Potential of Cation Exchange Resin as a Carrier for Anaerobic Consortia in Biohydrogen Fermentation

Abstract

Cation exchange resin (CER) has been reported to promote sludge fermentation. However, previous studies have typically focused on the effects of CER on sludge properties to enhance fermentation, and the role of CER as a biocarrier for anaerobic consortia during fermentation has been overlooked. Thus, in this study, the potential of gel-type and macro-reticular type CERs to serve as biocarriers in fermentation was investigated. A significant number of anaerobes appeared to be attached to the surfaces of CER during 2-day fermentation. However, an extended fermentation time negatively affected the attachment of anaerobic consortia, suggesting that CER may be a suitable carrier for short-term fermentation processes, such as biohydrogen fermentation. Electrochemical analyses revealed that the electron transfer capacities of CER with attached anaerobes were enhanced after both 2-day and 28-day fermentation periods, with the macro-reticular type CER exhibiting higher electron transfer capacity than the gel-type CER. Fermentation experiments using mixing model substrates with macro-reticular and gel-type CERs with attached anaerobes showed that the macro-reticular type CER was more beneficial for biohydrogen fermentation than the gel-type CER. Further analyses of microbial communities revealed that hydrogen-producing bacteria (i.e., Caloramator, unclassified_f_Caloramatoraceace, and Sporanaerobacter) were more likely to adhere to the macro-reticular type CERs. This outcome confirmed that macro-reticular type CERs have significant potential as a carrier for anaerobic consortia to promote the generation of hydrogen and volatile fatty acids. These findings are expected to provide a reference for using materials as biocarriers to enhance the biohydrogen fermentation of sludge.

1. Introduction

With the continuous enhancement of urban sewage treatment capacity, the production of sewage sludge has also increased substantially [1]. In China, the yield of sludge exceeded 76 million tons (based on 80% moisture content) in 2021 [2], and the total global sludge production is projected to increase from 75 million tons (dry matter) in 2013 to approximately 103 million tons by 2025 [3]. Sludge can be viewed as both a pollutant and a resource. Specifically, sludge contains harmful substances, such as heavy metals, pathogens, and organic contaminants, as well as numerous beneficial biological organic substances, such as proteins, polysaccharides, and lipids [4,5,6]. Fermentation is a promising technology to transform sludge organics into high-value, high-energy products such as volatile fatty acids (VFAs), hydrogen, or methane [1,7].

Recently, cation exchange resin (CER), an insoluble, low-cost, and recyclable material, has been introduced to treat sludge to induce sludge hydrolysis, thereby improving the fermentation performance [8,9,10]. In general, extracellular polymeric substances (EPSs) are critical for the stability of sludge flocs and the integrity of microbial cells [9,11]. The negatively charged functional groups in the organic components of EPSs allow them to bind with multivalent cations, thereby maintaining their structure [11]. Thus, removing the cations, especially metal ions, from sludge can disrupt the structure of EPSs, facilitating the release of organic substances and enhancing the efficiency of the subsequent anaerobic bioconversion of sludge. CER-mediated ion exchange enables reactions with multivalent metals in sludge (e.g., Ca²⁺ and Mg²⁺), disrupting the binding interactions between the multivalent metals and EPSs. This disruption destroys the EPS structure and induces cell lysis, leading to the accelerated disintegration and hydrolysis of sludge [9,10,12]. For example, Pang et al. [9] reported that the addition of CER to sludge released more biodegradable organic substances through the cyclic release and removal of multivalent metals (e.g., Ca²⁺, Mg²⁺, Fe³⁺, and Zn²⁺), promoting the subsequent VFA production. The authors also demonstrated that the concentrations of soluble chemical oxygen demand and VFAs after fermentation using regenerated CERs were similar to those using original CERs, proving the high reusability of CER [13]. In a previous study, CER-mediated ion exchange also provided the direct driving force for the effective contact between bacteria and organic particulates, resulting in a 104.7% increase in hydrogen production, with the fermented sludge separated from the CER–sludge mixture exhibiting higher potential for methane production [8]. Notably, existing work has primarily focused on the influence of CER-mediated ion exchange reactions on the sludge fermentation performance. CER has been demonstrated to have a polymer matrix and porous structure [14]. Rosato et al. observed differences in colonisation dynamics and microbial communities on different polymers [15], while Flemming suggested that anion exchange resin could benefit biofilm formation because of its large surface area and porous structure [16]. These findings indicate the potential of CER as a biocarrier for bacterial adhesion and growth. However, previous studies have not characterised CER post-fermentation or determined whether anaerobic consortia can adhere to the CER surface.

