Presented at the WISA 2000 Biennial Conference, Sun City, South Africa, 28 May - 1 June 2000 An evaluation of volatile suspended solids as a true measure of metabolic activity in activated sludge AP Degenaar, DD Mudaly, A Manganyi and F Bux Centre for Water and Wastewater Research, Technikon Natal, PO Box 953, Durban,4000, South Africa Abstract The measurement of the active biomass fraction within activated sludge is an important consideration for advancing the design and kinetic description of the process. The use of mixed liquor volatile suspended solids (MLVSS) reading as a measure of biomass is convenient but includes both endogenous and inert volatile solids present in activated sludge. With the advent of improved molecular techniques such as fluorescent in situ hybridization (FISH) it has now become possible to measure directly the metabolically active fraction of activated sludge mixed liquor. The objective of this study was to measure the active biomass fraction of conventional VSS determinations and to investigate VSS as a true reflection of biomass at different sludge ages. Activated sludge samples with sludge ages of 10, 15, 20 and 30 days, were harvested from a defined laboratory-scale sequencing batch reactor (SBR). VSS analyses were compared with FISH results at different sludge ages. The probe specific for all bacteria, EUB338, was used simultaneously with the DNAintercalating dye 4', 6' - diamidino-2-phenylindole (DAPI). A change in sludge age showed a marked change in the biomass active fraction which was not reflected by the VSS readings. It was found that due to its specificity, the application of rrna oligonucleotide probes gave a more accurate reflection of viable biomass than VSS analysis.
Introduction Engineers have traditionally used MLVSS as an indication of biomass within activated sludge systems. Although the MLVSS parameter has the advantage of fitting directly into mass balance equations, it has the disadvantage of not providing a true indication of the viable biomass present. This parameter represents not only active biomass but also endogenous and inert residue (originating from the influent) present in the sludge (Wentzel et al., 1998) The use of molecular techniques based on DNA or ribosomal RNA sequence analysis has been widely accepted as new techniques for the specific enumeration of bacteria due to its high level of sensitivity and specificity. Ribosomal RNA targeted oligonucleotide probes allow for the direct detection of individual cells. Therefore, they are a suitable method for determinative studies in microbiology (Amann et al., 1990). The objective of this study was to determine the true active bacterial fraction present in samples of activated sludge with different cell retention times using both MLVSS and FISH analyses. Results obtained were compared to evaluate the efficiency of VSS/MLVSS as compared to FISH. Materials and Methods Sampling Fours days after commencing the various sludge ages, a sample of mixed liquor was harvested from a laboratory-scale SBR treating edible oil effluent. Samples were fixed for FISH analysis at sludge
ages of 10, 15, 20, and 30 d. Fresh samples were used in the case of VSS/MLSS analysis. Cell fixation Mixed liquor samples were centrifuged at 5000 g for 10 minutes. In order to remove excess untreated oil, the pellet was resuspended in phosphate buffered saline (PBS), centrifuged a second time and the supernatant removed. This procedure was repeated until the pellet was relatively free of all residual oil and organics. Cells were fixed in fresh paraformaldehyde (4% m/v in PBS) for one hour and washed again with PBS to remove residual fixative. The fixed cell suspension was diluted to a final concentration of 50% (v/v) in ice cold absolute ethanol. Immobilization of fixed cells on slides A 5µl aliquot of fixed cell suspension was spotted on slides pre-coated with poly - L- lysine and spots were allowed to air dry. Cells were dehydrated through successive immersion in 60%, 80% and 96% (v/v) ethanol for three minutes each. Slides were stored dry at room temperature. Whole cell hybridization A moisture chamber equilibrated with hybridization solution consisting of 20% (v/v) formamide, 0.9 M NaCl, 20 mm Tris HCl and 0.01% SDS (ph 7.2) was used for whole cell hybridization. 10 µl of hybridization solution including 50 ng of probe was applied to each spot of cells and incubated for 1.5 h at 46 o C. Hybridization was stopped with 2 ml of wash buffer consisting 0.18 M NaCl, 20 mm Tris HCl and 0.01% SDS (ph 7.2) (Amann et al., 1990). Unbound probe was removed by immersing the slide in 50 ml of wash buffer for 20 mins at 48 o C.
DAPI staining After the post-hybridization wash, slides were washed with distilled water and dried at room temperature. Cells were stained with 10 µl DAPI (0.25 µg/ml) for 5 mins and washed with deionized water. Coverslips were mounted on the slides using one drop of antifading solution (Vectashield, Vector Laboratories Inc.). Membrane Filtration The concentration of cells in the activated sludge samples were counted directly by staining cells with DAPI on cellulose membrane filters ( Porter and Feig, 1980). Slight modifications to this method included mixing a fixed cell suspension with PBS to a final volume of 900 µl and bead beating the sample using 1 mm glass beads for 10 min. 0,1% (v/v) Nonidet, a non-ionic detergent was added and bead beating continued for a further 2 min. Staining was done in a filter tower by addition of 1 ml of a stock solution of DAPI (2.5 µg/ml) to a final concentration of 0.25 µg/ml. Cells were concentrated on a cellulose membrane through vacuum filtration. After washing with double distilled water, the filter was mounted on a microscope slide using antifading agent. Microscopy Slides were viewed using a Zeiss Axiolab microscope fitted for epifluorescence with a 50 W high pressure Mercury bulb and Zeiss filter sets specific for rhodamine (15) and DAPI (1). VSS/MLSS analysis 100 ml fresh activated sludge was centrifuged for 10 min. at 5000 g. MLSS and VSS were determined according to Standard Methods (1989).
