Methane (CH₄), an important greenhouse gas, exerts a substantial influence on global climate change. Rivers are crucial sites for methane production, but the complexity of environmental conditionsin rivers makes it difficult to conduct in-depth research on methane generation by river microorganisms with traditional methods. This article first outlines the basic classification of methanogenic microbes, their distribution and the pathways of methane formation such as the hydrogen nutrition metabolism pathway, the formic acid fermentation pathway, the acetic acid fermentation pathway, the methyl nutrition pathway, and the non-enzymatic reaction pathway. Then, it explores the eight environmental factors affecting the methane production process of river microorganisms: temperature, pH value, redox potential, flow rate and water level, water flow shear force, meteorological conditions, and vegetation type. Subsequently, an overview is provided of the application of molecular biology techniques including PCR technology and high-throughput sequencing, in the study of river microbial methane production. Finally, it points out the problems in current research on methane production by river microorganisms, which are the insufficiency in characterizing river environments, limitations in the application of PCR and sequencing technology, as well as the complexity of data interpretation and analysis. Moreover, it discusses future research on river methane emissions from three directions: the construction and application of river environmental models, the deeper application of molecular biology technology, and knowledge sharing and intelligent analysis, providing new ideas and methods for the exploration on river microbial methane production.

N,N-bis(carboxymethyl)-L-glutamate (GLDA) is an eco-friendly chelating agent that effectively extracts multivalent metal ions from waste activated sludge (WAS) flocs, which could potentially alter their structure. However, the effect of GLDA on the production of volatile fatty acids (VFAs) from WAS is not well known. Here, we demonstrate that pretreatment with GLDA at a concentration of 200 mmol per kg VSS results in a significant increase of 142% in extractable extracellular polymeric substances and enhances the total VFAs yield by 64% compared to untreated samples. We reveal GLDA's capability to mobilize organic-binding multivalent metal ions within sludge flocs. Specifically, post-pretreatment analyses showed the release of 69.1 mg L⁻¹ of Ca and 109.8 mg L⁻¹ of Fe ions from the flocs, leading to a more relaxed floc structure and a reduced apparent activation energy (10.6 versus 20 kJ mol⁻¹) for WAS solubilization. Molecular dynamic simulations further demonstrate GLDA's preferential binding to Fe³⁺ and Ca²⁺ over Mg²⁺. Our study suggests that GLDA pretreatment causes minimal disruption to reactor stability, thereby indicating the stability of microbial community composition. GLDA has emerged as a viable pretreatment agent for enhancing volatile fatty acids production from waste activated sludge.