Energy 294 (2024) 130736

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Energy
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The impact of salinity on biomethane production and microbial community

in the anaerobic digestion of food waste components
Xiaoman He a, Chen Deng b, *, Pengfei Li c, Wenbing Yu a, Huichao Chen a, Richen Lin a, d,
Dekui Shen a, **, Saeid Baroutian e, f, g
a
Key Laboratory of Energy Thermal Conversion and Control of Ministry of Education, School of Energy and Environment, Southeast University, Nanjing, 210096, PR
China
b
School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 210096, PR China
c
College of Mechanical and Electrical Engineering, Henan Agricultural University, Nongye Road 63, Zhengzhou, Henan, 450002, PR China
d
Civil, Structural, and Environmental Engineering, School of Engineering, University College Cork, Cork, Ireland
e
Department of Chemical and Materials Engineering, The University of Auckland, Auckland, 1010, New Zealand
f
Circular Innovations (CIRCUIT) Research Centre, The University of Auckland, Auckland, 1010, New Zealand
g
Ngā Ara Whetū Centre for Climate, Biodiversity, and Society, The University of Auckland, Auckland, 1010, New Zealand

A R T I C L E I N F O A B S T R A C T

Keywords: Food waste represents a valuable resource potential for the production of advanced biofuels, including bio­
Food waste methane. This study systematically assessed the impact of salt on biomethane production from different com­
Anaerobic digestion ponents of food waste. The highest biomethane yields of 258.89 ± 10.96, 337.52 ± 16.36, and 448.49 ± 23.375
Salinity
mL/g volatile solids (equivalent to biodegradability indexes of 75.75%, 88.92% and 92.66%) were achieved from
Biomethane
Organic components
cellulose-rich, starch-rich and protein-rich components, respectively, at a salt concentration of 3 g NaCl/L. The
biomethane yield was improved by 9.69–31.24% with low-concentration salt compared with no salt addition,
but it was inhibited by high salinity. Inhibition induced by high salinity (15 g NaCl/L) on biomethane production
followed the order of protein (15.50%) < starch (28.03%) < cellulose (42.92%). Co-digestion of different
components effectively mitigated the inhibitory effect of salt on biomethane production. Microbial community
analysis revealed that archaea were significantly affected by high salinity. With a high concentration of salt (15
g/L), acetoclastic methanogens (Methanosatea) predominated in the digestion of starch-rich components, whilst
hydrogenotrophic methanogens (Methaonmassiliicoccus and Methanobacterium) predominated in the digestion of
cellulose-rich and protein-rich components. Additionally, some salt-tolerant microorganisms (SC103, Thermo­
virga and Methanosarcina) were selectively enriched at high salinity.

1. Introduction sustainable management method for recovering biomass energy from

The quantity of food waste (FW) is progressively rising due to
accelerated population growth and enhanced living standards. China,
for instance, witnessed a significant rise in FW production, reaching
120.8 million tons in 2019, which is 1.55 times higher than the figures
reported in 2009 [1]. FW exhibits a high potential for resource recycling
due to its abundant availability of biodegradable organic compounds,
which can be further converted into biogas, H2, ethanol and high
value-added chemical products through fermentation, thermochemical
and chemical methods [2–4]. Among the disposal technologies avail­
able, anaerobic digestion (AD) is gaining increasing attention as a
FW [5]. AD effectively converts organic matter into valuable fuels (CH4
and H2) and biofertilizers, offering lower costs and reduced secondary
pollution compared to other methods [6,7].
AD of FW is a complex biochemical process in which all organic
components are digested, involving the coordination of a variety of
functional microbial communities in a single system. The performance
of AD is influenced by several key operating parameters (temperature,
pH, organic loading rate, C/N ratio, trace elements, salinity, and lipid
content) [8]. Methanogenic microorganisms, which play a crucial role in
AD, are particularly sensitive to environmental stresses associated with
these parameters. For example, the growth rate of methanogens would

* Corresponding author.
** Corresponding author.
E-mail addresses: chendeng@seu.edu.cn (C. Deng), 101011398@seu.edu.cn (D. Shen).

https://doi.org/10.1016/j.energy.2024.130736
Received 8 November 2023; Received in revised form 27 January 2024; Accepted 16 February 2024
Available online 17 February 2024
0360-5442/© 2024 Elsevier Ltd. All rights reserved.
X. He et al. Energy 294 (2024) 130736

Table 1 Table 2
Salt concentration in food waste sourced from different regions of China Characteristics of substrates and inoculum
Salt concentration in food waste Source Area Reference Parameters Starch Cellulose Protein SFW Inoculum

