Investigation of the microbial contribution to nutrient removal in an activated sludge wastewater treatment process
Report No 822/1/00
1. Background and motivation
Eutrophication is a natural ageing process which usually occurs in lakes and other quiescent bodies of water through the introduction of the plant nutrients, phosphorus and nitrogen, to the impoundment. Without human intervention, the process takes place over hundreds of years, but is greatly accelerated by various human activities in sensitive areas. Eutrophication of natural and man-made water impoundments is a problem encountered in many countries, including South Africa. However, problems experienced in South Africa which promulgate eutrophication are long storage times of dams and reservoirs, high summer temperatures and long daylight hours (Bolitho, 1976).
Wastewater entering a treatment plant is composed of many different and constantly changing substrates/pollutants which only mixed populations of microorganisms are capable of metabolising. The microbial community in wastewater treatment systems exist in unique and transient ecosystems, dependent upon contaminant type, load and the ambient oxidation reduction potential.
Although Koch's principles for other nutrient removal processes during biological wastewater treatment viz., nitrogen assimilation in biomass, nitrification and denitrification as well as ammonia release from organic nitrogen, have been satisfied, the same cannot be said for biological excess phosphorus removal processes (Jenkins and Tandoi, 1991). Although the process of biological excess phosphorus removal is well understood from a technical aspect, it remains difficult to achieve consistent and reproducible removal rates at full-scale due to our lack of understanding of the process from a biochemical and microbiological point of view (Satoh et al., 1996; Wang and Park, 1998). Since pure cultures which possess complete characteristics of polyphosphate accumulating organisms have not been isolated yet, the biochemical mechanism cannot be definitively described (Mino et al., 1998). Without understanding the correlation between the polyphosphate accumulating bacteria community structure within activated sludge mixed liquor (as well as the interactions between autotrophic and heterotrophic bacteria involved in nitrification and denitrifiers involved in simultaneous phosphorus uptake) and the wastewater plant's performance, reliable and efficient nitrification/denitrification biological excess phosphorus removal operations will remain difficult to design. In-depth microbial community analyses and descriptions of the vast interactions occurring amongst the bacteria involved in wastewater remediation are therefore integral to advance the science and efficiency of processes such as biological nutrient removal.
The process engineering and technology (modelling and implementation) and microbiological and biochemical disciplines have traditionally worked independently of one another. A consequence of this is that microbiologists can find it difficult to express information gathered in engineering terms which could otherwise be implemented to address the deficiencies of the existing steady-state design models and kinetic simulation models. Rapid developments in and application of molecular biological techniques have made a considerable contribution to the understanding of biological nutrient removal activated sludge systems. These techniques also provide quantitative data which is a prerequisite for the successful modelling of these systems. Integration of the engineering and microbiological/biochemical disciplines should remain the focus activity area in activated sludge research for the near future. To this end, a new multi-institutional Water Research Commission project has been initiated in which both engineering and microbiological-based research groups are participating.
2. Statement of objectives
The initial research proposal submitted to the Water Research Commission was entitled:
"INVESTIGATION AND COMPARISON OF THE MICROBIAL CONTRIBUTION TO NUTRIENT REMOVAL IN ACTIVATED SLUDGE AND TRICKLING FILTER WASTEWATER TREATMENT PROCESSES"
The aim of the original project was to establish a relationship between the microbial composition of activated sludge and trickling filter processes and the three primary functions of biological wastewater treatment viz., carbon, nitrogen and phosphorus removal.
The objectives of the proposed research programme were as follows:
With the consensus of the Water Research Commission Steering Committee, it was decided to alter the title and objectives of the original proposal slightly. These changes would therefore produce a project of more significance to the activity area of biological nutrient removal and be more in line with national and international trends.
