quality, climate change, and

resources. This approach eliminates problems shifting from one aspect or process to

another; thus it can be employed to investigate the overall environmental impacts

caused by the AD of HW. This methodology has long been applied to investigate the

environmental performance of coupled waste management systems and biogas

production. Chen et al., (2012) employed a life-cycle energy and environmental

assessment approach to scrutinize the environmental performance of a biogas–digestate

utilization system in China. In another study conducted by Arafat et al.,(2015) the

environmental impacts of five municipal solid waste treatment processes with energy

recovery potential were explored. Likewise, Chen and Chen (2013) made effort to

evaluate the coupling of biogas projects to traditional agrosystems from a life-cycle

5
perspective. In another study conducted by Gao et al.,(2017) LCA was employed to

compare the current human excreta sanitation technology with comprehensive schemes

of Chinese rural toilets, including standard flushing, rainwater harvesting flushing

systems, urine separation and composting systems. Although distinct management

practices for HW valorization in China are comprehensively explored from LCA point

of view, the environmental benefits of an optimized HW anaerobic digestion system has

not been scrutinized.

The aims of the present study were (1) to assess the process stability and optimal

conditions of HW anaerobic digestion in a continuous experiment with step-wise

increasing influent feedstock concentration; (2) to gain a better understanding of the

inhibiting factors and the changes in the microbial community of both bacterial and

archaeal OTUs under different states; (3) to analyze the environmental footprint for

anaerobic treatment of human waste using China as an example. To the best of our

knowledge, this is the first attempt to simultaneously investigate the microbial

dynamicity and its dependence on feeding load in a continuous AD system treating HW

as mono feedstock. Moreover, the environmental benefits of an optimized HW anaerobic

digestion system from LCA point of view was taken into account to show its possible

opportunities as a sustainable renewable energy system.

2. Material and methods

2.1 Human waste anaerobic digestion Semi-continuous trial

HW, obtained from an aqua privy (Changping, Beijing, China), was used as the

6
feedstock for semi-continuous assay. Continuous stirred tank reactor (CSTR) of 10 L

total volume (9 L working volume) was used and the inoculum was collected from an

anaerobic digester treating pig manure. The properties of HW and inoculum were shown

in Table 1. Over 87% of the solid HW was organic and was amenable to anaerobic

digestion. HW had a high proportion of crude protein and low carbon to nitrogen ratio

(C/N) of 8.73.

The experiment was divided into three phases denoted as Phase 1 (3%TS, 1.06

gVSL-1d-1), Phase 2 (4%TS, 1.40 gVSL-1d-1) and Phase 3 (5%TS, 1.75 gVSL-1d-1). The

operational parameters of each phase are presented in Table 2. Gas volume was

measured by a gas flow meter (LMF-1, Jinzhiye instrument equipment co. LTD, Beijing,

China) and gas samples were daily taken to determine the biogas composition. Liquid

samples were obtained every three days for analysis of total ammonia nitrogen (TAN),

free ammonia (FAN), volatile fatty acids (VFAs), pH and soluble chemical oxygen

demand (SCOD). Moreover, the cation concentration including potassium (K), sodium

(Na), calcium (Ca), magnesium (Mg), manganese (Mn), ferrum (Fe), copper (Cu)and

aluminum (Al) were measured at specific time points during each phase, i.e. Phase 1 on

day 70, denoted as S1 (steady-state), Phase 2 on day 109, denoted as S2A (steady-state

before inhibition) and on day 145, denoted as S2B (steady-state after inhibition) and

finally, Phase 3 on day 174, denoted as S3 (inhibition state).The stable biogas

production with a daily variation of lower than 10 % for a period of at least 6 days was

defined as the steady-state (De Francisci et al., 2015). The above mentioned indices, e.g.,

7
pH, VFAs, ammonia, SCOD and cation concentrations, under different IFC were

measured to assess the process performance and consequently obtain the optimal

operational conditions of AD treating HM. The average methane production during the

steady-state of each phase was used to represent the optimal methane production of

different IFC conditions. Due to the fact that any parameter under sub-optimal condition

affects microbial activity and consequently methane production potential, methane

production could be regarded as an appropriate measure to evaluate whether or not the

process is working under optimal condition.

2.2 Analytical methods

2.2.1 Physicochemical analysis

TS, VS, pH, and total Kjeldahl nitrogen (TKN) were determined according to the

standard methods (APHA, 2005). Crude fat was detected by the Soxhlet extraction

method. Crude protein was estimated by multiplying the total organic nitrogen by 6.25.

