Municipal solid waste (MSW) valorization is constrained by the mismatch between heterogeneous feedstock properties and the operating requirements of individual conversion technologies. This review synthesizes integrated thermochemical and biochemical pathways and treats MSW valorization as a system design problem organized around carbon cascading, in which each carbon fraction is routed to the process able to convert it and performance is governed by interactions between processes rather than by individual process yields. Four hybrid routes are examined: gasification-syngas fermentation, pyrolysis-anaerobic digestion, hydrothermal processing-anaerobic digestion, and reverse configurations in which digestion precedes thermochemical conversion. Across these routes, intermediate quality rather than process yield governs feasibility. Tar (1-150 g/Nm3), ammonia (up to 14,000 ppmv), and sulfur species must be reduced by two to three orders of magnitude before downstream bioconversion, relocating the critical engineering problem from the reactor to the interface. Environmental and economic evidence indicates that integration benefits are real but conditional. Coupling anaerobic digestion with pyrolysis approximately doubled the climate mitigation of standalone digestion, yet the advantage is not uniform across impact categories, and one configuration achieved the greatest climate benefit while increasing freshwater ecotoxicity 52-fold. Reported production costs for integrated routes ($0.31-1.95/L ethanol) exceed those of standalone biochemical conversion, though integrated systems convert carbon that cheaper routes cannot access, and scale and product value influence viability more than process configuration. Integration is therefore justified by feedstock coverage and interface management rather than by coupling processes as such, and full-chain demonstration on real MSW remains the principal research challenge.
Converting non-food cellulosic sugars into programmable amylose offers a route to higher-value carbohydrate materials. α-Glucan phosphorylase (αGP) controls glucose-1-phosphate (G-1-P)-dependent chain elongation in cellobiose-to-amylose cascades. Here, Nicotiana attenuata αGP (NicαGP) was engineered by integrating AlphaFold3-based structural modeling, ProteinMPNN, EVcouplings, structural priors, and DynaMut2 filtering. Of 916 candidate substitutions, 37 were selected for experimental validation, and combinatorial screening identified L226Y/V437L/F751H as the optimal triple mutant, with 2.10-fold higher relative activity than the same-batch wild-type (WT) control and clear positive epistasis. The mutant further shifted the optimal temperature from 40 to 45 °C, increased thermal transition midpoint from 56.2 to 58.8 °C, and enhanced catalytic efficiency toward both maltotetraose and G-1-P. In the Clostridium thermocellum cellobiose phosphorylase (CtCBP)-NicαGP cascade, L226Y/V437L/F751H reached 38.86 ± 2.06 % conversion at 72 h versus the previously reported WT value of 35.84 ± 1.04 % at 84 h, increasing apparent productivity by 26.5 %. Docking, CAVER, and molecular dynamics (MD) analyses suggested that these gains may be associated with optimization of structural regions surrounding the conserved catalytic core. This work provides an engineered αGP for amylose synthesis from cellobiose and supports peripheral regulatory engineering as a practical strategy for improving phosphorylase-based biocatalytic cascades.
Ammonium persulfate (APS)-assisted hydrothermal pretreatment (HP) is an emerging method for xylan depolymerization, yet its potential for integrated valorization of all biomass fractions remains underexplored. This study aimed to selectively produce xylo-oligosaccharides (XOS) and xylose from corncob via controlled APS-HP, evaluate XOS bioactivity, and ferment xylose hydrolysate to xylonic acid without detoxification, while assessing enzymatic digestibility of solid residues. Under 0.5 % APS at 150 °C for 90 min, an optimal XOS yield reached 40.90 % (based on xylan in corncob), whereas 1 % APS gave a maximal xylose yield of 56.34 %. The XOS hydrolysate at 50 mg/L improved Chinese cabbage (Brassica rapa L. var. pekinensis) seed germination, plant height, root elongation, and fresh biomass, revealing its agricultural bioactivity. Notably, the xylose hydrolysate without detoxification was converted to xylonic acid with a yield of 90.32 % within 24 h, and even a 2.15-fold concentrated hydrolysate achieved 90.36 %, demonstrating process robustness. Solid residues from XOS and xylose production exhibited excellent enzymatic digestibility, with glucose yields of 96.89 % and 99.03 %, respectively, at 20 FPU/g glucan. Preliminary techno-economic analysis and qualitative life cycle assessment are used to assess economic theoretical basis for practical industrial production. Collectively, this study presents a novel integrated biorefinery strategy enabling sequential production of bioactive XOS, fermentable xylose, and highly digestible solid residues from corncob via APS-HP, offering a promising route for comprehensive lignocellulosic carbohydrates utilization without requiring a separate detoxification step.