Considering these aspects, this study was aimed at exploring the ability of CER to serve as a carrier for anaerobic consortia. We selected two common types of CERs, gel-type and macro-reticular type, for fermentation experiments. The changes in the microbial adhesion, pore structure, and electrochemical properties of CERs separated from sludge after 2-day and 28-day fermentation experiments were characterised. Moreover, the microbial community structures of anaerobic consortia on the surface of CERs and fermented sludge were analysed. Additionally, fermentation experiments of the mixture of separated CERs and model substrates were conducted to evaluate the activity of the anaerobic consortia adhered on CERs. This study provides a reference for characterising the microbes adhered to potential biocarriers.

2. Materials and Methods

2.1. Main Characteristics of Sludge and CER

Sewage sludge, as substrate, was collected from a wastewater treatment plant in Shanghai, China, and stored at 4 °C prior to use. The total solids (TS) content, volatile solid (VS)/TS ratio, and pH values of the sludge were 2.2 ± 0.2%, 63.4 ± 2.7%, and 6.9 ± 0.3, respectively. The inoculum, sourced from a laboratory-scale mesophilic anaerobic sludge digester (37 ± 0.5 °C), had a TS content of 2.3 ± 0.3%, VS/TS ratio of 38.4 ± 1.4%, and pH of 7.1 ± 0.2.

Two types of CERs were used in this study, both purchased from Zhengguang Industrial Co., Ltd., Hangzhou, China. The sodium-type CER (001*7) was labelled as CER-1, and the hydrogen-type CER (D113) was designated as CER-2. The basic characteristics of CER-1 and CER-2 are listed in

Table 1. The basic characteristics of cation exchange resin (001*7, sodium type).

Parameter	Value	Parameter	Value
CER type	Gel-type	Wet apparent density	0.77~0.87 g/mol
Cation form	Na+	Wet true density	1.250~1.290 g/mol
Basic polymer	Styrene-Divinylbenzene copolymer	Particle size range	(0.315~1.250 mm) ≥ 95%
Functional group	-SO3H	Effective particle size	0.400~0.700 mm
Moisture content	45.00~50.00	Exchange capacity (mass)	≥4.5 mmol/g
pH range	1–14	Exchange capacity (volume)	≥1.90 mmol/mL

and

Table 2. The basic characteristics of cation exchange resin (D113, hydrogen type).

Parameter	Value	Parameter	Value
CER type	Macroporous-type	Wet apparent density	0.72~0.80 g/mol
Cation form	H+	Wet true density	1.140~1.200 g/mol
Basic polymer	Acrylic copolymer	Particle size range	(0.315~1.250 mm) ≥ 95%
Functional group	-COOH	Effective particle size	0.400~0.700 mm
Moisture content	45.00~52.00	Exchange capacity	≥10.80 mmol/g
pH range	4–14	Exchange capacity	≥4.40 mmol/mL

, respectively. These two CERs are commonly used to treat sludge for promoting fermentation [8,9,10]. The procedures for washing and activating CER-1, activating CER-2, and converting hydrogen-type CER-2 to sodium-type CER-2 can be found in our previous studies [8,9].