Carbohydrate was calculated by subtracting the amount of crude protein and crude fat

from dry biomass. Liquid samples were centrifuged at 4000 rpm under room

temperature for 10 min prior to chemical analysis. The concentration of SCOD and TAN

were measured by potassium dichromate method and salicylic acid spectrophotometry,

respectively. The FAN concentration was determined according to the TAN

concentration, temperature and pH using the following Eq.(1) (Hansen et al., 1998) :

NH 3 10 − pH
= (1 + 2729.92
) −1 Eq. (1)
TAN −( 0.09018 +
T (k )
)
10
where NH3 is the concentration of free ammonia in mg/L, TAN is the total ammonia

8
 nitrogen concentration (mg/L), pH is the pH value of the effluent, and T(k) is the

temperature (Kelvin).

VFA concentrations were determined using a high performance liquid

chromatography (LC-10A, Shimadzu Corporation, Kyoto, Japan) according to Li et al.

(2017). Cation concentrations were measured by Puni Company (Beijing, China).

2.2.2 Biogas composition and methane production potential

Methane content was analyzed by a gas chromatograph (SP-6890, Rainbow

Chemical Instrument Co., LTD, China) according to Lu et al. (2017). The theoretical

methane potential (Bo) was calculated based on the main organic content as described

by Eq.(2) (Ebner et al., 2016). The practical biomethane potential (Bpr) was calculated

on a basis of VS added (mLCH4 gVS-1). Thus, biodegradability can be calculated by the

ratio of Bpr and Bo.

= 415 × Carbohydrates % + 496 × Proteins % + 1014 × Lipids % Eq. (2)

2.2.3 Microbial analyses

Samples for microbial analyses were obtained from the inoculum (S0), Phase1 (S1,

on day 70), Phase 2 (S2A, on day 109 and S2B, on day 145) and Phase 3 (S3, on day

174).

The DNA extraction, PCR amplification and sequencing were performed by Majorbio

(Shanghai, China) with the same procedure done as previously described (Li et al.,

2017). Sequencing of the 16S rRNA amplicons was performed by Illumina Miseq

platform.

9
 Raw sequences were processed using CLC Workbench software (V.8.0.2) with

Microbial genomics module plug in (QIAGEN, Bioinformatics, Germany). Trimming

and chimera crossover filtering procedure was performed using the default parameters

of the software to obtain the high-quality reads. Clustering and phylogenetic assignment

of OTU was done with multiple sequence comparison using Greengenes v 13_5

database as a reference (clustered at 97%). Consensus sequences of OTUs were

manually verified by BLAST (16S ribosomal RNA database and Nucleotide collection

database) to obtain the taxonomical assignment. Alpha diversity was displayed using the

numbers of identified OTU and Shannon index. Beta diversity (PCoA) was performed

by Bray-Curtis matrix. The highly abundant OTUs (bacteria>1%, archaea>0.5%) were

presented to evaluate the changes of microbial relative abundance with IFC shift, and

were represented as heat maps by Multi experiment viewer software (MeV 4.9.0)

(Saeed et al., 2006). Raw sequences were deposited in Sequence Read Archive (SRA)

database of NCBI under the BioProject PRJNA485583 with accession numbers

SAMN09809784 to SAMN09809788. Data analysis for correlation between microbial

abundance and biochemical parameters was carried out in the correl function of

Microsoft Excel.

2.3 Environmental footprints

LCA was carried out to investigate the magnitude of environmental

savings/impacts caused by the AD of HW in China. LCA was performed taking into

account all mandatory steps issued by ISO 14040 guidelines (ISO, 2006) and

10
recommendations presented by Reference Life Cycle Data System (ILCD) Handbook

(Wolf et al., 2010) to correctly account the environmental impacts. The functional unit

was decided to be one tonne of HW volatile solids (7.8 m3 HW with a TS of 14.56%)

entering to biogas plants and anaerobically digested to produce biogas. The produced

biogas was assumed to be applied in different downstream processes, like CHP,

upgrading as transportation fuel, etc. The intended application of digestate remaining

after AD process was assumed to use on farmlands as soil amendment following a

sterilization step at 70 for one hour to kill all pathogens inside. The method

“IMPACT2002+” was used to aggregate the inventory data into related impact

categories and damage categories.

