Effects of Iron-Loaded Biochar on the Anaerobic Co-Digestion of Food Waste and Sewage Sludge and Elucidating the Mechanism Thereof

Abstract

The inhibition of volatile fatty acid (VFA) production is an important factor affecting biogas (methane) production in the anaerobic co-digestion systems comprising food waste and sewage sludge. In this study, batch experiments were conducted at medium temperature (36 ± 0.5 °C), during which the biogas production index and material–liquid characteristic parameters of the anaerobic digestion systems containing different concentrations of iron-loaded biochar (Fe-BC) were monitored. The cumulative biogas production data were analyzed using a modified Gompertz kinetic model to determine the effect of the Fe-BC on biogas production in the anaerobic co-digestion system. Studies have shown that addition of Fe-BC does not significantly influence the hydrolysis and acidification stages of anaerobic co-digestion, but does have a significant effect on promoting methanogenesis by alleviating the accumulation of VFAs and improving both the buffer capacity of the system and the efficiency of substrate-to-biogas conversion. When the Fe-BC concentration was 16 g·L⁻¹, the cumulative biogas production reached 329.42 mL·g-VS⁻¹, which was 49.7% higher than the blank group, and the lag period was 3.55 d, which was 42% shorter than the blank group. Mechanistic studies have shown that Fe-BC increased the concentration of coenzyme F₄₂₀ and the conductivity of the digestate in the co-digestion system, which increased the activity of methanogens in the anaerobic digestion system, thereby promoting methanogenesis.

1. Introduction

Food waste (FW) and sewage sludge (SS) are important components of urban organic solid waste. FW accounts for 50–60% of the total domestic waste in China, with an annual output of 108 million tons (300,000 tons/day). In addition, with an annual increase of 10% [1], sludge production has increased to 19 million tons/year [2]. Anaerobic digestion is considered to be one of the most suitable processes for the treatment and management of FW because it is highly sustainable and results in the concomitant production of biogas [3]. Furthermore, anaerobic digestion is a sustainable method of FW disposal because it can generate energy while preventing the FW from polluting the environment, thereby achieving sustainable social development [4].

The organic content of FW is as high as 90%. During the anaerobic digestion process, volatile fatty acids (VFAs) accumulate, resulting in the acidification of the medium that leads to the production of low-quality biogas due to rapid hydrolysis of carbohydrate. However, SS has a much lower organic content, 50–70% lower conversion rate, and longer start-up time, all of which greatly limit the efficiency of SS treatment and, therefore, energy recovery [5]. Previous studies have demonstrated that the co-digestion of FW and SS can alleviate the accumulation of VFAs during anaerobic digestion, improve the buffering capacity, and increase microbial activity of the system, thereby increasing methane production [6]. Liu et al. [4] co-digested FW and sludge and found that the pH of the digestion system was buffered between 7.5 and 8.5, and the weak alkaline environment avoided excessive acidification. They used a mixing ratio of 1:1 FW and sludge to help balance the carbon nitrogen ratio of the substrate, improve the pH buffering capacity, and improve the mass transfer effect of the substrate, which was enabled by a strong synergistic effect between the two substrates. However, the methane production in the co-digestion system did not reach the theoretical production, and the stagnation period was long. Therefore, it is necessary to further explore methods or modifications thereof to improve the gas production performance and stability of the anaerobic co-digestion of FW and sludge.

Studies have demonstrated that adding Fe, Co, Ni, and other trace elements could improve the performance of anaerobic digestion systems [7]. As a trace element necessary for the growth and reproduction of micro-organisms, iron is an indispensable component of micro-organisms by serving as a cofactor of enzymatic reactions. Thus, supplementing x with Fe can, in theory, improve the metabolism of anaerobic micro-organisms and enhance the activity of methanogens. Gamal K. Hassan et al. [8] have reported the development of a novel Cu@Fe₃O₄ core–shell nanostructure (NS) that resulted in a 3-fold higher production of biogas at a concentration of 20 mg/L compared to Fe₃O₄ nanoparticles at a concentration of 40 mg/L. Zhan et al. [9] added Fe⁰ and Fe₃O₄ to a sludge anaerobic digestion system, and their results show that methane production increased by 70% compared with the control, and the activity of coenzyme F₄₂₀ increased by 32.3%. Liang et al. [10] compared with the effects of Fe0, magnetite, and biochar on the performance of the anaerobic co-digestion of sludge and FW, the results of which show that Fe0 resulted in the highest cumulative methane production (394.0 mL·g-VS⁻¹) compared with the other two supplements.

