Development of a Primary Sewage Sludge Pretreatment Strategy Using a Combined Alkaline–Ultrasound Pretreatment for Enhancing Microbial Electrolysis Cell Performance

Abstract

Ultrasound and combined alkaline–ultrasound pretreatment (AUP) strategies were examined for primary sewage sludge (SS) disintegration and were utilized to evaluate the degree of solubilization (DS). Further, the pretreated primary SS was operated in microbial electrolysis cells (MECs) to maximize methane production and thereby improve the reactor performance. The highest DS of 67.2% of primary SS was recorded with the AUP. MEC reactors operated with the AUP showed the highest methane production (240 ± 6.4 mL g VS_in⁻¹). VS (61.1%) and COD (72.2%) removal in the MEC ALK-US showed the best organic matter removal efficiency. In the modified Gompertz analysis, the substrate with the highest degree of solubilization (AUP) had the shortest lag phase (0.2 ± 0.1 d). This implies that forced hydrolysis via pretreatment could enhance biodegradability, thereby making it easy for microorganisms to consume and leading to improved MEC performances. Microbial analysis implicitly demonstrated that pretreatment expedited the growth of hydrolytic bacteria (Bacteroidetes and Firmicutes), and a syntrophic interaction with electroactive microorganisms (Smithella) and hydrogenotrophic methanogens (Methanoculleus) was enriched in the MECs with AUP sludge. This suggests that the AUP strategy could be useful to enhance anaerobic digestion performance and provide a new perspective on treating primary SS in an economical way.

1. Introduction

Due to the increase in the population and wastewater treatment plants, the generation of sewage sludge has increased. Because of stringent rules regarding effluent quality, the management of sludge is a critical issue which accounts for about half of the overall cost of wastewater treatment plants. Primary sewage sludge, a by-product of municipal wastewater treatment plants (WWTPs), has a higher level of biodegradable organic compounds than secondary sludge and typically contains 97~98% water [1]. Anaerobic digestion (AD) is widely considered as a preferred approach to treat the sludge adequately by reducing its volume to produce energy. AD process consists of hydrolysis, acidogenesis, acetogenesis, and methanogenesis [2]. The production of biogas can be considered a valuable renewable energy resource with economic value. Together, the solubilization of complicated floc structures and the conversion of high-molecular-weight intracellular biopolymers into lower molecular compounds (i.e., VFAs and soluble substances) of easily degradable organics through hydrolysis is a rate-limiting step in the AD process [3]. One of the ways to accelerate hydrolysis is by pretreating the waste sludge. The aim of the pretreatment is to disrupt the cell wall/membrane and cause cell lysis to occur. Various physical, chemical, thermal, biological, and combined pretreatments were studied to enhance the hydrolysis rate, digestion efficiency, and methane production [4].

Ultrasound pretreatment is a widely used pretreatment method for disrupting microbial cells in sewage sludge [5]. Hydromechanical shear forces through ultrasonic cavitation disrupt microbial cell walls and sludge flocs, releasing the soluble substances [6]. Alkaline pretreatment is another chemical pretreatment method that is mainly used to solvate and swell the sludge at a high pH, making the uneasily biodegradable cellular material more accessible for enzymatic reactions [7]. Combined pretreatment methods have been studied to allow for a lower energy input than that needed for a single pretreatment and to increase solubilization through a synergistic effect [8]. In the combined alkaline and ultrasound pretreatment (AUP) mechanism, the alkaline pretreatment weakens the cell walls via saponifying, making them more susceptible to lysing processes, such as micro-cavitation in the ultrasound pretreatment [9]. AD with the ultrasound pretreatment showed an 8.58~31.43% higher methane yield than AD without any pretreatments [10]. The combined alkaline and ultrasound pretreatment exhibited 7.8 to 18.9% higher solubilization, which was higher than that in the individual pretreatment methods. [11]. Therefore, the AUP would be a better option to treat the primary sludge because of the synergistic effects of alkaline and ultrasound pretreatments. The effects of pretreatment methods are highly different in AD depending on the characteristics of the substrates and the pretreatment type [12]. Furthermore, the studies highlighting the impact of different pretreatments upon either the solubilization efficiency or the digestion performance of primary sewage sludge are limited. Thus, the selection of pretreatment methods and the optimization of pretreatment conditions should be standardized and evaluated in terms of economic and environmental concerns.