This review seeks to compile Polyhydroxyalkanoates (PHAs) degradation studies published over the past 20 years. It highlights the effect of physical properties, such as crystallinity and molecular weight, on the decomposition rate of these molecules. Both biotic processes, mediated by bacteria, fungi, and enzymes, as well as abiotic processes, such as hydrolysis and thermal degradation, are analyzed. A repertoire of diverse microorganisms, including their metabolic pathways and enzymes for PHA breakdown, is presented. Furthermore, this review presents the decomposition of PHAs in various environments, such as soil and seawater, highlighting their potential as a sustainable alternative. Finally, the resulting degradation products are described, emphasizing their potential applications in medicine and industry. Although degradation of PHAs has been extensively studied through these years, several knowledge gaps remain undisclosed, including the degradation of diverse polyester monomers. PHAs comprise numerous monomer compositions with variable properties, which present opportunities for different applications but pose a challenge in their degradation. The reader of this review can extract useful information for both the production of PHAs and their potential applications.
Chemo-biological valorization of CO2 into value-added chemicals, such as the biodegradable plastic monomer glycolic acid (GA), represents a promising frontier in sustainable manufacturing. However, the realization of this synergistic loop is hindered by the low carbon efficiency of C1 bioconversion. Herein, we address this challenge by engineering an efficient methanol bioconversion platform that selectively upgrades CO2-derived methanol into GA. To address metabolic redox constraints inherent to methanol assimilation that severely limit carbon yield, we reprogrammed Komagataella phaffii by constructing a cofactor-neutral biosynthetic route that couples NADPH generation directly to GA formation. Combined with the elimination of competing carbon sinks, this design eradicates the redox constraints that typically limit C1 assimilation. As a result, carbon flux toward GA surged by 4.6-fold while maintaining cellular fitness. Translating this rewired module to a 5-L bioreactor establishes a benchmark, achieving a titer of 41.2 g/L and the highest reported yield of 0.41 g/g for methanol-only bioconversion. Life-cycle assessment (LCA) reveals a 59.0% lower global warming potential (GWP) relative to fossil-based routes. This study establishes a high-efficiency methanol bioconversion platform for GA production, completing the CO2-to-GA loop and providing a sustainable route for the utilization of CO2-derived carbon resources.
Efficient pretreatment that enables selective fractionation, structural preservation, and coordinated downstream utilization remains a major challenge for lignocellulosic biorefineries. Herein, an alkaline deep eutectic solvent composed of triethylbenzylammonium chloride and ethanolamine was adopted for the integrated biorefinery of bamboo shoot shells. The pretreatment afforded 92.14-95.20 % delignification and 89.61-94.00 % hemicellulose removal across four feedstocks, while largely preserving cellulose integrity. Meanwhile, the recovered lignin retained substantial β-O-4 linkages, showed reduced condensation, narrowed molecular-weight distribution, and enhanced chemical reactivity, indicating its suitability for downstream valorization. Owing to the improved accessibility of the cellulose-rich residues, enzymatic hydrolysis delivered glucose yields of 92.06-96.70 % after 48 h. In addition to enzymatic saccharification, the fractionated components were further upgraded into lignin-containing cellulose nanofibrils, lignin nanoparticles, and nitrogen-doped carbon dots from the recovered spent solvent, demonstrating downstream utilization within one process. Overall, this work adopted an alkaline DES-based pretreatment strategy that integrates efficient fractionation, high enzymatic digestibility, and cascade valorization of major biomass components, demonstrating the potential of this strategy for integrated lignocellulosic biorefining.
