Anaerobic Granular Sludge Research Articles

Development of an internal two-stage upflow anaerobic reactor integrating biostimulation strategies to enhance the degradation of aromatic compounds in wastewater from purified terephthalic acid and dimethyl terephthalate manufacturing processes

In this study, we aimed to establish high-rate biological treatment of purified terephthalic acid (PTA) and dimethyl terephthalate (DMT) wastewater that minimizes the inhibitory effects of high concentration benzoate and acetate. To achieve this, we developed a novel bioreactor system and biostimulation strategy. An internal two-stage upflow anaerobic (ITUA) reactor was operated with (i) a packed bed containing green tuff medium underlying (ii) a compartment seeded with anaerobic granular sludge. Ethylene glycol was amended to stimulate syntrophic interactions. Continuous operation of the system for 1,026 days achieve an organic removal rate of 11.0 ± 0.6 kg COD/m3/d. The abundance of aromatic degraders significantly increased during operation. Thus, we successfully developed a high-rate treatment system to treat wastewater from the PTA/DMT manufacturing processes by activating syntrophs in an ITUA reactor.

High-performance internal circulation anaerobic granular sludge reactor for cattle slaughterhouse wastewater treatment and simultaneous biogas production

This research investigates the efficacy of a high-performance pilot-scale Internal Circulation Anaerobic Reactor inoculated with Granular Sludge (ICAGSR) for treating cattle slaughterhouse wastewater while concurrently generating biogas. The primary objective is to assess the efficiency and performance of ICAGSR in terms of organic pollutant removal and biogas production using granular anaerobic sludge. The research methodology entails operating the ICAGSR system under ambient conditions and systematically varying key parameters, including different Hydraulic Retention Times (HRTs) (24, 12, and 8 h) and Organic Loading Rates (OLRs) (3.3, 6.14, and 12.83 kg COD/m³. d). The study focuses on evaluating pollutants’ removal and biogas production rates. Results reveal that the ICAGSR system achieves exceptional removal efficiency for organic pollutants, with Chemical Oxygen Demand (COD) removal exceeding 74%, 67%, and 68% at HRTs of 24, 12, and 8 h, respectively. Furthermore, the system demonstrates stable and sustainable biogas production, maintaining average methane contents of 80%, 76%, and 72% throughout the experimental period. The successful operation of the ICAGSR system underscores its potential as a viable technology for treating cattle slaughterhouse wastewater and generating renewable biogas. In conclusion, this study contributes to wastewater treatment and renewable energy production by providing a comprehensive analysis of the ICAGSR system’s hydrodynamic properties. The research enhances our understanding of the system’s performance optimization under varying conditions, emphasizing the benefits of utilizing ICAGSR reactors with granular sludge as an effective and sustainable approach. Identifying current gaps, future research directions aim to further refine and broaden the application of ICAGSR technology in wastewater treatment and renewable energy initiatives.

A new strategy for accelerating recovery of anaerobic granular sludge after low-temperature shock: In situ regulation of quorum sensing microorganisms embedded in polyvinyl alcohol sodium alginate

Low-temperature could inhibit the performance of anaerobic granular sludge (AnGS). Quorum sensing (QS), as a communication mode between microorganisms, can effectively regulate AnGS. In this study, a kind of embedded particles (PVA/SA@Serratia) based on signal molecule secreting bacteria was prepared by microbial immobilization technology based on polyvinyl alcohol and sodium alginate to accelerate the recovery of AnGS system after low temperature. Low-temperature shock experiment verified the positive effect of PVA/SA@Serratia on restoring the COD removal rate and methanogenesis capacity of AnGS. Further analysis by metagenomics analysis showed that PVA/SA@Serratia stimulated higher QS activity and promoted the secretion of extracellular polymeric substance (EPS) in AnGS. The rapid construction of EPS protective layer effectively accelerated the establishment of a robust microbial community structure. PVA/SA@Serratia also enhanced multiple methanogenic pathways, including direct interspecies electron transfer. In conclusion, this study demonstrated that PVA/SA@Serratia could effectively strengthen AnGS after low-temperature shock.