On the other hand, microbial electrolysis cells (MECs) have been reported as an effective approach for enhancing the AD process by elevating the energy recovery, which is called MEC-AD [13]. MEC-AD showed higher methane productivity by consuming a small amount of electrical power, thereby adjusting the microbial community structure and promoting electron transfer; thus, it is considered to be a promising approach for capturing the energy in waste biomass in an ecofriendly manner [14]. MEC-AD can rapidly degrade highly concentrated organic matter, toxic materials, and non-degradable matter and accelerate methane production through this bioelectrochemical redox reaction [15]. Theoretically, this reaction process needs the help of an additional applied voltage (0.14 V), which is much lower than that needed for water electrolysis (1.23 V) [16]. However, in practice, more than 0.14 V is supplied due to various overpotentials [17]. Typically, the impact of the applied voltage application (0.6–1.2 V) has led to significant enhancements in methane production of 30–200% from various wastes [18,19]. A slight voltage application induces vital reactions, and as an outcome the significant growth of DIET-capable electroactive bacteria (EAB), which leads to the conversion of organic matter into protons, electrons, and carbon dioxide at the anode. These converted materials are then transferred to the cathode, where they are utilized by electromethanogenic archaea (EMA) to generate methane by electromethanogenesis [20]. It has been reported that the composition of methane in MEC-AD can be up to 86% due to using carbon dioxide as an electron acceptor [21]. Moreover, analysis of microbial communities in MEC-AD bioreactors has been emphasized to better understand the correlation between the microbial community and enhanced methane production. A few MEC-AD papers were reported in the pretreatment research to maximize biogas production. However, only limited papers were published using primary sewage sludge to compare the ultrasound and alkaline–ultrasound pretreatments in their ability to enhance MEC-AD performance and microbial community change. Liu et al. reported that an MEC supplied with 0.6 V and fed alkaline-pretreated secondary sludge (pH 9–10) produced 40% more hydrogen and 50% more methane than a raw sludge-fed MEC [22]. For hydrogen production, Hu et al. reported that an MEC supplied with 0.5 V and fed with ultrasound-pretreated secondary sludge (3 w/mL energy density) produced 368% more hydrogen than a raw WAS-fed MEC [23]. Joicy et al. reported that 44.8% more methane was produced in an MEC fed with an alkaline–thermal pretreatment (pH 10, 95 °C) and an applied voltage of 0.6 V [24]. Previous studies showed that the sludge pretreatment can enhance methane or hydrogen production compared to one without pretreatment during MEC operation. Therefore, this implies that the pretreatment actively disintegrates the substrate, thereby improving the bioelectrochemical redox reaction and enhancing performance. Nevertheless, previous research has been limited with regard to the WAS pretreatment since primary sewage sludge contains higher organic content and toxic materials than WAS. Additionally, there is a lack of studies comparing the effects of single or combined pretreatments with MEC studies using pretreated primary sewage sludge as a substrate and analyzing their effects on the structure of microbial consortia.

This study aims to develop an effective pretreatment strategy to increase the solubilization efficiency of primary sewage sludge and improve the operating performance of MEC-AD reactors by feeding pretreated substrates. In this study, the effect of different ultrasound exposure times and alkaline solution dosages on solubilization efficiency were evaluated to identify the most effective ultrasound and combined alkaline–ultrasound pretreatment conditions. Then, MEC-AD reactors were operated in batch mode by feeding the pretreated substrates. The bioelectrochemical methane production and organic removal rate were evaluated and the changes in the microbial community were identified to investigate the effect of the ultrasound pretreatment and combined alkaline–ultrasound pretreatment on the syntrophic oxidizing reaction and electromethanogenesis in MEC-AD.

2. Materials and Methods

2.1. Sludge Preparation and Disintegration Experiment

The anaerobically digested sludge and the primary sewage sludge used in this study were collected from the municipal wastewater treatment plant (WWTP) in the city of Jinju. Sludge samples were sieved using an 850 μm sieve after settling for 24 h at 4 °C (

Table 1. Characteristics of sludge samples.