2.2. Sludge Fermentation Experiments

The 2-day fermentation experiment was conducted using eight 500 mL serum bottles, each with a working volume of 400 mL. The substrate and inoculum were added to six bottles at a VS ratio of 5:1. Four bottles, labelled as experimental group-1 (EG-1), contained 4 g CER-1/g VS; and the remaining four bottles, labelled as experimental group-2 (EG-2), contained 4 g CER-2/g VS. The CER dosage was based on our prior study [8]. The pH of the mixture in each bottle was adjusted to approximately 6.0 using 1 M NaOH or HCl solutions. Each bottle was flushed with N₂ for 3–5 min to purge oxygen, sealed promptly, and placed in a water bath at 37.0 ± 1.0 °C with mechanical stirring at 120 rpm. After 2-day fermentation, the CER was separated from the fermented sludge by sieving the mixture through a screen mesh (ø 20 × 4.5 cm, 50 mesh). The separated CER (S-CER) from one bottle in each group was used for characterisation, while those from the other three bottles in each group were used for the subsequent fermentation experiments, as described in Section 2.4. The separated fermented sludge was used for microbial community analysis. The S-CER from this short-term fermentation was labelled as SS-CER.

The 28-day fermentation experiment was also conducted using eight 500 mL serum bottles, each with a working volume of 400 mL. The inoculum and CER were mixed directly, without substrates. The other procedures were the same as those in the 2-day fermentation experiment, except that the pH of the mixture in each bottle was adjusted to approximately 7.0 using 1 M NaOH or HCl solutions. The S-CER from this long-term anaerobic fermentation was labelled as LS-CER.

2.3. Characterisation of CER before and after Fermentation

Before characterisation, the S-CER was washed three times using 1× phosphate-buffered saline (PBS) to remove the loosely adhering biomass. The morphology of the CER and the anaerobes attached to the S-CER were identified using scanning electron microscopy (SEM, Zeiss, Zeiss Sigma 300, Oberkochen, Germany). The pretreatment of S-CER for SEM followed the protocol specified by Liu et al. [17]. The washed S-CER was fixed with 2.5% (v/v) glutaraldehyde and refrigerated at 4 °C overnight. Subsequently, the S-CER samples were successively dehydrated in 30%, 50%, 70%, 85%, and 90% ethanol solutions (once for each concentration) and a 100% ethanol solution (twice, each for 15 min), followed by freeze-drying for SEM analysis. The pore structure was determined through mercury intrusion porosimetry (Micromeritics, MicroActive AutoPore V 9600, Norcross, GA, USA). The moisture contents of the CER and S-CER were determined using a moisture meter (Ohaus, MB90ZH, Shanghai, China) at 45 °C.

2.4. Electrochemical Measurements

The electrochemical properties of the CER before and after fermentation were determined using an electrochemical workstation (CHI760E, Chenhua, China) with a three-electrode cell. The cell consisted of a vitreous carbon electrode (6 mm diameter), a Pt wire, and an Ag/AgCl electrode as the working, counter, and reference electrodes, respectively. A cyclic voltammetry (CV) test was conducted to analyse the electron transfer capacities (ETCs), electron acceptor capacities (EACs), and electron donor capacities (EDCs) of the CER and S-CER. For the tests, 1 g CER was mixed with 22 mL 1 × PBS (pH = 7.2–7.4) in the three-electrode cell, and CV measurements were conducted in the potential range of −1.2 to 1.0 V (vs. Ag/AgCl) at a scan rate of 40 mV/s. By integrating the current-time curve from CV curves, the number of transferred electrons is thereby quantified [18]. The ETC is composed of electron acceptor capacity (EAC) and electron donor capacity (EDC), and the relevant calculations are shown as the following equations [18,19]:

$$\mathrm{ETC}={\displaystyle \frac{\int \left({\mathrm{I}}_{\mathrm{red}}+{\mathrm{I}}_{\mathrm{ox}}\right)\mathrm{dt}}{\mathrm{F}·\mathrm{VS}}}$$

$$\mathrm{EAC}={\displaystyle \frac{\int {\mathrm{I}}_{\mathrm{red}}\mathrm{dt}}{\mathrm{F}·\mathrm{VS}}}$$

$$\mathrm{EDC}={\displaystyle \frac{\int {\mathrm{I}}_{\mathrm{ox}}\mathrm{dt}}{\mathrm{F}·\mathrm{VS}}}$$

where I_red (A) and I_ox (A) are the reductive and oxidative currents, respectively; F = 964,853.33 C/mol e⁻ and VS (g) is the mass of the added CER or S-CER.

