Summary: Long‐chain fatty acids ( LCFA ) can inhibit methane production by methanogenic archaea. The effect of oleate and palmitate on pure cultures of Methanosaeta concilii and Methanosarcina mazei was assessed by comparing methane production rates from acetate before and after LCFA addition. For both methanogens, a sharp decrease in methane production (> 50%) was observed at 0.5 mmol L ⁻¹ oleate, and no methane was formed at concentrations higher than 2 mmol L ⁻¹ oleate. Palmitate was less inhibitory than oleate, and M. concilii was more tolerant to palmitate than M. mazei, with 2 mmol L ⁻¹ palmitate causing 11% and 64% methanogenic inhibition respectively. This study indicates that M. concilii and M. mazei tolerate LCFA concentrations similar to those previously described for hydrogenotrophic methanogens. In particular, the robustness of M. concilii might contribute to the observed prevalence of Methanosaeta species in anaerobic bioreactors used to treat LCFA ‐rich wastewater.

Degradation of long-chain fatty acids (LCFAs) in methanogenic environments is a syntrophic process involving the activity of LCFA-degrading bacteria and hydrogen-utilizing methanogens. If methanogens are inhibited, other hydrogen scavengers are needed to achieve complete LCFA degradation. In this work, we developed two different oleate (C_18:1 LCFA)-degrading anaerobic enrichment cultures, one methanogenic (ME) and another in which methanogenesis was inhibited (IE). Inhibition of methanogens was attained by adding a solution of 2-bromoethanesulfonate (BrES), which turned out to consist of a mixture of BrES and isethionate. Approximately 5 times faster oleate degradation was accomplished by the IE culture compared with the ME culture. A bacterium closely related to Syntrophomonas zehnderi (99% 16S rRNA gene identity) was the main oleate degrader in both enrichments, in syntrophic relationship with hydrogenotrophic methanogens from the genera Methanobacterium and Methanoculleus (in ME culture) or with a bacterium closely related to Desulfovibrio aminophilus (in IE culture). A Desulfovibrio species was isolated, and its ability to utilize hydrogen was confirmed. This bacterium converted isethionate to acetate and sulfide, with or without hydrogen as electron donor. This bacterium also utilized BrES but only after 3 months of incubation. Our study shows that syntrophic oleate degradation can be coupled to desulfonation. IMPORTANCE In anaerobic treatment of complex wastewater containing fat, oils, and grease, high long-chain fatty acid (LCFA) concentrations may inhibit microbial communities, particularly those of methanogens. Here, we investigated if anaerobic degradation of LCFAs can proceed when methanogens are inhibited and in the absence of typical external electron acceptors, such as nitrate, iron, or sulfate. Inhibition studies were performed with the methanogenic inhibitor 2-bromoethanesulfonate (BrES). We noticed that, after autoclaving, BrES underwent partial hydrolysis and turned out to be a mixture of two sulfonates (BrES and isethionate). We found out that LCFA conversion proceeded faster in the assays where methanogenesis was inhibited, and that it was dependent on the utilization of isethionate. In this study, we report LCFA degradation coupled to desulfonation. Our results also showed that BrES can be utilized by anaerobic bacteria.

Long-chain fatty acids (LCFA) are common contaminants in municipal and industrial wastewater that can be converted anaerobically to methane. A low hydrogen partial pressure is required for LCFA degradation by anaerobic bacteria, requiring the establishment of syntrophic relationships with hydrogenotrophic methanogens. However, high LCFA loads can inhibit methanogens, hindering biodegradation. Because it has been suggested that anaerobic degradation of these compounds may be enhanced by the presence of alternative electron acceptors, such as iron, we investigated the effect of sub-stoichiometric amounts of Fe(III) on oleate (C18:1 LCFA) degradation by suspended and granular methanogenic sludge. Fe(III) accelerated oleate biodegradation and hydrogenotrophic methanogenesis in the assays with suspended sludge, with H₂-consuming methanogens coexisting with iron-reducing bacteria. On the other hand, acetoclastic methanogenesis was delayed by Fe(III). These effects were less evident with granular sludge, possibly due to its higher initial methanogenic activity relative to suspended sludge. Enrichments with close-to-stoichiometric amounts of Fe(III) resulted in a microbial community mainly composed of Geobacter, Syntrophomonas, and Methanobacterium genera, with relative abundances of 83–89%, 3–6%, and 0.2–10%, respectively. In these enrichments, oleate was biodegraded to acetate and coupled to iron-reduction and methane production, revealing novel microbial interactions between syntrophic LCFA-degrading bacteria, iron-reducing bacteria, and methanogens.

This work investigates the effects of increasing fraction of slowly biodegradable chemical oxygen demand (sbCOD) on the morphology, stability, and performance of aerobic granular sludge (AGS) used for wastewater treatment. A sequencing batch reactor (SBR) was supplied with synthetic wastewater containing acetate as readily biodegradable COD (rbCOD) and increasing concentrations of oleate as slowly biodegradable carbon source. The sbCOD fraction was gradually increased, reaching up to 50% of the total influent biodegradable COD (bCOD). Quantitative image analysis (QIA) revealed a significant shift in granule morphology and size distribution due to increasing sbCOD fractions. Larger granules (Deq >1.0 mm) become predominant due to the washout of smaller granules (Deq <1.0 mm), which evidenced deterioration in several structural parameters. In contrary, larger granules maintained stable compactness, robustness, and extent. These morphological and size distribution changes were concomitant with variations in reactor performance: total inorganic nitrogen (TIN) removal efficiency improved up to 94%, due to enhanced denitrification capacity, supported by the predominance of larger granules and increase in granules size at higher sbCOD fractions. In contrast, P-PO₄^3- removal efficiency declined, associated with the leakage of rbCOD to the aerobic phase, filamentous growth, and deteriorated sludge settling properties. These findings highlight the complex interactions between oleate characteristics, AGS morphology, and reactor performance, emphasizing the need for optimized strategies to mitigate process instability in AGS systems treating lipid-rich wastewater, ensuring sustainable and efficient wastewater treatment in real-world applications.