	ADseed a	Sludge b	UP c	AUP d
TS (g L−1)	25.65	44.12 ± 3.00	46.60	48.00
VS (g L−1)	17.20	29.13 ± 0.40	29.30	29.20
TCOD (g L−1)	20.74	44.88 ± 2.03	44.49	43.94
SCOD (g L−1)	1.25	1.47 ± 0.43	4.31	12.93
pH	8.3	5.78 ± 0.26	5.83	6.97
Alkalinity (mg L as CaCO3−1)	5945	710 ± 119	680	1610
TVFAs (mg L as HAc−1)	1030	784 ± 32	899	1328

^a Anaerobically digested seed sludge, ^b primary sewage sludge, ^c ultrasound-pretreated sludge, and ^d combined alkaline–ultrasound-pretreated sludge.

).

Before MEC operation, ultrasound (UP) and combined alkaline–ultrasound (AUP) pretreatment experiments were performed. The UP was conducted using the Q500 (Qsonica SONICATORS, Newtown, CT, USA) with a 19 mm tip at a 20 kHz frequency and 0.5 W mL⁻¹ energy density for 40, 60, and 80 min in the temperature-controlled ice bath. In the case of the AUP, the alkaline treatment was initially performed with a range from pH 7 to pH 10 using a 10 M NaOH solution, and was followed by the ultrasound treatment for 40, 60, and 80 min, which was conducted under the same energy density as the single ultrasound pretreatment. For MEC operation, the pH of AUP sludge was neutralized to pH 7 using a 6 M HCl acid solution (Table 1).

2.2. Reactor Construction and Operation

Four lab-scale anaerobic batch reactors (diameter: 12 cm; height: 22.5 cm; total volume: 2.54 L; effective volume: 1.7 L) were used in this study. The top of the reactor was installed with a motor connected to an impeller, a gas collection port, and a temperature sensor. Titanium rods were inserted into the side of the reactor to supply electricity to the electrode (Figure 1). Commercially available graphite fiber fabric (GFF; Samjung C&G Co., Pohang, Republic of Korea) was used for the electrodes in this study. To improve the electrode performance of the GFF, multi-walled carbon nanotubes (MWCNTs; Carbon Nano-material Technology Co., Ltd., Pohang, Republic of Korea) and Ni ions (NiCl₂; Sigma-Aldrich Co., St. Louis, MO, USA) were deposited on the GFF surface through the electrophoretic deposition (EPD) method [25]. Feng and Song reported that MWCNTs and Ni⁺ are commercially available materials and can enhance electrical conductivity and electrochemical properties [26]. After coating the electrode, both the anode and cathode were assembled in 7 pairs of electrode–separator–electrode (separator and electrode assembly (SEA)) [25]. Three MEC reactors were mounted with these electrodes, and one reactor was set as an AD. The gas collector for collecting the biogas from the reactor was filled with an oversaturated sodium chloride solution and adjusted to a pH of 2 or less.

Reactors were inoculated with anaerobically digested sludge and sewage sludge with a seed-to-feed volume ratio of 3:7 for microbial adaptation to the electrode surface. After inoculation, all reactors were fed with MEC seed sludge and sewage sludge in a volume ratio of 1:1. The operating temperature was 35 °C, the rotating speed was 100 rpm, and 0.6 V was applied to all MEC reactors (MEC, MEC US, and MEC ALK-US) using a power supply (OPM 93-4CH, ODA Technologies Co., Incheon, Republic of Korea). All of the reactors were sparged with nitrogen for 10 min to maintain anaerobic conditions before the start of the experiment.

2.3. Analytical Methods

The biogas volume was measured every day, and biogas composition was analyzed using a gas chromatograph (GC; Series 580, GOW-MAC Instrument Co., Ltd., Bethlehem, PA, USA) equipped with a thermal conductivity detector (TCD). Sludge samples were collected three times a week. TS, VS, COD_Cr, pH, alkalinity, and TVFAs were measured following APHA standard methods to examine water and wastewater [27]. The degree of solubilization (DS) of the pretreatment used is calculated as

$$\mathrm{DS} (\%)=\left(\frac{SCO{D}_{Pretreated}-SCO{D}_{raw}}{TCO{D}_{raw}-SCO{D}_{raw}}\right)\times {100}^{ }$$

(1)

The modified Gompertz model is calculated as

$$\mathrm{P}\left(\mathrm{t}\right)=P\exp \left[-\exp \left[\frac{R_{m}e}{P}\left(\lambda -t\right)+1\right]\right]$$

(2)

where $\mathrm{P}\left(\mathrm{t}\right)$ [mL] is the cumulative methane production at time t [d], $P$ [mL] is the predicted methane production potential, $R_m$ [mL d⁻¹] is the maximum methane production rate, λ [day] is the lag phase, and $e$ is a coefficient equal to 2.718282 [28].