Towards a circular bioeconomy to produce methane by co-digestion of coffee and brewery waste using a mixture of anaerobic granular sludge and cattle manure as inoculum

Coffee processing wastes, such as solid (pulp and husk) and wastewater, co-digested with industrial brewery wastewater, serve as excellent substrates for generating methane in the anaerobic digestion process. This study compared methane production using different compositions of cattle manure (CM) and granular sludge from an Upflow Anaerobic Sludge Blanket (UASB) reactor used in poultry wastewater treatment (GS). Four anaerobic batch reactors (500 mL) were assembled, A (50% CM and 50% GS), B (60% CM and 40% GS), C (70% CM and 30% of GS) and D (60% CM and 40% GS). Equal concentrations of substrates were added to all reactors: pulp and husk pretreated by hydrothermolysis (1 g L−1), coffee (10 g COD L−1) and brewery (1.5 g COD L−1) wastewaters. Assays A, B and C were supplemented with 2 g L−1 of yeast extract, except for assay D. The reactors were operated at 37 °C and pH 7.0. In assay B, the highest CH4 production of 759.15 ± 19.20 mL CH4 g−1 TS was observed, possibly favored by the synergistic interactions between cellulolytic bacteria Christensenellaceae_R-7_group and Methanosaeta archaea, as inferred by genes encoding enzymes related to acetoclastic methanogenesis (acetyl-CoA synthetase). Consequently, the electricity production potential of assay B (45614.08 kWh−1 year−1) could meet the energy demand of a farm producing coffee and beer, contributing to a positive energy balance concerning methane generation.

Enhanced Sb(V) removal of sulfate-rich wastewater by anaerobic granular sludge assisted with Fe/C amendment

Antimony (Sb) and sulfate are two common pollutants in Sb mine drainage and Sb-containing textile wastewater. In this paper, it was found that iron‑carbon (Fe/C) enhanced Sb(V) removal from sulfate-rich wastewater by anaerobic granular sludge (AnGS). Sulfate inhibited Sb(V) removal (S + Sb, k = 0.101), while Fe/C alleviated the inhibition and increased Sb(V) removal rate by 2.3 times (Fe/C + S + Sb, k = 0.236). Fe/C could promote the removal of Sb(III), and Sb(III) content decreased significantly after 8 h. Meanwhile, Fe/C enhanced the removal of sulfate. The 3D-EEM spectrum of supernatant in Fe/C + S + Sb group (at 24 h) showed that Fe/C stimulated the production of soluble microbial products (SMP) in wastewater. SMP alleviated the inhibition of sulfate, promoting AnGS to reduce Sb(V). Sb(V) could be reduced to Sb(III) both by AnGS and sulfides produced from sulfate reduction. Further analysis of extracellular polymeric substances (EPS) and AnGS showed that Fe/C increased the adsorbed Sb(V) in EPS and the c-type cytochrome content in AnGS, which may be beneficial for Sb(V) removal. Sb(V) reduction in Fe/C + S + Sb group may be related to the genus Acinetobacter, while in Sb group, several bacteria may be involved in Sb(V) reduction, such as Acinetobacter, Pseudomonas and Corynebacterium. This study provided insights into Fe/C-enhanced Sb(V) removal from sulfate-rich wastewater.

Microbial communities outperform extracellular polymeric substances in the reduction of hexavalent chromium by anaerobic granular sludge

The reduction of hexavalent chromium [Cr(VI)] by microbiota agents is a potential method for ecological remediation in heavily contaminated soil. This study investigated the contribution of microbial community and extracellular polymeric substances (EPS) to reduce Cr(VI) using lab-cultured anaerobic granular sludge (AGS). The AGS demonstrated an impressive reduction efficiency of 99.3% for Cr(VI) in soil, operating within an initial Cr(VI) concentration range of 500–3000 mg/kg. The distribution of typical electron acceptors, e.g., SO42-, NO3-, and Fe3+, had a negligible influence on microbial Cr(VI) reduction. The contribution of EPS on Cr(VI) reduction accounted for only 2.1% of the total Cr(VI) reduction, implying that Cr(VI) reduction driven by AGS was primarily caused by the direct effect of microbiota. High-throughput sequencing analysis confirmed that an increasing trend in the relative abundance of Cr reduction bacteria (Sphingopyxis: 0.40–5.54%, Thermonas: 0.41–5.70%, Edaphobaculum: 0.35–6.64%, etc.), and the stability (0.716–4.114) of microbial co-occurrence networks was significantly promoted by the end of the experiment. Materials characterization indicated that successful reduction of Cr(VI) to trivalent chromium [Cr(III)] and formed in Cr(OH)3, and functional groups, O-H, N-H, -CH, and -CH2, of AGS likely played a vital role in Cr(VI) reduction. Overall, these findings offer insights into the remediation technique of microbial Cr(VI) reduction in highly-contaminated soil based on the interaction network stability of the microbial community in AGS.

