To comprehensively assess the efficacy of employing the internal circulation (IC) anaerobic reactor for corn alcohol wastewater treatment and investigate its feasibility, this study focused on anaerobic digestion parameters, energy balance, and the composition of the prokaryotic microbial community. During the operation of the reactor, the hydraulic retention time was progressively reduced from 4.8 to 1.6 days while achieving an average organic loading rate of 12.46 kg chemical oxygen demand (COD)/(m3·d). Moreover, the removal rate of COD exceeded 98%, and the energy balance (ΔE) reached 10.29 kJ/g fed COD. The initial manifestation of organic acidosis in the reactor was a decline in gas production, which is primarily caused by propionic acid accumulation. The subsequent analysis revealed a high diversity of prokaryotes in granular sludge, with the predominant archaea primarily involved in methane production through the acetic acid pathway. The IC anaerobic reactor shows exceptional performance in treating corn alcohol wastewater by optimizing its operating conditions. Energy balance analysis confirmed the feasibility of the process. The findings of this study may offer valuable insights for optimizing control strategies and engineering applications.

  • The COD removal rate of the IC reactor exceeded 98%.

  • The production of biogas can serve as a crucial indicator for rapidly determining the occurrence of organic acidosis.

  • The implementation of the external circulation of the IC reactor enhanced system stability.

  • Methanosaeta is emerging as the predominant genus among methanogenic archaea.

To address the challenges of energy security and environmental pollution, numerous nations have sequentially established carbon neutrality objectives (Chen et al. 2022), advocated for the substitution of biomass fuels in lieu of petroleum-based energy sources, and guided the development of green and low-carbon economies (Manikandan et al. 2022). As a high-quality alternative liquid fuel and a fuel oil quality improver for vehicles (Mendiburu et al. 2022), fuel ethanol is helpful in reducing the emissions of greenhouse gases and other harmful pollutants, it has emerged as the most extensively employed renewable clean energy worldwide (Verger et al. 2022). China ranks third globally in fuel ethanol production (Wu et al. 2021), with corn being a primary feedstock for biofuel ethanol production (Han et al. 2022). The ethanol fermentation level typically ranges from 10 to 20% (v/v), resulting in the generation of a substantial quantity of distiller's grains following the distillation process. The production of 1 L ethanol results in the generation of approximately 10–15 L wastewater (Priyanka et al. 2022). The wastewater generated from alcohol production exhibits characteristics of high concentration, elevated temperature, and a low-pH value. The direct discharge of wastewater not only poses a significant threat to the environment and organisms but also leads to an inefficient utilization of water resources (Ratna et al. 2021). In order to address this issue, numerous domestic and international scholars have devised a range of wastewater treatment technologies for alcohol wastewater, among which the anaerobic digestion of alcohol wastewater emerges as a promising remediation approach. The high concentration of organic matter present in alcohol wastewater can be effectively converted into biogas, a sustainable energy source that can be utilized to power the ethanol production process (Wang et al. 2018a).

The process of anaerobic digestion involves the decomposition of organic matter by anaerobic or facultative anaerobic bacteria, resulting in the production of methane, water, carbon dioxide, and other compounds under anaerobic or anoxic conditions. The process encompasses four sequential stages: hydrolysis, acidogenesis, acetogenesis, and methanogenesis (Yadav et al. 2022). The process of anaerobic digestion not only facilitates the degradation of organic waste and contributes to environmental protection but also exhibits remarkable energy conversion efficiency and chemical oxygen demand (COD) removal rate. In the early stage of the development of anaerobic digestion technology, the ordinary digestion tank was the first generation of anaerobic bioreactor. Despite its ability to degrade organic matter, the sludge retention time (SRT) remained equivalent to the hydraulic retention time (HRT), resulting in a low anaerobic digestion efficiency for organic matter (He et al. 2019). To enhance the treatment efficiency and extend the SRT, the second-generation reactor, exemplified by the up-flow anaerobic sludge blanket (UASB) reactor, was developed and implemented (He et al. 2019). The UASB technology effectively prolongs the SRT and offers a smaller reactor volume capable of withstanding higher organic loads. However, certain limitations persist, including unstable sludge granulation (Mao et al. 2015) and susceptibility to toxicity (Mainardis et al. 2020). Therefore, a two-stage reaction zone was established by integrating two UASB reactors, while an internal circulation (IC) anaerobic reactor with a riser, downcomer, and three-phase separator was employed (Li & Li 2019). The IC possesses a distinctive dual-layer structure and is equipped with an internal gas–liquid circulation system. Combined with its elevated height-to-diameter ratio, it exhibits remarkable mass transfer characteristics and exceptional performance (Chen et al. 2021). The IC reactor enhances mass transfer efficiency through IC and exhibits superior tolerance to a higher organic loading rate (OLR). Implementing a two-stage sludge retention strategy facilitates the enhancement of effluent quality, representing a hallmark characteristic of third-generation anaerobic reactors. The research and application of the IC reactor in the treatment of organic wastewater have been extensively documented. The leachate from a municipal solid waste incineration plant was effectively treated using a full-scale IC reactor, demonstrating excellent treatment performance (Wang et al. 2018b). The OLR of IC was in the range of 21.06–25.16 kg COD/m3·d, exhibiting a remarkable COD removal efficiency ranging from 89.4 to 93.4%. Moreover, the biogas production reached an impressive yield of 0.42–0.50 m3/kg COD. The external circulation IC exhibited a shorter start-up time and demonstrated superior performance in terms of COD removal rate, gas production rate, and OLR tolerance compared to the expanded granular sludge blanket reactor (EGSB) when treated with high-salinity and high-concentration organic wastewater (Lu et al. 2022). Several scholars have studied the treatment strategies for organic acidosis in the IC reactor, including extending HRT, diluting influent with external circulation, implementing intermittent influent, substituting NaOH with quicklime addition, and other relevant measures to restore the normal operation of the reactor (Guan et al. 2019). While numerous studies have been conducted on the treatment of various types of organic wastewater using IC reactors, limited research and reports exist regarding the treatment of high-concentration and low-pH corn alcohol wastewater.