3. Results and discussion

3.1 Operation performance under different IFCS

3.1.1 Methane yield and biodegradability

The highest methane yield (327±21 mL (gVS)-1) of HW was obtained at Phase 1

with IFC of 3%TS (Fig.1a). The obtained methane yield corresponded to 68.4% of the

theoretical maximum and was in accordance with previous studies, which determined

the methane yield from real human waste digestion to be in the range of 124-338 mL

(gVS)-1（Table 2） (Singh et al., 1993; Meher et al., 1994; Park et al., 2001; Gomaa and

Abed, 2017; Lalander et al., 2018).

By increasing the IFC to 4%TS (Phase 2), the AD process was relatively stable

during day 76-122 with an average methane yield of 303±18 mL (gVS)-1. Nevertheless,

11
the process was not robust and on day 149 the methane production rate dropped by

approximately 50% with a simultaneous increment of the ammonia concentration

(Fig.1a and 1b). The imbalance was also evident by the poor degradation efficiency of

the organic matter as recorded by the increasing SCOD concentration of effluent

(Fig.1d). In Phase 3, due to the reduced methane production, intermittent feeding was

applied and fluctuation of methane production (<160 mL (gVS)-1) occurred. Meanwhile,

the biodegradability rate decreased to 25.3% of the theoretical maximum.

3.1.2 Process parameters changes

Ammonia (TAN and FAN) concentration was one of the most important

parameters for AD treatment of HW, since HW has high crude protein content and low

C/N ratio (Table 1). TAN and FAN concentrations during the start-up stage were very

low and stable having average values of 1007 and 24 mgL-1, respectively (during day

51-75, Phase 1). The VFA concentration was relatively low (0.54±0.09 gL-1 on average

from day 51-75) and the pH was maintained within a narrow range between 7.3 and 7.5

(Fig.1c and Fig.2a), indicating an efficient operation of the reactor. The TAN

concentration was remarkably increased with the elevated IFC (Fig.1b). Specifically, the

TAN and FAN concentration were rapidly increased to approximately 1500 and 100

mgL-1, respectively (day 127, Phase 2). Since the urea in HW was hydrolyzed to

ammonium carbonate, the pH of the anaerobic reactor increased leading to a

concomitant increment of FAN (Park et al., 2001). The biogas production changed

susceptibly along with FAN increase at Phase 2, resulting in an unstable AD process

12
lowering the methane yield. The results were in line with the previous studies that FAN

concentrations ranging from 80 to 150 mgL-1 have been reported to deteriorate the AD

process (Yenigün and Demirel, 2013). Additionally, a sharp increase of total VFA

concentration occurred (during day 127-150) along with the increasing FAN

concentration. Moreover, the profile of individual VFA altered after the increment of

IFC increased to 4%TS. From day 76-105, acetic acid (29% on average) and butyric

acid (61% on average) were the primary VFA. An interesting shift is that formic acid

started increasing significantly being among the dominant VFA molecules. After day

105, formic acid concentration was temporarily elevated to approximately 900mgL-1 on

day 127 and then was stabilized at 400mgL-1. From day 126-150, formic acid (41% on

average) and butyric acid (45% on average) were the main individual VFA, acetic acid

accounted for 14% of the total VFA (Fig.2a). Acetate and CO2/H2 are well-known

precursors of the methane formation. The absence or inactivation of formic

hydrogenlyase or microorganisms may limit the breakdown process of formic acid to H2

and CO2 (Zoetemeyer et al., 1982).

Regarding the cation concentrations, potassium ion (K+) (from 471 to 707 mgL-1)

and sodium ion (Na+) (from 275 to 741 mgL-1) might affect the overall process

efficiency (Fig.2b). Besides K+, all other ions were found to be at concentrations which

were lower than the inhibitory threshold reported in a previous study (Romero-Güiza et

al., 2016). The concentration of K+ (< 400 mgL-1) have been reported to be

advantageous for the AD process and to result in enhancement of methane production

13
(Chen et al., 2008); In the current study, the K+ concentration was increased remarkably

with IFC shift and became higher than 700mgL-1. VFA accumulation was observed with

high K+ concentration (Chen and Cheng, 2007), which was in line with our finding,

implying its adverse effect on the AD process. In addition, the optimal Na+

concentration for mesophilic acetoclastic and hydrogenotrophic methanogens was

reported to be 230 and 350mgL-1, respectively (Chen et al., 2008). Thereby, the

moderate Na+ concentration in Phase 1 (S1) was beneficial for achieving a

well-performing AD process. However, at higher IFCs, the Na+ concentration increased

to 623mgL-1(S2B) and 741mgL-1(S3), which has probably negatively affected the AD

process as indicated by the lower methane production and unstable process. These

results indicate that variation in FAN, K+ and Na+ concentration in the AD process may

be the significant factors influencing the process performance.

