WO1995012687A1

WO1995012687A1 - In situ measurement of bacterial growth in wastewater treatment processes

Info

Publication number: WO1995012687A1
Application number: PCT/AU1994/000666
Authority: WO
Inventors: Peter Charles Pollard
Original assignee: CRC for Waste Management and Pollution Control Ltd
Current assignee: CRC for Waste Management and Pollution Control Ltd
Priority date: 1993-11-01
Filing date: 1994-11-01
Publication date: 1995-05-11
Anticipated expiration: 1996-05-01

Abstract

A method of measuring the rate of growth of bacterial cells in wastewater samples is described. In the method of the invention, labelled thymidine added to a sample of wastewater is incorporated into DNA by the dividing cells in the sample. The method permits quantification of the rate of production of new bacterial biomass in situ.

Description

IN SITU MEASUREMENT OF BACTERIAL GROWTH IN WASTEWATER TREATMENT PROCESSES

TECHNICAL FIELD

This invention relates to the measurement of bacterial growth in wastewater. In particular, the invention relates to the measurement of DNA synthesis in dividing bacterial cells present in wastewater BACKGROUND ART

Micro-organisms decompose organic matter and remove inorganic nutrients from wastewater. The measurement of bacterial growth is important for determining kinetic constants when modelling degradation processes. Yet, in the monitoring and control of wastewater treatment processes, there is no accurate test to measure bacterial cell multiplication rates. In studies of batch activated sludge, for example, the known methods of measuring bacterial growth often lead to false interpretation of results.

Furthermore, most of the many bacterial species in wastewater cannot be cultured outside the treatment environment. Consequently, little is known about the function, community structure or dynamics of bacteria in the wastewater - that is, In situ. Even for those bacteria that are cultivable, many can exhibit a range of phenotypes and activities in response to different growth environment conditions that are unlike those in sttu.

The rates of substrate removal and decrease in endogenous biomass ultimately depend on the active microbial fraction of the wastewater. The methods most commonly used to measure microbial activity in wastewater are ATP levels, plate counts, oxygen uptake rates and the activity of dehydrogenase enzymes. While measurement of ATP concentrations offers the best estimate of activity, ATP synthesis is not specific to cell division as it is also involved in catabolic and anabolic processes of the bacterial cell.

A parameter currently used to determine whether or not bacterial cell multiplication will take place, particularly during exogenous substrate removal, is an indirect measure based on the ratio of the initial substrate concentration to the initial biomass, expressed as X^ . However, there is much speculation in the literature as to the relevance of this ratio. It is no more than an attempt to identify when bacteria are rapidly multiplying. To optimise the control of wastewater treatment by modelling microbiological degradation processes, accurate measurement of bacterial activity is required for determining kinetic constants.

In wastewater treatment systems, increases in bacterial biomass is marked by bacterial cell division which is often referred to as bacterial growth. The creation of new bacterial biomass, in the form of proteins, carbohydrates, lipids, RNA and DNA, culminates in the cell dividing once DNA synthesis is completed. Hence, bacterial growth rates could be measured by the rate that new DNA is synthesised.

It is known that bacterial cell division can be measured by the rate that thymidine is incorporated into bacterial DNA, (see, for example, Pollard and Moriarty Applied and Environmental Microbiology 48, 1076-1083, 1987). The principle of this technique is that exogenous radioactively labelled thymidine is rapidly and efficiently incorporated into bacterial DNA as the bacteria replicate. Thus, the rate that thymidine is incorporated into the bacterial DNA can be directly correlated to the rate of bacterial division. The method is a sensitive measure of bacterial growth rates at a molecular level. But, as yet, the technique has not been applied to the wastewater industry or in water quality assessment.

SUMMARY OF THE INVENTION It is an object of this invention to provide a method of measuring bacterial growth in wastewater by measuring the incorporation of labelled thymidine into DNA of dividing cells.

In one aspect, this invention provides a method of measuring bacterial growth in wastewater, the method comprising the steps of: i) obtaining a sample of biomass -containing wastewater; ii) mixing methyl-labelled thymidine with the sample; iii) incubating the mixture obtained in step (ii) to allow incorporation of methyl -labelled thymidine into newly synthesised DNA; iv) separating labelled DNA from methyl-labelled thymidine precursor; and v) determining the rate of methyl-labelled thymidine incorporation into DNA to determine the rate of bacterial cell division. In another aspect, this invention provides a method of detecting the presence of. or measuring growth of. particular bacteria in wastewater. the method comprising the steps of: a) obtaining a sample of biomass -containing wastewater; b) mixing radioactively-labelled thymidine with the sample; c) incubating the mixture obtained in step (b) to allow incorporation of radioactively-labelled thymidine into newly synthesised DNA; d) separating labelled DNA from radioactively-labelled thymidine precursor and lenaturing said labelled DNA; e) extracting DNA from a culture of known bacteria; f) denaturing the DNA obtained in step (e); g) mixing the denatured labelled DNA from step (d) with the denatured DNA of step (f); and h) determining the amount of radioactivity present in re- associated DNA to det. ct the presence of, or measure the growth rate of, said particular bacteria. In yet another aspect, the invention provides a kit for use in measuring ba - ^αrial growth in wastewater, the kit comprising: thyl-labelled thymidine;

for termination of cell division; and mally reagents and /or apparatus for the separation of methyl - l . Jlled thymidine precursor from DNA containing methyl -labelled thymidine phosphate.