2.4. 16S rRNA Gene Sequencing and Taxonomic Assignment

Microbial samples for four reactors were taken from the bulk solution in the reactors. DNA was extracted from the sludge samplesusing the AccuPrep genomic DNA extraction kit (Bioneer, Daejeon, Republic of Korea).

The amplicon sequencing was conducted following the manufacturer’s instructions (Illumina, San Diego, CA, USA) using the oligomers containing the Illumina overhang adapter sequence as well as the following 16S rDNA region-specific sequence as amplicon primers by annealing at 58 °C. The target regions were V3–4 (518F and 805R) and V4-5 (787F and 1059R) for bacteria and archaea, respectively [29,30]. The Illumina Nextera XT index kit was used to PCR-index of purified amplicons. The Taq DNA polymerase kit (Solgent, Daejeon, Republic of Korea) was used for library construction. After purification, the library was quantified, pooled, and combined with the PhiX control (Illumina, San Diego, CA, USA). The library was paired-end (150 bp × 2) sequenced using the iSeq 100 platform.

The paired reads were merged and further processed as described in the literature [31]. More than 90,000 (bacteria, 97,000 on average) or 37,000 (archaea, 116,000 on average) filtered reads were obtained from each sample. Operational taxonomic units (OTUs) were defined at a 97% sequence identity cutoff using the VSEARCH algorithm [32]. Taxonomic assignment was conducted using the online RDP classifier (https://rdp.cme.msu.edu/classifier/, accessed on 11 April 2023).

3. Results and Discussion

3.1. Sludge Solubilization Experiment

Pretreatment experiments with the ultrasound pretreatment (UP) and alkaline–ultrasound pretreatment (AUP) were performed (Figure 2). In the UP, the DS was 2.9% at 40 min, while being 5.5% at 60 min and 5.7% at 80 min. In a previous study, it was noted that the sludge flocs were disrupted through hydromechanical shear forces and the oxidation by H⁺ and OH^- radicals, thus causing the intracellular biomaterials (nucleic acid, proteins, etc.) to be released [33]. However, in the case of the AUP, the alkaline pretreatment was firstly performed by raising the pH from 7 to 10 for 2 h, which was followed by the ultrasound pretreatment with a 0.5 W mL⁻¹ energy density and sonication time from 40 to 80 min. Accordingly, at pH 7 with a sonication time from 40 min to 80 min, the DS increased from 7.2% to 22.4%, whereas at pH 8, the DS was 18.9% at 40 min and increased to 23.6% at 60 min and 32.6% at 80 min. However, for the strong AP at pH 9, the DS was drastically increased to 35.6% at 40 min, and was 44.1% at 60 min and 54.2% at 80 min. Comparatively, in the case of the AUP at pH 10, the DS escalated from 40.7% at 40 min to 63.1% (60 min) and to 67.2% (80 min). Generally, with the alkaline pretreatment, the saponification process happens via OH^- ions, and the organic substances in the cell are solvated [34]. The results indicate that the strong base condition easily induced the particulates of organics to swell, making them more susceptible to enzymatic action. After being swelled by the alkaline pretreatment, the cell membranes/walls were easily disintegrated by the shear forces generated by the ultrasound pretreatment [11]. Based on the DS, it is eminent that through the combined pretreatment (AUP), the intracellular materials are released more in comparison with the single pretreatment method (UP). As the DS increases, the elution amount of substances in the sludge cells increases, making it easier for microorganisms to access organic matter. In previous studies, various pretreatment methods were applied to enhance the solubilizing of sewage sludge (

Table 2. Effect of various combined pretreatment methods on sludge solubilization results.