3.2 Microbial community dynamics

The sequencing depth for all samples reached a stable plateau, indicating that

enough reads were obtained to cover the whole microbial diversity (Fig. S1). Alpha

diversity, represented as number of OTUs and Shannon index, showed the highest

values in the inoculum (S0) and decreased after feeding with HW. This result is

confirmed by the diversity covering the most abundant OTUs (bacteria>1%) that the

diversity was 57% in S0 and increased up to 85% after feeding with HW (Table S1).

This means that only some of the initial microbial members proliferated in the HW

anaerobic digestion environment. Due to the increased IFC, more organic matter entered

14
the reactor resulting in gradually increasing ammonia, VFA and SCOD values (Fig.1

and Fig.2a). The bacterial Shannon index increased at stable phase and then decreased

after the appearance of inhibition. The archaeal diversity based on the Shannon index

had a distinct decrease with increasing IFC probably due to the lower adaptation ability

to environmental conditions of archaea compared to bacteria. Moreover, PCoA

demonstrated a clear variation in microbial community composition with the increasing

IFC (Fig. S2).

3.2.1 Archaeal community composition

The results showed a large difference in archaeal community structure in inoculum

(S0) and subsequent process with the increased IFC (Fig.3a and Fig 4). Overall, the

archaea composition in inoculum (S0) was represented by 19 OTUs (>0.5%) that were

assigned to two phyla Crenarchaeota (21%) and Euryarchaeota (77%). With reactor

operation time, the relative abundance of Crenarchaeota decreased. The high abundance

of Crenarchaeota in S0 could be explained since the inoculum used was derived from a

biogas reactor fed with pig manure. In fact, Crenarchaeota was found to be

predominant in swine sludge obtained from an anaerobic treatment lagoon

(Cardinali-Rezende et al., 2012).

In addition, increasing IFC caused a shift of dominance from acetoclastic

methanogen (i.e. Methanosaeta) and hydrogenotrophic methanogens (i.e.

Methanothermobacter) towards sole acetoclastic methanogen (i.e. Methanosaeta)

(Fig.3a). Gomaa and Abed (2017) also found a clear shift towards an acetoclastic

15
dominant methanogenic community during the AD treatment of HW, but the specific

genus was different from our study, probably due to the differences in physiological

characters and operation conditions. The predominant genus was assigned to

Methanosaeta. Overall, it increased in relative abundance by approximately 2 folds

compared to S0 along with the IFC shift. Notably, Methanosaeta soehngenii 1 (100%

BLAST identity) was the most highly represented methanogen during the experiment

(78% on average). During the whole process, the acetate concentration was at low level

with neutral pH (Fig. 2a), favoring a high relative abundance of Methanosaeta

soehngenii 1, due to the higher growth rates of Methanosaeta compared to

Methanosarcina at low acetate concentrations (Padmasiri et al., 2007). Methanosaeta

soehngenii predominates in anaerobic reactors (Fernández et al., 2008; Lin et al., 2012)

and it can use acetate as a sole growth substrate with high affinity (Jetten et al., 1992).

The most abundant hydrogenotrophic methanogen (uncharacterized WSA2 group)

was affiliated to Methanobacteriales. The relative abundance of WSA2 group increased

by 2 folds from S0 to S1 and decreased remarkably from S1 to S3. Interestingly,

Methanobacteriales sp. 3 was only found at high relative abundance in S1 (36%). When

the IFC increased, its relative abundance sharply decreased to 7.20% for S2A and then

was less than 1% for S2B and S3. Its relative abundance was positively correlated with

the methane production（0.81）(Fig. 5). However, metabolic functions of WSA2 are

currently unknown because no cultured isolates have been reported up to date (Kuroda

et al., 2015). Nevertheless, Chouari et al. (2005) detected WSA2 at high relative

16
abundance in an anaerobic mesophilic sludge digester using formate- or

hydrogen-containing media, and suggested that members of this group may have

hydrogenotrophic metabolism. The high concentration of formic acid in 6th and 7th HRT

of the whole process also verified this finding (Fig. 2a). With the IFC shift, the total

relative abundance of hydrogenotrophic methanogens decreased and the dominant

hydrogenotrophic methanogens changed. Methanobacterium ferruginis 8,

Methanobrevibacter oralis 9 and Methanobrevibacter smithii 13, known as

hydrogenotrophic methanogens (Kuroda et al., 2015; Wong et al., 2013), strongly

increased in abundance (up to 6% in total). The composition of hydrogenotrophic

methanogens probably was affected by the pH, ammonia, K+ and Na+ concentration

(Fig. 5).

