1. Introduction
According to FAO estimates, approximately 1.3 billion tons of food waste (FW) are generated annually across the entire food supply chain [
1]. Between 2010 and 2016, global food waste accounted for 8–10% of all man-made greenhouse gas emissions, leading to an annual loss of approximately USD 1 trillion [
2]. Current traditional practices, such as incineration and landfill, help release some stress from garbage siege; however, a series of problems require urgent attention, including the further cost of waste disposal, the lack of land space, groundwater pollution by leachate, and the emission of greenhouse gases that need further treatment [
3]. As a major component of municipal solid waste [
4], FW also promotes the growth of various pathogens, risking harm to human health [
5]. Therefore, developing appropriate countermeasures to tackle FW is emerging as a key issue associated with sustainable development and the bioeconomy concept [
6]. Anaerobic digestion (AD) is now widely accepted as the most effective technology for energy production and adds value to agronomic organic waste [
7], while simultaneously reducing secondary environmental pollution during the digestion process [
8,
9].
Regarding the characteristics of FW, the crude lipid and crude protein contents are in the ranges of 22.8–31.45% and 14.71–28.64%, respectively, and the main component is carbohydrate [
10]. The reported hydrolysis rates for municipal organic waste are in the order of lipids < proteins < carbohydrates [
11]. This implies that lipid hydrolysis is the rate-limiting step of the whole anaerobic process for FW. The high lipid content can also result in the accumulation of lipid dross in the digester, which adversely affects organic matter usage by methanogens and equipment cleanliness. Accordingly, a technology that is highly efficient and requires only mild reaction conditions, with no secondary pollution pretreatment, is urgently needed. In the present study, lipase is investigated as an efficient catalyst for hydrolyzing FW lipids into free long-chain fatty acids (LCFAs), which are further converted to hydrogen and acetate by acetogenic bacteria (β-oxidation process), and finally to methane by methanogenic archaea [
12]. Lipase addition (LA) is harmless to anaerobic treatment processes, and its contribution to biochemical oxygen demand in the waste stream is negligible [
13]. The biomethane production (1704 mL) of the hydrolysis products of crude lipid in food waste by enzymatic pretreatment was enhanced by 26.9–157.7% [
14]. The amount of biogas produced has been proven to be larger when biomass is subjected to thermal preprocessing [
15]. Hydrothermal pretreatment (HTP) is a thermal treatment that requires no extra drying [
16], and it promotes the dissolution of recalcitrant organic compounds by decomposing cell membranes efficiently at an appropriate temperature and residence time [
17]. The application of HTP has focused on lignocellulosic substrates [
18], sewage sludge [
19], lipid-rich wastewater [
20], and babassu oil processing [
21]. Wang et al. reported that use of thermal pretreated food waste halved the time needed to produce the same quantity of methane in comparison with fresh food waste [
22]. Therefore, this study proposed the HTP method as a technique for effectively shortening the FW hydrolysis time by changing FW properties, resulting in improved efficiency of the subsequent AD process. Subjecting FW to HTP benefitted the two-stage fermentative hydrogen and methane co-production, which exhibited an increase of 31.9% compared with untreated FW (387.9 mL/gVS). However, new methods are still required to process rich lipids that remain stable in hydrothermally pretreated FW [
23]. Therefore, the subsequent lipase addition after HTP might also lead to further biomass decomposition.
