Acetotrophic Activity Facilitates Methanogenesis from LCFA at Low Temperatures: Screening from Mesophilic Inocula

Abstract

The inoculum source plays a crucial role in the anaerobic treatment of wastewaters. Lipids are present in various wastewaters and have a high methanogenic potential, but their hydrolysis results in the production of long chain fatty acids (LCFAs) that are inhibitory to anaerobic microorganisms. Screening of inoculum for the anaerobic treatment of LCFA-containing wastewaters has been performed at mesophilic and thermophilic conditions. However, an evaluation of inocula for producing methane from LCFA-containing wastewater has not yet been conducted at low temperatures and needs to be undertaken. In this study, three inocula (one granular sludge and two municipal digester sludges) were assessed for methane production from LCFA-containing synthetic dairy wastewater (SDW) at low temperatures (10 and 20°C). A methane yield (based on mL-CH₄/g-COD_added) of 86-65% with acetate and 45-20% with SDW was achieved within 10 days using unacclimated granular sludge, whereas the municipal digester sludges produced methane only at 20°C but not at 10°C even after 200 days of incubation. The acetotrophic activity in the inoculum was found to be crucial for methane production from LCFA at low temperatures, highlighting the role of Methanosaeta (acetoclastic archaea) at low temperatures. The presence of bacterial taxa from the family Syntrophaceae (Syntrophus and uncultured taxa) in the inoculum was found to be important for methane production from SDW at 10°C. This study suggests the evaluation of acetotrophic activity and the initial microbial community characteristics by high-throughput amplicon sequencing for selecting the inoculum for producing methane at low temperatures (up to 10°C) from lipid-containing wastewaters.

Figure 1

Methane production (mL) at (a) 20°C and (b) 10°C from different inocula (GS: granular sludge; RD: Rahola Digestate; VD: Viinikanlahti Digestate) incubated with no substrate (Blk), acetate (Ac), and synthetic dairy wastewater (SDW).

Figure 2

Residual volatile fatty acid (VFA) concentration at 10°C with synthetic dairy wastewater (SDW) and acetate at the end of the 200 d experiment (GS: granular sludge; RD: Rahola Digestate; VD: Viinikanlahti digestate).

Figure 3

Mass balance of COD at (a) 20°C and (b) 10°C in the assays with different inocula (GS: granular sludge; RD: Rahola Digestate; VD: Viinikanlahti digestate) and with the substrate synthetic dairy wastewater (SDW) or acetate.

Figure 4

Relative abundance (%) of the bacterial and archaeal classes found in the 16S rRNA amplicon libraries in the samples during the 200 d experimental period from different inocula (GS: granular sludge; RD: Rahola Digestate; VD: Viinikanlahti Digestate) incubated at 10°C and 20°C with no substrate (Blk), acetate, and synthetic dairy wastewater (SDW). Detailed information on the sample names is shown in Table S1.

Figure 5

Relative fraction (%) of the archaeal genera belonging to the classes Methanobacteria and Methanomicrobia found in the 16S rRNA amplicon libraries in the samples during the 200 d experimental period from different inocula (GS: granular sludge; RD: Rahola Digestate; VD: Viinikanlahti Digestate) incubated at 10°C and 20°C with no substrate (Blk), acetate, and synthetic dairy wastewater (SDW). Classes are mentioned within brackets. Detailed information on the sample names is shown in Table S1.

Figure 6

Relative fraction (%) of the bacterial genera belonging to the classes Deltaproteobacteria and Clostridia found in the 16S rRNA amplicon libraries in the samples during the 200 d experimental period from different inocula (GS: granular sludge; RD: Rahola Digestate; VD: Viinikanlahti Digestate) incubated at 10°C and 20°C with no substrate (Blk), acetate, and synthetic dairy wastewater (SDW). Classes are mentioned within brackets. Detailed information on the sample names is shown in Table S1.

Figure 7

Heat map showing the clustering and relative abundance of the bacterial and archaeal classes that formed 99.9% of the microbial community in the assays inoculated with VD, RD, and GS and fed with no substrate (blank), acetate, or synthetic dairy wastewater (SDW) at 10°C and 20°C. Symbols + and o indicate the kingdom bacteria and archaea, respectively, on the y-axis. Symbols ▲, ▼, and ■ represent the inoculum used in the assays as VD, RD, and GS, respectively, on the x-axis. Detailed information on the sample names is shown in Table S1.