Inoculum selection influences the biochemical methane potential of agro-industrial substrates

Abstract

Obtaining a reliable estimation of the methane potential of organic waste streams in anaerobic digestion, for which a biochemical methane potential (BMP) test is often used, is of high importance. Standardization of this BMP test is required to ensure inter-laboratory repeatability and accuracy of the BMP results. Therefore, guidelines were set out; yet, these do not provide sufficient information concerning origin of and the microbial community in the test inoculum. Here, the specific contribution of the methanogenic community on the BMP test results was evaluated. The biomethane potential of four different substrates (molasses, bio-refinery waste, liquid manure and high-rate activated sludge) was determined by means of four different inocula from full-scale anaerobic digestion plants. A significant effect of the selected inoculum on the BMP result was observed for two out of four substrates. This inoculum effect could be attributed to the abundance of methanogens and a potential inhibiting effect in the inoculum itself, demonstrating the importance of inoculum selection for BMP testing. We recommend the application of granular sludge as an inoculum, because of its higher methanogenic abundance and activity, and protection from bulk solutions, compared with other inocula.

Figure 1

Ultimate methane yields (ml CH₄ g⁻¹ VS) of the (A) molasses, (B) bio-refinery waste, (C) liquid manure, and (D) A-sludge substrates. Error bars show standard deviations, and different letters (A, B and C) indicate significant differences according to ANOVA and subsequent multiple comparison by a Tukey hsd test at the 5% significance level.

Figure 2

Methane yield curves of the molasses (), bio-refinery waste (), liquid manure (), and A-sludge () substrates for the (A) OBW, (B) MAN, (C) BREW and (D) ENG inocula. Error bars show standard deviations.

Figure 3

Methane yield curves of the liquid manure (), and A-sludge () substrates incubated without the addition of an inoculum. The molasses and bio-refinery waste substrates were not included in the figure, because of these substrates did not show any indigenous methanogenic activity. Error bars show standard deviations.

Figure 4

Real-time PCR results of the microbial community in the different inocula, showing (A) total bacteria () and total methanogens () and (B) the methanogenic populations Methanosaetaceae (), Methanosarcinaceae (), Methanobacteriales (), and Methanomicrobiales (). Error bars show standard deviations.

Figure 5

Real-time PCR results of the methanogenic community, showing the Methanosaetaceae (), Methanosarcinaceae (), Methanobacteriales (), and Methanomicrobiales (). Relative abundances are presented at the beginning (initial inoculum, big graphs) and at the end of the BMP test (after 35 days, small graphs) for the (A) OBW, (B) MAN, (C) BREW, and (D) ENG inocula for the different substrates.