Table 2 Evaluation of purification procedures and their modifications by fluorescence microscopy Procedure Cell aggregates present Maximum cell aggregate size1) Abiotic particles present Abiotic particles covered with cells 1-C1-S1-H1-F1 yes +++ yes no 1-C1-S1-H2-F1 yes ++ yes no 1-C2-S1-H1-F1 yes ++ yes no 1-C2-S1-H2-F1 yes + yes no 1-C2-S2-H1-F1 no – yes no 1-C2-S2-H1-F2 no – no no 2-C1-S1-H1 yes +++ yes yes 2-C1-S1-H2 yes +++ yes yes 3-C1-S1-H1 yes +++ yes yes 3-C1-S1-H2 yes ++ yes yes 3-C1-S2-H1 yes ++ yes yes 3-C1-S2-H2 yes + yes yes 3-C2-S1-H1 yes +++ yes yes 3-C2-S1-H2 yes
++ yes yes 3-C2-S2-H1 yes ++ yes yes 3-C2-S2-H2 yes ++ yes yes 3-C3-S1-H1 yes ++ yes yes 3C3-S1-H2 yes ++ yes yes 3-C3-S2-H1 yes ++ yes yes 3-C3-S2-H2 yes + yes yes 4-C1-H1 yes +++ yes yes 5-C1-S1-H1 yes +++ yes yes 5-C1-S2-H1 yes +++ yes yes 5-C1-S1-H2 yes ++ yes yes 5-C1-S2-H2 see more yes ++ yes yes 5-C2-S1-H1 JQ1 yes +++ yes yes 5-C2-S2-H1 yes +++ yes yes 5-C2-S1-H2 yes ++ yes yes 5-C2-S2-H2 yes + yes yes 6-C1-S1-H1 yes ++ yes yes 1) +++ = ≥ 52 μm2; ++ = ≥ 24 μm2; + = ≥ 6 μm2; - = no cell aggregates. The size of cell aggregates was determined by microscopic field analyses using an ocular micrometer at 630× magnification. One field covered an area of 5.76 μm2. Denomination of procedures is according to Table 1. The optimal combination is given in italics. Overall, the purification procedure 1 using the detergent sodium hexametaphosphate
provided the best results concerning the disbandment of cell aggregates and biofilms and the elimination of organic and inorganic particles from the biogas reactor GSK2245840 samples with a minimal cell loss during purification procedure. The final power of ultrasonic
treatment and the sodium hexametaphosphate concentration for procedure 1 without filtration (1-C2-S2-H1-F1) was 60 W (60 sec) and 0.5% (w/v), respectively, which finally resulted in an almost complete recovery of cells from particles and disbandment of cell aggregates (Table 2). After repeated detergent from and ultrasound treatment for a maximum of five times all supernatants were pooled and centrifuged at 8,000 × g for 20 min to collect all cells in a pellet and subsequently re-suspended in one fold concentrated phosphate buffered saline (1× PBS). A microscopic validation of this cell suspension showed a contamination with plant fibers and other inorganic particles which were free of cells, but made the samples unusable for analysis by Flow-FISH. Therefore a final vacuum filtration using a filter with a pore size of 12-15 μm was conducted. The cell loss resulting from filtration seemed to be negligible as the control experiment using E. coli cultures treated with procedure 1-C2-S2-H1-F2 revealed (Figure 1B). Figure 2 shows exemplary microscopic images of the application of purification procedure 1-C2-S2-H1-F2 using two different samples from the UASS biogas reactor (UASS-1 and UASS-2).