2.5. Fermentation Experiments of the Mixture of S-CERs and Model Substrates

The S-CERs were mixed with 200 mL of a model substrate solution (4 g/L l-glutamate and 1 g/L glucose) in a 250 mL serum bottle. The pH of the mixture was adjusted to approximately 7.0 using 1 M NaOH or HCl solutions. Subsequently, N₂ was introduced into the bottles for 2–3 min to remove oxygen for creating an anaerobic environment. The bottles were then rapidly sealed and placed in a water bath at 37.0 ± 1.0 °C with mechanical stirring at 120 rpm. The fermentation experiment lasted for six days. Liquid samples were extracted on days 0 and 6 for VFA measurement using a gas chromatograph (Shimadzu GC-1020 plus, Kyoto, Japan). Aluminium foil gas-collecting bags were used to collect the produced biogas, and its composition was determined using a gas chromatograph (INESA, GC112A, Shanghai, China).

2.6. Microbial Community Analysis

The S-CER samples were frozen at −80 °C for subsequent microbial community analysis. DNA was extracted from the sludge samples or frozen S-CER using a FastDNA Spin Kit for Soil DNA (MP Biomedicals, Santa Ana, CA, USA). The quality and concentration of the DNA were determined through 1.0% agarose gel electrophoresis. The primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′)/806R (5′-GGACTACHVGGGTWTCTAAT-3′) and 524F10extF (5′-TGYCAGCCGCCGCGGTAA-3′)/Arch958RmodR (5′-YCCGGCGTTGAVTCCAATT-3′) were used for the amplification of bacterial and archaeal 16S rRNA gene amplification, respectively. The PCR products were extracted from 2% agarose gel and purified. Purified amplicons were pooled in equimolar amounts and paired-end sequenced using an Illumina MiSeq PE300 platform. The resulting sequences were quality filtered with fastp (0.19.6) and merged using FLASH (pairs v1.2.11). The high-quality sequences were denoised, and denoised sequences are usually called amplicon sequence variants (ASVs). The taxonomic assignment of ASVs was performed using the Naive bayes consensus taxonomy classifier implemented in Qiime2 and the SILVA 16S rRNA database (v138). Bioinformatic analysis was performed using the Majorbio Cloud platform https://cloud.majorbio.com (accessed on 5 June 2023). Based on the ASV information, rarefaction curves and alpha diversity indices, including observed ASVs, Chao1 richness, Shannon index and Good’s coverage, were calculated with Mothur v1.30.1.

3. Results and Discussion

3.1. Characterisation of CER before and after Fermentation

Figure 1 shows the changes in the properties of the CER after 2-day and 28-day fermentation. As shown in Figure 1a, no material adhered to the surface of the raw CER. However, after fermentation, anaerobic consortia were visibly attached to the surface of the S-CERs, indicating the potential of the CER to function as a biocarrier during fermentation. Interestingly, the quantity of attached microbes on both S-CER-1 and S-CER-2 after 28-day fermentation was significantly less than that after 2-day fermentation, under the same magnification. This suggests that an extended fermentation period did not enhance microbial attachment on the CER, regardless of its type. Figure 1b shows the moisture content of CER-1 and pore structure distribution of CER-2 before and after fermentation. The moisture evaporation rate, derived from the moisture content results, was used to indirectly evaluate the changes in the pore structure of CER-1 after fermentation. The results showed that the moisture evaporation rates of SS-CER-1 and LS-CER-1 were both lower than that of raw CER-1, attributable to the attached microbes. CER-1, being a gel-type resin, lacks physical pores (capillary pores) and only has chemical pores. Thus, its moisture evaporation rate is independent of capillary forces. Moisture in CER-1 likely exists in a free state, which evaporates easily, resulting in a higher moisture evaporation rate. Conversely, the anaerobic consortia attached to the CER originate from sludge flocs that contain both free water and bound water. Bound water is immobilised on the solid phase through chemical bonding, physical adsorption, and mechanical trapping in the micro- and macro-capillaries, making it difficult to remove [20]. Hence, the moisture evaporation times for SS-CER-1 and LS-CER-1 were prolonged, and their evaporation rates were lower. Among the three groups, SS-CER-1 had the longest evaporation time and lowest rate, suggesting a reduction in the amount of attached microbes on CER-1 after 28-day fermentation. In the case of CER-2, the porosity of raw CER-2 was the highest, suggesting that the anaerobic consortia attached to its surface negatively influenced its porosity. Notably, similar to CER-1, the porosity increased with the fermentation time, indicating a larger amount of attached microbes during the 2-day fermentation. These results support the conclusions derived from the SEM measurements. A potential reason for this phenomenon is that the functional groups of CER are negatively charged, which might be unfavourable for bacterial adhesion to the CER surface during long-term fermentation [21]. Additionally, the exchange reactions between the CER and multivalent cations in sludge during long-term fermentation promoted the removal of divalent cationic ions (e.g., Mg²⁺ and Ca²⁺), enhancing electrostatic repulsion between the negatively charged bacterial surfaces and the CERs with functional groups of the same charge [22].