The addition of carbon-based materials, such as biochar [11], Prussian blue [12], graphene [13], and activated carbon [14], have been shown to improve the gas production performance and buffering capacity of anaerobic digestion systems. Biochar has the advantages of a high porosity, large specific surface area, abundant organic functional groups, low cost, and abundant and easy-to-obtain raw materials [15]. Several researchers have studied the synergism between Fe and biochar in promoting the anaerobic co-digestion of FW and sludge. Zhang et al. [16] added an appropriate amount of Fe-BC to a sludge-based anaerobic digestion system to reduce the concentration of free ammonia, increase the alkalinity, improve the stability of the digestion system, and increase the abundance and activity of methanogens, which led to a 115.39% increase in methane production. However, there are no relevant reports on the ability of Fe-BC to improve the efficiency of methane production through the consumption of volatile fatty acids in anaerobic co-digestion systems comprising FW and sludge. Studying the mechanism by which Fe-BC can promote the anaerobic co-digestion of FW and sludge is important for realizing the treatment and reduction of organic solid waste and guiding the resource utilization of the waste treatment product.

In this study, we analyzed the effect of the concentration of Fe-BC on the performance of an anaerobic co-digestion system comprising FW and sludge. We expected that the effect of Fe-BC in the combined anaerobic digestion of FW and SS could provide a reference for the engineering application of Fe-BC.

2. Materials and Methods

2.1. Food Waste, Sludge and Fe-BC

To ensure the uniformity of the FW and the reliability of the experimental results, the FW was prepared with a ratio of vegetables: meat: rice: vegetable oil of 50:15:31:4; this FW served as the substrate [17]. The prepared FW was crushed into pieces with particle sizes of 1–5 mm and stored at −20 °C. The SS was acquired from the sewage treatment plant in Huaxi District, Guiyang City and naturally precipitated. The supernatant was removed for use. Several basic parameters of the FW and SS are shown in

Table 1. Parameters of the FW and SS used in this study.

Metric	Food Waste	Sludge
pH	6.32	7.54
TS (%)	91.52 ± 2.24	20.70 ± 1.56
VS (%)	85.42 ± 3.14	7.72 ± 0.60
VS/TS (%)	92.79 ± 1.72	38.46 ± 1.61
Protein (g/L)	190.24 ± 2.12	-
Polysaccharide (g/L)	150.25 ± 3.24	-
TCOD (g/L)	445.42 ± 3.21	15.53 ± 2.14
TAN (mg/L)	2020.63 ± 3.2	-

.

The Fe-BC was prepared by mixing biochar (2.0 g) in 200 mL of a 1 mol/L NaOH solution for 2 h, after which Fe₂Cl₃·6H₂0 was quickly added, and the mixture was stirred for an additional 1 h. Following this, the mixture was placed in an airtight aging box for 2 days at a constant temperature of 40 °C. Fe-BC was washed with deionized water and then dried in a vacuum drying oven at 105 °C for 24 h.

2.2. Experimental Design and Procedure

A series of batch experiments were conducted to study the effect of the concentration of Fe-BC on the biogas production performance of the anaerobic co-digestion system. In the first batch of experiments, 40 mL of food waste and 210 mL of inoculated sludge were added to six 330 mL serum bottles. Then, Fe-BC was added to the serum bottles to achieve concentrations of 0, 4, 8, 16, and 24 g·L⁻¹, which were marked as Fe-BC0, Fe-BC4, Fe-BC8, Fe-BC16, Fe-BC24; Fe-BC0 served as the blank group. A second batch of experiments was conducted analogous to the first batch, except that Fe0 (0.95 g·L⁻¹) and biochar (15.05 g·L⁻¹) were added individually; this sample was denoted as Fe+BC16. The serum bottles were flushed with ultrapure nitrogen gas for 5 min to ensure an anaerobic environment, after which the pH of the system was adjusted to 7.2–7.5 with either 1 mol/L NaOH solution or HCl solution, depending on the initial pH. The serum bottles were then connected to a gas collecting bottle through a rubber tube, and the gas that evolved during the experiment was collected using the drainage method. The acidic gases, such as CO₂ and H₂S in the biogas, were absorbed by bubbling the gaseous mixture through a 3 mol/L NaOH solution. After absorption, the composition of the gas could be considered as mostly CH₄. The digestion reactors were kept in a constant temperature water bath at 36 ± 1 °C and shaken manually once a day. All experiments were performed in triplicate in parallel.