In comparison with the hydrolytic bacteria, methanogens are more sensitive to

environmental fluctuation, e.g. saline concentration (Wang et al., 2018). Thereby, the

ammonia, K+ and Na+ concentration increased significantly with the elevated IFC,

which might be the main reason affecting the microbial community dynamics and

further affecting process efficiency and reactor stability. In previous studies, ammonia

and Na+ were among key factors shaping microbial community structure (Lee et al.,

2018). The highest toxicity effects have been reported for H2/CO2 utilizing

microorganisms (Chen et al., 2008). Acclimation of methanogens to high concentrations

of sodium over prolonged periods as well as co-digestion with other feedstock are a

practical solution to deal with above-mentioned problem. Similarly, as an important

17
physiological factor in methanogens, K+ at low concentrations (less than 400 mg/L)

stimulates AD process, its toxicity may endanger microbial activity under high

concentration (Chen et al., 2008; Wu et al., 2016).

3.2.2 Bacterial community composition

Considering the bacterial community, clear differences in relative abundance were

observed among the same microbes in different samples. Bacteroidetes (21%),

Firmicutes (12%), WWE1 (Cloacimonetes) (7%), Proteobacteria (7%) and Chloroflexi

(3%) were the dominant phyla in S0. WWE1 (45%) and Candidatus Cloacimonas were

found to be the most abundant phylum and genus in S1, respectively. By increasing the

IFC from 3%TS to 5%TS, the total relative abundance of Bacteroidetes and Firmicutes

increased from 27% to 74% of the total bacterial community. Since many members of

Bacteroidetes and Firmicutes are associated with protein degradation, it is believed that

they had played a central role in protein and /or amino acid degradation. Their

increasing relative abundance resulted in high ammonia concentration, even more, VFA

accumulation, which was in line with the result of Antwi et al. (2017a).

Within the bacterial community, the two OTUs (Prolixibacteraceae sp. 2 and

Prolixibacteraceae sp. 5) belong to Bacteroidetes, whose total relative abundance

increased from 6% in S1 to 22% in S2B and then decreased to nearly 7% in S3. They

were 90% similar to Tangfeifania diversioriginum, which was also found as fermentative

bacteria in an upflow anaerobic sludge blanket reactor (Antwi et al., 2017b).

Tangfeifania diversioriginum was involved with carbohydrate or cellulose fermentation

18
with its optimum growth condition of mesophilic temperature and neutral pH; however,

it cannot grow in the absence of Na+ (Liu et al., 2014). The remarkable variation in

relative abundance of the two OTUs implied that Tangfeifania diversioriginum was Na+

tolerance and its threshold was lower than 700 mgL-1 (Fig.2b). The other three OTUs

(Sedimentibacter sp. 7, Sedimentibacter sp.19 and Sedimentibacter sp. 20) assigned to

Firmicutes were found to increase their relative abundance in the stable stage (S1-5%,

S2A-12%), however, their relative abundance was decreased during the unstable

process of the reactor (S3-3%) (Fig.4). These three OTUs were identified as

Sedimentibacter saalensis (>95% BLAST identity), owning the ability to utilize

pyruvate or amino acids with the main products of acetate and butyrate (Breitenstein et

al., 2002). Moreover, several Clostridium species (i.e. Ruminococcaceae sp.29) also can

produce butyric acid as an end product of fermentation (Jang et al., 2014). That is

probably the reason that butyric acid made up a high relative content among the other

VFA (Fig.2a).

WWE1 was present at high level only in S1, with the most abundant OTU being

assigned as Candidatus Cloacimonas sp. 1 (45%). Representatives of this phylum seem

not to be specifically related to HW degradation, as they have also been previously

detected in digesters operating with other substrates (Sun et al., 2016). WWE1 was

found to be able to grow by fermenting amino acids along with hydrogen and butyrate

production (Jankowska et al., 2018), which could be considered as a

hydrogen-producing syntrophic bacterium (Lee et al., 2018). In this study, the

19
concurrent high abundance of Candidatus Cloacimonas sp. 1 and hydrogenotrophic

methanogen WSA2 group (Methanobacteriales sp.2, Methanobacteriales sp.3) indicates

the existence of a potential syntrophic interaction between the two unknown microbes.