Enhancing biohydrogen production from xylose through natural FeS2 ore: Mechanistic insights

In this study, we investigated the advantages of utilizing natural FeS2 ore in the context of dark fermentative hydrogen production within a fermentation system employing heat-treated anaerobic granular sludge with xylose as the carbon source. The results demonstrated a significant improvement in both hydrogen production and the maximum rate, with increases of 2.58 and 4.2 times, respectively. Moreover, the presence of FeS2 ore led to a reduction in lag time by more than 2–3 h. The enhanced biohydrogen production performance was attributed to factors such as the intracellular NADH/NAD+ ratio, redox-active components of extracellular polymeric substances, secreted flavins, as well as the presence of hydrogenase and nitrogenase. Furthermore, the FeS2 ore served as a direct electron donor and acceptor during biohydrogen production. This study shed light on the underlying mechanisms contributing to the improved performance of biohydrogen production from xylose during dark fermentation through the supplementation of natural FeS2 ore.

Formation/characterization of humin-mediated anaerobic granular sludge and enhanced methanogenic performance

This study presents a novel method for accelerating the granulation of methanogenic anaerobic granular sludge (AnGS) in an upflow anaerobic sludge blanket (UASB) reactor using solid-phase humin (HM). The results demonstrated that HM-mediated AnGS (HM-AnGS) formed rapidly within 50 days. The increase in particle size, settling velocity and mechanical strength was attributed to the rapid granulation of the HM-AnGS. The maximum methane yield of the HM-AnGS was 5-fold higher than that of the control group. This is consistent with the findings, which showed that HM-AnGS had 3.2–3.4 times more methyl-coenzyme M reductase (Mcr) activity and 2.4–2.9 times more adenosine triphosphate (ATP) than control groups. Molecular analyses indicate that HM most likely accelerated interspecies electron transfer (IET) in HM-AnGS (e.g., from Enterococcus to Methanosaeta). Furthermore, the HM-AnGS was effective in recovering energy from actual slaughterhouse wastewater.

Study on treatment of chemical plant wastewater by using a integrated anaerobic–aerobic bioreactor

ABSTRACT In this work, a new type of integrated anaerobic aerobic bioreactor (IAAB) was formed by optimizing the structure of the anaerobic baffle bioreactor and aerobic biological contact oxidation pool. IAAB was used to treat the production wastewater of a chemical plant. The whole process was divided into two stages of domestication and stable operation. With increasing the proportion of chemical wastewater, the total amount of extracellular polymeric substances increased, and the ratio of protein to polysaccharide decreased. Dehydrogenase activity of anaerobic granular sludge in each compartment increased about 1.5 times with time, and decreased about 42% along the flow direction from the first compartment to the third compartment. The coenzyme F420 increased about 4 times with time, and increased about 1.2 times along the flow direction. The IAAB showed excellent removal rate of chemical oxygen demand (85%), ammonia nitrogen (94%), nitrobenzenes (98%).

Mechanistic insight into the aggregation ability of anammox microorganisms: Roles of polarity, composition and molecular structure of extracellular polymeric substances