The substrate utilization rate of the anaerobic digestion process is intricately linked to the microbial diversity present in anaerobic sludge. Consequently, analyzing microbial diversity serves as a pivotal approach to investigating the stable operation of anaerobic digestion systems. The dominant bacteria at various heights in the IC anaerobic reactor for brewery wastewater treatment were Proteobacteria, Bacteroidetes, and Chloroflexi (Chen et al. 2021). In the study of hydrogen production from beer wastewater, the IC reactor exhibited the highest relative abundance of prokaryotes, specifically Firmicutes and Proteobacteria. Moreover, Xylella and Saccharibacteria microorganisms were found to significantly enhance COD removal and volatile fatty acid (VFA) conversion (Li et al. 2022). Some scholars comprehensively elucidated the functional characteristics of alcohol waste mash in anaerobic digestion from a microbial perspective, emphasizing the crucial interplay between microbial structure, metabolic activity, and environmental factors (Li et al. 2023). These studies have demonstrated the pivotal role of microbial composition and species in each stage of anaerobic digestion, facilitating a comprehensive understanding of the microscopic characteristics through elucidating changes in the microbial community structure and the metabolic activity during alcohol wastewater treatment.

The purpose of this experiment was to investigate the performance of the IC anaerobic reactor in the continuous anaerobic digestion of corn alcohol wastewater (CAW) and to comprehensively analyze the functioning of the IC system, encompassing biogas production, degradation characteristics of COD, VFAs, and enhancement of sewage water quality. The energy balance calculation method was employed to quantitatively assess the energy input, output, and recovery during the operation of the IC system, aiming to investigate its potential for achieving positive net energy and enhancing overall energy efficiency. The visual observation of sludge particles was followed by an analysis of prokaryotic diversity. The objective of this study was to conduct a comprehensive analysis of the treatment of CAW using IC, aiming to provide valuable insights for the application and advancement of this technology.

Experimental device

The experimental device was a transparent organic glass IC anaerobic reactor, with a height diameter ratio of 10 : 1. The reactor had an effective volume of 3.15 L. Sampling ports were positioned at various heights within the IC, facilitating the circulation of temperature-controlled water to an intricately coiled PVC hose located outside the reactor for optimal heat preservation. The temperature in the reactor was controlled at 30 and 35 °C, and the accuracy of the temperature controller was ±1 °C. The schematic diagram of the experimental device is shown in Figure 1.
Figure 1

Schematic diagram of the IC anaerobic reactor.

Figure 1

Schematic diagram of the IC anaerobic reactor.