It is known that syntrophic relationships between microorganisms producing and

consuming hydrogen are crucial to ensure a stable and efficient AD process (Carballa et

al., 2015). Thereby, a stable AD process and high methane production were obtained in

S1 (Fig.1). Moreover, the IFC shift had a strong impact on the presence of the two

OTUs. The relative abundance of Candidatus Cloacimonas sp. 1 was significantly

decreased (< 2% in S2A, S2B and S3), probably because of the increasing ammonia

concentration and low ammonia tolerance of WWE1. This result was in accordance with

a previous study, which found that the relative abundance of WWE1 group decreased

with increasing ammonia concentration (Tian et al., 2018). The results in this study also

indicate that its relative abundance was affected by the variations of ammonia, VFA and

cation concentration (i.e. Na+, K+) (Fig.5).

3.2.3 Process stability and microbial community

The anaerobic microbiome composition and interactions among the microbial

members are affected by various environmental parameters, such as substrate

composition, temperature and feeding load (Carballa et al., 2015). In the current study,

three major phyla (Bacteroidetes, Firmicutes and WWE1) had predominantly

manifested in S1 with total relative abundances of 72% (Table S5). It is interesting that

Bacteroidetes and Firmicutes replaced WWE1 along with IFC increase. The increased

20
relative abundance of Bacteroidetes and Firmicutes indicated that the two phyla had a

strong ability to adapt to different operational conditions. In addition, a specific

hydrogen-producing bacteria (Candidatus Cloacimonas sp. 1) was found to be

negatively correlated with the pH (-0.91), ammonia (-0.89), Na+ (-0.92) and K+ (-0.84)

concentration (Fig.5). The two OTUs (Prolixibacteraceae sp. 2 and Prolixibacteraceae

sp. 5) identified as Tangfeifania diversioriginum had significant difference in relative

abundance under stable and unstable stage, which was correlated to the change of Na+

concentration discussed above. Interestingly, the OTU Marinilabiliaceae sp.3

(Maribacter polysiphoniae with 88% BLAST identity) belongs to Bacteroidetes and

Syntrophomonadaceae sp.6 (Dethiobacter alkaliphilus with 90% BLAST identity)

affiliated to Firmicutes were dominated in the most inhibitory period (Phase 3)(Fig.3c

and Fig.4). The two OTUs were both found to have stronger Na+-tolerant ability than

the dominant OTUs in the stable period (Nedashkovskaya et al., 2007; Sorokin et al.,

2008).

Similarly, the archaeal community is steered by the reactor environment. Ammonia,

Na+ and K+ concentrations were the primary factor influencing the archaeal community

structure and methane production (Fig.5). Methanosaeta and the uncharacterized WSA2

group were the main archaea in the stable stage of HW. Previous study also found the

uncharacterized WSA2 group and Methanosaeta primarily in archaeal sequences during

AD process (Nelson et al., 2011). However, the relative abundance of the methanogens

might decrease as shown by the methane reduction, and WSA2 was more sensitive to

21
ammonia, Na+ and K+ concentration in comparison with Methanosaeta.

3.3 Environmental footprints

According to Mizuta and Shimada (2010), the energy demand of a conventional

waste water treatment plant without sludge incineration ranges from 0.30 to 1.89

kWh/m3. In contrast, human wastes, if undergone AD process, produce biomethane

which can substitute petrol and other fossil fuels. Take China as an example, according

to the statistical data shown in Figure 6 and Table S2, the daily HW production in China

approximates to 2083 kt (on a wet basis) equals to 267 kt VS (National Bureau of

Statistics of China, 2018). If undergone AD process, 122 kt biogas could be potentially

produced on a daily basis. The produced biogas has an energy content of approximately

2.56E+6 GJ. Different downstream processes are possible for biogas as a renewable

source of energy: 1) Combustion in CHP plants to generate heat and electricity, 2)

Combustion in a boiler to generate heat, 3) Combustion in an engine motor to generate

electricity, 4) Upgrading to biomethane and being used as transportation fuel. The

environmental savings achieved from biogas production, as shown by Tsapekos et al.

(2019), highly depend on downstream processes and substitution choices.

The results of simplified LCA showed that the AD of HW has a huge potential to

decrease greenhouse gas (GHG) emissions since biological treatment of 1 tonne of HW

volatile solids would bring about a net saving of -272 kg CO2,eq in Climate change

damage category. This net saving would be achieved if the produced biogas was

combusted in CHP plants and substituted for mixed electricity production in China.

