Report No. 934/1/01

MAY 2001



Eutrophication of South Africa's natural waters is accelerated by human activities which result in the discharge of the nutrients nitrogen (N) and phosphorus (P) (Lilley et al., 1997). Eutrophication is marked by large visible blooms of algae, which makes water treatment difficult. Nitrogen can be fixed by algae in the water and therefore, phosphorus is the element that should be considered to minimise eutrophication (Lilley et al., 1997).

The prevention of eutrophication can be achieved by removing phosphate from wastewater by chemical or biological methods (Lilley et al., 1997). Biological phosphate removal has gained support since chemical removal is very expensive and increases the salt and mineral concentrations of the effluent (Slim, 1987).

The activated sludge process has been designed and operated for excess phosphate removal to reach effluent concentrations of 0.5 to 1.0 mg phosphate per litre (Barnard, 1975; Fuhs and Chen, 1975). However, many activated sludge systems fail, necessitating chemical addition. This has mainly been attributed to the lack of knowledge of the role of microorganisms and optimum conditions for their growth in order to facilitate biological phosphorus removal.

Research has indicated that there were no differences amongst the bacterial community structures of different activated sludge zones or amongst different activated sludge systems (Momba, 1995; Ehlers 1997). Hence, phosphorus removal cannot be attributed to the activity of a single population, but rather to the combined activity of all the populations in the microbial community. Momba and Cloete (1996) indicated that an increase in biomass resulted in an increase in phosphorus removal. This raised the question whether wastewater treatment systems could be bioaugmented in order to increase biomass.

Bioaugmentation involves the use of specially selected and adapted microorganisms for the biodegradation of wastewater (Oellerman and Pearce, 1995). The objective is not to replace the existing biomass, but to supplement it for improved efficiency. Its use could enhance the degradative potential of indigenous microbial populations to avoid predation, nutrient competition and biomass inactivation.

The application of bioaugmentation in wastewater treatment was originally the result of efforts to solve operational problems, such as shock loads in treatment plants (Oellerman and Pearce, 1995). According to De Haas (1999), results from the commercial application of bioaugmentation products, particularly in wastewater treatment systems facing operational problems, have tended to be positive. However, laboratory research investigations have contradicted these results. A number of bioaugmentation products do not perform as claimed by the suppliers (Oellerman and Pearce, 1995). Cases were reported with little or no advantage of bioaugmentation on the improvement of treatment works (Yu and Hung, 1992). It was concluded that no significant improvement in process performance can be achieved with bioaugmentation (De Haas, 1999). As an alternative, the biomass could be increased in a number of different ways.

An alternative to the application of commercial biosupplements would be to use total biomass from activated sludge systems, which optimally remove P. The use of such biomass would be inexpensive and the acclimation period shorter than that for commercially available biosupplements. Biomass could be augmented subsequent to system failure as a result of either toxic shock or biomass depletion.


The objectives of this study were:

  1. To determine the effect of bioaugmentation on phosphorus removal in laboratory experiments by adding commercially available bioaugmentation products.
  2. To determine the relationship between biomass and phosphate removal in different activated sludge systems.
  3. To evaluate the following:
    1. Anaerobic sludge biomass as supplement
    2. Return sludge biomass as supplement
    3. Aerobic sludge biomass as supplement
    4. Bioaugmentation biomass as supplement
  4. To determine the P removal capacity of a system based on biomass.
  5. To determine the effect of bioaugmentation on phosphorus removal in a conventional activated sludge system by adding biosupplements and/or anaerobic sludge etc. in order to increase the biomass.
  6. To compare different methods for the determination of biomass in activated sludge systems.
  7. To investigate the locality and quantity of phosphorus associated with extracellular polysaccharides (EPS).


For determining the effect of bioaugmentation on phosphorus removal in laboratory experiments different concentrations of bioaugmentation product A (SA Biotech) (2 g.l-1,' 6 g.l-1, 8 g.l-1, 10 g.l-1, 20 g.l-1, 40 g.l-1, 80 g.l-1, 100 g.l-1, 160 g.l-1, 200 g.l-1) were used as inocula in sterile anaerobic mixed liquor medium. There was an initial relationship between phosphate uptake and biomass, which thereafter was negatively affected by the phosphate concentration of the bioaugmentation product. At concentrations exceeding 80 g.l-1 of mixed liquor, no phosphorus was removed, instead the phosphate concentration increased as a result of the phosphorus content of the bioaugmentation product. The product obtained from SA Biotech had a high phosphate content, making it unsuitable for bioaugmentation.

For determining the possibility of culturing a bioaugmentation product in a separate fermentation unit for addition to activated sludge, the growth of bioaugmentation product B (Amitek) and anaerobic activated sludge in sterile mixed liquor was compared. The hypothesis was to use an inexpensive substrate to grow these products in separate tanks to have a reserve of biomass which can then serve as biomass inoculum to increase the already existing biomass in the system. Therefore, a commercially available bioaugmentation product (Product B obtained from Amitek) as well as anaerobic sludge were evaluated for this purpose.