Close modal

Experimental materials

  • (1)

    The corn flour and water were combined in a mass ratio of 1:4 during the alcohol fermentation process. Following the fermentation, alcohol was separated through distillation, leaving behind a solid–liquid mixture as residual mash. The residual mash was placed in a filter bag with a pore size of 100 μm, and the solid–liquid separation was achieved through external extrusion. The liquid component obtained after the process of solid–liquid separation was designated as CAW. Subsequently, the obtained CAW was stored at a temperature of 4 °C within a refrigerator. The physical and chemical properties of the CAW were determined. The COD of the wastewater ranged from 38,000 to 52,000 mg/L, while the Kjeldahl nitrogen content varied between 58 and 116 mg/L. Additionally, the total phosphorus content fell within a range of 15–25 mg/L. Moreover, the concentrations of total suspended solids ranged from 23.11 to 29.45 g/L, whereas volatile suspended solid (VSS) concentrations were found to be between 19.06 and 25.27 g/L. Lastly, pH values were measured in a range of 3.8–5.

  • (2)

    The dewatered sludge obtained from the Luolong River water purification plant (Kunming, China) was utilized as the fermentation substrate, with the addition of pig manure biogas residue accounting for 30% of the sludge volume in a single batch. Once the sludge reached an optimal anaerobic digestion state, the regular supplementation of CAW was introduced into the fermentation system. After 3 months of acclimation, the inoculum was obtained. The moisture content was recorded as 91.89%, while mixed liquid suspended solids and mixed liquor VSSs (MLVSSs) were measured at 82.14 and 46.44 g/L, respectively, resulting in a ratio of 1.76:1.

  • (3)

    The buffer solutions employed in this study encompassed a 0.5 mol/L HCl solution and a 0.5 mol/L NaHCO3 solution.

Experimental methods

The inoculated sludge was filtered using a 1 mm pore-size sieve to remove large particles of impurities before being introduced into the reactor. The volume of the inoculum was 945 mL, which corresponded to one-third of the effective volume of the reactor. The remaining volume was diluted with water and supplemented with CAW, which was adjusted to a neutral pH level. The experiment employed a continuous feeding strategy, which encompassed two distinct stages: the initial phase involved gradually increasing the influent COD concentration, followed by a subsequent stage where the HRT was progressively reduced. The parameters analyzed and measured during the operation include the concentration of COD, biogas production, methane content in biogas, pH level, temperature, and VFAs content.

Analysis method

  • (1)

    Biogas production: A wet gas tank was used to collect the biogas produced by the reactor, and the wet gas flowmeter was used to record the daily biogas production.

  • (2)

    The methane content in biogas was quantified using a GC9790 II gas chromatograph manufactured by Fuli Company (China). The instrument was equipped with a TDX-01 stainless steel packed column and a thermal conductivity detector (TCD).

Chromatographic conditions: The column temperature was set at 105 °C, while the detector and injector temperatures were maintained at 140 and 110 °C, respectively. Nitrogen gas was used as the carrier with a flow rate of 30 mL/min.

  • (3)

    COD: The influent and effluent COD concentrations of the reactor were determined using an on-line monitor called COD maxII, produced by Hach Company (USA). The determination method employed was the potassium dichromate method.

  • (4)

    The content of VFAs in the reactor effluent was regularly determined using the GC9790 II gas chromatograph manufactured by Fuli Company (China). The instrument was equipped with a KB-FFAP capillary column and an FID detector.

Chromatographic conditions: The column temperature was set at 130 °C, while the detector and injector temperatures were maintained at 250 and 200 °C, respectively. Nitrogen gas served as the carrier with a flow rate of 20 mL/min. Additionally, an air-flow rate of 300 mL/min and a hydrogen flow rate of 40 mL/min were employed.

  • (5)

    The pH values of the influent and effluent were determined using a PHS-3C pH meter manufactured by Shanghai Leici Company (China).

  • (6)

    Toward the end of the experiment, two sludge samples were collected from the sampling port of the IC reactor. The first sample was filtered using a 2 mm pore-size sieve to isolate and document the granular sludge. Another sample was placed on a glass slide instead of using a sieve filter, and the shape of the sludge was observed using an optical microscope.

Methodology for energy balance analysis

The potential to achieve positive net energy in production is crucial for the technology's acceptance (Mata-Alvarez et al. 2000). The assessment of anaerobic digestion (AD) energy balance encompasses the quantification of input, output, and recovered energy. The input energy is partitioned into electrical and thermal components, encompassing the power consumption associated with the pumping of incoming and outgoing materials. The thermal energy input is utilized to elevate the temperature of the incoming water to the desired digestion temperature (35 °C) and compensate for heat dissipation through walls, floors, and lids, excluding losses via conduits (Puchajda & Oleszkiewicz 2008; Passos & Ferrer 2015).