22
Thereby, it can decrease dependency on fossil-based electricity and significantly

contribute to low-carbon energy system. A rough estimation based on daily HW

production showed that, the electricity production from HW would amount to 257

Gwh/day. If this electricity substitutes coal-based electricity which accounted for 66%

of the mixed electricity in China (West 2017), -142 kt CO2,eq. would be avoided on a

daily basis. Detailed analysis, as shown in Figure 7 and Table S3, demonstrated that

Guangdong, Henan, and Shandong had the highest contribution to the total carbon

reduction followed by Jiangsu and Sichuan. Such spatial differences are attributed to the

population density in different parts of China.

The produced biogas can also be upgraded to biomethane and used as a

transportation fuel to substitute petrol. The potential for daily biomethane production

amounts to 49.4 kt which can substitute 51.7 kt petrol/day. This substitution could lead

to a net saving of -54.8 kt CO2,eq. Since 2007, China is the leading country in terms of

greenhouse gas emissions. In other words, China is responsible for almost 30% of the

global greenhouse gas emissions (Boden et al., 2017). The results achieved herein

showed that biogas production from low-cost substrates, i.e., HW, can significantly help

alleviate the negative environmental impacts of fossil-based fuels. Apart from its key

role in climate change damage category, AD process treating HW positively contributes

to the bio-circular economy due to its considerable biomethane potential. It is worth

mentioning that the use of HW as a substrate for bioenergy production would decrease

the market price of final product, i.e., electricity, heat, fuel, etc, so contribute more to

23
the sustainable development. Having ignored the investment cost, the average cost for

agricultural biogas in China approximates 0.11 USD/m3, and its price is 0.22 USD/m3

(Gao et al., 2019). A low-cost substrate like HW can reduce the production costs and

increase the economic benefit of biogas production. Moreover, the LCA results

demonstrated that such a bioenergy system would also bring about environmental

savings in ecosystem quality and resource damage categories (Fig.8). The only damage

category with positive environmental impact was human health with a net impact of

8.2E-05 DALY/1 t VS showing that no saving would be achieved under this damage

category. The sterilization of digestate requires considerable amount of heat which

supplies from coal-based heat production at the background system. Thus sterilization

process contributed significantly to Human health damage category. The environmental

savings attained by the substitution strategy, i.e., biogas based electricity for

fossil-based electricity and digestate nutrients for N-based fertilizers, was not big

enough to compensate the environmental impacts caused by air emissions from coal

power plants. Sterilization as shown in Fig.8 has also significant contribution to other

damage categories, i.e., Climate change, Ecosystem quality, and Resources, but was not

big enough to dominate other impacts, thus net environmental savings were obtained. A

solar-based heating system can help decrease the impacts of sterilization process.

Although promising results have been achieved herein, the results showed that

scaled-up operation would also suffer some limitations. As discussed above, the

maximum methane yield was attained at IFC of 3% total solid which is reportedly low

24
for a medium and large-scale biogas plant because it would increase the required

volume of the digesters and corresponding water requirement for TS reduction.

However, the higher IFC would lead to ammonia inhibition, dropped biogas production,

and process failure. Co-digestion with carbon rich substrate such as straw would be a

viable option to avoid ammonia inhibition problems at high IFC. Moreover, nitrogen

recovery prior to anaerobic digestion can help reduce ammonia inhibition risks and

improve profitability of the process (Khoshnevisan eta al., 2019). The energy cost for

nitrogen recovery using electrochemical system varies from 9.97 to 13.9 kWh/kg

N(Christiaens et al., 2017; Luther et al., 2015), which is comparable with production of

nitrogen fertilizers via the conventional Haber-Bosch process at cost of 10.3 to 12.5

kWh/kg-N. Novel hybrid systems consisting of a submersible microbial desalination

cell and anaerobic digesters could also help alleviate ammonia inhibition risks during

AD of human wastes while recovering ammonia and producing electricity(Zhang et al.,

2015).

Digestate, remaining after AD of human waste, is another concern since many

countries have banned its direct used on farmlands due to the specific pathogens which

are dangerous for human health. Although pasteurization at 70 can kill most

pathogens and decrease the risk of infection, further research is still required to

guarantee safe agricultural production.

4. Conclusion

Anaerobic digestion of HW can provide a meaningful source of energy with the

25
highest methane production of 327±21 mLCH4 (gVS)-1) at low IFC. An increment of

IFC led to high concentration of free ammonia and cation concentration (K+, Na+),

which inhibited reactor performance lowering the methane production by 50%, as well

as affect the microbial community structure and dominance. Therefore, co-digestion

with carbon-rich feedstock might be a suitable and economical method for overcoming

the inhibiting effect in high IFC. The results of environmental assessment showed that

the AD of HW has considerable potential for GHG mitigation as 1 t of HW VS would

lead to a saving of -54 to -272 kg CO2,eq, depending on the downstream application. The

high energy demand for digestate sterilization before field application could one of the

serious concerns if supplied from fossil-based sources. Furthermore, further research is

needed to make sure long-term application of HW digestate on farmlands would not put

soil, human, and plant health at risk..