2–6 g/L, even reaches up to 10 g/L China [13] TS (%) 15.69 5.85 ± 15.05 ± 11.84 ± 5.05 ± 0.02
7− 12 g/L China [14] ± 0.10 0.05 0.14 0.14
8.0 g/L Shanghai (East China) [15] VS (%) 15.54 5.19 ± 13.87 ± 11.45 ± 2.10 ± 0.01
2~6.0 g/L Gansu Province (West China) [16] ± 0.10 0.05 0.13 0.14
reached ~12 g/L Anhui Province (Central China) VS/TS 99.20 88.63 97.14 96.69 41.57
2–5% (mass fraction basis) China [17] pH 5.01 ± 4.53 ± 6.54 ± 5.02 ± 7.85 ± 0.03
0.03 0.02 0.03 0.03
SCOD (g/L) 13.35 30.65 ± 14.00 ± 45.03 ± 1.07 ± 0.08
be significantly decreased when pH levels drop below 6.0, and the ac­ ± 0.93 0.83 0.66 2.11
tivity of methanogenic bacteria would be inhibited at pH levels either TAN (mg/ 19.91 857.46 ± 120.61 1155.41 ± 1195.18 ±
L) ± 2.55 15.91 ± 7.60 58.90 27.39
higher or lower than the range of 6.5–8.2 [9–11]. The concentration of FAN (mg/ – 0.02 ± 0.24 ± 0.07 ± 46.28 ±
minerals such as salt, is another important factor affecting the activity of L) 0.00 0.04 0.01 3.80
methanogens. Sodium chloride (NaCl), as an essential flavoring agent in C (%) 45.72 40.88 ± 50.27 ± 44.89 ± –
food processing, presents in FW unavoidably. Salt concentration in FW ± 0.08 0.08 0.07 0.08
H (%) 5.85 ± 5.48 ± 6.94 ± 6.05 ±
may vary with several factors, such as local dietary habits, food pro­
–
0.07 0.05 0.07 0.06
cessing methods, and waste management practices [12]. A summary of N (%) 1.70 ± 2.09 ± 13.72 ± 5.42 ± –
the reported salt concentration in food waste sourced from different 0.07 0.06 0.08 0.08
regions of China is shown in Table 1. The salt concentration in FW from O (%) 51.64 51.39 ± 28.35 ± 41.34 ± –
China typically ranged from 2 to 12 g/L [13–17].Sodium (Na) is an ± 0.24 0.25 0.14 0.03
C/N 26.89 19.60 3.67 8.28 –
essential element to maintain the normal metabolism of microbial cells
in the AD system, which can facilitate ATPase to catalyze the conversion Notes: TS: total solids, VS: volatile solids, SCOD: soluble chemical oxygen de­
of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) mand, TAN: total ammonia nitrogen, FAN: free ammonia nitrogen, SFW: Syn­
derived from the transmembrane gradient of Na+ [18]. Suitable con­ thetic food waste.
centrations of Na+ would increase the activity of bacteria and metha­
nogens, and improve the biodegradability of organic matter and the potential and biodegradability characteristics. Previous studies exam­
efficiency of methane production. The optimal sodium ion concentra­ ining the impact of NaCl on AD have primarily concentrated on estab­
tions for the growth of hydrogenotrophic methanogens and acetoclastic lishing the quantitative correlation between NaCl concentration and the
methanogens in the mesophilic AD system were documented as 350 production of VFAs and methane [12,17]. However, these studies have
mg/L and 230 mg/L, respectively [19]. Additionally, it has been re­ tended to overlook the diversity of FW components and its potential
ported that salt can also alleviate the inhibition of animal fats and influence on the AD process. The salinity-induced effect on the AD
vegetable oils on AD of FW [20]. However, a high-level of Na+ could performance of individual organic components in FW, particularly with
interfere with microbial metabolism because of the sharp increase in regard to the inhibitory threshold for biomethane production, lacks
osmotic pressure and cell dehydration, inhibiting methane production comprehensive investigation and understanding.
and even results in AD system failure [21,22]. Several studies have The novelty of this study lies in its pioneering investigation of the
indicated the dosage-depend inhibition of salt to AD. Lee et al. (2019) salinity-induced effects on digestion of key components of FW alongside
reported that Methanosaeta concilii exhibited suppressed methanogenic an exploration of variations in inhibition levels during AD in response to
activity at NaCl concentrations exceeding 3 g/L, with a calculated high salt concentrations. The objectives of this study are threefold: 1) to
half-maximal inhibitory concentration (IC50) for methanogenesis of 5.2 compare the biomethane production from AD of different organic
g Na+/L [23]. Another study demonstrated that 10.1 g-Na+/L can cause components under varying salt concentrations, 2) to evaluate the system
a 50% reduction in the biomethane production from anaerobic stability and substrate biodegradability of substrates by monitoring
co-digestion of cattle manure with macroalgal biomass [24]. The process parameters, and 3) to analyze the microbial community struc­
inhibitory concentration of salt in AD has shown considerable variation ture of bacteria and archaea under different salt stress conditions. This
in previous studies. This disparity can be attributed to various factors, study can potentially reveal the salinity-induced microbial responses
including the composition of FW, the varying sensitivity of microor­ during AD of FW. Moreover, it could serve as a valuable guide for en­
ganisms to salt, and potential antagonistic or synergistic effects with gineers, enabling them to optimize energy recovery from FW by
other cations [25]. adjusting the appropriate salt concentration and organic components
Several studies showed that the AD performance of FW can be within the digestion system.
affected by the organic composition of the substrate [26,27]. Lipids have
a higher theoretical biomethane potential of 1014 mL/g volatile solids 2. Materials and methods
(VS) than carbohydrates and proteins [28]. However, the accumulation
of long-chain fatty acids resulting from lipid hydrolysis can lead to in­ 2.1. Food waste and inoculum
hibition during AD, which may decrease nutrient transport efficiency
and impede methanogenic activity [29]. Therefore, in practical appli­ FW is predominantly sourced from households, canteens, and res­
cations, waste lipids from FW are commonly recycled through physical taurants, with its composition being subject to variations influenced by
and/or chemical methods after their separation [30–32]. In contrast, factors such as seasons, cultural practices, geographical regions, climate
carbohydrates and proteins can be easily hydrolyzed into glucose and conditions, and the economic status of the area [34]. A synthetic FW
amino acids, which are further converted into volatile fatty acids (VFAs) (SFW) was used in this study to ensure uniformity of substrate. This
and biogas by acidogens and methanogens [33]. Nonetheless, it has been involved selecting representative concentrations of starch, protein, and
demonstrated that acidification and ammonia inhibition are prone to cellulose based on the component analysis of Chinese FW [1]. The
occur during the AD of carbohydrate/protein-rich FW. This is attributed components were created as follows:
to the rapid hydrolysis of substrates, which hampers the activity of
methanogenic archaea, thereby disrupting the system stability [1]. The (1) Starch-rich components: rice, steamed buns and noodles;
various components of FW exhibit distinct biochemical methane (2) Protein-rich components: lean meat, beef meat, fish and chicken;
(3) Cellulose-rich components: vegetables, fruits and peel.