The advantage of using thymidine as a DNA precursor is that it is only used by bacteria for DNA synthesis and it is not involved in other metabolic pathways, as is ATP. Hence, the rate that labelled thymidine is incorporated into bacterial DNA can be directly correlated to bacterial cell division.

Essential to the application of the foregoing technique to the wastewater treatment industry is a knowledge of the conditions necessary to optimise the incorporation of the labelled thymidine into bacterial DNA.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of a preferred procedure used to isolate the bacterial DNA radioactively labelled with [methyl-³HJ thymidine from activated sludge sampled from a sequencing batch reactor. Figure 2 is a graph of the radioactivity (dpm) incorporated into bacterial DNA as a function of the volume of activated sludge with a constant amount of [methyl-³H] thymidine (25 Ci) over a period of 5 min.

Figure 3 presents a kinetic study of the incorporation of radioactivity into macromolecules of bacteria in the activated sludge. The volume of sludge and [methyl-³Hl thymidine was held constant - only the incubation time was varied. The radioactivity (dpm) in the bacterial DNA (hot acid- soluble material) and total macromolecules (non-diffused material) was estimated as described in Figure 1.

Figure 4 depicts a study of changing bacterial growth rates (cells h^"1 mL), in a sequencing batch reactor through a treatment cycle.

Figures 5 to 9 are plots of the number of new bacterial cells formed with respect to time in assays conducted on samples from various stages of the Brendale Biological Nutrient Removal wastewater treatment plant. The relationship between figures and sources of samples is as follows: Figure 5, aerobic tank; Figure 6, anaerobic tank; Figure 7, anoxic tank; Figure 8, secondary clarifier; and. Figure 9, prefermentor.

Figure 10 is a schematic representation of the steps involved in determining the growth rate of a particular bacterial species as a proportion of the total bacterial activity. Figure 11 presents the results of agarose gel electrophoresis to determine the molecular size range of DNA resulting from the alkaline extraction step used in the method of the invention.

BEST MODE AND OTHER MODES OF CARRYING

OUT THE INVENTION In the following description and claims, the term "wastewater" includes biofilms present in wastewater treatment systems as well as biofilms such as those occurring in food and pharmaceutical preparation processes and equipment.

In the method of this invention, labelled thymidine added to a sample of wastewater is incorporated into DNA by the dividing cells in the sample. The method relies on the fact that bacteria decompose organic matter to produce new bacterial biomass. The cell increases in size until its biomass doubles, then division occurs. Hence, bacterial growth is both an increase in the number of individual cells as well as biomass and is marked by the synthesis of new bacterial DNA and cell division. The method permits quantification of the rate of production of new bacterial biomass in situ.

In the method, pre treatment of the wastewater sample is not required. Hence, step (ii) of the method as outlined above is carried out using the sample taken directly from the wastewater. Labelled thymidine is advantageously added to the sample immediately after sampling to avoid changes in the biomass prior to commencing incubation. In the case of activated sludge, incubation is commenced within one minute of sampling.

The labelled thymidine must be methyl-labelled to avoid labelling of other DNA precursors via nucleotide interconversion. The methyl-label can be any label detectable after labelled DNA is separated from the precursor. Typically, the label is a radioactive label such as ¹ C or ³H. The preferred labelled thymidine is [methyl-³H] thymidine.

Incubation of the mixture prepared in step (ii) of the method is typically carried out at the temperature of the wastewater from which the sample was taken. Usually, regulation of temperature is not required.

The time of incubation can vary depending upon the nature of the wastewater. Typically, the incubation time is between 2 and 5 minutes. The optimal time of incubation for a particular type of wastewater sample can be determined from a plot of labelled thymidine incorporated versus time of incubation (Figure 3).

The incubation is advantageously terminated by adding a reagent which stops DNA synthesis. Such reagents include 80% ethanol, phenol, trichloroacetic acid and other protein denaturants. Preferably, the reagent also acts as a DNA precipitant. The preferred reagent for terminating the incubation is a solution of 80% ethanol including 10 mM thymidine in which solution DNA is not soluble but thymidine is soluble. The solution thus allows pelleting of the DNA after centrifugation of the mixture and removes most of the radioactively labelled thymidine that is not incorporated into DNA. Following termination of incubation, labelled DNA is separated from the remaining labelled thymidine precursor - step (iv) of the method. This step may be carried out immediately or at any other time provided that the labelled DNA is stored under conditions which prevent DNA breakdown.