In the energy balance calculation, the specific heat and density of the CAW were assumed to be 4.18 kJ/(kg·°C) and 1.0 g/mL, respectively. The heat of methane combustion was determined to be 35.8 kJ/L, while the initial temperature of the residual sludge was estimated based on an average experimental temperature of 22 °C. The temperature of the CAW was determined to be 22 °C following the experiments, whereas a temperature of 35 °C was observed in the AD reaction. The output energy values of the reactors were determined by employing the methane yield equation for calculation purposes.
formula
(1)
where E0 is the output energy (kJ/g fed COD), is the methane yield (m3 CH4/m3·d), V is the working volume of the digester (m3), ξ is the lower heating value of methane (35,800 kJ/m3 CH4), ηm is the energy conversion factor of methane (0.9), Q is the influent flow rate (m3·d−1), and CODin is the concentration of the substrate (g fed COD/m3).
Input electricity was estimated using Equation (2); input heat was determined using Equations (3)–(5):
formula
(2)
formula
(3)
formula
(4)
formula
(5)
where Ei,electricity is the input electricity (kJ/g COD), θ is the electricity consumed by pumping (1,800 kJ/m3), ω is the electricity consumed by mixing (300 kJ/m3reactor·d), Ei,heat is the input heat (kJ/g COD), Eh,r is input heat to raise the influent temperature to the digestion temperature, Eh,c is input heat to compensate for heat losses, ρ is the density of the influent (1,000 kg/m3), γ is the specific heat of the influent 4.18 kJ/(kg·°C), Td is the temperature in the anaerobic digester (35 °C), Ti is the temperature of the influent (°C), k is the heat transfer coefficient (W/(m2·°C)), and A is the surface area of the reactor.
The energy balance (ΔE), energy ratio, and energy conversion efficiency (ηs) were calculated using the following equations:
formula
(6)
formula
(7)
formula
(8)
where ΔE is the energy balance (kJ/g COD), ηs is the energy conversion efficiency (kJ/g COD), Re is the energy ratio, and Rs is the COD removal rate.

Prokaryotic biodiversity analysis

The activated sludge in the IC reactor was regularly collected, and the samples were sent to NovoGene for high-throughput sequencing analysis. Firstly, the DNA samples were extracted using either the cetyltrimethylammonium bromide (CTAB) or sodium dodecyl sulfate (SDS) method, followed by the assessment of DNA purity and concentration through agarose gel electrophoresis. The 16S (V3 + V4) region was amplified by the PCR using universal primers 341F and 806R with the Phusion® High-Fidelity PCR Master Mix containing GC buffer and a high-efficiency, high-fidelity enzyme from New England Biolabs. After amplification, the PCR products were mixed and purified. The Ion Plus Fragment Library Kit 48 rxns from Thermo Fisher was ultimately employed for library construction. Following Qubit quantification and library detection, the constructed library was subjected to sequencing using Thermo Fisher's Ion S5TMXL platform. The sample operational taxonomic units (OTUs), alpha diversity, and prokaryotic diversity were subjected to analysis.

Analysis of biogas production and COD removal

The biogas production and methane content were quantified during the experiment. Based on the feed volume and COD, the OLR was calculated, and the resulting data are organized and presented in Figure 2(a). Additionally, measurements of influent and effluent COD concentrations allowed for the calculation of the COD removal rate, which are then sorted and plotted to generate Figure 2(b).
Figure 2

The variation in biogas production and methane content with OLR in the IC reactor (a), while it presented the changes in the COD concentration and the removal rate of influent and effluent materials (b).

Figure 2

The variation in biogas production and methane content with OLR in the IC reactor (a), while it presented the changes in the COD concentration and the removal rate of influent and effluent materials (b).