Cultivation conditions are known to affect the structural organization of microbial cell walls. However, the impact of these structural variations on protein release following mild cell disruption remains largely unknown. Therefore, this study investigated the effects of cultivation conditions on the Saccharomyces cerevisiae cell wall. Its robustness and apparent protein permeability were assessed indirectly through, respectively, zymolyase susceptibility and intracellular protein release following hydrophobic deep eutectic solvent assisted membrane-permeabilization. More specifically, the influences of cultivation pH, glucose-limitation, growth phase and cultivation mode were evaluated. Shake-flask experiments demonstrated that cultivation conditions associated with different physiological states strongly affected cell wall structural organization, since a shift from glucose-limitation to glucose-abundance increased zymolyase susceptibility by 5-fold, and increased protein release by up to 3.8-fold. Therefore, the effects of growth phase and cultivation mode were further investigated in controlled bioreactors, resulting in more profound differences. Biomass harvested from exponentially growing batch cultures exhibited a 5.7-fold higher soluble protein release than low-rate fed-batch cultures, while zymolyase susceptibility increased by 19.4-fold. Moreover, strong correlations were found between physiological state, zymolyase susceptibility and protein permeation behaviour, suggesting that cultivation-dependent physiological states impact cell wall characteristics and intracellular protein release. Finally, apparent molecular size analysis demonstrated that the released material obtained from permeabilized biomass was enriched with components smaller than 100 kDa. Overall, this study demonstrates that cultivation-driven modulations in cell wall characteristics strongly affect mild disruption yields, thereby highlighting the importance of integrating upstream and downstream processes for efficient recovery of intracellular proteins.
Metal hyperaccumulators are promising plants for phytoextraction, but the root traits that help them grow toward metal-rich soil remain unclear. Metal-directed root foraging has been proposed as a unique trait of hyperaccumulators, yet inconsistent observations have obscured its functional significance. Here, we combined a meta-analysis of 128 published observations with split-root experiments in Sedum alfredii to evaluate the recurrence, environmental sensitivity, and physiological relevance of metal-directed root foraging. A meta-analysis showed a broad but context-dependent tendency for hyperaccumulators to allocate more roots to metal-enriched patches, and this response was positively associated with shoot metal accumulation and biomass. However, this response was strongly shaped by soil conditions, particularly soil texture and soil source. Split-root experiments further revealed a clear ecotype-specific response in hydroponics. The hyperaccumulating ecotype (HE) allocated more than 60 % of its root biomass to the Cd-enriched compartment, whereas the non-hyperaccumulating ecotype (NHE) showed reduced root allocation to this compartment. This ecotype-specific contrast was not fully retained in the tested soil system: Cd-directed foraging by HE was no longer detectable, whereas Cd avoidance by NHE persisted. These findings identify metal-directed root foraging as a recurrent but soil-dependent trait of hyperaccumulators. By showing that soil conditions can constrain the detectability of this trait, our study reconciles inconsistent observations and points to soil management as a potential strategy to strengthen root foraging and enhance phytoextraction in contaminated soils.