2
X. He et al. Energy 294 (2024) 130736

(4) SFW was prepared by collecting, crushing, mixing and homoge­

VSinitial -VSfinal
nizing starch-rich, protein-rich and cellulose-rich components in VSremoval (%) = × 100% (3)
VSinitial
a 2:1:1 total solids (TS) ratio to mimic a real FW according to the
characteristics of FW sourced from China reported in previous where VSinitial and VSfinal represent the VS of substrates at the beginning
studies [1,35–39]. and end of anaerobic digestion, respectively.
The inhibition degree was determined using Eq. (4) [43].
These components and SFW were stored at 4 ◦ C.
The inoculum sludge used in this study was originated from a full- Inhibition degree (%) =
YS,0 -YS,i
× 100% (4)
scale mesophilic continuously stirred digester in a wastewater treat­ YS,0
ment plant in Zhengzhou, China. To eliminate residual biodegradable
organic matter, the inoculum sludge was acclimated at 37 ◦ C under where YS,0 and YS,i denote the cumulative CH4 yield in AD of substrates
anaerobic conditions until no biogas production was detected for seven at NaCl concentration of 0 g/L and i (where i = 3, 5, 10, 15 g/L),
consecutive days prior to the biomethane potential (BMP) experiments. respectively.
Table 2 shows the physicochemical characterization of the different The theoretical biomethane potential (TBMP, mL CH4/g VS) of
components of the SFW and the inoculum. substrates was calculated according to the Buswell Equation (Eq. (5) and
(6)) based on the elemental composition data [44]. The BMP (mL CH4/g
VS) of substrates represents the measured methane yield in biomethane
2.2. Batch biomethane potential (BMP) test potential test. The biodegradability index (BI, %) was calculated as the
ratio of BMP to TBMP (Eq. (7)).
Batch AD was conducted to investigate the impact of salt on anaer­ ( ) ( ) ( )
bc 3 a bc3 ab c 3
obic methanogenesis. Glass bottles with a volume of 1000 mL (working Ca Hb Oc Nd + a- - + d H2 O → + - - d CH4 + - + + d CO2
42 4 2 848 28 4 8
volume was 800 mL) were used as digesters, each capped with a rubber
stopper. Following the volatile solids (VS) content detailed in Table 2 for + d NH3
the various components of the SFW and the inoculum, 5 g VS of FW and (5)
10 g VS of inoculum sludge were added to each digester. Subsequently, ( )
different amounts of NaCl were added to the corresponding digester to 22.4 × 2a + b8-4c-38 d × 1000
achieve NaCl concentrations of 0, 3.0, 5.0, 10.0, 15.0 g/L, based on the TBMP (mL / g VS) = (6)
salinity analysis of Chinese FW [13,40]. Each digester was connected to 12a + b + 16c + 14d
a 1000 mL gas bag via a latex hose. A blank trial containing only inoc­
BMP
ulum was used to adjust for biogas yield. Before initiating the experi­ BI(%) = × 100% (7)
TBMP
ments, nitrogen gas (99.9%) was purged into each digester for 5 min to
remove air and establish an anaerobic environment. The cultivation The modified Gompertz model (Eq. (8)) was empolyed to fit the
bottles were then placed in a constant temperature incubator set at 37 ◦ C cumulative methane production curve for anaerobic digestion.
for 25 days. To ensure thorough mixing, the bottles were manually { [ ]}
Rm e
agitated before each sampling event. Biogas was collected daily by H(t) = Hm × exp -exp (λ-t) + 1 (8)
Hm
changing the gas collection bag attached to the bottle to determine
biogas volume and methane concentration. Digestate samples were Hm
collected every three days for physicochemical analysis. Each trial was Tm = +λ (9)
Rm e
conducted in triplicate, and the average value and standard deviation
were reported. where H(t) refers to the yield of cumulative biomethane at a digestion
time (mL/g VS), Hm is the maximum biomethane yield (mL/g VS), λ is
the lag-phase time (d), Rm is the peak biomethane production rate (mL/
2.3. Analytical methods
g VS/d), Tm is the peak digestion time (d), e ≈ 2.7183.

The total solid (TS) and VS in the various components of the SFW,
inoculum and digestate were quantified based on the standard method
(APHA, 1998). The elemental composition (C, H, O, N and S) of FW was
analyzed using the Vario EL cube elemental analyzer. Methane content
of the biogas was determined using a gas chromatograph (GC9720Plus,
Fuli Instruments). Soluble chemical oxygen demand (SCOD) was
determined using the COD digester (Hach DBR200, America) and
portable spectrophotometer (Hach DR900, America). Total ammonia
nitrogen (TAN) was measured via Nessler’s reagent spectrophotometry
(HJ 535–2009). Free ammonia nitrogen (FAN, mg/L) concentration was
calculated using the following with equations (1) and (2) [41].
FAN (mg / L) =
TAN
1 + 10 (pKa-pH)
2729.92
pKa = 0.09018 +
T + 273.15
where pKa is the equilibrium constant; and T is the temperature (◦C).
2.4. Kinetic calculations
The VS removal rate (%) was defined as per Eq. (3) [42].
2.5. Microbial community analysis
To analyze the structure of microbial community in the AD system of
various components of the SFW under different salt concentrations (0, 3,
15 g/L), digestate samples were collected at the end of the experiments
and subjected to the high-throughput 16S rRNA gene sequencing. Mi­
crobial DNA extraction was performed using the FastDNA® Spin Kit for
Soil (MP Biomedicals, USA). Subsequently, the DNA fragments of the
microbial communities were amplified by polymerase chain reaction
(PCR). The primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-
GGACTACNNGGGTATCTAAT-3′) were utilized to target the V3–V4
(1) variable regions of the bacterial 16S rRNA genes, while primers
Arch519F (5′-CAGCCGCCGCGGTAA-3′) and Arch915R (5′-
GTGCTCCCCCGCCAATTCCT-3′) were employed to target the archaeal
(2) 16S rRNA V4–V5 variable region. Following PCR, purification and
quantification of the resulting DNA products were conducted using the
AxyPrep DNA Gel Extraction Kit (Axygen, USA) and Quantus™ Fluo­
rometer. Barcoded libraries were prepared and sequenced on an Illu­
mina platform (Miseq PE300, USA) by Major Bio-Medicine Technology
Co., Ltd (Shanghai, China). Operational taxonomic units (OTUs) were
clustered through UPARSE with a 3% divergence (97% similarity).
Classification of the final OTUs was conducted on the RDP Classifier