Close modal

The data presented that during the initial 59 days of the experiment, there was a consistent upward trend observed in daily biogas production. This can be attributed to the gradual increase in influent COD concentration from 2,200 to over 20,000 mg/L. During the microbial growth in the reactor, a significant portion of the organic matter present in the wastewater was effectively metabolized and utilized, resulting in a remarkable COD removal rate exceeding 95% after operating the reactor for a duration of 19 days. The methane content in biogas exceeded 50% by the third day of experimentation, and it exhibited sustained combustion after ignition. However, after the 59th day of experimentation, the reactor experienced organic acidosis, leading to a reduction in gas production by more than 40% on the 60th day compared to the previous day. Subsequently, the gas production exhibited a rapid decline, reaching only 0.4 L by the 62nd day. The concentration of COD in the effluent reached a peak of 10,930 mg/L on the 63rd day. The methane content in biogas decreased to 47% on the 63rd day, indicating a significant reduction in the metabolic activity of methanogenic microorganisms. In order to mitigate the state of complete imbalance, the influent COD concentration was rapidly reduced to 7,210 mg/L while simultaneously increasing the influent pH to prevent further exacerbation of organic acidosis. On the 64th day, external circulation was initiated with an effluent/influent ratio of 1. Once the COD removal rate reached 77.5% on the 68th day, it was advisable to gradually increase the influent COD concentration. The biogas production showed a significant increase after the 70th day, with the methane content in the biogas reaching 52% on the 66th day. The biogas production of the reactor increased proportionally with the rise in COD concentration in the feed while remaining stable within the corresponding OLR range. The reactor had essentially reverted to its state prior to organic acidosis by the 90th day. The influent COD concentration was subsequently maintained at approximately 20,000 mg/L, while the HRT gradually decreased from 4.8 to 1.6 d. Consequently, the maximum daily gas production reached 32.2 L/d, with the highest OLR recorded at 12.58 kg COD/(m3·d). Moreover, throughout this period, the COD removal rate remained consistently above 97%.

VFAs and pH analysis

The VFA types and concentrations in the reactor discharge were measured during the experiment, and the resulting data are utilized to generate Figure 3. The pH values of both the reactor influent and effluent were measured, and these measurements were used to construct Figure 4.
Figure 3

Variation of VFAs content in IC reactor effluent.

Figure 3

Variation of VFAs content in IC reactor effluent.

Close modal
Figure 4

Variations of pH in influent and effluent.

Figure 4

Variations of pH in influent and effluent.

Close modal

The monitoring of VFAs holds significant importance in the analysis of substrate degradation and reactor operation status, as VFAs serve as a crucial intermediate product of anaerobic digestion and an essential source of methanogens. The concentration of VFAs in the reactor effluent exhibited a slight increase on day 17, with acetic acid reaching a concentration of 162 mg/L. Compared to the majority of hydrolytic fermentation bacteria, methanogens exhibited a slightly slower utilization rate of acetic acid following an initial increase in OLR, owing to their relatively extended growth cycle. The syntrophic metabolic relationship between archaea and bacteria gradually stabilized, leading to the near-complete consumption of VFAs and a gradual increase in effluent pH. This observation also reflected the enhanced operational efficiency of the reactor. The average pH of the effluent from the IC reactor was 7.3 during the period from day 40 to 59, despite a low influent pH of only 4.8. However, organic acidosis abruptly manifested in the IC reactor on the 60th day. This was primarily evidenced by a decline in gas production, with the effluent pH decreasing from 7.1 on the previous day to 6.5 and gradually dropping below 6. On the 61st day, the effluent exhibited a VFA concentration of 1,177 mg/L. The concentration of VFAs reached 2,052 mg/L on the 63rd day, with acetic acid accounting for 51.2%, propionic acid accounting for 23.9%, and butyric acid accounting for 20.1%. During the period of 60–68 days, the inadequate conversion of VFAs by methanogenic microorganisms led to varying degrees of VFA accumulation. The decrease in effluent pH and increase in VFAs can indicate organic acidosis, but it should be noted that changes in effluent parameters may be delayed by the impact of HRT. This observation aligned with the occurrence of organic acidosis during the anaerobic digestion of food waste (Yu et al. 2021).

It was noteworthy that the accumulation of propionic acid often signifies an imbalance in the anaerobic digestion system, impeding electron transfer between microbial species (Yang et al. 2022). The disruption of the syntrophic metabolic relationship among microorganisms constitutes the primary cause of acidification. Given the intricate biochemical nature of anaerobic digestion, which involves a consortium of microorganisms, any disruptions in the metabolic activities of one or more microorganisms can have detrimental implications on the degradation of organic matter and conversion into methane. The operational efficiency of the reactor experienced a significant decline subsequent to the incident involving organic acidosis, leading to a temporary reduction in its capacity for COD conversion. By elevating the influent pH to optimize the reactor environment, the methanogenic microorganisms were placed within a relatively favorable neutral pH range. Coupled with a reduction in the OLR and the implementation of external circulation, it necessitated an extended duration for the complete restoration of the reactor. Due to the low initial pH of CAW, continuous external circulation was necessary for the IC reactor to maintain system stability. Diluting a portion of the influent with effluent helped mitigate the risk of organic acidosis in the reactor. After the gradual restoration of microorganisms in the reactor, no accumulation of VFAs was observed in the anaerobic system, and the effluent pH gradually recovered and surpassed 7. After day 68 of the experiment, despite a continued increase in the OLR, VFAs remained persistently at a low concentration level. The pH of the influent gradually decreased, eliminating the need for alkali adjustment liquid. During the period of 110–162 days, the average pH of influent was recorded as 4.2, while that of effluent was measured at 7.2. The overall performance of the IC reactor remained satisfactory, with the pivotal role played by external circulation in effectively diluting the influent.