Enhancing lake carbon sequestration is a vital nature-based climate solution, whereas the degradation of submerged macrophytes seriously threatens this potential. Plant growth-promoting rhizobacteria (PGPR) are capable of accelerating the stress recovery of submerged macrophytes and enhancing the greenhouse gas (GHG) absorption for single-species macrophyte. Here, four-species submerged macrophyte communities (Potamogeton wrightii, Myriophyllum spicatum, Vallisneria denseserrulata, and Hydrilla verticillata) were established under simulated underwater low-light conditions and inoculated with a high-efficiency PGPR consortium (Pseudomonas vancouverensis, Pseudomonas plecoglossicida, and Enterobacter ludwigii). Treatments included inoculation applied to 0, 1, 2, 3, and 4 species treatments, respectively. Over an annual timescale, inoculation of all four plant species resulted in the highest monthly biomass increment (19 % above control) and increased the system's CO2-equivalent sink by 336 %, yielding the lowest global warming potential (GWP, -7939.91 ± 96.73 g CO2-eq m-2 yr-1). These results indicate a strong synergy between growth promotion and carbon sink enhancement. CO2-equivalent uptake during the overwintering stage significantly exceeded that during the rapid growth stage, likely because low temperatures suppress biological metabolic activities while simultaneously enhancing physicochemical absorption, thereby enhancing net CO2 sequestration. Methodologically, the conventional 1-hour morning sampling overestimated CO2-equivalent fluxes by 3.54 to 6.28 times compared to 24-hour continuous monitoring. These findings demonstrate the dual capacity of PGPR to boost macrophyte productivity and GHG sink functions under low-light stress. The results underscore the necessity of 24-hour continuous sampling for accurate GHG flux assessment in aquatic ecosystems and provide a scientific basis for developing low-carbon lake restoration and carbon-neutral management strategies.
This eight-year study characterized trace compounds in grid-injected biomethane from methanization using a multi-analytical strategy combining broad thermal desorption gas chromatography-mass spectrometry (TD-GC/MS) screening with targeted quantitative methods. Between 2016 and 2023, 75 sampling campaigns were conducted at 40 French biomethane injection plants, covering diverse feedstocks and upgrading technologies. 530 organic compounds across nine chemical families were identified by TD-GC/MS. Of these, 65 calibrated compounds were quantified and 465 non-target compounds were semi-quantitatively estimated as order-of-magnitude concentrations. Most compounds occurred sporadically and at low levels: only 100 were detected in more than ten samples, whereas 207 appeared only once, with an average of 48 trace compounds per sample. Average concentrations were generally low, with only 13 % exceeding 100 µg/Nm3. Hydrocarbons were dominant, followed by oxygenated compounds, while terpenes, organic sulfur compounds, siloxanes, halocarbons and amines occurred less frequently. Chemometric analysis of 45 selected TD-GC/MS compounds showed broadly similar biomethane trace profiles, with no robust clustering by feedstock or upgrading technology. Targeted analyses quantified compounds of regulatory or operational relevance, including monoaromatic hydrocarbons, terpenes, sulfur species, ammonia, mercury and trace elements. Regulated sulfur species, ammonia, mercury and siloxanes remained below current injection requirements, and compounds shared with conventional natural gas were generally comparable to or lower than natural-gas reference levels. This information-rich dataset demonstrates the complementarity of analytical strategies and shows that grid-injected biomethane contains a chemically diverse but low-abundance trace-compound fraction, supporting secure grid integration and future monitoring, standardization and specification evolution.
Partial substitution of chemical fertilizers with organic amendments is a promising strategy to sustain soil productivity while reducing chemical inputs. However, the ecological mechanisms by which organic substitution and inorganic reduction reshape soil microbial community assembly, life‑history strategies, and nutrient cycling potential remain poorly understood. A 43-year field experiment with different proportions of organic fertilizer substituting for inorganic nitrogen was conducted. Microbial community structure, assembly processes, keystone taxa, and functional genes involved in nitrogen (N) and sulfur (S) cycles were investigated with high-throughput amplicon and metagenomic sequencing. Our results showed that organic substitution significantly reshapes microbial community composition, increasing community evenness while maintaining species richness. It significantly reduced the proportion of transient and persistent microorganisms while increasing intermittent taxa. Organic substitution significantly reduced (p < 0.05) the contribution of stochastic processes in soil microbes in comparison to those treated only with chemical fertilizers. This shift was accompanied by the enrichment of specific functional phyla such as Actinomycetota (class Thermoleophilia), Myxococcota, and Gemmatimonadota, which served as keystone species in co‑occurrence networks. Functionally, organic substitution significantly upregulated genes involved in organic nitrogen mineralization (glnAB&ureABC&gdhA&GLUL) and anaerobic ammonium oxidation (anammox), while downregulating nitrification, dissimilatory nitrate reduction to ammonium (DNRA), and assimilatory nitrate reduction. Likewise, organic substitution reduced organic sulfur mineralization and hydrogen sulfide production (sreAB) but enhanced assimilatory sulfate reduction. The findings of this study provide new insights into the ecological mechanisms through which organic substitution regulates soil microbiomes and nutrient cycling.
