Measurement of Microbial Numbers and Biomass Contained in Thermophilic Anaerobic Reactors

Abstract

This paper describes the determination of the microbial population in terms of the number, biomass, and composition of single‐ and two‐phase, laboratory‐scale thermophilic (55 °C) anaerobic reactors under steady‐state conditions. Epifluorescence microscopy with 4',6‐diamidine‐2‐phenylindole (DAPI) as fluorochrome was used to determine the total number of microorganisms in the reactors, and autofluorescence microscopy was used to determine the total methanogenic bacteria populations. The results obtained by the direct count methods were compared to the quantity of biomass contained in the system, which was determined by volatile suspended solids. The concentration of acidogenic bacteria was estimated by subtraction of the autofluorescence results from those of the DAPI epifluorescence microscopy. The viable bacterial population was determined by plating techniques using an anaerobic chamber. The total bacterial and methanogenic populations of single‐stage digesters increase when the hydraulic retention time decreases; nevertheless, the percentages of the principal bacterial groups (acidogenic and methanogenic) remain constant at 87% and 13%, respectively. In the two‐stage reactors, the percentages of the acidogenic and methanogenic groups are 99% and 26% of the total population in the acidogenic and methanogenic reactors, respectively. In the single‐stage reactors, biomass determinations can be used to estimate microbial concentrations, and vice versa, as there is a high positive correlation between microorganism concentration and biomass. The syntrophic relationship between the bacteria involved in the anaerobic process is a possible explanation for the low values of viable population obtained in the reactors studied. Nevertheless, there is a high correlation between direct counts by epifluorescence microscopy and viable plate counts for the combined system studied.