Energy balance assessment

In this study, the IC reactor was operated at a stable temperature of 35 °C, and the HRT was shortened to increase the OLR. The assessment of energy balance is calculated in Table 1.

Table 1

Energy conversion of the IC reactor under stable operation at 35 °C

StagesOLR (kg COD/(m3·d))E0 (kJ/g fed COD)Ei,heat+Ei,electricity (kJ/g fed COD)ΔE (kJ/g fed COD)Reηs
5.99 ± 0.19 15.24 ± 1.53 8.33 ± 0.27 6.90 ± 1.54 1.83 ± 0.19 37.24 ± 8.44 
7.56 ± 0.33 16.01 ± 1.46 7.24 ± 0.30 8.77 ± 1.51 2.22 ± 0.23 38.46 ± 6.66 
9.29 ± 0.35 15.94 ± 0.90 6.52 ± 0.01 9.42 ± 0.87 2.44 ± 0.12 33.47 ± 3.25 
11.36 ± 0.18 14.32 ± 0.66 5.73 ± 0.01 8.59 ± 0.09 2.50 ± 0.09 24.39 ± 1.98 
12.46 ± 0.02 15.79 ± 0.54 5.49 ± 0.01 10.29 ± 0.54 2.87 ± 0.10 23.72 ± 1.37 
StagesOLR (kg COD/(m3·d))E0 (kJ/g fed COD)Ei,heat+Ei,electricity (kJ/g fed COD)ΔE (kJ/g fed COD)Reηs
5.99 ± 0.19 15.24 ± 1.53 8.33 ± 0.27 6.90 ± 1.54 1.83 ± 0.19 37.24 ± 8.44 
7.56 ± 0.33 16.01 ± 1.46 7.24 ± 0.30 8.77 ± 1.51 2.22 ± 0.23 38.46 ± 6.66 
9.29 ± 0.35 15.94 ± 0.90 6.52 ± 0.01 9.42 ± 0.87 2.44 ± 0.12 33.47 ± 3.25 
11.36 ± 0.18 14.32 ± 0.66 5.73 ± 0.01 8.59 ± 0.09 2.50 ± 0.09 24.39 ± 1.98 
12.46 ± 0.02 15.79 ± 0.54 5.49 ± 0.01 10.29 ± 0.54 2.87 ± 0.10 23.72 ± 1.37 

Table 1 demonstrates the consistent stability of the IC reactor across all intervals, indicating a continuous and sustained energy output throughout the entire system. The net energy value ΔE was positive, and both the energy ratio Re and the energy conversion efficiency ηs exhibited positive values. The HRT was divided into five stages and progressively shortened, with a corresponding gradual increase in the OLR for each stage. In the fifth stage, with an average OLR of 12.46 kg COD/(m3·d) and a HRT of 1.6 days, the energy balance (ΔE) of the IC system reached 10.29 kJ/g fed COD. The proliferation of prokaryotes facilitated the energy recovery of the IC system. With the influent COD remaining relatively stable, a higher amount of substrates entered the IC per unit time, resulting in a significant enhancement of anaerobic digestion efficiency. As the conversion of COD to methane increased, there was a gradual increase in the ΔE of the IC system. The good performance of the IC system at a higher OLR can be inferred from an energy output perspective.

Formation of granular sludge

On the 150th day of operation, a portion of the anaerobic sludge was extracted from the sampling port located in the middle of the IC reactor. The sludge was photographed and recorded under a magnification of 40 times, as shown in Figure 5.
Figure 5

Images depicting granular sludge.

Figure 5

Images depicting granular sludge.

Close modal
Figure 6

Prokaryotic biodiversity statistics (a) show the bacterial community structure at the phylum level, (b) show the bacterial community structure at the genus level, and (c) show the archaeal community structure at the genus level.

Figure 6

Prokaryotic biodiversity statistics (a) show the bacterial community structure at the phylum level, (b) show the bacterial community structure at the genus level, and (c) show the archaeal community structure at the genus level.

Close modal

The figure is illustrated in the granular sludge in IC, which is characterized by a diameter ranging from approximately 2 to 4 mm. The granular sludge exhibited predominantly ellipsoidal appearance geometry, which is characterized by uniform and delicate particles with the minimal presence of rigid impurities. The microscopic examination revealed variations in the size of granular sludge particles in the absence of sieve filtration; however, a majority of smaller sludge particles exhibited an ellipsoidal shape. The successful cultivation of granular sludge served as a crucial foundation for ensuring the stable operation of the reactor.