The chemical characteristics of extracellular polymeric substances (EPS) of anammox bacteria (AnAOB) play a crucial role in the rapid enrichment of AnAOB and the stable operation of wastewater anammox processes. To clarify the influential mechanisms of sludge EPS on AnAOB aggregation, multiple parameters, including the polarity distribution, composition, and molecular structure of EPS, were selected, and their quantitative relationship with AnAOB aggregation was analyzed. Compared to typical anaerobic sludge (anaerobic floc and granular sludge), the anammox sludge EPS exhibited higher levels of tryptophan-like substances (44.82–56.52 % vs. 2.57–39.81 %), polysaccharides (40.02–53.49 mg/g VSS vs. 30.22–41.69 mg/g VSS), and protein structural units including α-helices (20.70–23.98 % vs. 16.48–19.32 %), β-sheets (37.43–42.98 % vs. 25.78–36.72 %), and protonated nitrogen (Npr) (0.065–0.122 vs. 0.017–0.061). In contrast, it had lower contents of β-turns (20.95–27.39 % vs. 28.17–39.04 %). These biopolymers were found to originate from different genera of AnAOB. Specifically, the α-helix-rich proteins were mainly derived from Candidatus Kuenenia, whereas the extracellular proteins related to tryptophan and Npr were closely associated with Candidatus Brocadia. Critically, these EPS components could drive anammox aggregation through interactions. Substantial amounts of tryptophan-like substances facilitated the formation of β-sheet structures and the exposure of internal hydrophobic clusters, which benefited the anammox aggregation. Meanwhile, extracellular proteins with high Npr content played a pivotal role in the formation of mixed protein-polysaccharide gel networks with the electronegative regions of polysaccharides, which could be regarded as the key component in the maintenance of anammox sludge stability. These findings provide a comprehensive understanding of the multifaceted roles of EPS in driving anammox aggregation and offer valuable insights into the development of EPS regulation strategies aimed at optimizing the anammox process.

Methane Production from Poly(lactic Acid) (PLA) and Polyhydroxybutyrate (PHB) Biodegradable Plastics with Anaerobic Granulated Sludge

- In this study, using Methanosarcina barkeri DSM 800 and Methanococcus vannielii DSM 1224 methanogenic bacteria under mesophilic (38 ± 1oC) and thermophilic (58 ± 1oC) conditions in anaerobic granulated sludge taken from Pakmaya Yeast Factory in Izmir, Turkey; Methane production from biodegradable plastics with poly(lactic acid) (PLA) and polyhydroxybutyrate (PHB) was investigated. Effect of different operating parameters, increasing biodegradation times (from 10 days to 500 days), different inoculumsubstrate ratios (ISRs) (16, 8, 4, 2, 1) and increasing biochemical methane potential (BMP) times (between 10 day and 500 days) for the production of methane gas from PLA and PHB biodegradable plastics in anaerobic granular sludge waste; Methanosarcina barkeri DSM 800 and Methanococcus vannielii DSM 1224 methanogenic bacteria were operated during the anaerobic digestion process under anaerobic conditions at mesophilic (38 ± 1oC) and thermophilic (58 ± 1oC) experimental temperatures. PLA biodegradable plastics were operated at optimum pH=7.6. PHB biodegradable plastics were carried out at optimum pH=8.1. Predicting the biodegradation behavior of PLA and PHB biodegradable plastics with BMP tests; It is found that the ISR parameter plays a very important role. This study showed that temperature plays a key role in the aging of microorganisms (Methanosarcina barkeri DSM 800 and Methanococcus vannielii DSM 1224 methanogenic bacteria) during anaerobic digestion, the degradation of bioplastic materials (PLA and PHB) and the degradation of their natural properties. The increase in temperature from mesophilic conditions to thermophilic conditions increased the activities of methanogenic bacteria such as Methanosarcina barkeri DSM 800 and Methanococcus vannielii DSM 1224. The maximum cumulative CH4(g) production was measured at 630 NL CH4 / kgVS for PHB biodegradable plastics in anaerobic granulated sludge with inoculum culture (the mixture of Methanosarcina barkeri DSM 800 and Methanococcus vannielii DSM 1224 methanogenic bacteria), at ISR=16 value, after 100 days, at pH=8.1 and at 58±1oC, respectively. The maximum 97% biodegradation efficiency was observed for PHB biodegradable plastics after 100 days, at pH=8.1 and at 58 ± 1oC thermophilic conditions, respectively.