2.3. Analytical Methods

2.3.1. Chemical Analysis

The total solids (TS) and volatile solids (VS) of the substrate were determined by the gravimetric method [18]. The substrate was centrifuged (TG16G, China, Changzhou, Jintan Gaoke Instrument Factory) at 8000 rpm for 10 min, after which the supernatant was removed, and the pH of the supernatant was measured using a portable pH meter (PHB- 4, Shanghai Leiche) for BET (US, Mike 2406); SEM (US, ZEISS); and ICP (US, Agilent 5110). The polysaccharides content was determined by anthrone-sulfuric acid spectrophotometry [19]. The protein content was determined by the Coomassie brilliant blue method.

The concentrations of VFAs were determined colorimetrically by UV–vis spectrophotometry. The sample solution containing the VFAs were reacted with ethylene glycol in the presence of heat to form the corresponding esters, which reacted with carboxylamines to form hydroxamic acids. In the presence of a ferric reagent, the hydroxamic acids were converted into a brownish-red ferric-hydroxamic acid complex, whose color depth was proportional to the concentration of the starting VFA. The concentration of coenzyme F₄₂₀ was determined by UV–vis spectrophotometry (752 UV-Vis Shanghai Youke) [20]. The daily gas production and cumulative gas production were measured by passing the evolved gas through a 3 mol/L NaOH solution, and the volume of the discharged NaOH solution was recorded every 24 h and expressed as mL·g-VS⁻¹.

2.3.2. Kinetics Analysis

A modified Gompertz model [6] (Equation (1) below) is often used to understand the variation in the substrate consumption and biogas production over time.

$$B_{\left(t\right)}=f_d·\mathit{exp}\left(-\mathit{exp}\left[\frac{R_m·e·\left(\lambda -t\right)}{f_d}+1\right]\right)$$

(1)

In Equation (1), B_(t) is the cumulative biogas production (mL·g-VS⁻¹) at a given time (t), f_d is the cumulative biogas production (mL·g-VS⁻¹), λ is the stagnation period (d), R_m is the maximum biogas yield (mL·g-VS⁻¹·d⁻¹), t is the digestion time (d), and e is Euler’s number (e = 2.7183).

2.3.3. Data Analysis

Statistical data analysis was performed using Excel. The software used for fitting kinetics and plotting was performed using Origin9. There are two replicates for each assay. Statistical analysis was performed by Lu Wenxu.

3. Results

3.1. Effects of Fe-BC on Combined Anaerobic Digestion of Food Waste and Sludge

3.1.1. Effects of Fe-BC on the Rate of Hydrolysis and Acidification Stages

Figure 1 shows the changes in the concentrations of protein, polysaccharides, and VFAs, as well as the pH, in the various anaerobic co-digestion systems with and without Fe-BC. The protein concentration first increased and then decreased (Figure 1a). The rapid dissolution of the substrate on the first day of anaerobic digestion led to an increase in the concentration of protein. However, from day 2 to 9, the protein concentration began to slowly decrease. During the digestion process, the protein was hydrolyzed into constituent amino acids, which were used by acid-producing bacteria to synthesize VFAs, which led to an increase in the protein concentration. The polysaccharides concentration dropped sharply from x to x after the first day, and then fluctuated for the remainder of the anaerobic digestion period, indicating that polysaccharides were more easily hydrolyzed into their corresponding monosaccharides than were the proteins during the anaerobic digestion process (Figure 1b). Polysaccharides can be used metabolically as carbon sources. In the early stage of the digestion, the concentration of polysaccharides significantly decreased, and the monosaccharide products were converted to biogas, resulting in peak gas production. Compared with the blank group, the Fe-BC did not increase the protein and polysaccharide concentrations in the biogas slurry, which indicates that the Fe-BC did not significantly promote hydrolysis during anaerobic digestion.