3
X. He et al.
Fig. 1. Effects of salt concentration on cumulative biomethane yield (A, B, C, D) in anaerobic digestion of different food waste components (A: Starch-rich; B:
Cellulose-rich; C: Protein-rich; D: Synthetic food waste, SFW-mix).
with a 70% confidence threshold.
2.6. Statistical analyses
The primary data were underwent statistical analysis using Microsoft
Excel 2019. Statistical differences between the means were analyzed by
one-way analysis of variance (ANOVA) using SPSS 26.0 (p < 0.05).
Statistical significance was assumed at p < 0.05. All figures were drawn
using Prism 8.0.
3. Results and discussion
3.1. Biomethane production
To investigate the effects of salt on the biomethane production in the
AD of various FW components, the biomethane production rate and
cumulative biomethane yield during the 25-day batch BMP test were
determined. As shown in Fig. 1 (A) and Fig.S1 (A), the cumulative
biomethane yield initially increased for the starch-rich FW as the salt
concentration increased from 0 to 3 g/L, then decreased as the salt
concentration further increased beyond 3 g/L. Similar increasing-
decreasing trends were observed with cellulose-rich, protein-rich, and
SFW-mix components with different inflection points of the salt con­
centration. The peak biomethane yield, attained at a salinity level of 3 g/
L, reached cumulative biomethane yields of 337.52 ± 16.36 mL/g VS for
the starch-rich components, 258.89 ± 10.96 mL/g VS for the cellulose-
rich components, 448.49 ± 23.37 mL/g VS for the protein-rich com­
ponents, and 324.03 ± 16.01 mL/g VS for the SFW-mix. Compared to
4
Energy 294 (2024) 130736
the control without salt addition, cumulative biomethane yields from
the reactors added with 3 g NaCl/L increased by 9.69%, 31.24%,
21.42%, and 35.29%, respectively. These findings suggest that low
concentrations of salt can enhance methane production. For instance,
Zhang et al. achieved a maximum biomethane yield of 243.06 mL/g VS
in the digestion of kitchen waste at a NaCl concentration of 4.0 g/L [45].
Nevertheless, as the salinity increased further, the inhibition of bio­
methane production became evident in the digestion systems, and the
extent of inhibition varied depending on the specific feeding substrates
(Fig. S1). In starch-rich components, cumulative biomethane yield re­
ductions of 12.55%, 26.18%, and 28.03% occurred at NaCl concentra­
tions of 5, 10, and 15 g/L, respectively, compared to the control without
NaCl addition (Fig. 1A). This inhibition may be attributed to microbial
activity loss due to cell damage and reduced activity of key enzymes
(such as CoAT and F420) under high-salt conditions [14,46]. Compared
with the starch-rich components, the AD of cellulose and protein-rich
components showed higher resistance to salt inhibition on biomethane
production. As seen in Fig. 1B and C, the cumulative biomethane yield
for the AD of cellulose and protein-rich components started to decline
when the salinity reached 15 g/L and 10 g/L, respectively. At a NaCl
concentration of 15 g/L, the biomethane production from cellulose-rich
components exhibited an inhibition degree of 42.92%. Similarly, the
inhibition degrees for protein-rich components in the AD system were
9.11% at 10 g/L NaCl and 15.50% at 15 g/L NaCl (Figs. S1B and C). This
underscores how high salt inhibiton effect on the AD of FW varries by
the chemical nature of organic fractions. These results are consistent
with the observations of Li et al. [47], whereby different components
may contribute to varying degrees of inhibition under high salt
X. He et al. Energy 294 (2024) 130736

Table 3
Kinetic analysis of food wastes fractions anaerobic digestion at different salt concentrations
Conditions Experimental data Kinetic model parameters