Magnetite alleviating calcification of anaerobic granular sludge (AnGS): Electron transfer enhancement and ion competition

Calcification accompanied by deactivation of anaerobic granular sludge (AnGS) is a continuing challenge for high calcium wastewater treatment. The interaction between Ca2+ and extracellular polymeric substances (EPS) is a precondition for this problem. In this study, magnetite for activity recovery and calcification alleviation simultaneously of AnGS under high calcium stress was investigated. The results showed that, in the presence of magnetite, the relative biogas production increased by 13.2 % with the higher activities of key enzymes involved in methanogenesis. Methanosarcina turned into the dominant methanogens, and syntrophic bacteria such as Chloroflexi, Synergistota were enriched, which indicated the enhancement of electron transfer by magnetite, supported by an 18 % increase of the electron transfer system (ETS) activity. Further characterizations of AnGS suggested that the granule calcification was alleviated with a final decrease of 13–40 % calcium content of AnGS with particle size of 1–2.5 mm. Besides, calcium was partially substituted by iron in the EPS, and the secretion of EPS especially proteins decreased. Batch tests demonstrated the competition between Fe2+ dissolved from magnetite and Ca2+, which interfered the interaction between Ca2+ and EPS, so the granule calcification was prevented. Therefore, magnetite played a pluripotent role in the alleviation of granule calcification and deactivation in situ via (1) enhancing electron transfer, and (2) blocking the complex between Ca2+ and EPS. This study provides a novel insight into the application of conductive metal materials in biological wastewater treatment systems suffering from high calcium attack.

Coupling extracellular glycan composition with metagenomic data in papermill and brewery anaerobic granular sludges

Glycans are crucial for the structure and function of anaerobic granular sludge in wastewater treatment. Yet, there is limited knowledge regarding the microorganisms and biosynthesis pathways responsible for glycan production. In this study, we analysed samples from anaerobic granular sludges treating papermill and brewery wastewater, examining glycans composition and using metagenome-assembled genomes (MAGs) to explore potential biochemical pathways associated with their production. Uronic acids were the predominant constituents of the glycans in extracellular polymeric substances (EPS) produced by the anaerobic granular sludges, comprising up to 60 % of the total polysaccharide content. MAGs affiliated with Anaerolineacae, Methanobacteriaceae and Methanosaetaceae represented the majority of the microbial community (30–50 % of total reads per MAG). Based on the analysis of MAGs, it appears that Anaerolinea sp. and members of the Methanobacteria class are involved in the production of exopolysaccharides within the analysed granular sludges. These findings shed light on the functional roles of microorganisms in glycan production in industrial anaerobic wastewater treatment systems.

Extracellular polymeric substances as paper coating biomaterials derived from anaerobic granular sludge

Recovering extracellular polymeric substances (EPS) from waste granular sludge offers a cost-effective and sustainable approach for transforming wastewater resources into industrially valuable products. Yet, the application potential of these EPS in real-world scenarios, particularly in paper manufacturing, remains underexplored. Here we show the feasibility of EPS-based biomaterials, derived from anaerobic granular sludges, as novel coating agents in paper production. We systematically characterised the rheological properties of various EPS-based suspensions. When applied as surface sizing agents, these EPS-based biomaterials formed a distinct, ultra-thin layer on paper, as evidenced by scanning electron microscopy. A comprehensive evaluation of water and oil penetration, along with barrier properties, revealed that EPS-enhanced coatings markedly diminished water absorption while significantly bolstering oil and grease resistance. Optimal performance was observed in EPS variants with elevated protein and hydrophobic contents, correlating with their superior rheological characteristics. The enhanced water-barrier and grease resistance of EPS-coated paper can be attributed to its non-porous, fine surface structure and the functional groups in EPS, particularly the high protein content and hydrophobic humic-like substances. This research marks the first demonstration of utilizing EPS from anaerobic granular sludge as paper-coating biomaterials, bridging a critical knowledge gap in the sustainable use of biopolymers in industrial applications.

Adsorption Characteristics between Different Sizes of Microplastics and EPS Fractions of Anaerobic Granular Sludge

Adsorption Characteristics between Different Sizes of Microplastics and EPS Fractions of Anaerobic Granular Sludge

Enhancing methane production and interspecies electron transfer of anaerobic granular sludge by the immobilization of magnetic biochar