VFAs are products of hydrolysis and acidification and act as both substrates of methanogenesis and stability indicators of anaerobic digestion. During the anaerobic digestion of our study, VFAs accumulated rapidly in the early stage, reaching a peak value, and then gradually decreased to lower levels after being gradually consumed and utilized in the later stage of anaerobic digestion (Figure 1c). This suggested that VFAs were produced during the hydrolysis stage and accumulated during the acidification stage. The highest concentrations of VFAs were 19.6891 g·L⁻¹ and 13.983 g·L⁻¹ in the Fe-BC0 and Fe-BC16 groups, respectively, which were high enough concentrations to be toxic to the micro-organisms within the systems and inhibit the anaerobic digestion process. During the digestion period, the concentrations of VFAs in the addition group were lower than those in the blank group, indicating that the addition of Fe-BC promoted the conversion of VFAs to methane, alleviated the accumulation of VFAs, and improved the buffer capacity of the anaerobic co-digestion system. The changes in the pH of the anerobic digestion systems were negatively correlated with the accumulation of VFAs (Figure 1d). In the Fe-BC groups, the pH was higher than in the blank group, and the time it took for the pH to return to the optimal range (6.8–7.8) was shorter than that of the blank group, which might have been due to the generation of iron oxides and carbonic acid in the Fe-BC-supplemented systems. The dissolution of salts, for example, has been known to increase the buffering capacity of the system against acid shocks [21].

3.1.2. Effects of Fe-BC on Gas Production in the Anaerobic Co-Digestion Systems

Biogas production is an important parameter to measure the performance and energy recovery efficiency of anaerobic digestion systems. Therefore, we measured the effects of the Fe-BC concentration in the anaerobic co-digestion system on biogas production, and the results are shown in Figure 2. All five Fe-BC groups reached peak gas production on the first day of anaerobic digestion, after which the gas production decreased before finally ceasing on the third day due to over-acidification of the digestion system (Figure 2a). This was likely because the carbohydrates in the FW were rapidly hydrolyzed into intermediate products, such as VFAs, whose rate of consumption was slower than the rate of production, resulting in a large accumulation of VFAs in the system, causing the system to acidify. In the Fe-BC0 digestion system, the daily gas production peaked on days 9 and 25 (16.97 mL·g-VS⁻¹ and 18.85 mL·g-VS⁻¹, respectively). In the Fe-BC16 system, the peak daily gas production occurred on days 9 and 14 (45.63 mL·g-VS⁻¹ and 30.42 mL·g-VS⁻¹, respectively). Based on these results, Fe-BC not only increased the rate of gas production but also accelerated the time when the gas production peak occurred. As shown in Figure 2b, the cumulative gas productions of the Fe-BC0, Fe-BC4, Fe-BC8, Fe-BC16, and Fe-BC24 systems were 220.7, 255.3, 268.6, 329.4, and 232.9 mL·g-VS⁻¹, respectively. In addition, the systems supplemented with Fe-BC all exhibited higher gas production rates than the blank group due to the large specific surface area and high porosity of the Fe-BC, which provided micro-organisms with specific sites to be enriched and fixed, promoted their growth and metabolism, and ultimately promoted biogas production. The enzymatic activity of methanogens promotes direct interspecies electron transfer (DIET) efficiency and improves anaerobic digestion performance [22]. Zhou et al. found that polyaniline enriched the microbial community, such as Clostridium sp. and Methanosaeta sp., by promoting DIET [23].

The cumulative biogas production in the Fe-BC4, Fe-BC8, Fe-BC16, and Fe-BC24 systems was 15.9%, 21.8%, 49.7%, and 5.5% higher, respectively, than the blank group. Jiang et al. [14] added orange peel biochar (1.5 g/g VS) to an anaerobic co-digestion system comprising sludge and FW and observed a cumulative methane production of 250.8 mL· mL·g-VS⁻¹. However, when 24 mL·g-VS⁻¹ Fe-BC was added, the cumulative biogas production decreased. There were likely two reasons for this result. First, a high Fe-BC concentration made it easy for the Fe-BC to coagulate and agglomerate, which increased the solid content of the anaerobic digestion system. This reduced the mass transfer efficiency, which reduced the microbial activity and deleteriously affected the performance of the anaerobic digestion system. Second, the iron content in this system (Fe-BC24) was 1423 mg/L, which was high enough to damage the bacterial cell membrane and inhibit the activity of the micro-organisms [24].