"INVESTIGATION OF THE MICROBIAL CONTRIBUTION TO NUTRIENT REMOVAL IN AN ACTIVATED SLUDGE WASTEWATER TREATMENT PROCESS"
The use of trickling filters for nutrient removal was not investigated as it is not common practice to use this technology in wastewater treatment plants in KwaZulu-Natal any longer. The research project was therefore designed to:
Within the context of improving the microbiological knowledge of biological nutrient removal, three core areas of activity were defined and selected for in-depth investigation. These included (i) biological excess phosphorus removal; (ii) biological nitrogen removal (nitrification and denitrification processes) and; (iii) the implementation and use of molecular techniques such as fluorescence in situ hybridization and dot blot hybridizations to conduct quantitative and qualitative studies of the bacteria involved. Research protocols and results of these three sub-activity areas will be reported and discussed individually for ease of reading.
3. Summary of results
Biological phosphorus removal
The experimental procedure for biological phosphorus removal assays was conducted in two phases: (i) laboratory, where samples of mixed liquor were obtained from Darvill Wastewater Works and, (ii) pilot plant, where an enhanced culture of polyphosphate accumulating organisms was developed. During phase 1 of experimentation, mixed liquor samples were taken from the aerobic zone of Darvill Wastewater Works and cultivated on solid media. Isolates were screened for their ability to accumulate polyphosphate through successive anaerobic/aerobic incubation and only those isolates displaying the desirable phosphorus transformation patterns i.e., anaerobic phosphorus release and aerobic phosphorus uptake, were subsequently identified. Identification of the various isolates was achieved using the Gram stain, various qualitative biochemical tests and the Analytical Profile Index 20NE identification system. Significant findings during biological excess phosphorus removal experimentation on a full-scale wastewater works included:
An enhanced culture of polyphosphate accumulating organisms, at pilot-scale, was developed during phase 2 of experimentation by incrementally increasing the acetate fraction whilst concomitantly decreasing the settled sewage fraction to the feed influent of a laboratory unit. The pilot plant, modelled on the 3-stage Phoredox process, was seeded with mixed liquor obtained from a non-nutrient removing (perhaps apart from coincidental nitrification) single aerobic full-scale activated sludge plant (Amanzimtoti Wastewater Works, KwaZulu-Natal South Coast) in order to primarily investigate population dynamics and to monitor changes (if any) in the bacterial community structure when subjected to conditions conducive to the biological excess phosphorus removal mechanism. Community analyses of both activated sludge systems were performed using both conventional cultivation and molecular techniques which involved ribosomal ribose nucleic acid-targeted fluorescently-labeled oligonucleotide probes. Significant findings when conducting cultivation-dependant microbial community analyses on an enhanced biological excess phosphorus removal culture at pilot-scale were:
In situ probe hybridization investigations
The total bacterial population of both full-and pilot-scale biological excess phosphorus removing activated sludges was found to be underestimated by at least three orders of magnitude by cultivation-dependant methods. In addition, cultivation-dependant techniques have been found to overestimate the gamma Proteobacterial subclass and underestimate the alpha and beta Proteobacteria.
Biological nitrogen removal
Findings confirm the existence of a diverse community of heterotrophic bacteria involved in nitrogen removal during wastewater treatment of which Pseudomonas spp. show significant involvement. Numerous heterotrophic bacteria were found to be capable of nitrification.
With respect to denitrification, the ordinary heterotrophic fraction within the Darvill Wastewater Works mixed liquor was found to comprise five distinct functional groups, four of which interactively contribute to denitrification occurring in the system and one group that are non-denitrifying.
The ordinary heterotrophic fraction is therefore more complex than currently accepted and needs to be amended for more accurate modelling of denitrification kinetics in nitrification/denitrification biological excess phosphorus removal processes.
Numerous heterotrophic bacteria were also found capable of phosphorus uptake under anoxic conditions utilising nitrates instead of oxygen.
4. Meeting the objectives
It was generally agreed that the research objectives, as formulated and detailed at the beginning of the Executive Summary, were successfully achieved. An in-depth microbiological survey of both a full-and pilot-scale nitrification/denitrification biological excess phosphorus removal activated sludge system was performed. Both cultivation-dependant and molecular in situ techniques were applied to obtain an adequate description of the physiologically significant microbial community involved in biological phosphorus removal, heterotrophic nitrification and denitrification. Autotrophic nitrifiers were not investigated during the course of this study due to difficulties in their isolation and excessively long growth rates. Of great significance during this study was the establishment of a molecular biology laboratory and application of the fluorescence in situ hybridization technique to obtain both qualitative and quantitative information regarding the microbial community in biological nutrient removal systems. Comparisons between various cultivation-dependant identification techniques were not performed due to the potential excessive costs in the purchase of software and consumables. All isolates were subsequently identified using the Analytical Profile Index.