Supplementation of conductive materials has been proved to be a promising approach for enhancing microbial interspecies electron transfer (IET) in anaerobic digestion systems. In this study, magnetic bamboo-based biochar was prepared at temperatures of 400–800 °C via a ball milling/carbonization method, and it immobilized in mature anaerobic granular sludge (AGS) aimed to enhance methane production by improving the IET process between syntrophic microbial communities in the AGS. Results showed that the AGS with magnetic biochar immobilization demonstrated increased glucotrophic and acetotrophic methane production by 69.54–77.56 % and 39.96–54.92 %, respectively. Magnetic biochar prepared at 800 °C with a relatively higher Fe content (0.37 g/g magnetic biochar) displayed a stronger electron charge/discharge capacity (36.66 F/g), and its immobilization into AGS promoted methane production most. The conductivity of AGS increased by 52.13–87.32 % after incorporating magnetic biochar. Furthermore, the extracellular polymeric substance (EPS) of AGS showed an increased capacitance and decreased electron transfer resistance possibly due to the binding of magnetic biochar and more riboflavin secretion in EPS, which could contribute to the accelerated IET process in the inner AGS. In addition, the immobilization of magnetic biochar could promote the production of volatile fatty acids by 15.36–22.50 %. All these improvements may jointly lead to the enhanced methane production capacity of AGS. This study provided a fundamental understanding of the role of incorporated magnetic biochar in AGS in promoting anaerobic digestion performance.

Roles of entrapped bubbles in methanogenic granules under oscillating pressure: Respiration and embolization for intra-granular transport

Anaerobic granular sludge plays a pivotal role in the treatment of concentrated organic wastewater. However, previous studies on intra- granular transport have generally overlooked lung-like respiration that expedites transport in response to fluctuating pressure. This study explored the activities of calcified and normal granules under simulated hydrostatic pressure oscillations. The results revealed a significant enhancement in the bioactivity of calcified granules under oscillating pressure, contrasting with the comparatively lower bioactivity observed in normal granules. The hypothesis posited that the gas pockets in calcified granules facilitated respiration as the functional structure. The presence of tiny bubbles exhibited a propensity for inducing clogging, thereby diminishing the capillary connectivity essential for substrate diffusion. The proposed respiration and embolization concepts decipher the distinct roles of entrapped bubbles in the granular bioactivity across diverse fluid states. This study offers valuable insights into the impact of fluidization on microscopic transport within granule-based bed reactors.

Rapid start-up sulfur-driven autotrophic denitrification granular process: Extracellular electron transfer pathways and microbial community evolution

Sulfur-driven autotrophic denitrification (SAD) granular process has significant advantages in treating low-carbon/nitrogen wastewater; however, the slow growth rate of sulfur-oxidizing bacteria (SOB) results in a prolonged start-up duration. In this study, the thiosulfate-driven autotrophic denitrification (TAD) was successfully initiated by inoculating anaerobic granular sludge on Day 7. Additionally, the electron donor was successfully transferred to the cheaper elemental sulfur from Day 32 to Day 54 at the nitrogen loading rate of 176.2 g N m−3 d-1. During long term experiment, the granules maintained compact structures with the α-helix/(β-sheet + random coil) of 29.5–40.1 %. Extracellular electron transfer (EET) pathway shifted from indirect to direct when electron donors were switched thiosulfate to elemental sulfur. Microbial analysis suggested that thiosulfate improved EET involving enzymes activity. Thiobacillus and Sulfurimonas were dominant in TAD, whereas Longilinea was enriched in elemental sulfur-driven autotrophic denitrification. Overall, this strategy achieved in-situ enrichment of SOB in granules, thereby shortening start-up process.

Role of extracellular polymeric substances in selenite and tellurite reduction by waste activated and anaerobic sludge

This study investigated the (simultaneous) reduction of selenite and tellurite by the Extracellular Polymeric Substances (EPS) of waste activated and anaerobic granular sludge. The EPS reduced the oxyanions to their...

A review on efficient technologies for fish canning wastewater treatment

Food industries stand out as significant contributors to industrial wastewater production. The canning industry, with its diverse production processes, presents a challenge in treating its wastewater due to its distinctive qualitative characteristics. This study reviews the methods employed for the treatment of fish canning wastewater and assesses their efficiency. A search protocol identified 67 articles, and ultimately, 25 were selected for review. The findings highlight that the crucial qualitative parameters in fish canning wastewater are oil and salt, presenting significant challenges in its treatment. Oil concentrations were reported up to 26,800 mg/L, and salt concentrations up to 30,000 mg/L. Commonly utilized processes in the studies encompassed aerobic and anaerobic granular sludge, as well as the SHARON/anammox system. While biological processes constitute the primary approach in fish canning wastewater treatment, addressing challenges such as high turbidity and quality variations requires the use of salinity-adapted and integrated processes.