Analysis of prokaryotic biodiversity

Alpha diversity

Throughout the research process, three samples were collected: the inoculum, the sludge from an IC reactor operating for 63 days, and that from a reactor operating for 100 days. The Alpha diversity analysis indices (Shannon, Simpson, Chao1, ACE, and Goods coverage) were determined for the three samples from the IC reactor under a 97% consistency threshold and are presented in Table 2.

Table 2

Alpha index statistics

Sample nameObserved speciesShannonSimpsonChao1ACEGoods coverage
Inoculum 952 6.593 0.970 1,142.667 1,135.570 0.996 
Sludge 63d 891 6.602 0.955 957.128 954.072 0.998 
Sludge 100d 857 6.887 0.977 914.594 920.371 0.998 
Sample nameObserved speciesShannonSimpsonChao1ACEGoods coverage
Inoculum 952 6.593 0.970 1,142.667 1,135.570 0.996 
Sludge 63d 891 6.602 0.955 957.128 954.072 0.998 
Sludge 100d 857 6.887 0.977 914.594 920.371 0.998 

The observed species index revealed that the inoculum exhibited the highest number of observed species, while the two samples from the IC reactor showed a comparable but lower diversity compared to the inoculum. The Simpson index is indicative of community evenness, reflecting the differences in abundance between members. A value closer to 1 indicates a higher proportion of dominant flora within the total biological flora (Simpson 1949). The Shannon index serves as an indicator of the community's richness and evenness, thereby reflecting the intricate microbial composition. A higher value denotes a greater complexity within the community (Jiang et al. 2020). The Shannon index of the three samples exhibited minimal variation, while the Simpson index surpassed 0.95, indicating a relatively stable dominance of their respective flora within the overall biological composition. Chao1 and ACE indices serve as indicators of species richness based on OTUs. A higher value of Chao1 or ACE indicates greater community richness (Ma et al. 2013). The Chao1 and abundance-based coverage estimator (ACE) indices of samples collected on day 100 from the IC reactor exhibited a slight decrease compared to those obtained on day 63, and both were significantly lower than those observed in the inoculum samples. The ‘Goods coverage’ of the samples was found to be greater than 0.99, indicating that the sequencing depth effectively captures nearly all prokaryotic microbial community information present in the sample.

Distribution of prokaryotic communities

The performance of anaerobic digestion is intricately linked to the composition and structure of the microbial community, which can be classified into two domains: bacteria and archaea. Bacteria play a predominant role in hydrolysis, acidification, and hydrogen and acid production during anaerobic digestion, while archaea are primarily responsible for methanogenesis. The bacteria and archaea in the sludge samples were taxonomically classified at both the phylum and genus levels, as shown in Figure 6.

  • (1)

    Phylum level analysis

The relative abundance of Cloacimonetes in the inoculum was 10.35%, whereas a significant decrease in the relative abundance of Cloacimonetes was observed in the two sludge samples from IC. This decrease can potentially be attributed to the dynamic nature of the IC reactor environment. Furthermore, the relative abundance of Bacteroidetes and Proteobacteria exhibited an increase due to their sustained hydrolysis of organic matter in CAW while concurrently maintaining a favorable growth state. With the increase in OLR, a greater abundance of substrates and intermediates became available for conversion. Bacteroidetes and Proteobacteria possess the ability to hydrolyze carbohydrates into acetic acid and propionic acid, thereby facilitating their growth in environments with a higher OLR. The relative abundance of Firmicutes decreased compared to the inoculum. Firmicutes exhibited a thicker cell wall and were characterized by a high content of peptidoglycans, enabling them to form endospores and display remarkable resistance to extreme conditions, particularly acid stress. The IC system exhibited stable operation at a temperature of 35 °C and maintained an equilibrium state without organic acidosis, resulting in a decrease in the abundance of Firmicutes.

The bacterial phyla Firmicutes and Bacteroidetes were found to be predominant in both anaerobic digestion processes, exhibiting a diverse array of extracellular hydrolases capable of degrading macromolecular organic matter and facilitating the production of VFAs during the hydrolysis stage of anaerobic digestion. The comparison was made between the hydrolytic bacterial populations (represented by the relative abundance of Firmicutes and Bacteroidetes) in two samples from an IC reactor. The order of IC 63d > IC 100d > inoculum suggested that during organic acidosis, there was a higher proportion of fermentation hydrolysis bacteria, which aligned with the accumulation of VFAs in the discharge.