5. Contribution to state-of-the-art
Upon initiation of this research project, fluorescence in situ hybridization technology had not yet been applied to any South African activated sludges. Microbial community analyses appeared to be largely confined to culture-dependant techniques. Although routinely applied at an international level, it appeared after an intensive literature search that fluorescence in situ hybridization had not yet been applied to continuous systems designed to stimulate biological excess phosphorus removal either. The majority of these articles seemed to be confined to the use of sequencing batch reactors and/or full-scale systems. Quantitative data provided by molecular based assays can also be used by the engineering and technology disciplines to improve existing activated sludge models and to address the deficiency of the active biomass concept within these models. Based on these points, it was felt that the current project positively contributed to the state-of-the-art in wastewater treatment both locally and abroad.
6. Recommendations for future research
7. Technology transfer
i) Capacity building The following students received relevant qualifications as a result of this project:
|M. Tech. (Biotechnology)||- B Atkinson
- G Drysdale
- D Mudaly
|B. Tech. (Biotechnology)||- M Sidat
- N Lacko
- M Chetty
- J Moulds
- W Stevens
|Experiential||- R Pieterse
- A Degenaar
ii) Publications and conferences
Atkinson, BW (1999) Identification of polyphosphate accumulating bacteria from and pilot and full-scale nutrient removal activated sludges. Master's dissertation, Department of Biotechnology, Technikon Natal.
Drysdale, G, Bux, F and Kasan, HC (1999) Denitrification by heterotrophic bacteria during activated sludge treatment. Water SA 25(3): 357-362.
Sidat, M, Bux, F and Kasan, HC (1999) Laboratory-scale investigation of biological phosphate removal from municipal wastewaters. Water SA 25(4): 459-462.
Sidat, M, Bux, F and Kasan, HC (1999) Polyphosphate accumulation by bacteria isolated from activated sludge. Water SA 25(2): 175-180.
Mudaly, DD, Atkinson, BW and Bux, F (2000) Microbial community profile of a biological excess phosphorus removal activated sludge system using a cultivation-independent approach. Water SA 26(3):343-352.
Atkinson, BW, Bux, F and Kasan, HC (1997) Characterisation and function of phosphate accumulating bacteria present in an activated sludge plant modeled upon the NDBEPR JHB system. South African Society for Microbiology Conference, University of Natal, 15 - 17 October.
Atkinson, BW, Bux F and Kasan, HC (1998) The contribution of Acinetobacter spp. and other poly-P organisms to biological phosphorus removal operations. The Water Institute of Southern Africa Biennial Conference and Exhibition, Baxter Theater, Cape Town, 4-7 May.
Atkinson, BW, Bux, F and Kasan, HC (1999) Enhancement of polyphosphate accumulating bacteria in an activated sludge system. African International Environmental Protection Symposium, Imperial Hotel, Pietermaritzburg, 4-8 July.
Atkinson, BW, Mudaly, DD and Bux, F (2000) Pseudomonas spp. and anoxic phosphorus uptake in a biological nutrient removal activated sludge system. BlOY2K Biotech SA 2000 Conference, Rhodes University, Grahamstown, 23-28 January.
Atkinson, BW, Mudaly, DD and Bux, F (2000) Incidence of Pseudomonas in a biological nutrient removal activated sludge system performing anoxic phosphate uptake. WISA 2000, Sun City, 28 May - 1 June.
Atkinson, BW, Mudaly, DD and Bux, F (2000) Contribution of Pseudomonas spp. to phosphorus uptake in the anoxic zone of an anaerobic-anoxic-aerobic continuous activated sludge system. lst World Congress of the International Water Association, Paris, France, 3-7 July.