No.	Types of Sludge	Pretreatment		DS (%)	Reference
		Methods	Conditions
1	WAS a	Electrical + alkaline	pH 12.2 + 20 V 40 min	54.7	[37]
2	WAS a	Alkaline + thermal	pH 11 18 h + 90 °C 1.5 h	53.1	[24]
3	WAS a	Alkaline + microwave	pH 11 + 38,400 kJ/kg TS	65.9	[36]
4	WAS a	Alkaline + ultrasound	pH 12 0.5 h + 24 kJ g TS−1	36.9	[38]
5	WAS a	Ultrasound + alkali	0.5 kW L−1 10 min + pH 10 0.5 h	35.0	[35]
6	Primary sewage sludge	Alkaline + ultrasound	pH 10 2 h + 0.5 W mL−1 80 min	67.2	This study

^a Waste-activated sludge.

). In the research of [35], an alkaline pretreatment was performed on WAS using a NaOH solution up to a pH of 10 after 0.5 kW L⁻¹ of ultrasound pretreatment and reached a DS of 35%. Reference [36] reported that a WAS pretreatment using 38,400 kJ kg⁻¹ TS of a microwave treatment after a 1 M NaOH treatment showed a DS of 65.9%. Overall, it was proved that a primary sewage sludge pretreatment can increase the DS. Our study showed an even higher DS after the AUP (pH 10 and 80 min) that was 1.02~1.92 times higher compared to that in previous studies [24,35,36,37,38]. This suggests that the disintegration performance was enhanced by the synergistic effect whereby ultrasound more effectively disintegrated the organic matters after the strong alkaline pretreatment. Among all pretreatment conditions, the UP at 80 min and AUP at pH 10 and 80 min were regarded as the most efficient single and combined pretreatment conditions, respectively. Therefore, the primary sewage sludge pretreated with these two conditions were applied in the subsequent MEC operation as the substrates.

3.2. Organic Matter Removal Rate

After the pretreatment experiment, anaerobic batch reactors (AD and MECs) were operated. Between the AD and MECs (without pretreatment), the organic matter removal rate was higher in MECs compared to AD (Figure 3). It might be that the exoelectrogenic bacteria (EAB) in the anode oxidize organic substances via bioelectrochemical oxidation, and methanogenic archaea produce methane via hydrogenotrophic methanogenesis in the cathode [39,40]. In MECs, as the DS increased through pretreatment, the MEC performance was also enhanced simultaneously. VS removal was highest in the MEC ALK-US (AUP) at 61.1%, which was followed by a VS removal of 52.4% in the MEC US (UP) and 52.0% in the MEC. In the case of the COD removal rate, a similar tendency as VS removal was shown. The MEC ALK-US showed the highest COD removal rate of 72.2%, which was followed by a COD removal rate for the MEC US at 62.8% and the MEC at 59.1%. Hydrolysis was forced via sewage sludge pretreatment, and acidogenesis and acetogenesis processes were accelerated [41]. Therefore, the organic removal rate of sewage sludge was enhanced after the pretreatment. Interestingly, a higher organic removal rate was observed in the MEC with the AUP than that with the UP. This implies ssthat the synergistic effect of the two pretreatments enabled more effective degradation and the release of soluble substances, allowing microbial consumption to be easy and resulting in higher organic matter removal in the combined pretreatment than in the single pretreatment [9].

3.3. Biogas Production and Modified Gompertz Model

AD produced methane noticeably from the seventh day of operation, whereas in MECs the methane production was noted instantly after the operation started (Figure 4). This implies that the electroactive bacteria (EAB) at the anode oxidize various organic compounds and sugars to produce metabolites and the electromethanogenic archaea (EMA) at the cathode produce methane [42]. The HRT of the three microbial electrolysis cell reactors was 13 days; however, the HRT of AD was prolonged to 19 days. The lowest methane production was 2846 mL L⁻¹ in AD, but the methane production showed enhancement in the MEC reactors, being 53.7~98.8% higher than in AD. In the methanogenesis process, methane is mainly produced through acetoclastic methanogenesis in AD, and hydrogenotrophic methanogenesis is the main methanogenesis reaction in MECs [43]. At mesophilic conditions, it is reported that acetoclastic methanogenesis takes 3~5 days to convert methane; however, in hydrogenotrophic methanogenesis, hydrogen and carbon dioxide are converted to methane within a day. Thus, the bioelectrochemical reaction in MECs accelerates the methane production rate [44]. For this reason, the MEC showed faster and higher methane production than AD due to the difference between their major methanogenic pathways, i.e., acetoclastic methanogenesis for AD and hydrogenotrophic methanogenesis for an MEC [45]. The MEC with pretreated sludge produced more methane than the MEC without pretreated sludge as the DS increased. The highest methane production was observed in the MEC ALK-US of 240 mL g VS_in⁻¹, and was followed by the MEC US at about 204 mL g VS_in⁻¹, whereas the lowest methane production was noted in the MEC at 193 mL g VS_in⁻¹. This might be due to the physicochemical pretreatment, which disrupts extracellular polymeric substances (EPSs) and microbial cell flocs of sewage sludge and increases substrate solubility, resulting in increasing biodegradability in which methanogens are easy to consume (Table 1) [4]. This implies that the biodegradability of the substrate was increased when the combined pretreatment was applied compared to the single pretreatment, resulting in microorganisms in MECs that can easily digest and produce methane [46].