The labelled DNA is separated from labelled thymidine precursor using any of the techniques or apparatus known in the art. For example, the separation can be carried out by dialysis, electrophoresis, gel filtration, resin binding chromatography and the like. Apparatus suitable for separation of labelled DNA from precursor include centrifugal dialysis cartridges, centrifugal filters, DNA extraction cartridges, cell harvesters and the like. After separation, the amount of precursor thymidine incorporated into the labelled DNA is determined by measuring the amount of label in the DNA. Measurement is made using equipment appropriate to the nature of the label. For example, if the precursor thymidine is radioactively labelled, the amount of label in the DNA can be measured by liquid scintillation spectrometry.

If the method of separating labelled DNA from precursor thymidine also results in the extraction of other macromolecules, limitation of labelling to DNA can be verified by the separation of DNA from the other macromolecules using known methods. The growth rate of, or presence of, bacterial species in the active fraction of the biomass can be established by hybridisation of labelled DNA with DNA from cultures of known bacteria. In a typical process, hybrid DNA molecules are isolated after a single strand of labelled DNA anneals to a complementary strand from DNA of the known species. The amount of label in the hybrid DNA as a proportion of the total labelled DNA indicates the proportion of bacteria corresponding to the known species in the active fraction. Application of the method of the invention in this manner allows tn situ growth rates of particular bacterial species to be determined.

A preferred embodiment of the method of the invention is shown schematically in Figure 1. In that scheme, a wastewater sample is mixed with [methyl-³Hl thymidine within 1 minute of sampling. After incubation, the reaction is stopped with 10 mM thymidine in cold 80% ethanol and stored at -10°C for a minimum of 15 minutes. The ethanolic mixture is centrifuged at β.OOOfif for 15 minutes and the pellet taken up in 2 ml of 1 M NaOH/ 10 mM thymidine. After heating the resuspended pellet at 65°C for 5 minutes the solution is cooled and centrifuged at 6,000g for 15 minutes. The supernatant from the last mentioned centrifugation is dialysed and the dialysed material recovered as total labelled macromolecules which in most instances will represent labelled DNA. This is particularly the case when short incubation periods, such as less than 5 minutes, are used. However, DNA can be separated from other macromolecules as indicated in Figure 1.

Advantageously, the following conditions are used for in situ assays using [methy-³H] thymidine to measure the rate of growth of bacteria in wastewater: short incubation times of isotope with wastewater (typically about 5 min);

50 Ci of isotope at a low specific activity (preferably less the 2 Ci/mmol); small volume of wastewater (about lmL); assay should be carried out within about 10 min of sampling wastewater; and care must be taken not to alter the wastewater micro-environment.

In the application of the method of this invention to wastewater, it is possible that labelled thymidine incorporated into bacterial DNA may have to compete with non-labelled thymidine. Sources of thymidine include the salvage and de noυo biosynthetic pathways, the latter leading to the synthesis of thymidine monophosphate that is incorporated into bacterial

DNA that subsequently dilutes out the amount of labelled thymidine incorporated. This dilution may need to be accounted for or the de noυo biosynthetic pathway blocked with high concentrations of exogenously supplied thymidine or inhibitors such as halogenated deoxyuridylate compounds. Isotope dilution is discussed for example, by Pollard and

Kogure in Australian Journal of Marine and Fresh Water Research 44, 155-

172, 1993. For use in the method of the invention, a kit is provided comprising at least methyl-labelled thymidine and a reagent for stopping the incubation.

In accordance with the preferred embodiment described above, the kit contains a portion of [methyl-³H] thymidine and a portion of 10 mM thymidine in 80% ethanol. The kit may also include reagents for carrying out separation of the labelled DNA from precursor thymidine or apparatus for effecting separation such as those referred to above.

So that the invention may be more fully understood, examples of the method follow.

Example 1 Measurement of cell division in activated sludge This example illustrates the determination of the optimal sample volume for measuring cell division in activated sludge. Activated sludge samples were taken from a bench-scale sequencing batch reactor. The volume of the reactor was varied between 4.5 and 6.0 L and was intermediately operated. The feed consisted of raw wastewater after grit removal. The feed was collected from the Brendale Municipal Sewage Treatment Plant, Brendale, Queensland, Australia.

[Methyl-³^ thymidine was used as the labelled thymidine and was purchased from ICN, Australia, at a concentration of 37 MBq/ 1 mL (1.0 mCi/ 1 mL) in a sterile aqueous solution with a specific activity of 1.3 TBq mmol^"1 (35 Ci mmol^"1). Incubations were started by adding 25 μL of the stock [methyl-³H] thymidine to a small sample (1 to 5 mL) of activated sludge from the sequencing batch reactor. After 5 minutes, the incubation was stopped by adding cold 80% ethanol containing 10 mM thymidine and stored at -10°C. For zero time controls, ethanol solution was added immediately after the isotope was mixed with the sludge.

