Effect of increasing organic loading rate on hydrolytic enzyme activities and microbial communities during anaerobic digestion of energy crops

A step-by-step strategic adaptation of anaerobic sludge for high-solids digestion of fruit and vegetable waste

ABSTRACT Dry anaerobic digestion has emerged as a promising alternative for treating and recovering energy from organic solid waste, including fruit and vegetable waste. This study investigated the progressive adaptation of a mesophilic anaerobic sludge, previously used to treat brewery effluents (2.4% Total Solids), to operate under high-solids conditions (13.8%), using the dry fraction of fruit and vegetable waste as the sole substrate. Acclimation was conducted in a mixing reactor at 35°C, with gradual replacement of sucrose by the dry fraction of fruit and vegetable waste and sequential increase in organic concentration from 0.1 to 6.0 kg COD m-3. The adaptation was evaluated through physicochemical parameters, a Specific Methanogenic Activity test, and metagenomic analysis of the microbial community. The results demonstrated a significant biological restructuring, with a reduction in alpha diversity but an increase in functional speciation, reflecting greater resilience to the operational stresses of dry anaerobic digestion. The taxonomic succession showed higher relative abundances of the phyla Proteobacteria and Bacteroidota, and, with adaptation, Nitrospirae, Chloroflexi, and Synergistota stood out. Furthermore, enrichment of the methanogenic archaea genera Methanothrix and Methanobacterium was observed, increasing from 0.13 to 28.9% and from 0.0 to 2.2%, respectively, indicating methane production via complementary acetoclastic and hydrogenotrophic pathways. In Specific Methanogenic Activity assays, the adapted sludge showed an increase of up to 254% in actual methane production compared to the non-adapted inoculum. This increase in methanogenic capacity, coupled with the robust conversion of volatile fatty acids, confirms the effectiveness of the gradual adaptation strategy for dry anaerobic digestion systems. These results contribute to the consolidation of reproducible protocols for inoculum conditioning in the treatment of complex organic waste under high organic concentrations.

Application and Mechanisms of Biochar in Anaerobic Digestion: Towards Process Resilience and Waste Valorization

Anaerobic digestion (AD) is widely used for organic waste stabilization and bio-energy recovery, but its performance is often constrained by process instability, slow syntrophic metabolism, and sensitivity to acidification, ammonia, organic overloading, and inhibitory contaminants. Biochar has emerged as a promising strategy to enhance AD, with benefits extending beyond increased methane yield. This review examines how biochar properties, including pore structure, surface functional groups, alkalinity, and electrical conductivity, regulate different AD stages and improve process stability. Biochar provides microbial habitats, buffers pH, adsorbs inhibitors, accelerates volatile fatty acid conversion, and facilitates interspecies electron transfer. The effects of biochar dosage, feedstock type, reactor configuration, and operational conditions on digestion performance and resource recovery are critically evaluated. Broader contributions to organic waste valorization are also discussed. Future research should prioritize tailored biochar design, standardized characterization, long-term validation in continuous reactors, techno-economic analysis, and life-cycle assessment. These efforts are essential for translating laboratory findings into reliable and sustainable applications for organic solid waste treatment and resource recovery.

Mechanistic Insights from C/N Ratio and Biodegradability on Methane Yield and Microbial Dynamics in High-Solids Anaerobic Digestion

High-solid anaerobic digestion (HS-AD) is one of the most efficient and popular solid-waste-treatment and energy-recovery technologies. However, it is significantly influenced by composition and substrate characteristics, with the carbon-to-nitrogen ratio (C/N ratio) and biodegradability being important. In this study, the substrate was adjusted and compounded to investigate the methanogenesis performance with the substrate C/N ratio (2.82–82.72) and biodegradability (refractory and easily degraded) variation during HS-AD. The results showed that the highest methane yield (MY) was achieved with white meat (273.02 mL/g-VS), which was 2.22 times higher than that of substrates with higher C/N ratios. However, this enhanced methane productivity was accompanied by elevated total ammonia nitrogen (TAN) concentrations, which substantially increased the risk of system instability. For a high C/N ratio (>50:1), the difference between the C/N ratio and biodegradability had little influence on MY, and under a low C/N ratio (<5:1), the methane production rate was higher. For low C/N substrates, acetic (14.53–76.82%) accounted for the highest total volatile fatty acids (VFAs) during HS-AD, and for high C/N ratio substrates, propionic (46.44–82.66%) had a higher proportion (r = 0.94). An increased C/N ratio decreased total ammonia nitrogen (TAN) and alkalinity (p < 0.05). Variations in the C/N ratio and biodegradability led to differences in the microbial composition.