AD and MECs were analyzed using a modified Gompertz plot (

Table 3. Experimental methane yield and modified Gompertz parameters of four reactors during the operation.

	Yield (mL g VSin−1)	Rm e (mL g VSin−1 d−1)	λ f (day)
AD a	122 ± 8.4	10.2 ± 2.7	5.6 ± 0.3
MEC b	193 ± 11.8	34.7 ± 3.0	0.4 ± 0.4
MEC US c	204 ± 9.4	38.3 ± 1.4	0.4 ± 0.2
MEC ALK-US d	240 ± 6.4	40.0 ± 2.1	0.2 ± 0.1

^a Anaerobic digestion, ^b microbial electrolysis cell, ^c microbial electrolysis cell fed ultrasound-pretreated sludge, ^d microbial electrolysis cell fed combined alkaline–ultrasound-pretreated sludge, ^e maximum methane production rate, ^f lag phase.

). The maximum methane production rate (R_m) of MECs was 3.3~4.0 times higher than that of AD (10.2 mL g VS_in⁻¹ d⁻¹). In MECs, the lowest R_m was noted at around 34.7 mL g VS_in⁻¹ d⁻¹ in the MEC, and was followed by the MEC US whose R_m was 1.1 times higher than that of the MEC, at about 38.3 mL g VS_in⁻¹ d⁻¹. The MEC ALK-US was about 40.0 mL g VS_in⁻¹ d⁻¹, being 1.2 times higher than that of the MEC, indicating that the methane generation was positively related to the pretreatment on sewage sludge. The lag phase of the AD reactor was the highest of around 5.62 days and was reduced to 0.43 days in the MEC and 0.37 days in the MEC US. It was further lessened in the MEC ALK-US to 0.23 days. Typically, the lag phase is regarded as the acclimatization of the anaerobic microbial community to the new environment. MECs are amiable as the polarized electrodes that serve as extracellular electron donors and acceptors, adapting the electroactive microorganisms in reactors such as EAB and EMA [44,47]. The pretreatment shortens the lag phase of hydrolysis that is bottlenecked by slow bacterial hydrolysis by effectively solubilizing substrates into VFAs. Moreover, the AUP with the highest DS further shortens the hydrolysis phase through the synergistic effect of the saponification of alkaline and the hydrodynamic shear force of the ultrasound pretreatment. Based on this, in the future, it is necessary to conduct an economic analysis between the continuous operation of MECs with the pretreated substrate and electricity from methane through combined heat and power (CHP), the recycling of chemical reagents, and the value-added utilization of digestate [48].

3.4. Microbial Taxonomy Analysis

Bacterial and archaeal 16S rRNA gene sequencing was performed to investigate in the microbial community changes in the reactors (AD, MEC, MEC US, and MEC ALK-US). At the phyla level, the predominant bacterial populations in all reactors were Bacteroidetes, Firmicutes, Cloacimonetes, Proteobacteria and Spirochaetes. The relative abundance of Proteobacteria which was the most dominant phyla in AD (28.5%), was decreased after voltage application (25.5% in MEC) and substrate pretreatment (16.4% in MEC US and 14.2% in MEC ALK-US). On the other hand, phylum Bacteroidetes and Firmicutes showed increase in their relative abundance when voltage and substrate pretreatment were applied. Bacteroidetes, the second dominant phylum in AD (18.9%), increased its relative abundance to 21.5% in MEC, 22.0% in MECUS and 24.7% in MEC combine. Similarly, the relative abundance of Firmicutes was 7.3% in MEC, 16.7% in MECUS and 17.4% in MEC combine which were higher than that of AD (6.2%). This result implies that Proteobacteria in AD is mainly involved in hydrolysis stage which is the first step of AD process whereas Bacteroidetes and Firmicutes are more responsible for the later steps such as acidogenesis and/or acetogenesis than Proteobacteria [49]. A sludge pretreatment that produces VFAs might have enhanced Firmicutes, a fermenting bacteria, by degrading the VFAs. [50].