The isotope incorporated into newly synthesised bacterial DNA was separated from the unincorporated [methyl-³H] thymidine using a non¬ destructive dialysis technique (Figure 1) essentially as described by Pollard (Journal of Microbiological Methods 8, 91-101, 1987), the entire disclosure of which is incorporated herein by cross-reference. A change to the procedure described in the foregoing reference was that the pellet from ethanol precipitation was resuspended in 1.0 M NaOH and heated at 65°C for 5 minutes rather than 0.3 M NaOH with heating at 109°C for 30 minutes. The optimum sludge volume for the assay was determined from a plot of the radioactivity incorporated into DNA against the sludge volume (mL). There was a linear (R = 0.95) relationship between the amount of isotope incorporated and the sludge volume up to 1.5 mL (see Figure 2). One mL of sludge was found to be an acceptable combination of sludge volume and isotope as it fell within the linear portion of this relationship.

Example 2 Optimal incubation time In this example the optimal time of incubation of sludge with labelled thymidine was determined. One mL samples of activated sludge from the sequencing batch reactor were combined with 25 mL of [methyl-³H] thymidine for various times. Following termination of the assay with 80% ethanol/ 10 mM thymidine, the pellet from centrifugation was dialysed as described in Example 1.

The dialysed alkali extract (2.5 mL) was assayed for radioactivity then acidified with 125 μ\ of 100% (w/v) trichloroacetic acid at 90°C for 15 min in a water bath. The solution was cooled and centrifuged (6,000g) for 15 min to remove all precipitated material. The clear supernatant was again assayed for radioactivity (labelled newly synthesised bacterial DNA - Figure

1). The period [methyl-³HJ thymidine can be incubated with activated sludge can be determined from this kinetic study. If the incubation period is too long, macromolecules other than DNA become radioactively labelled. This is shown by the divergence, after 5 minutes, of the two plots presented in Figure 3. After 5 min the label was found in DNA and other macromolecules. The labelling of other macromolecules may lead to an overestimate of bacterial division rates. Accordingly, 5 min is the maximum incubation time for the activated sludge used in this example.

Example 3 Calculating Bacterial Growth Rates To calculate bacterial growth rates, the radioactivity incorporated into bacterial DNA must be converted into the increase in the number of new bacterial cells. Knowing the specific activity ( Ci nmol^"1 thymidine) of the [methyl-³Hl thymidine the radioactivity (dpm) can be converted into the number of moles of thymidine incorporated over the incubation period (2.2 x 10⁶ dpm = 1 Ci). For each mole of thymidine (deoxythymidine) incorporated one mole of each of the other three nucleotides (deoxyadenosine, deoxycytidine and deoxyguanosine) is also incorporated into DNA. Thus the total number of moles of nucleotides incorporated into DNA is four times that of thymidine. In this way the total number of moles (or weight) of nucleotides incorporated into bacterial DNA can be calculated. The average weight of bacterial DNA is 3.5 fg per bacterial cell, dividing this value into the weight of all the nucleotides incorporated allows the number of all newly synthesised bacterial DNA molecules to be determined. There is one bacterial cell per DNA molecule. This is the theoretical basis for converting the rate that bacterial DNA is radioactively labelled, with [methyl-³HJ thymidine, into bacterial growth rates.

Both theoretically and empirically determined multiplication factors are used to convert the number of moles of [methyl-³H] thymidine incorporated into DNA to the increase in the number of bacterial cells per unit time (bacterial growth rates); there is good agreement between these factors. A review of the conversion factors in the literature shows a mean value of 2 x

10⁹ bacterial cells are synthesised per nmole of [methyl-³H] thymidine incorporated into DNA. So bacterial growth rates in wastewater can simply be calculated from the number of moles of labelled thymidine (radioactivity; dpm) incorporated into DNA with the following equation:

Radioactivity(dpm) x 60 min x [2xl0⁹ cells, mol]

Bacterial growth rate =

(cells h^"1 mL sludge^"1)

[2.2 x 10⁶1 x 5 min x [Specific Activity Ci.nmol^"1]

The data from Figure 2 were converted to bacterial growth rates and the results of this conversion are presented in Table I.

TABLE I

Sludge Volume Radioactivity in Bacterial growth rates

(mL) DNA (dpm) x 10⁸ (cells h^_1 mL^"1)

0.25 38,292 0.358

0.50 64, 153 0.600

0.75 63,140 0.590

1.00 68,599 0.641

1.25 82.674 0.773

1.50 92.685 0.867

1.75 90,565 0.847

2.00 109,535 1.024

2.50 105,721 0.988

3.00 104,332 0.976

4.00 123,007 1.150

6.00 147,466 1.379

Example 4 Monitoring Growth Rates

Bacterial growth rates in relation to various stages of operation of the sequencing batch reactor referred to in Example 1 were assessed. One mL samples of sludge were taken from the reactor at various times and incubated for 5 minutes with [methyl-³H] thymidine as detailed in Example 1. Labelled DNA was separated from thymidine precursor as also detailed in Example 1. Bacterial growth rates were determined as described in the previous example. The results obtained are presented in Figure 4.