TBMP (mL/g VS) BMP (mL/g VS) BI (%) Hm (mL/g VS) Rm (mL/g/d) λ (d) Tm (d) R2

(A) Starch-rich
0 g/L NaCl 379.54 ± 2.89 307.70 ± 5.53 81.07 309.70 ± 2.05 76.11 0.62 2.12 0.9909
3 g/L NaCl 337.52 ± 16.36 88.92 339.12 ± 2.01 75.14 0.67 2.33 0.9934
5 g/L NaCl 269.09 ± 7.85 70.90 270.41 ± 1.27 58.04 2.02 3.74 0.9974
10 g/L NaCl 227.15 ± 7.14 59.84 225.62 ± 2.04 50.30 3.93 5.58 0.9933
15 g/L NaCl 221.45 ± 9.39 58.35 213.90 ± 2.60 44.04 6.39 8.18 0.9923
(B) Cellulose-rich
0 g/L NaCl 341.77 ± 2.93 197.27 ± 5.53 57.72 196.30 ± 1.19 64.66 2.94 4.05 0.9957
3 g/L NaCl 258.89 ± 10.96 75.75 259.48 ± 1.31 57.83 1.66 3.31 0.9966
5 g/L NaCl 231.80 ± 13.36 67.82 229.98 ± 1.45 54.99 3.91 5.45 0.9967
10 g/L NaCl 238.31 ± 9.16 69.73 235.12 ± 1.90 62.65 4.87 6.25 0.9950
15 g/L NaCl 112.60 ± 5.31 32.95 111.89 ± 0.48 53.50 8.49 9.26 0.9990
(C) Proteins-rich
0 g/L NaCl 484.01 ± 2.24 369.36 ± 19.38 76.31 365.42 ± 3.20 59.22 0.68 2.95 0.9899
3 g/L NaCl 448.49 ± 23.63 92.66 451.27 ± 2.54 64.73 1.47 4.04 0.9970
5 g/L NaCl 401.31 ± 25.63 82.91 412.47 ± 4.59 42.87 3.93 4.59 0.9953
10 g/L NaCl 335.71 ± 15.07 69.36 337.18 ± 4.31 41.25 5.50 4.31 0.9942
15 g/L NaCl 312.10 ± 5.10 64.48 309.77 ± 4.09 32.86 5.69 4.09 0.9950
(D) SFW-mix
0 g/L NaCl 403.60 ± 1.73 239.51 ± 8.01 59.34 240.51 ± 1.37 60.53 3.20 4.66 0.9968
3 g/L NaCl 324.03 ± 16.01 80.28 324.50 ± 0.91 76.03 1.03 2.60 0.9987
5 g/L NaCl 269.85 ± 13.05 66.86 268.20 ± 1.83 49.15 2.10 4.11 0.9953
10 g/L NaCl 234.85 ± 12.37 58.19 232.87 ± 1.66 57.18 3.91 5.41 0.9956
15 g/L NaCl 212.48 ± 6.30 52.65 211.08 ± 1.00 51.50 4.90 6.41 0.9984

Note: TBMP: theoretical biomethane potential, BMP: biomethane potential, BI: biodegradability index, Hm: maximum biomethane yield, Rm: peak biomethane pro­
duction rate, λ: lag-phase time of biomethane production, Tm: peak digestion time, SFW-mix: Synthetic food waste.

Fig. 2. Effects of salt concentration on biomethane production rate (A, B, C, D) in anaerobic digestion of different food waste components (A: Starch-rich; B:
Cellulose-rich; C: Protein-rich; D: Synthetic food waste, SFW-mix).

