Activated sludge as inoculum improves methane production and community functionality during the anaerobic digestion of mixed agave wastes

Magdaleno Caballero-Caballero, Fernando Chiñas-Castillo, Agustín Vidal Gómez-Guerrero, Idania Valdez-Vazquez, Rafael Alavez-Ramirez, José Luis Montes Bernabe

Research output: Contribution to journalArticlepeer-review


Agave plants are receiving increasing attention due to a wide range of products obtained from them. Besides, Agave processing generates lignocellulosic biomass (leaves and bagasse) and vinasses, all of them are wastes with a high organic matter content, which are suitable for methane production. However, Agave bagasse has been identified among the most recalcitrant lignocelluloses for biomethanization, while vinasses typically present low biodegradability indices. This study is aimed to improve methane production testing the inocula activated sludge (AS), pig manure (PM), and a mixture of them (M) at substrate-to-inoculum (S/I) ratios of 0.25, 0.50, 1.0, and 2.0, in terms of g volatile solids (VS)/gVS. The substrate consisted of a mixture of leaves, bagasse, and vinasses from the mezcal production. The study also analyzed microbial composition (bacteria and archaea) before and after anaerobic digestion and correlated performance with species abundance. AS reactors reached the highest methane production of 498 ± 67 mL (166 mL/gVS), followed by PM and M reactors that produced 188 ± 39 mL each (63 mL/gVS), all at a 0.25 S/I ratio. At a 0.50 S/I ratio or higher, the methane production stopped due to an insufficient quantity of microorganisms which were active during the process. AS reactors maintained the pH between 6.8 and 7.5 at all S/I ratios with a negligible volatile fatty acid accumulation. On the contrary, PM and M reactors led to volatile fatty acid accumulation as high as 12.2 g/L, so pH became acidic, ranging from 4.9 to 5.8. AS reactors contained the highest alpha diversity. The archaeal community in AS reactors consisted of Methanosarcina, Methanobrevibacter, and Methanospirillum. Unlike, Methanobrevibacter was the predominant genus in PM/M reactors. Pseudomonas and Clostridium were the predominant genera in the bacterial communities of AS reactors and PM/M reactors, respectively. The methane production positively correlated with Methanosarcina (r = 0.79) in AS reactors. On the contrary, the volatile fatty acid accumulation positively correlated with Methanobrevibacter (r = 0.57), Clostridium (r = 0.99), and Turicibacter (r = 0.96) in PM/M reactors. In sum, the AS inoculum at a 0.25 S/I ratio provided the proper quantity and type of microorganisms (such as Methanosarcina) and also the buffer capacity for improving notably the start-up of an anaerobic digester which treated the mixed agave wastes.

Original languageEnglish
JournalBiomass Conversion and Biorefinery
StateAccepted/In press - 2022


  • Bagasse, Methanosarcina
  • Spearman correlation
  • Substrate-to-inoculum ratio
  • Vinasses


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