It can be seen from Figure 4 that changes in growth rate occur at each of the different stages of treatment and that process changes lead to a gradual change of microbial activity.

Example 5

Bacterial Biomass

The maximum specific growth rates (μ,^ of bacteria is an important parameter used to model population dynamics and kinetics in wastewater treatment processes. This parameter is defined as the bacterial growth rate divided by the bacterial biomass. Example 3 shows how In situ growth rates can be determined. The following is an example of how bacterial biomass can be measured on the same wastewater used to determine bacterial growth rates. Formaldehyde solution (30 μl, 36%, v/v) was added to the sub-sampled activated sludge ( 1 mL) at the same time that 1 mL was taken to measure bacterial growth rates (see Figure 1). Biomass samples were stored at 4°C. The sample was then blended with an ultra-turrax (Janke and Kunkel, Ika Werk, Staufen i., Breisgau, West Germany) for 1 min. The bacteria were stained with acridine orange and collected onto an irgalan black stained-nuclepore polycarbonate filter (0.2 μm pore size) and counted with an epifluorescence microscope.

To calculate bacterial production in terms of carbon, the bacterial cell volumes were also estimated with the epifluorescence microscope. With a stage micrometer and ocular grid, an estimate was made of the cell diameter of the bacteria and the cell volume calculated. The cell volumes were converted to bacterial biomass, in terms of carbon, with the value of 0.220 fg C μm^~3 (Bratbak and Dundas, Applied and Environmental Microbiology 48, 755-757, 1984). Example 6

Effect of specific activity of radioactively-labelled thymidine on the determination of growth rates Experiments were conducted to see what effect the specific activity of the radioactively-labelled thymidine used in assays according to the invention has on the determination of bacterial growth rates. As noted above, isotope may be diluted which would result in the underestimation of growth rates.

In these experiments, bacterial cells dividing per mL were plotted against the time of incubation of the wastewater sample with isotopically- labelled thymidine. Three time courses were developed in each experiment: one for each of three different specific activites of [methy-³H] thymidine used - 2, 4 and 35 Ci/mmol. The slope of each of these plots was a measure of the rate of bacterial growth.

The samples used for bacterial growth rate measurement were obtained from the Brendale treatment plant referred to above (see Example 1) which is a Biological Nutrient Removal (BNR) plant operating in a UCT (University of Cape Town) configuration. In this plant, influent wastewater passes from a pre-fermentor (settler) to anaerobic, anoxic and aerobic tanks then finally into a secondary clarifier tank. Bacterial growth was measured in samples from each of these tanks. Assays were carried out generally as described in Example 1 with 50 Ci of [methy-³H] thymidine used per assay.

Kinetic plots of [methy-³H] thymidine incorporation into DNA of growing cells for each stage of the Brendale BNR plant are shown in Figures 5 to 9. The results presented in the figures show that there is a linear relationship with a correlation coefficient greater than 0.9 between the amount of labelled thymidine incorportated and the period of incubation. The incorporation of isotope by dividing bacteria does not appear to be limited by the rate that labelled thymidine diffuses through solid material in wastewater. Figure 5 shows that in the aerobic tank samples from the Brendale BNR plant, for example, [methyl ^H] thymidine was rapidly and efficiently incorporated into bacterial DNA. This is evident from the point at which plots intersect the vertical axis indicating the absence of a lag phase before radioactively-labelled thymidine was incorporated into the DNA of dividing cells.

The highest rates of bacterial growth were obtained using [methyl ³H] thymidine of low specifc activity: 2 and 4 Ci/mmol (see Figures 5 to 9). At these specific activities bacterial growth rates were generally similar, indicating that isotope dilution had been either eliminated or minimised by inhibition of de noυo synthesis of thymine. Most, if not all, of the [methyl ³H) thymidine at a specific activity of 2 Ci/mmol was utilised by the growing bacteria.

However, at a specific activity of 35 Ci/mmol estimates of bacterial growth rates were sometimes more than an order of magnitude lower than that determined at 2 or 4 Ci/mmol (see Figures 7 and 8). This suggests that at high specific activities - that is, specific activities greater than 4 Ci/mmol - in situ isotope dilution causes an underestimate of bacterial growth as de noυo thymine synthesis dilutes the labelled thymidine that is incorporated into bacterial DNA. Best estimates of bacterial growth will be obtained using radioactively-labelled thymidine at a specific activity of 2 Ci/mmol or lower.

Example 7

Measurement of bacterial growth rates in various wastewater treatment systems In this example, the method according to the invention was applied to samples from a variety of wastewater treatment systems. Brief details of the systems in which bacterial growth rates were measured follow. Biological nutrient removal system

Biological nutrient removal (BNR) wastewater treatment systems are generally configured such that the activated sludge system includes one or more non-aerated zones with a single sludge system and includes one secondary clarifier in which settled sludge is separated for recycling through the other zones. The samples used for bacterial growth rate measurement were obtained from the Brendale treatment plant referred to above (see Example 6).