5
X. He et al.
Fig. 3. Effects of salt concentration on soluble chemical oxygen demand (SCOD) in anaerobic digestion of food waste components (A: Starch-rich; B: Cellulose-rich;
C: Protein-rich; D: Synthetic food waste, SFW-mix).
condition (12 g NaCl/L). One reason for the difference in the degree of
inhibition appears to be related to variations in the hydrolysis process
for different components of FW. As for cellulose-rich components, the
complex lignocellulose structure leads to a lower hydrolysis rate than
that of starch-rich components [48,49]. This slower hydrolysis allows
the anaerobic microbes involved in the digestion of cellulose-rich
components more time to adapt to the high salinity environment, as
evidenced by the longer lag time (λ) estimated according to the Modified
Gompertz model (Table 3). In the case of protein-rich components, the
hydrolysis of proteins produces higher levels of ammonia. This increased
ammonia content can help alleviate the drop in pH due to the accu­
mulation of VFAs and prevent acidification inhibition during AD [26],
and the inhibitory effect of salt on protease activity is also lower than
that of α-glucosidase [43]. In addition, it is noteworthy that the SFW-mix
group presented a lower degree of inhibition compared with the
single-component groups (Fig. S1D). This phenomenon implies that the
anaerobic co-digestion can effectively mitigate the inhibition of high
salinity on biomethane production from AD of the mono organic
component. As reported previously, Zhao et al. (2017) found that the
methane yield in the co-digestion of FW and waste activated sludge was
higher than that in mono digestion of FW at different salinity [12].
The salinity-induced effects on the biomethane production rate from
FW are presented in Fig. 2. Overall, the addition of NaCl to a certain
level (such as exceeding 3 g/L) weakened and delayed the maximum
peak values of biomethane production rate in comparison to the control
group (0 g NaCl/L). As shown in Fig. 2B, there was a peak in biomethane
production rate of 70.58 ± 8.21 mL/g VS/d on the fourth day in
6
Energy 294 (2024) 130736
cellulose-rich components with no salt addition, and it significantly
decreased to 53.83 ± 4.33 mL/g VS/d on the tenth day with the addition
of NaCl at 15 g/L (p < 0.05). Similarly, the addition of NaCl at con­
centrations ranging from 3 g/L to 15 g/L caused a reduction of 45.05%–
61.09% in the peak production rate of the protein-rich components.
Furthermore, the peak time was also delayed by 1–7 days compared to
the control group (Fig. 2C). These results confirm that high salinity leads
to a decrease in the rate of biomethane production and an elongation of
the lag phase time. The calculated kinetic parameters, obtained by
fitting the modified Gompertz model, are presented in Table 3. At higher
salinity levels, the maximum biomethane yield (Hm) and peak bio­
methane production rate (Rm) exhibited a decline, indicating a reduc­
tion in the overall efficiency of biomethane production. Concurrently,
the lag time (λ) and peak digestion time (Tm) appeared to be extended,
suggesting a delay in the onset and completion of biomethane produc­
tion, in agreement with Hou et al. (2021) who found that the maximum
methane production rate (RCH4, max) dropped from 122.7 to 52.65 mL/
g VSadded/d and the λ prolonged from 7.52 d to 20.71 d when the salt
concentrations increased from 0 to 20 g NaCl/L [43]. This observation
indicated that longer start-up times are required under higher salt
conditions.
3.2. Biodegradation of organic substrates
The organic substrate degradation rate serves as a crucial indicator
reflecting the overall performance of AD. Organic matter degradation
includes the dissolution of solid organics and utilization of soluble
X. He et al.
Fig. 4. Effects of salt concentration on VS removal rate in anaerobic digestion of food waste components (A: Starch-rich; B: Cellulose-rich; C: Protein-rich; D:
Synthetic food waste, SFW-mix).
organic matter by microorganisms. The effects of salt on the solubili­
zation of different FW components are presented in Fig. 3. As can be
seen, initially SCOD in all reactors increased during the early stage of AD
(0–3 days), peaking on the third day before declining with prolonged
digestion time. Notably, the addition of NaCl amplified peak SCOD
concentrations across all components. For instance, in the AD of starch-
rich components, the maximum SCOD concentration significantly
increased (p < 0.05)from 3252.5 ± 222.5 mg/L to 5790.0 ± 10.0 mg/L
when NaCl concentration increased from 3 to 15 g/L. This phenomenon
suggests that high concentrations of salt may accelerate the dissolution
of organic substrates by increasing the osmotic pressure and inducing
cell lysis [50]. Additionally, high salinity could inhibit the utilization of
soluble organic matter by methanogens, leading to SCOD accumulation.
Previous studies have noted that NaCl addition accelerated protein and
carbohydrate release in waste activated sludge digestion systems, with
soluble substrate yield rising alongside NaCl dosage [14,50]. Notably,
the addition of NaCl in the reactors led to a slower SCOD concentration
decline post-third day and higher residual SCOD concentrations in the
digestate. These findings are in line with the results of biomethane
production, suggesting the suppression of AD by high salt (Fig. 1). This
observation was consistent with the findings from Zhao et al. (2017) that
the NaCl improved the hydrolysis but inhibited the process of meth­
anogenesis [12].
In addition to SCOD, the removal rates of VS were monitored
throughout the entire AD process (Fig. 4). The highest VS removal rate of
91.37%, 80.73%, 95.31 and 82.35% were achieved from starch-rich,
cellulose-rich, protein-rich components and SFW-mix with salt
7
Energy 294 (2024) 130736
addition of 3 g/L, respectively. However, a significant decline in the VS
removal rate was noted across different components when the salt
concentration exceeded 10 g/L. This indicated that high salt concen­
trations adversely affect the efficiency of VS removal during AD. In
comparison to scenarios with no NaCl addition, the reduction in VS
removal rate with the addition of 15 g NaCl/L was 25.33%, 43.50%,
18.22% and 12.69% for starch-rich, cellulose-rich, protein-rich com­
ponents and SFW-mix, respectively. These findings aligns perfectly with
that of the biodegradability index (BI) (Table 3) and biomethane yield
(Fig. 1). This correlation suggests that low salt concentrations may
enhance the dissolution of organic substrates and improve their
biodegradation. Conversely, high salt concentrations can impede mi­
crobial activity and restrict the utilization of soluble organic matter,
leading to a decline in the biodegradation rate and biogas production.
3.3. Stability of anaerobic digestion system
The pH, TAN and FAN are the key parameters affecting the stability
of AD process. The variations in pH values for different FW components
under different NaCl concentrations are shown in Fig. 5. Initially, all
digesters experienced a drop in pH during the first 3 days due to acidi­
fication, followed by a gradual increase as the organic acids were
consumed by the methanogens [51]. Notably, the system pH value
exhibited a more pronounced decline during the start-up stage with the
increase in salt concentrations. For example, the pH values significantly
decreased from the range of 7.63 ± 0.03–7.52 ± 0.01 to 7.00 ±
0.03–6.47 ± 0.01 for starch-rich components with the addition of NaCl
X. He et al.
Fig. 5. Effects of salt concentration on pH in anaerobic digestion of food waste components (A: Starch-rich; B: Cellulose-rich; C: Protein-rich; D: Synthetic food waste,
SFW-mix).
(p < 0.05) (Fig. 5A). This phenomenon may be attributed to the pro­
motion of high salt on the solubilization of organic substrates (Fig. 3).
Conversely the pH decrease in the AD system of protein-rich fractions
was notably attenuated during the initial stage, maintaining values
above 7 throughout the experiment across all tested salinity levels. This
could be attributed to the buffering effect of ammonia nitrogen [52].
This was evident from the stable concentrations of TAN shown in
Fig. 6C, where the concentrations of TAN levels significantly increased
in all reactors containing protein-rich components during the first 6 days
before stabilizing. In contrast, carbohydrate-rich components exhibited
varying degrees of reduction in TAN concentration during the first 3
days (Fig. 6A and B)likely due to nitrogen source consumption for mi­
crobial growth [42]. Although the TAN concentration did not signifi­
cantly differ at different salinities, the FAN concentration in the
added-salt reactor was relatively low (Fig. S2), possibly due to the
lower pH induced by salt according to equation (1) [53]. However, it is
noteworthy that the maximum concentrations of TAN and FAN, reach­
ing 1995.33 mg/L and 215.46 mg/L respectively, were obtained in the
protein-rich components with the addition of NaCl at 3 g/L and 0 g/L
(Fig. 6C–Fig. S2C), which were lower than the inhibition concentration
(2000 mg/L and 300 mg/L) to methanogens [54,55]. These results
suggest that ammonia has no significant inhibitory effect on biomethane
production under salt stress.
8
Energy 294 (2024) 130736
3.4. Microbial community structure
3.4.1. Microbial diversity and richness
To gain more insights into the response mechanism of AD perfor­
mances among different FW components under salt stress, the compo­
sition and diversity of microbial community in the digestion system
under different salt concentrations were investigated via 16S rRNA high-
throughput sequencing. As illustrated in Table 4, concerning the bac­
terial community, the Shannon, ACE and Chao1 values in the cellulose-
rich and protein-rich components under different salt concentrations
showed no significant difference. However, high salinity (15 g NaCl/L)
led to reduced diversity and richness in both the starch-rich components
and SFW-mix groups compared to the control groups without salt
addition. Regarding the archaeal community, the diversity and richness
of all FW components groups gradually decreased with increasing NaCl
concentration, indicating significant inhibitory effects on archaeal
growth in AD due to high NaCl concentrations. This aligns with the
findings of Wang et al. (2017) who reported that the addition of NaCl to
a mesophilic anaerobic digester at a dosage of 20 g/L decreased the
number of OTUs, Chao 1 and Shannon index for archaeal communities
[15]. These results suggest that high salinity exerts a more potent
inhibitory effect on archaea compared to bacteria, which elucidates the
fundamental reason why biomethane production is suppressed under
high salt conditions. To further investigate the distinction of microbial
community compositions in different reactors, a detailed analysis of the
distribution of each microorganism at the phylum and genus level was
X. He et al. Energy 294 (2024) 130736