The sustainable treatment of surfactant-rich wastewater in anaerobic systems is often hindered by the biochemical recalcitrance of linear alkylbenzene sulfonate (LAS) and its inhibitory effects on methanogenesis. This study investigated controlled microaeration under two hydraulic retention time (HRT) and organic loading rate (OLR) regimes as a strategy to intensify LAS degradation and energy recovery in upflow anaerobic sludge blanket (UASB) reactors. Four configurations were evaluated: R1 (anaerobic, 8-h HRT), R2 (microaerated, 8-h HRT), R3 (anaerobic, 16-h HRT), and R4 (microaerated, 16-h HRT). Results demonstrated that under strictly anaerobic conditions, operating at a 16-h HRT and the corresponding lower OLR resulted in higher chemical oxygen demand (COD) removal than at 8-h HRT (87.4% vs. 65.7%). In contrast, microaeration played an important role in enhancing surfactant biotransformation. R4 achieved the highest LAS removal (64.0 ± 6.2%), significantly outperforming the strictly anaerobic R3. The 16-h HRT/lower-OLR configuration provided operational conditions that may have favored the progression of successive LAS biotransformation steps, including oxygenase-mediated ω-oxidation and the transformation of aromatic compounds under microaerated conditions. Notably, R4 exhibited a remarkable specific methane yield of 0.28 ± 0.01 LCH4/gCODapp, doubling the efficiency of the other reactors. Microbial analysis indicated that microaeration was associated with a more diverse consortium in which potentially oxidative bacteria (Smithella and Thauera) coexisted with acetoclastic methanogens (Methanosaeta). These findings indicate that microaeration, when combined with the longer HRT tested, enhanced LAS degradation and process performance compared with the 8-h condition, highlighting the potential of this strategy for process intensification in treatment plants.
Straw return is widely used to enhance soil organic matter in black soil regions; however, low temperatures severely constrain the microbial degradation of straw. In this study, a wild fungal strain Talaromyces stipitatus with robust hydrolytic activity under cold conditions was isolated from black soil. A xylanase, TsTA1, secreted by this fungus, was shown to be active at low temperatures. Through molecular engineering, a series of TsTA1 mutants were generated, and three variants, M142Y, I161V, and F215Y, were demonstrated have substantially (1.5 to 2-fold) increased catalytic efficiency compared to the wild-type enzyme. Structural analyses indicated that the M142Y and F215Y substitutions enhance activity by introducing additional hydrogen bonding with the substrate. Interestingly, the I161V mutation markedly altered the pH range in which the enzyme is active, conferring alkaline pH tolerance. These findings enhance the understanding of straw decomposition by cold-adapted microbes and demonstrate a successful strategy for improving enzyme performance under challenging environmental conditions.