The biogas production process was analyzed using the modified Gompertz model, and the results are listed in

Table 2. The modified Gompertz parameters of the biogas production data.

	Rm/mL·(g·d)−1	λ/d	fd/mL·g-VS−1	Bactual/mL·g-VS−1	Deviation/%	R2
BC0	8.99	6.12	223.25	220.75	2.40	0.9909
BC4	11.40	5.07	264.60	255.30	3.28	0.9919
BC8	13.84	4.18	268.78	268.59	2.07	0.9944
BC16	19.62	3.55	333.01	329.42	0.47	0.9945
BC24	13.07	4.22	239.92	232.94	1.29	0.9965

. The stagnation period (λ) reflected the adaptability of micro-organisms to the anaerobic digestion system; the smaller the λ, the faster the anaerobic micro-organisms could adapt to the environment. Fitting of the numerical data to the model found that the λ of the Fe-BC4, Fe-BC8, Fe-BC16, and Fe-BC24 systems were shortened by 17.15%, 31.70%, 42.00%, and 31.05%, respectively, compared with the blank group. Based on these results, the Fe-BC not only accelerated the ability of the micro-organisms in the anaerobic co-digestion system to adapt to new environments but also shortened the stagnation period. Overall, the fitting results of the biogas production data to the modified Gompertz model were consistent with the changes in the biogas production (R² > 0.99).

Figure 3 compares the changes in the cumulative biogas production between the Fe-BC0, Fe+BC16, and Fe-BC16 systems. The peak cumulative biogas production rates in the Fe-BC16 and Fe+BC16 groups were 329.4 mL·g-VS⁻¹ and 265.08 mL·g-VS⁻¹, respectively, which were 49.7% and 20.5% higher, respectively, than the blank group (220.7 mL·g-VS⁻¹). While the presence of the BC had a deleterious effect on the peak cumulative biogas production, given that the biogas production in the Fe-BC16 system was greater than in the Fe+BC16 system, the peak cumulative biogas production in both systems was higher than in the blank group. This is because the concentration of the Fe-BC was 15.05 g·L⁻¹, which was larger than the inhibition threshold of the biochar addition (10 g·L⁻¹) studied by Yuan et al. [25].

Biochar may inhibit the growth of methanogens, especially at higher concentrations. Therefore, we studied the microscopic morphologies of biochar and Fe-BC by SEM, which are shown in Figure 4a,b, respectively. The surface morphology of the biochar was relatively uniform, honeycomb-like, and rich in pores, while the structure of the Fe-BC surface had collapsed, and the surface featured higher amounts of debris and was rougher. The rough surface could facilitate the colonization and growth of micro-organisms.

Table 3. Physicochemical properties of the biochar and Fe-BC.

	Specific Surface Area (m2/g)	Total Pore Volume (cm3/g)	Average Pore Size (nm)	Fe (%)
BC	3.5959	0.005511	6.1305	0.47
Fe-BC	64.5216	0.079635	4.9369	5.93

provides the physicochemical parameters of the biochar and Fe-BC obtained from the SEM images. The specific surface area of the Fe-BC was 64.5216 m²/g, and the total pore volume is 0.079635 cm³/g, which were 17.9-fold and 14.3-fold higher, respectively, than the corresponding parameters of the biochar. However, the average pore volume of Fe-BC decreased from 6.1305 nm to 4.9369 nm, which was caused by the collapse of the micropore structure during the loading of native biochar. The collapsed structure could promote the formation of biofilms, provide more sites and suitable growth environments for bacteria, improve the activity of micro-organisms, and reduce the mass transfer distance between anaerobic-dominant bacteria, which promotes DIET between micro-organisms [26]. However, the application of Fe+C microbatteries in anaerobic digestion is limited, and biochar and Fe0 are stratified due to the density difference of water. Fe0 nanoparticles are small in size, have weak van der Waals interactions and magnetic attraction between particles, can be easily passivated by agglomeration, cause clogging and slow flow, and gradually weaken the promoting effect [27]. Therefore, the addition of Fe-BC to the anaerobic digestion system had a more significant effect on the anaerobic co-digestion of the FW and SS than the combined addition of iron and carbon.

