Dynamic variation of the microbial community structure during the long-time mono-fermentation of maize and sugar beet silage

Abstract

This study investigated the development of the microbial community during a long-term (337 days) anaerobic digestion of maize and sugar beet silage, two feedstocks that significantly differ in their chemical composition. For the characterization of the microbial dynamics, the community profiling method terminal restriction fragment length polymorphism (TRFLP) in combination with a cloning-sequencing approach was applied. Our results revealed a specific adaptation of the microbial community to the supplied feedstocks. Based on the high amount of complex compounds, the anaerobic conversion rate of maize silage was slightly lower compared with the sugar beet silage. It was demonstrated that members from the phylum Bacteroidetes are mainly involved in the degradation of low molecular weight substances such as sugar, ethanol and acetate, the main compounds of the sugar beet silage. It was further shown that species of the genus Methanosaeta are highly sensitive against sudden stress situations such as a strong decrease in the ammonium nitrogen (NH₄(+)-N) concentration or a drop of the pH value. In both cases, a functional compensation by members of the genera Methanoculleus and/or Methanosarcina was detected. However, the overall biomass conversion of both feedstocks proceeded efficiently as a steady state between acid production and consumption was recorded, which further resulted in an equal biogas yield.

Figure 1

Highly temporal resolution of the kinetics of the biogas production rates over 4 days at OLR 2.0 g_VS l⁻¹ day⁻¹ for both the anaerobic digestion of maize silage and sugar beet silage. Shown are mean values including the standard deviation of the three parallel reactors per feedstock. “Feeding” indicate time-points of substrate addition.

Figure 2

Total Kjeldahl nitrogen (TKN) and ammonium nitrogen (NH₄⁺-N) concentration of the maize reactor digestate as well as the sugar beet reactor digestate over the entire experimental time as mean values including standard deviation of the three parallel reactors per feedstock.

Figure 3

Structure of the bacterial community involved in the biomethanation process of (A) maize silage and (B) sugar beet silage. Shown is the relative abundance of the detected terminal restriction fragments (TRFs) as a function of the percental fluorescence intensity of each individual TRF in relation to the total fluorescence intensity. Coloured bars symbolize TRFs in base pairs (bp), which were identified by 16S rRNA gene sequence libraries. Only TRFs with a relative abundance over 5 % in at least one sample are shown. Each sampling point is given as median value of three biological replicates (i.e. parallel-operated biogas reactors) and three technical replicates (i.e. three DNA extracts per reactor). Numbers in sampling point descriptors indicate the duration of continuous fermentation in days.

Figure 4

Structure of the archaeal community involved in the biomethanation process of (A) maize silage and (B) sugar beet silage. Shown is the relative abundance of the detected terminal restriction fragments (TRFs) as a function of the percental fluorescence intensity of each individual TRF in relation to the total fluorescence intensity. Coloured bars symbolize TRFs in base pairs (bp), which were identified by 16S rRNA gene sequence libraries. Each sampling point is given as median value of three biological replicates (i.e. parallel-operated biogas reactors) and three technical replicates (i.e. three DNA extracts per reactor). Numbers in sampling point descriptors indicate duration of continuous fermentation in days.

Figure 5

Correlation between the abundance of TRF-428bp (related to Methanoculleus) and TRF-108bp (related to Methanosaeta) in (A) the maize reactors and (B) the sugar beet reactors. Only samples indicated by a full black dot were considered for correlation. R² is the correlation coefficient.