Sequencing batch reactor (SBR)

Bacterial growth was measured in an intermittently aerated and decanted activated sludge treatment system. After primary screening of municipal wastewater, a single vessel was used for biological oxidation, settling followed by decanting of the activated sludge, and incorporated nitrification and denitrification processes. The different processes of wastewater treatment were separated in time rather than spatially. Bacterial growth rates were measured in the activated sludge of the aerobic and anaerobic stages of the SBR. The SBR used in this study was a bench- scale system maintained in the Department of Chemical Engineering at The University of Queensland, St. Lucia, Queensland, Australia. The SBR feed was raw sewage from a municipal treatment works. The treatment system is described in detail by Ho (Ph.D. Thesis, Department of Chemical Engineering, The University of Queensland, 1994). Biofilms

Biofilms represent complex bacterial communities. They are found in areas as diverse as wastewater treatment, where their growth is encouraged, to the food and medical industries where biofilms are a health risk. There is a need to be able to measure microbial growth in biofilms of these diverse areas.

In this study, bacterial growth was measured on the biofilm of particles of a fluidised bed reactor. This was a laboratory scale reactor used to remove heavy metals from wastewater. The reactor was constructed and is maintained in the Department of Microbiology at The University of Queensland. The reactor has been described in detail by Sly et al. [Water June 1993, pp 38-40). The results presented below in Table II show that all the bacterial growth measured with thymidine is on the particle surfaces of the biofilm. Anaerobic wastewater treatment (digester) system Anaerobic wastewater treatment processes involve the anaerobic microbial conversion of substrate to methane and carbon dioxide. The systems are applied to both industrial and municipal waste.

The study was carried out on fruit and vegetable processing wastewater of the Golden Circle Cannery at Northgate, Queensland, Australia. The wastewater treatment was in a two-stage high-rate anaerobic treatment system. The first stage consisted of an equalisation tank (sometimes called an acidification or fermentation tank) while the second treatment stage was an up flow anaerobic sludge blanket reactor (UASB). The UASB has been described by Lawson [Journal of the Australian Water and Wastewater Association 1992, pp 29-30). Bacterial growth rates were measured in both the first and second stage reactors. Industrial wastewater treatment

This system was an activated sludge process used to treat the toxic waste of coke ovens at BHP's Port Kembla Steelworks, New South Wales, Australia. The influent source is wastewater from coal, water generated in the coking process and from cooling water. The influent contained ammonia, cyanide, phenol, and oils. The wastewater treatment process included both biological and chemical methods, with most of the COD being removed by bacterial activity (biologically). Bacterial growth rates were measured in the first of a series of three oxygen-aerated activated sludge reactors. The system has been described in detail by Paris and Jell [Water 1992, pp 23-28). Oxidation ditch system

Bacterial growth rates were measured in an oxidation ditch system. Wastewater was sampled from the aeration section of the oxidation ditch of the municipal sewerage treatment plant at Logan City Council, Loganholme, Queensland, Australia. Oxidation ditches (carousels) have been described, for example, by Eckenfelder [Industrial Water Pollution Control McGraw-Hill Book Company, New York, 1989). Nitrification: measuring the growth rate of nitrifying bacteria

A series of experiments were carried out to measure growth rates of four cultures of autotrophic, ammonia-oxidising bacteria ("Nitrosomonas") and two nitrite oxidisers ("Nitrobacter"). The isolation and identification of these bacteria, which seem to be important for the removal of nitrogen from wastewater, has been described by Allison and Prosser [Identification Methods in Applied and Industrial Microbiology Vol. 29, 1992). The cultures were grown and maintained in the Department of Microbiology and Immunology at the University of NSW, Kensington, New South Wales, Australia. Growth rates of these bacteria were measured while the cultures were in log phase.

Advanced constructed wetland

A study was made of wastewater treated in an artificially constructed wetland. Wetland influent was secondary treated effluent of an intermittently operated activated sludge plant (West Byron treatment plant, Byron Shire Council, New South Wales, Australia). Decanted liquid passed through a circulation (settlement) pond passed directly into the artifical wetland without sterilisation. These were advanced constructed wetlands, that were designed to provide a low-cost, low maintenance solution to the problem of nutrient removal from wastewater. Macrophytes of the wetland were grown with their roots permanently submerged in water. Bacterial growth was measured in the biofilm on the roots and in the root zone, and in the interstitial and effluent water. General assay conditions

Assays on samples from the above wastewater systems were performed generally as described in prececeding examples. For the results presented below in Table II, each assay used 50 Ci of [methyl-³H] thymidine at a specific activity of 2 Ci/mmol. Calculating bacterial growth rates and other parameters

Bacterial growth rates were determined as described above in Example 3. The number of bacteria in wastewater samples was determined with epifluorescence microscopy. One mL of wastewater was fixed with 200 μL of

25% gluteraldehyde. This mixture was cooled on ice and blended using a small head ultra- turrax for 1 min. A 200 μL portion of the blended mixture was diluted to 5 mL with sterile Milli RQ water and sonicated for 1 min. Between 1 and 0.2 mL was sampled for counting on irgalan black-stained polycarbonate filters which were left in a 2% solution of acetic acid and rinsed in Milli RQ water just prior assay. All samples were photographed using a Leitz microscope. The conversion factor for the epifluorescence microscope is 5.25 x 10⁶ per square in graduated ocular (x 12) x objective (x 100). This method is described in detail by Bitton et al. (Water Research 27,

1113-1118, 1993).