Different volumes (2.5 ml and 10 ml) of the bioaugmentation product and anaerobic sludge were inoculated in sterile mixed liquor medium (250 ml and 90 ml), Nutrient broth (90 ml) and sterile mixed liquor added with nutrients (sodium acetate, magnesium sulphate and potassium nitrate) respectively. The experiments were conducted under aerobic and anaerobic conditions at room temperature (21C). No growth of the microorganisms occurred when sterile mixed liquor medium was used, even when nutrients were added. The microorganisms were able to grow in the Nutrient Broth.

For determining the P removal capacity of a system based on biomass, aerobic sludge was used as biomass in sterile mixed anaerobic mixed liquor.

The increase in P removal with an increase in biomass indicated a direct relationship between biomass and P removal from mixed liquor. Our results are in agreement with that of Lemos et al. (1997) and others (Momba and Cloete, 1996; Muyima, 1995) who stated that a phosphorus free effluent could be obtained when there are enough phosphorus accumulating bacteria in a wastewater process. This suggested that the failure of EBPR under certain conditions could be due to insufficient biomass.

An investigation was done into the relationship between the MLSS and MLVSS fractions of activated sludge, as measures of total biomass, and phosphorus removal. In this study, by means of two independent experiments, differences in orthophosphate uptake ability of different activated sludges treated in exactly the same way were observed. Orthophosphate removal was consistently high with higher biomass concentrations as measured by TPC and ATP. This supports the notion that the viable biomass fraction of the MLSS is the key to orthophosphate removal by activated sludge. Orthophosphate uptake differed amongst systems in terms of wet sludge mass. In experiment 1 (constant wet mass of 40 g), the Centurion Wastewater Treatment Plant (WTP) showed, on average, the highest orthophosphate removal capacity (30.79 mg.g-1 initial MLSS), followed by the Baviaanspoort, Zeekoegat, Rooiwal and Daspoort WTPs with average orthophosphate removal capacities of 23.78, 20.17, 15.40 and 14.88 mg.g-1 initial MLSS. respectively. For experiment 2 (simulated MLSS values), for average orthophosphate uptake in terms of initial MLSS, the Centurion WTP performed best (9.19 mg.g-1), followed by the Baviaanspoort, Daspoort, Rooiwal and Zeekoegat WTPs with uptakes of 7.97, 4.60, 4.55 and 2.82 mg.g-1, respectively. For average orthophosphate uptake calculated in terms of initial MI,VSS, the same pattern was observed as that for initial MLSS and followed the same order of systems 13.48, 11.43, 6.28, 5.63 and 3.76 mg.g-1, respectively for Centurion, Baviaanspoort, Daspoort, Rooiwal and Zeekoegat. The different removal capacities observed were attributed to differences in the MLSS active biomass fraction of the different activated sludges. Results indicated that the MLSS and MLVSS fractions of activated sludge per se, are not good indicators of biomass in activated sludge. ATP proved to be a more reliable method for indicating biomass concentration than TPC. Although MLSS and MLVSS showed the same trend in orthophosphate removal. initial concentrations of these fractions did not correlate with ATP or TPC, indicating the unsuitability of these fractions as indicators of biomass in activated sludge. In addition, orthophosphate removal was consistently higher in the sludges with higher ATP and TPC values, indicating a relationship between biomass and orthophosphate removal.

Analysis of extracellular polysaccharides (EPS) of activated sludge was done by means of Scanning Electron Microscopy (SEM) combined with Energy Dispersive Spectrometry (EDS). Cell clusters with associated EPS on average contained between 57 and 59% phosphorus, while EPS alone contained on average between 27 and 30% phosphorus. Results suggest that phosphorus removal in activated sludge might be due not only to polyphosphate accumulating organisms (PAO), but also by EPS acting as a phosphorus reservoir.


Bioaugmentation product A caused an increase in the phosphate concentration of the mixed liquor in all our experiments and could therefore not be considered for increasing the biomass in activated sludge. Bioaugmentation product B also contained a high P concentration making it unsuitable for P-removal studies. Bioaugmentation product B and anaerobic sludge did not grow in the substrate used, except in Nutrient Broth.

When aerobic sludge was used as augmentation, an increase in biomass led to an increase in P removal. When calculating the quantity of P removal per g of wet sludge no significant difference was observed indicating that there was a direct relationship between P removal and MLSS for a specific system.

ATP proved to be a more reliable method for indicating the biomass concentration in activated sludge than TPC, due to the higher yield and a smaller standard deviation. The MLVSS showed the same trend in orthophosphate removal as the MLSS, although always somewhat lower, due to it being the volatile fraction of the MLSS. Neither initial MLSS, nor MLVSS concentrations correlated with ATP and/or TPC, indicating that MLSS and MLVSS were not good indicators of biomass in activated sludge. Orthophosphate removal was consistently higher in the sludges with higher ATP and TPC values, indicating a relationship between biomass and orthophosphate removal.

Results of EDS suggest that phosphorus removal in activated sludge might be due not only to PAO, but also by EPS acting as a phosphorus reservoir.


Further biomass studies should include the physical, chemical and biological characterization of MLSS for different activated sludge systems.