Crude glycerol (CG) is the main byproduct of the biodiesel industry from the transesterification of vegetable oil, animal fat, or used kitchen oil with alcohol, accounting for about 10% of the initial feedstock weight. Due to their low cost, sodium and potassium hydroxide are principally implicated in the alkali-based transesterification and introduce heavy metals into CG [
24]. As a complex mixture, CG contains glycerol, ethanol, water, salt, heavy metals, free fatty acids, unreacted monoglycerides, diglycerides, triglycerides, and methyl esters [
25]. There is an oversupply of CG containing a large amount of impurities, and its purification is difficult and expensive. Therefore, CG is treated as waste in many areas of industry, resulting in a waste of resources. The concept of mixing FW with CG has been proposed because the high water content of FW could act as a solvent for CG. Nuchdang [
26] reported that the AD of acid-treated glycerol in a synthetic medium had a maximum methane yield of 0.32 L g
−1 at standard temperature and pressure, with chemical oxygen demand (COD) removal achieved at an organic loading rate (OLR) of 1.6 g COD L
−1 d
−1. Astals et al. [
27] reported an increase of about 400% in biogas production under mesophilic conditions when pig manure was co-digested with 4% glycerol, on a wet basis, compared with mono-digestion. Nartker et al. [
28] showed that biogas and consequent energy production were significantly increased by a 25% glycerol loading within an anaerobic co-digestion process using primary sewage sludge.
Developing a system that is sustainable, and capable of handling the large amounts of organic waste currently produced in urban and rural areas with high efficiency, is a major current challenge. Therefore, this study aimed to improve the anaerobic digestion of FW by conducting LA, HTP, and their combination (HL) as pretreatments and co-digestion with different ratios of CG, and to evaluate the fermentation quality of these pretreatments and co-digestion methods.
2. Materials and Methods
2.1. Materials and Sample Preparation Procedures
Anaerobic sludge used as the inoculum was obtained from Hokkaido No.1 farm and stored at 52 °C, with its characteristics shown in
Table 1. The raw materials used as substrates were FW collected from the central restaurant of Hokkaido University and CG derived from the transesterification process during biodiesel production provided by Revo International Co., Ltd. (Kyoto, Japan). The FW was minced, homogenized using a blender, and then stored at −4 °C before use, with its characteristics shown in
Table 1.
The lipase used in the present study was sourced from Pseudomonas fluorescens and purchased from Amano Enzyme Inc. (Nagoya, Japan). The optimal growth conditions were pH 7–8.5 and a temperature of 50–60 °C, and the enzyme activity was 20,000 U/g.
2.2. Pretreatment Methods
2.2.1. Lipase Addition for Food Waste (LA)
Lipase (25 mg) was accurately weighed, dissolved in sodium chloride solution (10 g/L) to a final volume of 1 L, and cooled to below 10 °C. To obtain appropriate conditions for lipase application, the FW pH was adjusted to 8.0 using sodium carbonate solution (4 g/L). A 50% (w/w) lipase solution was then added to the FW, followed by incubation at 52 °C for 24 h, with no further pH adjustment during the subsequent process.
2.2.2. Hydrothermal Pretreatment (HTP)
HTP of FW was conducted in a 50 mL autoclave (PPY-CTRL, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). FW (30 g) and water (10 mL) were placed in the autoclave. The reactor was operated at 120 °C and 0.3 MPa, held for 60 min from when the autoclave reached the set temperature. The reactor was then cooled to ambient temperature. To generate little of furfural and hydroxymethylfurfural (HMF) during the hydrothermal process, the temperature at a relatively low severity (120 °C) was chosen [
29,
30]. As FW, which was stored in a refrigerator, had a certain viscosity that can act as a protective shield around the microbes, hydrothermal reaction conditions of 120 °C for at least 40 min were necessary to obtain sufficient sterilization [
31].
2.2.3. Combination of Hydrothermal Pretreatment and Lipase Addition (HL)
After HTP treatment of the FW (see
Section 2.2.2), 50% (
w/
w) lipase solution was added. The mixture was stirred evenly and then left to stand at 52 °C for 24 h.
2.3. Anaerobic Biodegradability Tests
Lipase addition and hydrothermal treatment were both expected to somewhat decrease the contents of total solids (TS) and volatile solids (VS). The VS removal rate was calculated using Equation (1) [
32].
These changes are shown in
Table 2 and were used to recalculate the feed amounts using Equation (2) to ensure that the OLR was equal to 1 g VS/kg inoculum/day. Experiment design regarding the proportions of the feed mixture is shown in
Table 3.