The amount of organic carbon required to support growth rates - that is, the biodegradable carbon - was also calculated. This figure was calculated on the assumptions that the cells were 30% efficient at converting substrate into their own biomass and that each cell contained 25 fg of carbon per cell (R.T. Bell in Handbook of Methods in Aquatic Microbiol Ecology, P.F. Kemp et al. eds, Lewis Publishers, London, 1993) using the following equation: ((cells/min)/mL) x 1000(L) x 60 (h) x 24 (d) x (100/30) x 25xl0^"15 x 1000(m³) =

Kg Carbon per day per m³ Results

Results obtained for each of the wastewater systems specified above are summarised in Table II below where biomass, growth rates, specific growth rates and bacterial doubling times are given with standard errors. Also listed is the amount of organic carbon required to support growth rates. TABLE II

Table II indicates that the method of this invention is applicable to the measurement of bacterial growth rates in a wide variety of wastewater systems.

Like the experiments described in Example 6, labelled thymidine was found to be immediately incorporated into the DNA of replicating bacteria for all wastewater systems for which results are presented in Table II.

Example 8 Measuringthe growth rate of particular bacteria in situ Figure 10 is a flow diagram which shows how in situ labelled DNA of particular bacteria can be extracted from a wastewater sample for identification and/or estimation of the actively replicating species present in the biomass. In this process, bacteria in wastewater are radioactively labelled as described and exemplified above. The bacteria of interest, the "known bacteria", are cultured in the laboratory. In parallel, DNA of the known bacteria and from the wastewater bacteria is extracted as described above (see Figure 1 and Example 1). This process also provides denatured DNA for the hybridisation step. The single stranded DNA of the known bacteria is mixed with the single stranded DNA of the bacteria in the wastewater sample using standard DNA-DNA reassociation techniques. Preferably one of the DNA components of the mixture is immobilised on a solid support such as hydroxylapatite or a membrane (see for example J.L. Johnson in Methods for General Bacteriology, P. Gerhart et al., eds., American Society for Microbiology, Washington, D.C., pp 655-682, 1994). If the bacteria under investigation replicated in the initial wastewater thymidine assay, reassociated DNA will become radioactively labelled (Figure 10). This activity divided by the total activity, determined with the standard thymidine assay, can be multiplied by the overall rate of bacterial growth to estimate the in situ rate of growth of the particular bacteria under investigation.

To assess whether the labelled DNA resulting from the alkali extraction procedure depicted in Figure 1 would be suitable for use in the hybridisation process outlined in Figure 10, the molecular size (bp) range of labelled DNA was determined by agarose gel electrophoresis. The DNA of bacteria in wastewater from the aerobic and anoxic compartments of the Brendale BNR treatment plant was radioactively labelled with [methyl Η] thymidine as described above. After alkali extraction and dialysis (Figure 1), preparations were buffered with 0.1 volumes of 3 M sodium acetate buffer then stored at -70°C for three days. DNA was finally harvested by precipitation from ethanolic solution and taken up in Tris- acetate/EDTA buffer at pH 6.7 for agarose gel electrophoresis.

Electrophoresis was carried out as described, for example, by Sambrook et al. (Molecular Cloning: a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989), the entire contents of which is incorporated herein by cross-reference. Restriction endonuclease-digested lambda DNA was used for molecular size standards. Following electrophoresis, DNA was visualised by ethidium bromide staining, the gel cut into strips corresponding to each lane thereof, and the gel strips further segmented into 0.5 cm pieces. Individual pieces were melted in 1 mL of water and combined with 10 mL of scintillant (Ready Safe) for liquid scintillation determination of the radioactivity in each gel piece.

The results of the electrophoretic analysis are presented in Figure 11. The figure shows where the lambda standards appeared on the gel and also the distance from the origin of the radioactivily labelled DNA fragments. The radioactively labelled DNA predominantly comprised fragments between 260 and 1000 base pairs (Figure 11). These sizes are large enough to be used in the DNA-DNA reassociation procedure described in Figure 10. INDUSTRIAL APPLICABILITY

The method of the invention is applicable to the measurement of bacterial growth in the following wastewater processes:

Activated sludge

Sequencing batch reactors Biological nutrient removal (BNR) plants

Continuous compartmentalised systems

Anaerobic fermentors and treatment systems

Methanogenic reactor

Anaerobic sludge digesters Wetland systems

Biofilms

Pond wastewater treatment systems

The method can also be used for assessing water quality thereby replacing an E. coli count with a measure of whole bacterial population activity.