Results and discussion Table 1 includes the EUB:DAPI ratios for the activated sludge samples taken at various sludge ages. EUB:DAPI ratios vary from 55% (30 d sludge age) to 75% (10 d sludge age) of the total biomass present. This indicates that 55% of the total cells present in the 30d sludge and 75% of the total cells present in the 10d sludge were metabolically active bacterial cells (Table 1). These metabolically active cells contained sufficient rrna molecules to bind to the probe and produce a detectable signal. Previous studies have shown that a good correlation exists between growth rate and cellular rrna content (Schaechter et al., 1958; Delong et al., 1989). This makes use of rrna directed oligonucleotide probes an appropriate tool for detecting metabolically active cells within the sludge mixed liquor. A decrease in EUB:DAPI ratios was observed as the sludge age increased indicating lower metabolic activity of bacterial cells (Table 1). This was also shown by a decrease in viable cell numbers. These observations were not supported, however, by VSS/MLSS analyses which remained relatively constant throughout the various sludge ages (Table 1). A decrease in total cell counts (viable and non-viable cells) was also observed as sludge age increased (Table 1). In a batch reactor at extended sludge ages, it can be expected that the process of cell death and lysis increasingly contribute to decreasing cell numbers and activity. Figures 1 and 2 show a noticeable decrease in fluorescence between the 10d and 30 d sludge ages, indicating a decrease in probe target number and metabolic activity of the biomass present.
Table 1. Comparison of percentage VSS, total cell counts, EUB/DAPI ratios and cell viability at different sludge ages. Sludge age (d) VSS/MLSS (%) Total cell counts (x10 8 cells/ml) EUB/DAPI (%) Viable cells (x10 8 cells/ml) 10 75 9.0 75 6.70 15 73 8.16 70 5.60 20 74 6.33 66 4.10 30 73 5.92 55 2.75 Figure 1. Photomicrograph of 10 d activated sludge stained with DAPI (left panel) and hybridized with probe EUB338 (right panel). Photomicrographs represent identical fields (100 ).
Figure 2. Photomicrograph of 30 d activated sludge stained with DAPI (left panel) and hybridized with probe EUB338 (right panel). Photomicrographs represent identical fields (100 ). Table 2 expresses the number of viable cells per mgvss for various sludge ages. Theoretical predictions are supported by experimental results showing a 61 and 40% decrease in viable biomass per mgvss between a sludge age of 10 and 20 d and 20 and 30 d, respectively. If VSS was assumed to represent cell viability in the mixed liquor, the slight increase in VSS as sludge age increased could lead to the assumption that active cell numbers increase proportionally with sludge age (Table 2). It is likely that an increase in endogenous and inert material contributed to the increase as it is evident from the EUB:DAPI ratios that cell viability and metabolic activity (determined as strength of signal) are inversely proportional to sludge age (Table 2).
Table 2. Expression of viable cell numbers/mg VSS at different sludge ages. Sludge age VSS EUB/DAPI Viable cells Viable cells (d) (g/l) (%) (x10 8 cells/ml) (x10 11 )/mg VSS 10 1.88 75 6.70 3.6 15 2.15 70 5.60 2.6 20 2.06 66 4.10 2.0 30 2.3 55 2.75 1.2 Conclusions These results show at a molecular level that VSS determinations, due to their inability to distinguish between the various volatile fractions in an activated sludge system, are not ideal to base bacterial cell counts upon. Due to differing fluorescent signals, metabolic activity can also be correlated with sludge age. There is of further significance, the possibility that a molecular approach can be adapted to quantitatively determine the various bacterial fractions ie., heterotrophic, autotrophic and polyphosphate accumulating organisms, in activated sludge mixed liquors. Acknowledgements The researchers wish to thank the National Research Foundation (NRF) for funding this project and the Centre for Water and Wastewater Research (CWWR) for the use of their facilities.
References Amann R.I., Binder B.J., Olsen R.J., Chrisholm S.W., Devereaux R. and Stahl D.A. (1990). Combination of 16s rrna targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56: 1919-1925. Delong E.F., Wickham G.S., and Pace N.R. (1989). Phylogenetic stains : ribosomal RNA-based probes for the identification of single microbial cells. Science 243:1360-1363. Porter,K.G and Feig,Y.S. (1980). The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25(5): 943-948. Schaechter M.O., Maaloe O. and Kjeldgaard. (1958). Dependancy on medium and temperature of cell size and chemical composition during balanced growth of Salmonella typhimurium. J. Gen. Microbiol. 19: 592-606. Standard Methods for the Determination of Water and Wastewater (1989). 17 th edn. American Public Health Association, Washington DC. Wentzel M.C., Ubisi M.F. and Ekama G.A. (1998). Heterotrophic active biomass component of activated sludge mixed liquor. Water Sci. Tech. 37 (4-5): 79-87.