Figure 2 shows the electrochemical properties of CER before and after fermentation. As shown in Figure 2a, the CV curves of the CERs before and after fermentation were significantly different, indicating the presence of anaerobes on the surface of CERs after fermentation. The EAC, EDC, and ETC values of these CERs were calculated to evaluate the anaerobes attached to the surface of S-CERs. As illustrated in Figure 2b, the EAC, EDC, and ETC values of the LS-CERs were higher than those of the raw CERs, regardless of the CER type, while those of the SS-CERs showed no significant increase, indicating the higher electron transfer potential of LS-CERs. Electron transfer reflects the electron motion in the redox reactions occurring in microbial metabolism [23]. Thus, the enhanced electron transfer potential was attributable to the attachment of microbes to the surface of CERs (Figure 1). However, the SEM measurements and pore structure distribution results showed that the quantity of microbes adhered to SS-CERs was larger than that for the LS-CERs. It has been reported that the EPSs surrounding a cell slow charge transfer, thereby hindering the electron transfer by microbes [24]. Thus, the higher ETC of LS-CERs might be due to the removal of a larger amount of cation ions in sludge during 28-day fermentation of sludge, which could have disrupted the EPSs, thereby accelerating the electron transfer by microbes. The ETC values of the macro-reticular type CER were higher than those of the gel-type CER, regardless of fermentation time, suggesting that the macro-reticular type CER provides a more suitable environment for microbial growth, likely because of its larger pore diameter.

In summary, during the short-term fermentation of sludge, CER can serve as an effective biocarrier for hydrogen or VFA production. However, CER has limited effectiveness as a biological carrier during the long-term fermentation of sludge, which may explain why the introduction of CER into sludge fails to enhance methane production.

3.2. Fermentation Potential of the Anaerobic Consortia Attached to S-CERs

To further evaluate the fermentation potential of the anaerobic consortia attached to the S-CERs, 6-day fermentation experiments were conducted using S-CERs mixed with the model substrate, and the results are shown in Figure 3. As shown in Figure 3a, after 6-day fermentation, the VFA yields in the SS-CER-1 and SS-CER-2 groups were 42.6 mg COD/L and 226.1 mg COD/L, respectively. In comparison, the VFA yields in the LS-CER-1 and LS-CER-2 groups were significantly higher (Figure 3b). This difference in VFA yield likely occurred because the 2-day fermentation was a short-term process in which the fermentation system was in its initial phase and the microbial metabolism was limited. Conversely, during the 28-day fermentation, the inoculum was directly mixed with the CER, resulting in enhanced microbial metabolism. Figure 3 also shows that the VFA production in the SS-CER-2 group was significantly higher than that in the SS-CER-1 group, whereas the performance of LS-CER-2 was inferior to that of LS-CER-1. During the 2-day fermentation, the macro-reticular type CER performed better due to its advantageous structure. In contrast, during the 28-day fermentation, despite the higher ETC of the microbes attached to the separated macro-reticular type CER (Figure 2), the lower richness of the bacterial community led to reduced VFA production (Table 1). Additionally, no methane was detected during the 6-day fermentation of the LS-CERs and model substrates, suggesting minimal presence of methanogens or low methanogen activity levels.