Furthermore, the method can replace BOD as a predictor of influent and effluent substrate quality on bacterial activity in waste treatment and can be used in relation to the modelling of wastewater systems by the direct measurement of the following parameters:

Maximum specific growth rates (μ_m )

Bacterial doubling time (t

Biodegradability;

With regard to biodegradability - that is, the ability of bacteria to decompose material in wastewater - the decomposition process is the main link between the biodegradable substrate and the removal of the organic and inorganic compounds from wastewater. Bacterial growth rates and nutrient removal from wastewater treatment processes are inextricably linked. Ultimately the growth rate of the bacteria is dependent on the supply and quality of oxidisable substrates in the feed. In wastewater, if the bacterial production (measured in terms of carbon) and input of organic carbon are similar, after taking into account losses due to bacterial respiration, all the substrate input is biodegradable. Conversely, if bacterial production is substantially lower than the input of organic carbon from the inputs, it is more refractive or contains toxic material to the microbial community in the system.

The method of this invention can also be used for the monitoring of bacterial growth rates to improve control of wastewater treatment processes.

DNA synthesis does not occur in non-growing cells so the direct measurement of bacterial division under in situ conditions also allows increase in cell biomass to be distinguished from cell division. For example, increases in cell biomass without cell division could indicate the synthesis of microbial polymers, such a poly-β-hydroxy butyrates.

In wastewater treatment systems the rate of substrate removal depends on the active microbial fraction. Engineers often describe and quantify the relationship between substrate (waste) removal and bacterial growth with models based on simple Michaelis-Menten substrate saturation kinetics - that is, the Monod equation: μ = μ x (s / ( s + κj) where: μ = bacterial specific growth rates = rate of bacterial growth/number of bacteria μ,_^ = maximum specific growth rate S = substrate concentration

K, = "half saturation constant"

Bacterial growth (μ) is a key parameter in these models. Yet, in the wastewater industry, there has been no direct measurement of the dynamics of the many diverse bacterial communities. Using the labelled thymidine method in accordance with the present invention, the specific growth rate of bacteria can be directly measured in situ because it is defined as the rate of bacterial growth divided by the number of bacteria.

Claims

1. A method of measuring bacterial growth in wastewater, the method comprising the steps of: i) obtaining a sample of biomass-containing wastewater; ii) mixing methyl-labelled thymidine with the sample; iii) incubating the mixture obtained in step (ii) to allow incorporation of methyl-labelled thymidine into newly synthesised DNA; iv) separating labelled DNA from methyl-labelled thymidine precursor; and v) determining the rate of methyl-labelled thymidine incorporation into

DNA to determine the rate of bacterial cell division.

2. The method according to claim 1, wherein said methyl-labelled thymidine is radioactively-labelled thymidine.

3. The method according to claim 2, wherein said methyl-labelled thymidine is [methyl-¹ C] thymidine.

4. The method according to claim 2, wherein said methyl-labelled thymidine is [methyl-³H] thymidine.

5. The method according to claim 4, wherein said [methyl-³H] thymidine has a specific activity of less than 4 Ci/mmol.

6. The method according to claim 5, wherein said [methyl-³H] thymidine has a specific activity of about 2 Ci/mmol.

7. A method of detecting the presence of, or measuring growth of, particular bacteria in wastewater, the method comprising the steps of: a) obtaining a sample of biomass-containing wastewater; b) mixing radioactively-labelled thymidine with the sample; c) incubating the mixture obtained in step (b) to allow incorporation of radioactively-labelled thymidine into newly synthesised DNA; d) separating labelled DNA from radioactively-labelled thymidine precursor and denaturing said labelled DNA; e) extracting DNA from a culture of known bacteria; f) denaturing the DNA obtained in step (e); g) mixing the denatured labelled DNA from step (d) with the denatured DNA of step (f); and h) determining the amount of radioactivity present in re-associated

DNA to detect the presence of, or measure the growth rate of, said particular bacteria.

8. The method according to claim 7, wherein said radioactively-labelled thymidine is [methyl-¹ C] thymidine or [methyl-³H] thymidine.

9. A kit for use in a method comprising the steps of: i) obtaining a sample of biomass-containing wastewater; ii) mixing methyl-labelled thymidine with the sample; iii) incubating the mixture obtained in step (ii) to allow incorporation of methyl-labelled thymidine into newly synthesised DNA; iv) separating labelled DNA from methyl-labelled thymidine precursor; and v) determining the rate of methyl-labelled thymidine incorporation into

DNA to determine the rate of bacterial cell division; wherein said kit comprising methyl-labelled thymidine and a reagent for termination of cell division.

10. The kit according to claim 9, wherein said methyl-labelled thymidine is [methyl-¹⁴C] thymidine or [methyl-³H] thymidine.

11. The kit according to claim 9, wherein said reagent for terminating cell division is a solution of 10 mM thymidine in 80% ethanol.

12. The kit according to claim 9, further comprising reagents and/or apparatus for the separation of methyl-labelled thymidine precursor from DNA containing methyl-labelled thymidine phosphate.