Chetty, M, Mudaly, DD and Bux, F (2000) Enhancement of activated sludge for the direct detection of polyphosphate accumulating organisms. WISA 2000, Sun City, 28 May - 1 June.
Degenaar, AP, Mudaly, DD, Manganyi, A and Bux, F (2000) An investigation of volatile suspended solids as a measure of activated sludge biomass. BlOY2K Biotech SA 2000 Conference, Rhodes University, Grahamstown, 23-28 January.
Degenaar, AP, Mudaly, DD and Bux, F (2000) The determination of viable biomass in activated sludge. WISA 2000, Sun City, 28 May - 1 June.
Drysdale, GD, Bux, F and Kasan, HC (1998) Evaluation of the role of heterotrophic bacteria in denitrification during activated sludge treatment. The Water Institute of Southern Africa Biennial Conference and Exhibition, Baxter Theater, Cape Town, 4-7 May.
Drysdale, GD, Bux, F and Kasan, HC (1999) Interactive microbial denitrification in a biological nutrient removal process. African International Environmental Protection Symposium, Imperial Hotel, Pietermaritzburg, 4-8 July.
Drysdale, GD, Kasan, HC and Bux, F (2000) Assessment of nitrite denitrification behaviour by denitrifying heterotrophic organisms in a NDBEPR activated sludge system. WISA 2000, Sun City, 28 May - 1 June.
Drysdale, GD, Kasan, HC and Bux, F (2000) Assessment of denitrification by the ordinary heterotrophic organisms in a NDBEPR activated sludge system. lst World Congress of the International Water Association, Paris, France, 3-7 July.
Mudaly, DD, Bux, F and Kasan, HC (1998) The application of a fluorescently labeled rRNA targeted oligonucleotide probe for the in situ identification of bacteria in activated sludge. The Water Institute of Southern Africa Biennial Conference and Exhibition, Baxter Theater, Cape Town, 4-7 May.
Mudaly, DD, Moulds, J, Bux, F and Kasan, HC (1998) Dual staining of activated sludge with DAPI and a kingdom level 16S rRNA targeted oligonucleotide probe. South African Society for Microbiology 10th Biennial Congress, International Convention Centre, Durban, 5-8 July.
Mudaly, DD, Bux, F and Kasan, HC (1999) Determination of bacteria predominating in a pilot-scale enhanced biological phosphate removal activated sludge process using a cultivation independent approach. African International Environmental Protection Symposium, Imperial Hotel, Pietermaritzburg, 4-8 July.
Mudaly, DD, Atkinson, BW and Bux, F (2000) Combined approach of FISH and dot-blots for a study of bacteria predominating in full-and pilot-scale EBPR activated sludge processes. The COE Symposium on Establishment and Evaluation of Advanced Water Treatment Technology Systems Using Functions of Complex Microbial Communities, University of Tokyo, Tokyo, Japan, 6-8 March.
Mudaly, DD, Atkinson, BW and Bux, F (2000) 16S rRNA in situ probing for the determination of the family level community structure implicated in enhanced biological nutrient removal. 1st World Congress of the International Water Association, Paris, France, 3-7 July.
Sidat,M,Bux,FandKasan,11C (1997) Laboratory scale investigation to improve biological phosphate removal from wastewater. South African Society for Microbiology Conference, University of Natal, 15-17 October.
Stevens, W, Drysdale, GD and Bux, F (1999) An assessment of the nitrifying behaviour of heterotrophic bacteria in a biological nutrient removal system. 12th Annual Symposium of SASM (Natal Branch), MI, Sultan Technikon, Durban, 15 October 1999.
Stevens, WE, Drysdale, GD and Bux, F (2000) An assessment of the potential for heterotrophic nitrification in biological nutrient removal systems. BlOY2K Biotech SA 2000 Conference, Rhodes University, Grahamstown, 23-28 January.
Stevens, WE, Drysdale, GD and Bux, F (2000) The potential of heterotrophic bacteria to nitrify in a biological nutrient removal system. WISA 2000, Sun City, 28 May - 1 June.
8 Archiving of data
All raw and processed data collected from this research project will be archived and made available upon request at Technikon Natal.