These findings suggest that the two types of CERs were more beneficial for bacteria with acidification functions. The gel-type CER may be more suitable for long-term fermentation, while the macro-reticular type CER may be more effective as a biological carrier in short-term fermentation processes.

3.3. Microbial Community Structure of the Anaerobic Consortia on the Surface of CERs

3.3.1. Microbial Richness and Diversity

Table 3. Results of alpha diversity analysis of the bacterial community.

	ACE	Chao1	Shannon	Simpson
SS-CER-1	861.00 ± 38.11	861.00 ± 41.36	5.47 ± 0.13	0.014 ± 0.0028
LS-CER-1	1015.77 ± 43.12	1014.09 ± 52.86	5.41 ± 0.37	0.015 ± 0.0042
S-EG-1	1464.00 ± 46.39	1464.00 ± 58.77	5.36 ± 0.22	0.019 ± 0.0042
L-EG-1	1690.44 ± 44.45	1662.88 ± 44.26	5.16 ± 0.14	0.037 ± 0.0099
SS-CER-2	1513.00 ± 51.48	1492.88 ± 38.48	5.75 ± 0.45	0.010 ± 0.0028
LS-CER-2	538.56 ± 35.90	534.96 ± 31.80	3.67 ± 0.09	0.090 ± 0.042
S-EG-2	1672.04 ± 65.74	1636.21 ± 46.50	5.24 ± 0.42	0.022 ± 0.0056
L-EG-2	1664.15 ± 56.08	1646.19 ± 49.16	5.62 ± 0.51	0.014 ± 0.0057

and

Table 4. Results of alpha diversity analysis of the archaeal community.

	ACE	Chao1	Shannon	Simpson
LS-CER-1	102.31 ± 17.41	102.00 ± 14.14	2.33 ± 0.47	0.20 ± 0.028
LS-CER-2	173.05 ± 18.46	171.65 ± 7.99	2.48 ± 0.49	0.27 ± 0.042
L-EG-1	205.00 ± 35.35	205.00 ± 26.7	2.62 ± 0.31	0.21 ± 0.057
L-EG-2	224.06 ± 34.03	222.50 ± 27.58	3.09 ± 0.41	0.11 ± 0.028

present the results of the alpha diversity analysis of anaerobes attached to the surface of S-CERs and sludge samples after fermentation. Alpha diversity reflects the richness and diversity of microbial communities [25]. The Chao1 and ACE indexes are typically used to assess the richness of a microbial community, while the Shannon and Simpson indexes indicate its diversity. In terms of the bacterial community, the ACE, Chao1, and Shannon indexes of SS-CER-2 were higher than those of SS-CER-1, and the Simpson index of SS-CER-2 was lower than that of SS-CER-1 (Table 3), demonstrating that the bacterial species attached to SS-CER-2 were more abundant and diverse than those attached to SS-CER-1. In contrast, the ACE, Chao1, and Shannon indexes of LS-CER-2 were considerably lower than those of LS-CER-1, and the Simpson index of LS-CER-2 was approximately six times that of LS-CER-1. These findings indicated that the abundance and diversity of the bacterial species attached to SS-CER-2 were lower than those on SS-CER-1, leading to the differences in VFA production (Figure 3). In the case of the archaeal community (Table 4), the ACE, Chao1, and Shannon indexes of LS-CER-2 were higher than those of SS-CER-1, indicating more abundant and diverse archaeal species on LS-CER-2. However, no methane was detected during the 6-day fermentation of the mixture of LS-CER-2 and model substrates. This suggests the low activity of the attached archaea, possibly due to the inhibitory effect of CER on methane production during long-term fermentation [10].