Fig. 6. Effects of salt concentration on total ammonia nitrogen (TAN) in anaerobic digestion of food waste components (A: Starch-rich; B: Cellulose-rich; C: Protein-
rich; D: Synthetic food waste, SFW-mix).

Table 4
Alpha diversity index for bacterial and archaeal community of digesters under different salt concentrations.
Digesters Ace Chao1 Shannon Simpson Coverage

Bacteria S0 1107.4725 1109.7339 4.5421 0.0289 0.9924

S3 1011.8718 1017.4825 3.8689 0.0605 0.9922
S15 983.99193 1003.8000 3.8124 0.0632 0.9922
C0 1093.4697 1132.4054 4.5541 0.0348 0.9925
C3 1187.3594 1152.0446 4.6020 0.0367 0.9917
C15 1108.7345 1114.2047 4.4932 0.0329 0.9923
P0 1137.0161 1133.2880 4.2842 0.0422 0.9916
P3 1037.6445 1042.7949 3.6413 0.0906 0.9917
P15 1046.5837 1046.3810 4.3186 0.0376 0.9926
SFW0 1183.7028 1242.5091 4.4055 0.0353 0.9910
SFW3 1056.6583 1036.4889 4.1562 0.0452 0.9925
SFW15 966.3078 988.7273 3.7727 0.0565 0.9924
Archaea S0 716.9732 688.8571 3.0285 0.1669 0.9965
S3 657.6678 654.9630 2.6742 0.2167 0.9965
S15 599.6678 600.2000 2.1736 0.2935 0.9966
C0 799.5510 795.9406 3.1908 0.1583 0.9963
C3 695.5456 692.4946 3.0579 0.1853 0.9972
C15 648.7548 529.5000 1.3930 0.5298 0.9968
P0 726.2557 716.5556 3.2898 0.0980 0.9964
P3 666.7312 689.9565 3.0553 0.1380 0.9965
P15 539.6328 551.6604 2.4599 0.2130 0.9969
SFW0 741.2971 731.9406 2.8645 0.2071 0.9963
SFW3 764.9022 764.2609 3.0731 0.1693 0.9963
SFW15 646.7009 553.3171 1.2548 0.5859 0.9968

Notes: S: Starch-rich; C: Cellulose-rich; P: Protein-rich; SFW: Synthetic food waste, SFW-mix; 0: 0 g NaCl/L; 3: 3 g NaCl/L; 15: 15 g NaCl/L.