5. Acknowledgments

This work was supported by National Key Research and Development Program

(2018YFD0800803) and open fund of Jiangsu Key Laboratory of Anaerobic

Biotechnology and Key Laboratory of Nonpoint Source Pollution Control, Ministry of

Agriculture, P.R.China. We also thank Bill & Melinda Gates Foundation. Na Duan

would like to thank for the financial support from China Scholarship Council.

Appendix A. Supplementary data

E-supplementary data of this work can be found in online version of the paper.

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34
Figure captions:

Fig. 1 Process performance of the HW anaerobic digestion (a-methane yield and organic

loading rate (OLR), b-TAN and FAN concentration, c-pH value, d-SCOD of the effluent).

Fig. 2 Time course of VFA concentration (a) and cation concentration (b).

Fig. 3 Changes of the relative abundance of all microbial samples (a-archaea on genus level;

35
b-bacteria on phylum level; c- bacteria on genus level)

Fig. 4 Relative abundances (%) (a-archaea and c-bacteria) and the corresponding folds change

(b-archaea and d-bacteria) in response to IFC shift

Fig.5 Correlation between the microbial abundance and biochemical parameters (a-

bacteria and b-archaea)

Fig.6 The trend in daily human waste production in top 6 producers in China

Fig.7 Potential electricity production from human waste and their associated carbon

dioxide mitigation in different provinces in China

Fig.8 Environmental impacts of human waste anaerobic digestion

Table 1 Properties of human waste and inoculum

Parameter Human waste Inoculum

Total solid (TS)(%w) 14.56±0.36 1.73±0.04
Volatile solid (VS)(%TS) 87.93±0.99 55.49±0.58
Crude protein (%TS) 33.88±0.91 —
Crude fats (%TS) 14.33±0.54 —

36
 Carbohydrate (%TS) 39.73±1.37 —
C/N ratio 8.73 —

—: no analysis, w: wet base.

37
 Table 2 Comparison of methane production and organic matter removal with literatures

Feedstock HRT Temperature Methane SCOD

Substrate Phase Reactor Average Bpr Source
concentration (d) (oC) content (%) removal (%)
1 3%TS(0-75d) 327±21 mLgVS-1 63.4±1.8 80.4
4%TS(76-122d) 303±18 mLgVS-1 63.0±2.5 81.1
HW 2 CSTR 25 35 This study
4%TS(123-150d) 210±31 mLgVS-1 60.8±3.2 68.7
3 5%TS(151-175d) 121±26mLgVS-1 61.3±3.8 69.9
HW / / Batch / 37 338.5 mLgVS-1 60 / Lalander et al., 2018
HW / / Batch / 37 208±89 mLgVS-1 / / Gomaa et al., 2017
HW / 2.60gVSL-1d-1 ASBR 10 35 166-181 mLgVS-1 72.1-72.4 66-67 Park et al., 2001
HW / 0.235gVSL-1d-1 FAR 30 15 124 mLgVS-1 57 97 Meher et al., 1994
HW / 10%TS FDR 20-25 20 0.71-0.86LL-1d-1 60-63 54 Singh et al., 1993

HW: human waste; ASBR: Anaerobic sequencing batch reactor; FDAR: Floating dome anaerobic reactor; FAR: Fixed-dome anaerobic reactor; FDR:

Floating-dome reactor

38
Fig.1

39
Fig.2

40
Fig.3

41
Fig.4

42
Fig.5

43
Fig.6

44
Fig.7

45
Fig.8

Human health Ecosystem quality

0.0008 300
200
0.0004 100

PDF/m2/yr
DALY

0
0
-100

-0.0004 -200
-300
-0.0008 -400

Climate change Resources

600 8000
400
4000
200
MJ primary
kg CO2 eq

0 0

-200 -4000
-400
-8000
-600
-800 -12000

46
Highlights:

1. The highest methane production (327±21 mL (gVS)-1) was obtained at low feeding

load

2. Free NH3, Na+, and K+ concentration affect overall process and microbial dominance

3.Potential syntrophic interaction between WWE1 and WSA2 contributed stable process

4. Anaerobically treating human waste can achieve GHG mitigations as -54 to -272 kg

CO2,eq
Declaration of interests

☐ √The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: