Assessment of Methane Production and Microbial Community Dynamics in One and Two-Stage Semi-continuous Anaerobic Systems Treating Orange Peel Waste and Crude Glycerol

Abstract

Agro-industrial wastes such as orange peel waste (OPW) and crude glycerol (CG) can be used in anaerobic digestion (AD) to mitigate environmental impacts while generating methane as a renewable biofuel. Brazil produces significant amounts of these residues, highlighting the need for sustainable management strategies. This study evaluated the performance of single and two-stage anaerobic systems in the co-digestion of OPW and CG under mesophilic conditions (35–37 °C) for 60 days, with increasing organic loading rate (OLR). The two-stage system achieved higher cumulative methane yields (2703 NmL CH₄/gVS) compared to the single-stage system (2012 NmL CH₄/gVS). However, in the two-stage configuration, the acidogenic stage was associated with a pronounced accumulation of VFAs in the methanogenic reactor (SCM2), reaching concentrations up to 60,000 mg/L, which negatively affected methane production, despite the absence of detectable D-limonene inhibition. Additionally, increasing the OLR by 20% and the daily addition of alkalizing agents to control pH were detrimental to methane generation. Microbial analysis revealed the predominance of hydrolytic and acidogenic genera after day 29, such as Enterobacter (16% in SCM1 and 3% in SCM2), Proteiniphilum (35% in SCM1 and 27% in SCM2), and Fermentimonas (48% in SCM2 on day 60), indicating an incomplete methanogenic phase. Overall, the results suggest that, under the evaluated conditions, the inclusion of an acidogenic stage upstream of methanogenesis did not result in a clear enhancement of methane production.

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