Laboratory batch anaerobic tests were conducted in Schott Duran bottles as reactors, each with a working volume of 1.0 L and fed with 0.2 kg of sludge as the inoculum. All the reactors were fed as the ration of 1.54 g VSsubstrate/VSinoculum. The feedstock for the group labeled “Raw” was raw FW for mono-digestion, while the feedstocks for the other three groups were FW pretreated by lipase addition (LA group), hydrothermal processing (HTP group), and a combination of these two methods (HL group). The co-digestion group contained CG added in proportions of 5%, 10%, and 15% (denoted as CG5, CG10, and CG15). After flushing with nitrogen for 3 min to remove oxygen, all reactors were capped, sealed, and kept in an incubator (MIR-153, SANYO Electric Co., Ltd., Osaka, Japan) at 52 °C (thermophilic condition) for a hydraulic retention time (HRT) of 21 days.
2.4. Analytical Methods
Element analysis (CE440, Exeter Analytical, Inc., Coventry, UK) was performed to determine the carbon and nitrogen content. Generated gas was collected in gas bags, and its volume was measured using a wet gas meter (W-NK, Shinagawa Corp., Tokyo, Japan). The CH
4 contents of the gas samples were further characterized using a gas chromatograph (GC-4000, GL Science, Tokyo, Japan) equipped with a flame ionization detector. In this study, evaluation of methane production was based on corrected methane yields according to standard temperature and pressure. The daily methane volume was normalized (T = 0 °C, P = 1 bar (1 bar = 105 Pa)) according to Equation (3)
where V
N is the volume of the gas under standard conditions (NL), V is the volume of the biogas (NL), P
w is the water vapor pressure as a function of ambient temperature (mmHg, 1 mmHg ≈ 133.322 Pa), and T is the ambient temperature (°C).
Alkalinity was determined according to standard methods [
33]. The concentrations of volatile fatty acids (VFA) and total ammonia nitrogen (TAN) in the digestate were assessed with a titration method using a BUCHI Distillation Unit Type B-323 (BUCHI Corp., Tokyo, Japan). The free ammonia nitrogen (FAN) concentration was calculated using Equation (4) [
34]:
where [NH
3] is the free ammonia concentration and K
b is the dissociation constant (34.4 × 10
−10 at 52 °C).
TS and VS were determined by drying wet samples at 105 °C for 24 h, followed by incineration at 600 °C for 3 h. The pH of each sample was determined using a pH meter. Each measurement was performed in triplicate, and the mean result was calculated. The composition of the liquid phases of FW following pretreatments was analyzed using high-performance liquid chromatography (HPLC, Agilent 1260 Infinity, Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a Shodex SUGAR SH 1821 column (Showa Denko, K.K, Tokyo, Japan) and an Optilabrex 1260 GPC differential refractive index detector. VFA components in the digestate were analyzed by HPLC equipped with a Shodex RSpak KC811 column (Showa Denko KK, Tokyo, Japan). The mobile phase was 0.1% H3PO4 at a flow rate of 0.9 mL/min, with a reflective index detector and a column temperature of 40 °C used.
2.5. Modified Gompertz Model
The modified Gompertz model [
35], shown in Equation (5), was used for curve fitting of the biogas and methane production values. The kinetic constants for anaerobic digestion (AD) under different treatment conditions were determined, the dynamic process was simulated, and the biogas and methane production potential of all groups was quantitatively analyzed. The fitting of this model was achieved using Origin 2020b software (OriginLab Corporation, Northampton, MA, USA). Data obtained from all experimental groups were checked for goodness of fit with the model and evaluated using Pearson correlation coefficients (SPSS Statistics, IBM, Armonk, NJ, USA).
where H is the cumulative methane production (NL/g VS) recorded at time t (d), P is the methane potential (NL/g VS), R
m is the maximum methane production rate (NL/g VS d), e is exp (1) = 2.718, and λ is the lag-phase period (d). The fitness of this model was evaluated using analysis of variance (ANOVA), and the significance (
p-value) was considered according to a 95% confidence level.