A New Approach to Understanding Enhanced Biological Phosphorus Removal

Treatment of wastewater is an essential process that is performed in all parts of the world. Each one of us typically produces more than 200 litres of wastewater per day. What happens to this wastewater? In an industrialised country like Britain the wastewater is collected for treatment and is then discharged into either a river or a coastal region. The treatment ensures that our rivers are not transformed into toxic soups and that most of the coastal waters remain safe for swimming. Presently our water treatment systems operate well to remove dangerous microorganisms and remove most of the organic and solid materials. Some components are more difficult to remove, such as nutrients like nitrogen and phosphorus. These nutrients cause damage to natural water systems such as rivers and coastal waters, as they encourage unwanted microbial growth, such as algae. This can damage the ecology of these waters and transform clear waters into green microbial soups. If a wastewater treatment facility is designed and operated in a particular manner, microorganisms (bacteria) in these systems can be encouraged to take up the phosphorus (P) and remove it from the wastewater. This is called biological P removal. It is the future aspiration of modern governments (e.g. the EU) that wastewater treatment facilities are improved and operated for this sustainable biological P removal. There are in fact many treatment facilities that already operate for biological P removal around the world. However, the performance of the biological systems is sometimes variable, and improvements in the performance and reliability would result in savings in the operation and construction of these systems. To achieve improvements in the biological systems we need to be able to understand how the bacteria carry out the P removal. There have been many investigations to gain understanding of these systems over the past 35 years. However, many of these investigations are flawed as they are studying the wrong bacteria, the ones that grow easily in the laboratory, and not the ones that grow well in the wastewater treatment systems and perform the P removal. Thankfully, modern methods to analyse DNA and protein directly in these systems are now being used to gain understanding of what the bacteria are doing. By analysing the DNA directly in the system we can now identify the bacteria important for the P removal. This has been a recent important achievement. Recently, the US government has invested heavily into understanding the bacteria of these systems, as they have obtained large amounts of DNA sequence from P removing systems (this is somewhat similar to whole genome sequencing programmes, such as the sequencing of the human DNA). This information will inform us of the genes that are present in these systems. It is important now to study the proteins of these systems. Proteins are produced by the bacteria, and are the molecules involved in carrying out the work, such as the reactions that result in the P removal. In our laboratory we operate small-scale wastewater treatment reactors that are performing biological P removal. A main part of this study is to analyse the proteins that are produced by the bacteria as they carry out the P removal. In these laboratory reactors we can alter the P removal performance and observe how the levels of the different proteins may vary. With this approach we will associate particular proteins with the biological P removal process. This information will enable us to put together an improved picture that explains how the bacteria are carrying out the P removal. This is a very important process for the water companies that treat the wastewater. Engineers and microbiologists are very interested to improve the understanding and details of the bacterial process, as they strive to develop strategies to improve the biological P removal performance in the wastewater treatment systems.

Enhanced biological phosphorus removal (EBPR) is an activated sludge system that operates for P removal. This is a desirable wastewater treatment process. Excess P causes eutrophication in receiving water bodies, which is an increasing concern given population increases and climate change. This biological process is favored over chemical P removal as it is economical, sustainable and minimises environmental impact. While many EBPR systems are successfully operated around the world, improvements in the performance and reliability would result in savings in the operation and construction of these. Over the next few years the European Water Framework Directive will require upgrading of many UK wastewater treatment facilities for P removal. Thus, there is considerable interest to understand the details of the microbial processess of EBPR. This project will gain detailed insight of the microbial processes important to EBPR, by novel application of environmental proteomics and metagenomics. EBPR systems are operatated in a particular configuration. The influent wastewater mixes with the bacterial sludge and the mixture then passess through anaerobic and aerobic reactor zones. In EBPR certain biological transformations occur, for example, there is a huge phosphate release in the anaerobic zone, followed by a larger uptake of phosphate in the aerobic zone. It is seen that certain types of bacteria are selected for in EBPR and these are polyphosphate accumulating organisms (PAO). There has been considerable research effort to understand the microbiology of EBPR. However, poor progress is due to the unculturble nature of the PAO. It is only recently confirmed that uncultured organisms of the Rhodocyclus group are important to EBPR. A big question then is, how to make progress and improve the poor understanding of the EBPR details? Laboratory scale reactors can be operated for extremely high performance EBPR. These are then a useful research tool as the EBPR transformations are more enhanced and the microbial community is dominated by Rhodocyclus type PAO. Recently, a DNA sequencing project, performed by the Joint Genome Institute, has nearly completed a shotgun sequencing from two EBPR laboratory-scale reactors. These sludges are also dominated by the same Rhodocyclus-type PAO organisms and this sludge genome data is extremely timely for the research proposed here. This study will operate finely tuned EBPR reactors and correlate analyses of the EBPR performance with proteome and mRNA expression data. Proteome analyses are already performed on activated sludge samples in our laboratory. We will continue analyses by 2-dimensional polyacrylamide gel electrophoresis, and by multidimensional protein identification technology (MudPIT) to examine protein expression from the high performance EBPR systems. Protein identification will be obtained by mass spectrometry analyses and searching the EBPR sludge genome data as well as the larger sequence databases (MASCOT). The EBPR reactor performance will be incrementally varied and data on microbial community composition, biochemical transformations, protein and mRNA expression will be compiled. Metagenomic libraries of EBPR sludge DNA will be constructed and screended for genes and function that are relevant to EBPR. Bioinformatic examination of sub-genomic regions in the vicinity of genes inferred to be involved in EBPR (as inferred from these studies) will be performed to detect possible operons, infer function, and detect regulatory elements. With this approach we aim to associate particular proteins with EBPR. This will enable us to put together an improved model explaining how the bacteria carry out the P removal. Engineers and microbiologists are very interested to improve the understanding and details of the bacterial process, as they strive to develop strategies to improve the biological P removal performance in the wastewater treatment systems.