Bioelectrochemically Enhanced Anaerobic Digestion of Brewery Wastewater at Ambient Temperature: Performance, Methane Yield, and Energy Efficiency.

Bioelectrochemically-enhanced anaerobic digestion (BEAD) has emerged as a promising approach to improve methane production and process stability during the treatment of high-strength industrial wastewater. In this study, a continuous upflow BEAD reactor was operated for 365 days to evaluate its performance in treating real brewery wastewater at ambient temperature (22-24 °C). The effects of organic loading rate (OLR; 1-7 g sCOD/L∙d), hydraulic retention time (HRT), and applied potential (1.2 V) on organic matter removal, volatile fatty acid (VFA) degradation, methane production, and energy efficiency were systematically investigated and compared with conventional anaerobic digestion (AD). The BEAD system achieved high chemical oxygen demand (COD) removal efficiencies (>92%) at OLRs up to 5 g sCOD/L∙d and demonstrated superior degradation of complex VFAs, particularly propionic and butyric acids. Methane yield increased with increasing OLR, reaching a maximum of 0.35 LCH₄/g sCODRemoved at an OLR of 7 g sCOD/L∙d. Compared with AD, BEAD enhanced methane production by 16-30% at higher OLRs while maintaining stable biogas quality (≈74-75% CH₄). Although shortening HRT at high OLRs reduced overall performance, BEAD consistently outperformed AD. The system exhibited high energy efficiency (~1000%), suggesting it could serve as a robust, highly energy-efficient candidate for brewery wastewater treatment in cold regions.

A Review on Effects of Sulfate in Anaerobic Digestion and Effect of Auto Generative Pressure on Biogas Production

Effect of Inoculation Ratio on the Anaerobic Co-Digestion of Intensive Dairy Farm Wastewater and Sewage Sludge: Gas Generation, VFA Composition, and Process Stability

Intensive dairy farm wastewater (DFW) poses significant environmental challenges due to its high organic loading and complex composition. Anaerobic co-digestion with sewage sludge (SS) offers a promising strategy for simultaneous pollutant removal and bioenergy recovery. However, the optimal inoculation ratio for maximizing both methane production and volatile fatty acid (VFA) accumulation remains unclear for liquid DFW following a solid–liquid separation. This study investigated the effects of three SS addition ratios (0%, 15%, and 45%) on anaerobic co-digestion performance. Daily methane production, cumulative yield, VFA composition, pH, electrical conductivity (EC), ammonium nitrogen (NH4+-N), and chemical oxygen demand (COD) were monitored over 26 days. The 45% SS treatment (S45) achieved the highest cumulative methane yield (2882.60 mL), representing 35.1% and 7.2% increases over S0 and S15. Modified Gompertz modeling confirmed S45 attained the highest methane potential (3056.8 mL) and production rate (569.8 mL/d), with the shortest lag phase (14.47 d). S45 also reached the highest total VFAs peak (2209.02 mg/L) on day 3, advancing acidification by 3 days. Process stability was maintained across all treatments (pH 6.94–8.44), with S45 showing the earliest pH recovery and lowest NH4+-N accumulation. COD removal in S45 exceeded S0 by 25.1% at day 26. These findings indicate that 45% SS addition optimally balances methanogenic performance, acidogenic efficiency, and process stability in DFW anaerobic co-digestion.