9
X. He et al.
Fig. 7. Taxonomic composition of bacteria at the phylum level (A) and archaea at the genus level (B). Phyla and genera with lower abundances than 1% are classified
into “Others”. (S: Starch-rich; C: Cellulose-rich; P: Protein-rich; SFW: Synthetic food waste, SFW-mix; 0: 0 g NaCl/L; 3: 3 g NaCl/L; 15: 15 g NaCl/L).
performed in Fig. 7 and Fig. S3.
3.4.2. Bacterial community
The composition and relative abundance of bacteria at the phylum
level in 12 reactors are shown in Fig. 7A. Following the batch test, the
dominant bacteria phyla in the 12 digesters were Firmicutes
(14.00–39.92%), Chloroflexi (5.92–20.17%), Actinobacteriota
(8.18–16.15%), Thermotogota (2.75–24.51%), Synergistota
(3.95–12.62%) and Caldatribacteriota (3.83–14.19%). However, the
relative abundances of these species in different reactors were signifi­
cantly influenced by the salinity and substrate types. Notable changes in
microbial community composition were observed with increasing salt
concentration. In the starch-rich and cellulose-rich components, the
relative abundance of Firmicutes increased from 22.90% to 24.62%–
34.13% and 38.16%, respectively. Conversely, in the SFW-mix group,
there was an increase in the relative abundance of Firmicutes from
23.09% to 39.92%. These findings suggest that the co-digestion system
could enhance the resistance of Firmicutes to high salt levels. It has been
reported that the phylum Firmicutes are mainly involved in the degra­
dation of macromolecular organic substrates (such as protein and lipids)
into VFAs, and they can produce endospores to protect against extreme
environments, and becoming the predominant bacteria [56].
Conversely, compared to the control without NaCl addition, the Chlor­
oflexi and Caldatribacteriota were enriched in carbohydrates fermenta­
tion (starch-rich and cellulose-rich components) under the high salt
conditions (15 g NaCl/L), whereas the opposite trend was exhibited in
10
Energy 294 (2024) 130736
protein-rich components and SFW-mix group. Chloroflexi and Calda­
tribacteriota are mainly responsible for the degradation of various com­
plex carbohydrates like glucose, cellulose and hemicellulose during AD
[57,58]. Notably, the relative abundances of Thermotogota increased
consistently across all substrate types as the NaCl concentration
increased from 0 to 15 g/L. This observation suggests that certain
salt-tolerant bacteria were selectively enriched to maintain hydrolysis
and acidification capacities in high-salt environments [45,59]. Further
comparison of the bacterial community composition from different re­
actors at the genus levels was observed in Fig. S3, the predominant
bacterial genera were SC103, Clostridium_sensu_stricto_1, Romboutsia,
ADurb.Bin120 Candidatus_Microthrix and Thermovirga. Among them, the
genus SC103 and Thermovirga, belonging to the Thermotogota and Syn­
ergistota phylum, respectively, were significantly enriched in all
salt-added groups. Ma et al. (2021) showed that the relative abundance
of SC103 and Thermovirga increased with elevated salinities [21].
Therefore, the enriched SC103 and Thermovirga may play a key role in
the hydrolysis of organic substrates under high-salt conditions.
3.4.3. Archaeal community
The analysis of relative abundances of the methanogenic archaeal
communities at genus level provided further insights into the effect of
salinity on biomethane production during anaerobic digestion of FW. As
shown in Fig. 7B, it was observed that the richness of archaeal com­
munities among all the digesters was lower than that of bacterial com­
munities. Specifically, Candidatus_Methanofastidiosum, Methanosatea,
X. He et al.
Methanobacterium, and Methaonmassiliicoccus were the predominant
genera in different digesters, all of which accounted for 57.80–82.85%
of the total archaeal sequences. While these four dominant archaeal
genera were common across all digesters, their relative abundance
varied significantly with changes in substrates and salinity. Candidatus
Methanofastidiosum, known for methane production from acetate, pro­
pionate and methylated compounds [60,61], was the most dominant
methanogen genus in starch-rich components digestion at the low
salinity (0 g/L and 3 g/L). However, its relative abundance decreased
significantly from 37.03% to 18.07% at the high salinity (15 g/L).
Additionally, the relative abundance of hydrogenotrophic Meth­
aonmassiliicoccus decreased by 85.73% at 15 g/L NaCl compared to the
control (0 g/L), indicating weakened methanogenesis through the
H2/CO2 pathway in starch-rich components under the high salt condi­
tions. Conversely, the obligate acetoclastic Methanosatea showed an
increase in the relative abundance with increasing salinity, and it
became the most predominant methanogen genus in starch-rich com­
ponents with the relative abundance of 50.35% at the NaCl concentra­
tions of 15 g/L. Methanosaeta, one of the most common methanogens in
AD systems, exhibited a high affinity for acetic acid and a low minimum
threshold [15]. Increasing relative abundance of Methanosaeta may be
associated with the composition of VFAs as more acetic acid was pro­
duced in the digestion of starch-rich substrates than in the digestion of
protein-rich and cellulose-rich substrates [62] This result was consistent
with the findings of Wang et al. [15], whereby the relative abundance of
Methanosaeta showed a significant increase in response to high salinity
stress in a mesophilic AD of 6 g/L starch. However, there are a opposite
trend was exhibited in cellulose-rich, protein-rich components and
SFW-mix group. The relative abundance Candidatus Methanofastidiosum
and hydrogenotrophic methanogenic archaea (Methanobacterium and
Methaonmassiliicoccus) were dramatically increased and became domi­
nant genera under high-salt conditions. On the contrary, the increasing
NaCl concentrations had a strong inhibitory effect on the acetoclastic
Methanosatea, which indicated that the methanogenic pathways shifting
from acetoclastic to hydrogenotrophic methanogenic pathways with
increasing salt concentration in the digesters of cellulose-rich, pro­
tein-rich components. It was noticed that the Methanosarcina was
selectively enriched at 15 g NaCl/L in all substrate types, which
demonstrated that the Methanosarcina could potentially adapt to
high-salinity environments. It has been reported that the Methanosarcina
was a salt-tolerant mixotrophic methanogenesis, which could counteract
the increased osmotic pressure caused by high salinity by accumulating
Nε-acetyl-β-Lysine in cell [45,59].
4. Conclusions
This study highlights the significant impact of salinity on biomethane
production from AD of FW which is greatly influnced by the chemical
composition of organic components. The highest biomethane produc­
tion of 448.49 ± 23.37 mL/g VS was observed in protein-rich compo­
nents at 3 g NaCl/L. The inhibition induced by high salinity (15 g NaCl/
L) followed the sequence of SFW < protein < starch < cellulose. Mi­
crobial community analysis revealed significant effects of high salinity
on archaea. In starch-rich components, high salinity led to an increase in
acetoclastic Methanosatea, while hydrogenotrophic Methanobacterium
and Methaonmassiliicoccus were enriched in the digestion of cellulose-
rich and protein-rich components. Further investigations are necessary
to elucidate the inhibition mechanism of high salt on anaerobic diges­
tion of food waste in future studies.
CRediT authorship contribution statement
Xiaoman He: Conceptualization, Investigation, Methodology,
Writing – original draft. Chen Deng: Methodology, Supervision, Vali­
dation, Writing – review & editing. Pengfei Li: Conceptualization,
Methodology. Wenbing Yu: Investigation, Validation. Huichao Chen:
11
Energy 294 (2024) 130736
Writing – review & editing. Richen Lin: Methodology, Supervision,
Validation, Writing – review & editing. Dekui Shen: Conceptualization,
Funding acquisition, Methodology, Supervision, Writing – review &
editing. Saeid Baroutian: Writing – review & editing.
Declaration of competing interest
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.
Data availability
Data will be made available on request.
Acknowledgments
This work was supported by the National Key Research and Devel­
opment Program of China (2020YFC1910000), National Natural Science
Foundation of China (Grant No. 52376172) and Jiangsu Provincial Key
Research and Development Program (No. BE2020114).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.energy.2024.130736.
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