3.3.2. Microbial Community Structure

Figure 4a shows the relative abundances of the main bacterial genera (top 15) in the attached microbes and sludge samples after 2-day fermentation. After 2-day fermentation, the dominant genera in both S-EG-1 and S-EG-2 (sludge samples) were Caloramator, unclassified_f__Caloramatoraceae, and Fervidicella. Caloramator is a typical hydrogen-producing bacterium [26], and unclassified_f__Caloramatoraceae belongs to the family Caloramatoraceae, which includes hydrolytic bacteria, with most strains capable of producing hydrogen [27,28]. Fervidicella can degrade macromolecular compounds during the hydrolysis stage, as reported in other studies on the CER-enhanced fermentation of sludge [29]. Regarding the attached microbes, Caloramator and unclassified_f__Caloramatoraceae were also the most abundant genera in both SS-CER-1 and SS-CER-2, indicating that the main hydrogen-producing bacteria were attached to the CERs. Additionally, Sporanaerobacter was notably enriched on the surface of SS-CER-2, with a relative abundance of 4.9%, more than twice that in S-EG-2. Sporanaerobacter is often enriched in hydrogen fermentation processes and is involved in hydrogen production [30,31]. Additionally, Sporanaerobacter has the potential for extracellular electron transfer, possibly explaining the slight increase in the EDC of SS-CER-2 compared with raw CER-2 (Figure 2).

Figure 4b shows the relative abundances of the top 15 bacterial genera in the attached microbes and sludge samples after 28-day fermentation. In the 28-day fermentation process, the bacterial community structures for L-EG-1 and L-EG-2 were significantly different. In L-EG-1, the dominant genera were norank_f__norank_o__Aminicenantales (17.5%), Thiobacillus (7.9%), and norank_f__norank_o__norank_c__JS1 (5.4%), while in L-EG-2, Petrimonas (10.2%), Thiobacillus (8.7%), and Tissierella (7.9%) were the dominant genera. These results indicate that with increased fermentation time, the effect of CER type on the bacterial community structure became more obvious. For the attached bacterial community, the dominant genera on the surface of LS-CER-1 were Smithella (12.0%), Bradyrhizobium (7.7%), and norank_f__Bacteroidetes_vadinHA17 (4.0%), while those on the surface of LS-CER-2 were norank_f__Desulfuromonadaceae (27.6%), Azoarcus (14.4%), and Stappia (7.3%). These results indicated that the difference in the bacterial community structures in sludge samples also led to differences in the attached bacterial community structure between LS-CER-1 and LS-CER-2. Smithella can dismutate propionate to acetate and butyrate, followed by the syntrophic β-oxidation of butyrate to acetate, and is also capable of extracellular electron transfer [32,33], thereby contributing to the high acetate production (Figure 3b) and the slight increase in EDC value (Figure 2b). Norank_f__Bacteroidetes_vadinHA17 can convert glucose into acetate, propionate, and H₂/CO₂ [34]. In the fermentation experiments involving the mixture of the LS-CERs and model substrates, glucose was used as one of the model substrates. Thus, the attachment of norank_f__Bacteroidetes_vadinHA17 on the surface of LS-CER-1 likely promoted the degradation of glucose into acetate. This observation, combined with the abovementioned results, suggests that the attachment of Smithella and norank_f__Bacteroidetes_vadinHA17 contributed to the high acetate yield during the fermentation of the mixture of LS-CER-1 and model substrates. Norank_f__Desulfuromonadaceae represents a sulphate-reducing and exoelectrogenic bacterium [35,36]. Azoarcus can degrade sugars and organic acids into acetate and butyrate and is capable of extracellular electron transfer [37,38]. Hence, the attachment of Azoarcus on LS-CER-2 resulted in the production of VFAs, especially acetate and butyrate, during the fermentation experiments of the mixture of LS-CER-2 and model substrates (Figure 3b). However, the relative abundance of Azoarcus was lower than the total relative abundance of Smithella and norank_f__Bacteroidetes_vadinHA17, which could explain the reduced VFA production in LS-CER-2 in the fermentation experiments of the mixture of the LS-CERs and model substrates. Additionally, the high abundances of norank_f__Desulfuromonadaceae and Azoarcus could likely account for the enhancement in ETC (Figure 2b). During the 28-day fermentation, the predominant methanogens in the two sludge samples were different. Methanosaeta, a typical acetoclastic methanogen [39], exhibited the highest relative abundance in L-EG-1, while hydrogenotrophic Methanobacterium dominated in L-EG-2. Interestingly, the predominant methanogens attached to the surfaces of LS-CER-1 and LS-CER-2 were Candidatus_Methanofastidiosum and Methanosaeta, respectively. Candidatus_Methanofastidiosum is restricted to methanogenesis through methylated thiol reduction [40]. Thus, both microporous and gel-type CERs exhibited a considerably different attached archaeal community structure compared with their free-state counterparts. However, no methane was detected in the fermentation experiments of the mixture of LS-CERs and model substrates, likely because the activities of the attached methanogens were too low to convert the produced acetate, methyl compounds, and/or H₂.