3.2. Effect of Fe-BC on DIET Efficiency

The changes in the coenzyme F₄₂₀ concentration and conductivity in the anaerobic digestion reactors are shown in Figure 5a,b. Biogas synthesis requires a variety of enzymes and coenzymes/cofactors, of which iron-dependent enzymes account for a large proportion. Coenzyme F₄₂₀ is a unique enzymatic factor utilized by methanogens as an electron transfer carrier and plays an important role in carbon dioxide reduction [28]. The concentration of coenzyme F₄₂₀ is often used as an indicator of methanogen activity in anaerobic digestion systems [29]. Figure 5a depicts the changes in the concentration of coenzyme F₄₂₀ for each anaerobic digestion reactor over time. After commencement of the digestion, the concentration of coenzyme F₄₂₀ in each reactor was relatively consistent; the concentration first increased and then decreased, except in the blank group, in which the concentration first decreased and then increased. The concentration of coenzyme F₄₂₀ in the Fe-BC16 reactor reached a maximum on day nine, when the methanogen activity was the strongest, and the test group reached the peak of gas production. The concentration of coenzyme F₄₂₀ in the other groups increased, while the concentration of coenzyme F₄₂₀ in the blank group decreased. This was because the accumulation of VFAs in the blank group inhibited the activity of methanogens, reducing the concentration of coenzyme F₄₂₀. The concentration of coenzyme F₄₂₀ peaked after a period of reaction in the experimental group with Fe-BC added, since Fe is an integral cofactor of many enzymes in methanogens involved in anaerobic digestion.

Conductivity is an important indicator of electron conductivity [30]. The higher the conductivity, the stronger the electrical conductivity of the system and the higher the electron transfer rate in the system. According to the DIET theory, anaerobic micro-organisms can use exogenous conductive materials for electron transfer, and conductive materials can enhance electron transfer efficiency in anaerobic digestion systems. As shown in Figure 5b, the electrical conductivity at the end of the anaerobic digestion was higher than in the beginning of the digestion. During anaerobic digestion, the electrical conductivity continued to increase, which might have been due to the increase in the ion concentration in the system due to the hydrolysis of macromolecular organic matter to produce acid and other electrolytes in the biochar. As the concentration of Fe-BC increased, the conductivity of the experimental group also increased. At the end of anaerobic digestion, the conductivity in the Fe-BC16 system was 9.59 mS·cm⁻¹, which was 26.4% higher than the blank group. Due to the large redox potential difference between Fe and BC, a large number of micro-galvanic cells were formed in the digestion matrix, which converted chemical energy into electrical energy, indicating that Fe-BC increased the electrical conductivity in the anaerobic digestion system. This result served as a foundation to build a DIET system [31]. The enhanced mechanism of Fe-BC on anaerobic digestion is shown in Figure 6.

4. Conclusions

(1) Adding Fe-BC to the anaerobic co-digestion systems of FW and SS led to an increase in the biogas production compared with the systems without Fe-BC. The best measurement was observed when the concentration of Fe-BC was 16 g·L⁻¹, in which the cumulative biogas production was 329.42 mL·g-VS⁻¹ (49.7% higher than the blank group).

(2) Based on the changes in the concentrations of proteins, polysaccharides, and VFAs in the anaerobic co-digestion system, Fe-BC had no significant effect on the hydrolysis and acidification stages of the anaerobic digestion process. However, Fe-BC had a significant strengthening effect on the methanogenesis stage.

(3) The conductivity of the anaerobic digestion system increased with increasing biogas production. When the concentration of the Fe-BC was 16 g·L⁻¹, the conductivity of the digestate was 9.59 mS·cm⁻¹ at the end of the anaerobic digestion duration, which was 26.4% higher than the blank group. In addition, Fe-BC increased the concentration of coenzyme F₄₂₀ in the anaerobic digestion system. The Fe-BC16 reactor had the highest concentration of coenzyme F₄₂₀ on the ninth day of digestion, which was 126.09% higher than the blank group. Therefore, Fe-BC improved the methanogen activity in the anaerobic digestion system and increased the methanogenesis performance.