The initiation of methanogenesis in refuse occurs under high volatile fatty acid (VFA) concentration and low pH (5.5 to 6.25), which generally are reported to inhibit methanogenic Archaea. One hypothesized mechanism for the initiation of methanogenesis in refuse decomposition is the presence of pH-neutral niches within the refuse that act as methanogenesis initiation centers. To provide experimental support for this mechanism, laboratory-scale landfill reactors were operated and destructively sampled when methanogenesis initiation was observed. The active bacterial and archaeal populations were evaluated using RNA clone libraries, RNA terminal restriction fragment length polymorphism (T-RFLP), and reverse transcription-quantitative PCR (RT-qPCR). Measurements from 81 core samples from vertical and horizontal sections of each reactor showed large spatial differences in refuse pH, moisture content, and VFA concentrations. No pH-neutral niches were observed prior to methanogenesis. RNA clone library results showed that active bacterial populations belonged mostly to Clostridiales, and that methanogenic Archaea activity at low pH was attributable to Methanosarcina barkeri. After methanogenesis began, pH-neutral conditions developed in high-moisture-content areas containing substantial populations of M. barkeri. These areas expanded with increasing methane production, forming a reaction front that advanced to low-pH areas. Despite low-pH conditions in >50% of the samples within the reactors, the leachate pH was neutral, indicating that it is not an accurate indicator of landfill microbial conditions. In the absence of pH-neutral niches, this study suggests that methanogens tolerant to low pH, such as M. barkeri, are required to overcome the low-pH, high-VFA conditions present during the anaerobic acid phase of refuse decomposition
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