Investigation of the microbial contribution to nutrient removal in an activated sludge wastewater treatment process
Report No 822/1/00

EXECUTIVE SUMMARY

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:

• microbiological survey at different sites of wastewater treatment at Darvill and Umbilo Wastewater Treatment Plants;
• simultaneous monitoring of plant parameters including chemical and biological oxygen demand, nitrogen, phosphorus, pH etc.;
• establishment of extent of correlation between microbial predominance and nutrient removal viz., carbon, nitrogen and phosphorus;
• comparison of treatment technologies i.e., trickling filter and activated sludge, with respect to the microbial community and nutrient removal; and
• construction of database of above correlations for future use in design and optimization of respective treatment processes.

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:

• conduct a microbiological survey of a full-scale nitrification/denitrification biological excess phosphorus removal plant in KwaZulu-Natal (Darvill Wastewater Works, Pietermaritzburg), concentrating on the various redox zones i.e., anaerobic, anoxic and aerobic;
• establish the extent of correlation between microbial dominance in a particular zone and the extent to which this dominance contributes to the removal of specific nutrients;
• increase current knowledge of microbial community structure-functionality;
• assess the removal capacity of specific, isolated bacteria in order to determine their individual contribution to the system as a whole;
• assess molecular techniques (fluorescence in situ hybridizations), as opposed to conventional cultivation techniques, to determine the microbial community structure; and
• compare various culture-dependent identification techniques such as the Analytical Profile Index, Microbact and Biolog assays using bacteria isolated from activated sludge.

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:

• cultivation results showed the unequivocal dominance of the gamma subclass of the Proteobacteria cluster (Proteobacteria contains the majority of the traditional Grain negative organisms);
• at genus level, the Pseudomonads appeared to dominate the polyphosphate accumulating bacterial community;
• although Acinetobacter spp. accumulated large quantities of polyphosphate from a phosphate enriched liquid medium, low recovery rates of this genus on solid agar media did not warrant its implication as the organism pivotal to phosphate uptake and removal from the activated sludge system in question.

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:

• Pseudomonas spp. appeared to dominate the bacterial community;
• plating resulted in the total dominance of the gamma subclass of Proteobacteria;
• results of this aspect of the study indicated the importance of the Proteobacteria to biological excess phosphorus removal operations. Surprisingly, the dominance of Acinetobacter spp. in biological excess phosphorus removing activated sludge was never demonstrated, even when using plating techniques.

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.

• the alpha and beta Proteobacteria were both shown to be functional groups implicated in full-and pilot-scale biological excess phosphorus removing systems, together comprising more than 60% of the active population;
• however, the overall community profile shows that the biological excess phosphorus removal population is quite diverse and it is unlikely that any one species dominates the process.
• the polyphosphate accumulating population of the total active bacterial community was found to be at percentages of 55% for an enhanced pilot plant and 35% for the full-scale biological excess phosphorus removal system;
• the total number of polyphosphate accumulating organisms required to remove one gram of soluble phosphorus was thereby estimated to be between 1-2 x 10¹¹ cells;
• the quantity of biomass (as metabolically active bacterial cells) to be associated with one gram of volatile suspended solids was determined at 1 x 10¹³ cells;
• the portion of the volatile suspended solids present as biomass only was estimated to be 15% for the full-scale biological excess phosphorus removal system investigated.

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.

• heterotrophic isolates demonstrated direct oxidation of ammonium to nitrates with little or no nitrite accumulation;
• different patterns of nitrification behaviour were noted amongst heterotrophic nitrifiers with some displaying potential nitrification/denitrification behaviour;
• Staphylococcus and Micrococcus spp. showed significant involvement in nitrification along with Streptococcus, Pseudomonas and Bacillus spp.

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.

• these groups were defined and characterised as true denitrifiers (bacteria capable of both nitrate and nitrite reduction), incomplete denitrifiers (bacteria that reduced nitrates to nitrites with no further reduction of the nitrites produced), incomplete-nitrite reducers (bacteria capable of both nitrate and nitrite reduction, however, exhibiting severe inhibition of nitrite reduction by nitrates),exclusive nitrite reducers (bacteria only capable of reducing nitrites) and non-denitrifiers (bacteria not capable of nitrate or nitrite reduction).
• 60.7% of the bacteria isolated were capable of nitrate reduction as compared to 32% which showed any capacity for nitrite reduction.

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.

• Serratia spp. and Vibrio spp. were the most efficient anoxic phosphorus accumulators isolated demonstrating 7.10 and 7.29mgP0₄-P/L removal, respectively;
• weak phosphorus accumulating bacteria were also identified;
• these organisms may not necessarily be weak phosphorus accumulators under aerobic conditions, but, were limited under anoxic conditions due to weak denitrification capacity.
• anoxic phosphorus release was also observed by some denitrifying heterotrophic bacteria;
• Pasteurella spp. released. a maximum of 5.91mgP0₄-P/L with concurrent reduction of 6.25 mgN0₃-P/L.

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

• As microbiologists, efforts to isolate phosphorus accumulating organisms from biological excess phosphorus removal sludges should continue. Although this will require a certain degree of innovation, the presence of viable but non-culturable bacteria in environmental ecosystems should not deter these efforts;
• Although the broad community profile (family and sub-group levels) of an ecosystem can be determined through application of fluorescence in situ hybridization technology, techniques involving cloning, sequencing and probe design must also be incorporated to adequately describe the microbial community profile at a generic and species level;
• In order to improve the quantitative potential of molecular techniques when conducting activated sludge studies, methods to allow for maximum floc dispersion should be investigated. Counting of sufficient numbers of microscopic fields will also be required when attempting to provide the raw data required to advance the predictive and descriptive capacity of current mathematical models for nitrification/denitrification biological excess phosphorus removal processes;
• Heterotrophic nitrification needs to be evaluated in situ in order to establish the involvement, if any, of heterotrophic bacteria in nitrification occurring during wastewater treatment;
• The kinetics of nitrate and nitrite reduction need to be determined for true denitrifiers, incomplete denitrifiers, incomplete-nitrite reducers and exclusive nitrite reducers in order to establish the contribution to denitrification by each group;
• A method needs to be established to determine the non-denitrifying and denitrifying fractions of the heterotrophic active biomass for more accurate modelling of denitrification behaviour in biological nutrient removal systems; and,
• The microbiology and biochemistry of anoxic phosphorus accumulation requires further investigation;
• Results of the Analytical Profile Index identification method need to be substantiated using additional biochemical tests and qualitative molecular techniques to allow more conclusive identification of bacterial isolates from environmental samples;
• Microbiologists/biochemists and process engineers must begin to communicate and not work independently of one another. The quantitative information which molecular biologists are now able to generate regarding active cell biomass will prove invaluable in improving the accuracy and predictive ability of the existing or new activated sludge mathematical models.

7. Technology transfer

i) Capacity building The following students received relevant qualifications as a result of this project:

ii) Publications and conferences

Publications:

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.

Presentations:

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. l^st 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. l^st 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 10^th 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. 1^st 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. 12^th 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.