  • (2)

    Genus level analysis

The inoculum exhibited significant dissimilarity in terms of prokaryotic diversity and community structure compared to the other two samples. These differences could potentially be attributed to disparities in both acclimation environment and internal conditions within the IC reactor. The acclimation process of the inoculum was conducted using a batch biogas fermentation method, which differed from the operational mode of gradually increasing OLR in an IC reactor. The similarity between samples collected on the 63rd and 100th day of the IC reactor exhibited a degree of resemblance, although notable disparities were observed in terms of community composition and relative abundance. The relative abundance of Anaerovibrio was 17.75% on the 63rd day, which decreased to only 5.77% by the 100th day. The abundance of Parabacteroides significantly increased from 2.07% at the 63rd day to 7.52% at the 100th day. The relative abundance of Desulfovibrio was 2.19% on day 63, which decreased to only 0.77% by day 100. The variations in the relative abundance of dominant genera and the composition of certain communities are manifested through the distribution of interspecies products and the anaerobic digestion status.

The analysis of archaeal community distribution in the reactor is linked to the metabolic transformation of VFAs and the operational status of the reactor. The study conducted by Liu et al. revealed that Methanosaeta exhibited dominance in both the inoculum and reactor sludge, aligning with our experimental findings (Liu et al. 2020). The study conducted by Qian et al. also revealed the significant contribution of Methanosaetaceae in the anaerobic digestion process of molasses-based alcohol wastewater (Qian et al. 2021). The sludge sample collected on day 63 was noteworthy as it coincided with the occurrence of organic acidosis in the IC reactor. Due to the inability of methanogens to metabolize VFAs into CH4 in a timely manner, the process of methane production was significantly impeded. The relative abundance of Methanosaeta was 1.68% on the 63rd day, and it increased to 2.18% in the sample taken on the 100th day due to the mitigation of organic acidosis and the continuous improvement of OLR. We found that Methanosaeta played a crucial role in converting acetate to methane and dominated as the main methanogenic archaea in various anaerobic reactors during CAW treatment (Ji 2019). Methanosarcina was a predominant methanogen in mesophilic anaerobic activated sludge; however, its relative abundance remained significantly low during the treatment of CAW. The findings were in line with the research outcomes reported by Li et al. (2016). The relative abundance of Methanobrevibacter increased to 0.25% in the 100th-day sludge sample from the IC reactor. Methanobrevibacter is a hydrogenotrophic methanogen that facilitates the efficient conversion of CO2 into CH4, thereby enhancing the energy efficiency of the reactor. As the operation time of the IC reactor increased, the OLR of the anaerobic digestion system undergoes continuous fluctuations, leading to dynamic changes in the composition of the prokaryotic community.

In this experiment, an IC anaerobic reactor was used to treat high-concentration and low-pH CAW. The reactor's performance was evaluated under mesophilic conditions, and the prokaryotic biodiversity in the sludge was assessed. The removal rate exceeded 98% for high-concentration wastewater with a COD of 20,000 mg/L. The OLR of the IC system reached a maximum value of 12.58 kg COD/(m3·d). The energy balance (ΔE) reached 10.29 kJ/g fed COD, demonstrating the significant potential of the IC system for efficient energy conversion. The occurrence of organic acidosis was observed during IC operation, primarily characterized by the accumulation of acetic acid, propionic acid, and butyric acid. The reactor was timely restored through manual intervention. The prokaryotic community structure varied in response to the operational conditions of the IC. The present study comprehensively examines the viability of employing an IC anaerobic reactor for treating CAW. Further investigation is recommended to analyze the ultimate performance of the IC system and investigate temporal and spatial variations in prokaryotes during long-term operation.

This work was supported by Yunnan Research Center of Biogas Engineering and Technology. This work was financially supported by the Special Basic Cooperative Research Programs of Yunnan Provincial Undergraduate Universities' Association (grant number 202001BA070001), Yunnan Province Special Project for International Science and Technology Cooperation (grant number 202003AF140001), and Virtual Simulation Experimental Teaching Project – Biogas Project Based on Waste (2021).

J.J. conceptualized the work, performed data curation, wrote the original draft, reviewed and edited the manuscript, and found resources. G.X. visualized the study, carried out methodology, and wrote the original draft. H.Y. and J.L. visualized the study. C.W. investigated the work. W.Z. and S.H. supervised the work. F.Y. did methodology and funding acquisition and administered the project.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.

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Author notes

Authors contributed equally.

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