Enhancing biogenic coalbed methane (CBM) production through algae-coal co-digestion has attracted increasing interest. However, the potential role of coal particles in mediating direct interspecies electron transfer (DIET) and the dominant methanogenic pathways in such systems remain unresolved. Laboratory-simulated coalbed bioreactors were established to investigate these questions, with stable isotope fingerprinting employed as an independent analytical tool to resolve methanogenic pathways beyond what community composition data alone can reveal. The co-digestion group with pre-activated inoculum (Group O) achieved the highest CH4 yield (1400.68 μmol/g COD) and effectively shortened the lag phase. Pre-activation selectively enriched the electroactive bacterium Clostridium alongside the methanogenic archaea Methanobacterium and Methanosarcina. Critically, the electroactive properties of Clostridium, acting in concert with coal particles, may have synergically contributed to DIET. Group O had the highest coenzyme F420 activity, which indirectly supports this result. The stable coexistence of Methanosarcina and Methanobacterium, uniquely established by inoculum pre-activation, reflected functional complementarity, with the former sustaining CH4 flux via acetoclastic methanogenesis and the latter maintaining redox balance through H2 consumption. Analysis of the apparent fractionation factor (αc) from stable isotope data suggested that the acetoclastic pathway dominated in methanogenesis in the co-digestion systems and that its contribution progressively strengthened during anaerobic digestion, supported by acetate consumption and enrichment of key acetoclastic genes (ackA, pta, cdh). These findings provide a mechanistic framework for in situ CBM bioaugmentation, in which co-digestion of algae with pre-activated inocula enriched in electroactive bacteria can simultaneously stimulate DIET potential and sustain acetoclastic methanogenic activity in coal seam environments.
Incorporating natural biological macromolecules to reduce reliance on petroleum-derived adhesives and valorize bio-wastes is a key path for achieving low-carbon manufacturing. However, the inherent hydrophobic barrier of cork restricts the interfacial wetting and penetration of traditional highly polar bio-based adhesives, severely limiting their mechanical performance. To overcome this bottleneck, this study used Chitosan (CS) as the raw material, Blocked Isocyanate (BI) as the efficient crosslinking agent to develop a low-carbon emission composite adhesive and prepared high-performance cork agglomerated materials. The average tensile strength of the materials prepared by this adhesive reached up to 2.21 MPa, which was much higher than the 1.4 MPa stipulated by the international standards ISO 7322:2014. The average initial compression ratio was 6.5%, the average residual compression ratio was 1.42%, the thermal conductivity ranged from 0.076 to 0.094 W/m·K, satisfying the standards of commercial cork flooring and insulation materials. The analysis results revealed that the amino and urethane groups of chitosan reacted with the isocyanate groups to form urea bonds and urethane bonds, achieving strong and durable bonding. The results from the "cradle-to-gate" life cycle assessment (LCA) confirmed that compared with commercial isocyanate adhesives, the CS/BI composite system maintained excellent bonding performance while reducing key environmental loads such as global warming potential (GWP), abiotic depletion potential (ADP), and acidification potential (AP) by 45.3%, 52.6%, and 81.6%, respectively. This study not only revealed the interface bonding mechanism of multi-component composite systems but also provided a promising application prospect for cork agglomerated materials.
Iron (Fe) plaques on rice roots are naturally occurring redox-active mineral interfaces and recognized hotspots for nitrous oxide (N2O) production in paddy rhizospheres. However, the role of trace manganese (Mn) naturally co-deposited within Fe plaques remains poorly understood. Here, we used experimentally induced Fe plaques and Mn-embedded Fe plaques as a controlled model to evaluate whether plaque-associated Mn modifies rhizosphere redox conditions and N2O production. Compared with Fe-only plaques, Mn-embedded Fe plaques showed a greater increase in N2O emissions and were associated with higher hydroxyl radical (•OH) production. Under illuminated conditions, quenching •OH with terephthalic acid reduced N2O emissions most strongly in the Mn-embedded Fe plaque treatment, supporting an association between plaque-associated •OH and light-enhanced N2O accumulation. Despite the shading-induced decrease in rhizosphere O2, N2O production remained higher in the Mn-embedded Fe plaque treatment than in the Fe plaque and control treatments. In anaerobic incubations, EDTA addition reduced the Mn-associated response, whereas exogenous Mn(II) increased N2O production. Metagenomic profiling provided supporting evidence for these responses, showing shifts in denitrification-related taxa and functional genes, including norB and nosZ. Together, these findings identify Mn incorporation into rice root Fe plaques as a previously overlooked factor modulating rhizosphere N2O production and highlight the need to consider trace-metal composition when evaluating the biogeochemical function of root plaques.