At the genus level in all reactors, there was a taxonomic abundance of the bacterial communities Rectinema, Candidatus Cloacamonas and Smithella (Figure 5a). Rectinema showed a relative abundance of around 15.0 and 20.3% in MECs with pretreated sludge and 11.4% in the MEC without pretreated sludge. In a previous study, Rectinema was shown as fermenting proteins, peptides, and carbohydrates to produce acetate, ethanol, and hydrogen [51]. This suggests that the hydrogen degraded from pretreated metabolites by Rectinema is further consumed by hydrogenotrophic methanogens in the MEC ALK-US, which increases methane production and maintains lower hydrogen partial pressure [12,52]. Candidatus cloacamonas was commonly present in all the reactors from 10.8 to 15.2%, as it can oxidize propionate into acetate and hydrogen through syntrophic propionate oxidation under methanogenic conditions [53,54]. Smithella, a well-known genus that converts propionic acid to acetic acid, hydrogen, and carbon dioxide through syntrophic propionate oxidation, was notably found at a higher relative abundance in the MEC ALK-US of 3.8%, possibly due to the enhanced degradation rate in alkaline–ultrasound-pretreated sludge [55,56]. It is understood that in MECs with pretreated sludge, EAB produce hydrogen, formate, and carbon dioxide more effectively, and EMA receive metabolites and thereby produce methane more actively through hydrogenotrophic methanogenesis.

At the phyla level of archaea, Euryarchaeota was predominant in all four reactors at more than 95%. At the genus level, the predominant genera in all reactors were Methanoculleus, Methanothrix, Methanospirillum, Methanomassiliicoccus, and Methanoregula (Figure 5b). Methanoculleus showed the highest relative abundance in the MEC ALK-US of 46.5%, which was followed by the MEC US (37.4%), AD (19.1%), and the MEC (15.3%). Methanoculleus is a genus of hydrogenotrophic methanogens that convert hydrogen, formate, and carbon dioxide into methane [57]. It has been reported to have a partnership with Smithella, and therefore, it is assumed that Smithella and Methanoculleus have a syntrophic connection, as they showed the highest relative abundance (3.8 and 46.5%, respectively) and methane production (240 mL g VS_in⁻¹) in the MEC ALK-US. The proportion of Methanothrix was similar in the MEC ALK-US (27.9%) and the MEC US (27.2%), increased in the MEC (44.9%), and was further enriched in AD (49.6%) [52]. Methanothrix, a representative acetoclastic methanogen, was lower in the MEC with pretreated sludge [58]. The enhanced methane production in MECs with pretreated sludge might be due to the EAB that stimulate the growth of hydrogenotrophic methanogens [59]. This implies that as the pretreated sludge produces more soluble substances, this is followed by the MEC operation accelerating the hydrogenotrophic methanogenesis reaction rather than acetoclastic methanogenesis, thereby enhancing methane production.

4. Conclusions

In this study, the preliminary experiment results showed around a 67.2% disintegration of waste sludge in the combined pretreatment (AUP). The MEC operation with the AUP showed the highest methane production of 240 mL g VS_in⁻¹ compared with the MEC with/without pretreated sludge. In addition, in the modified Gompertz model analysis, the MEC with the AUP showed the shortest lag phase of 0.2 ± 0.1 d. The MEC with the AUP showed the best VS and COD removal efficiency of 61.1% and 71.1%, respectively. The microbial community confirms that a syntrophic partnership between Smithella (degrading bacteria) and Methanoculleus (hydrogenotrophic methanogen) enriched the MEC operated with the AUP. Therefore, the combined alkaline–ultrasound pretreatment converted macromolecules into easily consumable soluble substances (i.e., VFAs), resulting in a shortened hydrolysis phase and higher methane production. Additionally, the population of electroactive microorganisms increased. In future studies, it is necessary to conduct an economic feasibility analysis to determine whether the pretreated substrate utilized in the continuous operation of MECs is cost-effective compared with a conventional AD system.