3.4. Implications and Future Considerations

Characterisation analyses of different CERs before and after sludge anaerobic fermentation suggest that CER can serve as an effective biocarrier, providing a suitable environment for the growth of hydrogen-producing bacteria that generate hydrogen and VFAs. In other words, CER can not only regulate the sludge structure but also serve as a biocarrier for anaerobic consortia during sludge fermentation. In addition, the suitability of CER types varies with the fermentation time. In this study, macro-reticular type CER was more effective for short-term fermentation (2-day fermentation), while the gel-type CER performed better during long-term fermentation (28-day fermentation). Overall, the fermentation time negatively affected the microbial adhesion to CER, indicating that both the activity and amount of attached microbes are significantly influenced by the CER type and fermentation time. Compared with the gel-type CER, the macro-reticular type CER emerged as the more suitable biocarrier in this study, potentially attributable to its macroporous structure. Therefore, further modification of CER could enhance its ability to attach microorganisms, thereby improving bioenergy recovery from sludge. These findings suggest that materials capable of ion exchange with the polymer matrix and those having a porous structure can effectively function as biocarriers in anaerobic fermentation processes of sludge.

When CER is used as a biocarrier during sludge fermentation, the attachment of microbes may reduce the available exchange sites on the CER surface, thereby weakening its ion exchange capacity. This capacity directly affects the extent of the disruption of the EPS structure, which is associated with the fermentation efficiency. Therefore, maintaining the CER exchange capacity and carrier function is a promising direction for future research. Future work could also focus on the modification of CER based on its polymer matrix and porous structure and exploring the performance of the modified materials. Additionally, the separation of CER from sludge through screens remains a problem that limits its engineering application. Thus, it is desirable to develop materials that can be easily separated and recycled, and magnetic ion exchange resin may be a viable choice in this context. In a previous study, we prepared magnetic porous microspheres (MPMs) as an excellent biocarrier for anaerobes [41]. These MPMs were characterised by a stable macroporous hybrid structure consisting of magnetic cores and polymeric shells, and the magnetism of MPMs facilitated their separation from sludge. The addition of MPMs increased methane production by approximately 100%, offering potential advantages in fermenting waste biomass (e.g., sewage sludge, kitchen waste, and agriculture waste) [41].

4. Conclusions

This study investigated the possibility of using CER to serve as a biocarrier during sludge fermentation and identifying the main microbial community of the anaerobes attached to the CER. During the fermentation of sludge with CER, microorganisms attached to the surface of the CER, but the amount of attached microorganisms was not proportional to the fermentation time. The ETC of the separated macro-reticular type CER was higher than that of the separated gel-type CER from both 2-day and 28-day fermentation processes, indicating the higher activities of anaerobes adhering to the macro-reticular type CER. In the 2-day fermentation of sludge, the microbes attached to the macro-reticular type CER were more abundant and diverse, and the attached top-three bacterial genera were all hydrogen-producing bacteria: Caloramator, unclassified_f _Caloramatoraceace, and Sporanaerobacter. During the 28-day fermentation, both types of CERs exhibited unfavourable conditions for the attachment of methanogens, resulting in VFA accumulation and inhibited methane production. These findings provide a theoretical basis for enhancing the hydrogen fermentation of organic wastes using exogenous materials as biocarriers.