Microalgal CO2 fixation is constrained by inorganic carbon utilization, photosynthetic capacity, and oxidative stress, and although electrical stimulation can modulate algal physiology, the effective current–density range remains unclear. Here, Scenedesmus quadricauda was cultivated under microcurrent densities of 0, 10, 20, 30, 40, 70, and 100 μA cm−2, with detailed analyses focused on 0–30 μA cm−2. Cultivation and physiological assays were performed with three biological replicates, and metabolomics with five per treatment. Microcurrent stimulation exhibited current–density dependence, with 20 μA cm−2 yielding the best performance. After 7 days, biomass reached 1.19 g L−1, 63.0% above the control, and the biomass-based apparent CO2 fixation rate increased to 0.30 g L−1 d−1. Chlorophyll a and b contents increased, and maximum photochemical efficiency of photosystem II was maintained. Carbonic anhydrase, ribulose-1,5-bisphosphate carboxylase/oxygenase, and acetyl coenzyme A levels were highest at 20 μA cm−2, indicating enhanced carbon assimilation and central carbon metabolism. This treatment promoted extracellular polymeric substance secretion while maintaining the lowest reactive oxygen species accumulation. 13CO2 tracing confirmed supplied CO2 as the dominant biomass carbon source, with 13C abundance exceeding 90% across treatments. In contrast, 30 μA cm−2 promoted lipid accumulation, whereas higher densities triggered stress responses and severe inhibition at 100 μA cm−2. Untargeted metabolomics further indicated that 20 μA cm−2 favored carbon assimilation, energy metabolism, and antioxidant protection, whereas 30 μA cm−2 shifted metabolism toward stress adaptation and lipid-related responses. These findings identify a moderate microcurrent-density window for enhancing growth-coupled CO2 fixation in S. quadricauda and guiding electro-assisted algal CO2 bioconversion.
The transition from transient to persistent foaming under organic overloading in waste activated sludge anaerobic digestion (AD) remains insufficiently understood. This study investigated foam evolution under increasing organic loading rates (OLRs) in a semi-continuous mesophilic digester treating thermal-alkaline-pretreated sludge, focusing on sludge-derived amphiphilic substances and their interfacial effects. Foam initiation occurred at an OLR of 5 g COD/(L·d), whereas persistent foaming was established at 7 g COD/(L·d). Increasing OLR promoted the accumulation of soluble extracellular polymeric substances, particularly proteins and humic-like substances, accompanied by a shift in protein composition toward higher hydrophobicity. The proportion of hydrophobic amino acids, mainly alanine and proline, increased from 29.8% to 35.7% during foam development, suggesting enhanced protein affinity for the gas-liquid interface. Humic substances contributed disproportionately to foam stabilization: 0.5 g/L achieved a foaming tendency comparable to 5.0 g/L sludge-derived proteins. This strong stabilizing effect was associated with lower critical micelle concentration, reduced surface tension, higher viscosity, slower drainage, and delayed bubble coarsening. Beyond physicochemical effects, foam-microbe feedback may occur: the hydrophobic niche favors foam-associated taxa (e.g., Thermovirga and Methanofastidiosum), which, though enriched primarily as an ecological response to overloading, may in turn influence foam stability. Persistent foaming in sludge AD results from organic overloading driving a transition to interfacial stabilization, where seemingly humic substances act as efficient foam stabilizers and hydrophobic amino acids contribute to the stabilization via interfacial adsorption. This mechanism informs a basis for OLR-based early warning and targeted foam control in full-scale digesters treating thermal-alkaline-pretreated waste activated sludge.
