PII: S0043-1354(00)00450-4

Wat. Res. Vol. 35, No. 7, pp. 16491658, 2001 # 2001 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/01/$ - see front matter

COMPARISON OF METHODS FOR DETERMINATION OF MICROBIAL BIOMASS IN WASTEWATER

JES VOLLERTSEN1*, ANDREAS JAHN1y, JEPPE LUND NIELSEN1, THORKILD HVITVED-JACOBSEN1 and PER HALKJR NIELSEN1
1

Environmental Engineering Laboratory, Aalborg University, Sohngaardsholmsvej 57, 9000 Aalborg, Denmark (First received 5 April 2000; accepted in revised form 13 September 2000)

Abstract}Microbial biomass in wastewater was determined by methods used in environmental microbiology and by a method used in wastewater engineering based on a conceptual model simulating fundamental microbial processes in wastewater from measured oxygen uptake rates. The methods originating from environmental microbiology are based on staining and counting of cells for the determination of total cell biomass (acridine orange and DAPI), physiological state of cells (LIVE/ DEAD1 BacLightTM ) and activity of cells (reduction of the redox dye CTC and microautoradiography). Depending on the staining method applied, cell biomasses yielded 1586% of the biomass dened by the model, and good correlations between cell biomass and model biomass were found. Cell biomass, oxygen uptake and acetate uptake were measured in wastewater, where acetate was added. Substrate uptake rates were found not to be proportional to the increases in cell biomass, suggesting that only a small fraction of the cell biomass was responsible for the main part of the substrate uptake. Despite the dierences found between cell biomass and model biomass, it was recommended to use the conceptual model as an engineering tool for simulation of microbial processes and wastewater quality changes. However, there should be a clear distinction between the terms model biomass, cell biomass and dierent activity measurements of cells. # 2001 Elsevier Science Ltd. All rights reserved Key words}wastewater, biomass activity, oxygen uptake rate, cell biomass.

INTRODUCTION

In sewer systems, the microbial biomass has a central role when simulating biological, chemical and some physical transformation processes (Bjerre et al., 1998). Application of the microbial biomass as a central wastewater component has allowed a conceptual model of in-sewer processes to be developed, validated and applied to solve engineering problems (Stemplewski et al., 1999). When modeling the consumption of electron donors and acceptors as well as when modeling hydrolysis of slowly biodegradable components, the biomass has successfully been used as a key model component (HvitvedJacobsen et al., 1998). In order to determine this model biomass, experimental techniques have been established, allowing it to be determined from oxygen uptake rates (OUR) during substrate non-limited growth. Furthermore, methodologies for the determination of the necessary model parameters as well

*Author to whom all correspondence should be addressed. Tel.: +45-963-58504; fax +45-981-42555; e-mail: jv@ civil.auc.dk y Now: Universita tsklinikum Freiburg, Zentrum Klinische Studien, Hugstetter Str. 55, D-79106 Freiburg, Germany

as validation techniques have been developed (Vollertsen and Hvitved-Jacobsen, 1999). As microbial biomass is a central model component, it is critical to distinguish between dierent uses of the term biomass. How the term biomass is understood depends on the denition and on the method used for its determination. In the conceptual model for simulation of wastewater transformation processes, biomass is dened as a calculated biomass based on measurement of the wastewater OUR, i.e. an activity-based biomass determination (Bjerre et al., 1995). In environmental microbiology, several biomass components are dened, e.g. total cell biomass, active cell biomass, living cell biomass and dead cell biomass (Christon, 1997). Total cell biomass includes all cells independent of their state of activity. Active cell biomass measures the biomass actually active in substrate consumption (Karner and Fuhrman, 1997). The correlation between the dierent measures for cell biomass and the conceptual understanding of biomass in the wastewater transformation models based on oxygen uptake activity has, however, not yet been established. Several methods for the determination of the total cell biomass and the active cell biomass are used in environmental microbiology. Total cell biomass is

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usually determined by DNA staining, e.g. DAPI and acridine orange, often combined with image analysis techniques for size measurements (Karner and Fuhrman, 1997). Also measurement of biomass components such as protein, lipids, ATP and DNA are used for the determination of microbial cell biomass (Christon, 1997). Active cell biomass is measured by methods relying on the metabolic activity of microorganisms: e.g. reduction of the tetrazolium salt CTC that has been used to identify the electron transport activity of actively respiring cells (Rodriguez et al., 1992). Another method to dierentiate between living and dead cells is to detect if their membrane system is intact (Molecular Probes, 1998). The microautoradiography (MAR) technique determines cells that actively take up radioactive labeled substrates, e.g. mixtures of amino acids, glucose or acetate (Carman, 1990; Andreasen and Nielsen, 1997). The objective of this study was to compare methods for determination of cell biomass and model biomass in wastewater. Cell biomass was determined by dierent microbiological methods and model biomass was calculated from OUR measurements.
METHODS

Fig. 1. Concept for microbial transformation of wastewater components. Electron donor and electron acceptor concentrations are measured in COD units.

found from measurements of the OUR alone (Bjerre et al., 1998; Kappeler and Gujer, 1992; Vollertsen et al., 1999; Vollertsen and Hvitved-Jacobsen, 1999). The concentration of XB during such substrate nonlimited growth conditions becomes a function of a maximum specic growth rate (mH ), a heterotrophic yield (YH ), a maintenance energy requirement rate constant (qm ) and the OUR (equation (1)) (Vollertsen and HvitvedJacobsen, 1999). Values for mH can readily be found from the OUR during substrate non-limited growth and are determined for each experiment. Values for YH and qm were not determined in this study, instead average values found by Vollertsen and Hvitved-Jacobsen (1999) for sewer solids were used (YH 0:65 and qm 1:6 d1). XB OURt 1 YH =YH mH qm 1

Municipal wastewater was sampled in combined sewers at two locations in Aalborg and one location in Frejlev, Denmark. Eleven samples were taken from dry weather runo and two samples were taken from combined wastewater during storm events. Analysis was initiated within 1 h after sampling. Samples were analyzed for total solids (TS), volatile solids (VS) and chemical oxygen demand (COD) according to APHA (1995). Acetate was determined on a Dionex ion chromatograph with a suppressed conductivity detector, 0.2 mM NaOH as mobile phase (1 ml/min) and an IonPac, AS11 column. Model biomass The model concept applied to simulate wastewater transformations in sewers has similarities to model concepts applied to simulate wastewater treatment processes. However, it is not identical to those concepts, as biomass growth conditions dier. The availability of substrate and the biomass concentration are main dierences. Compared to treatment systems, sewers contain high concentrations of readily biodegradable substrate and low concentrations of biomass. Often organic substrate concentrations are so high that}if oxygen is plentiful}the heterotrophic aerobic biomass will grow at rates close to or equal to its maximum specic growth rate. Hvitved-Jacobsen et al. (1998) and Vollertsen and Hvitved-Jacobsen (1998) formulated the concept, which denes the model biomass (XB ) as used in this investigation. The concept assumes that the microbial transformations under aerobic conditions can be described by the processes of heterotrophic growth, non-growth related substrate uptake of the heterotrophic biomass (i.e. maintenance energy requirement) and hydrolysis of 23 fractions of hydrolysable substrate (Fig. 1). When the DO concentration is above 0.51 g O2 m3 and the concentration of readily biodegradable substrate is above 510 g COD m3, the biomass growth rate stipulated in the concept simplies to a rst-order rate process in the biomass concentration. Based on known model parameters, the biomass concentration can under these conditions be

In this study, non-limiting electron donor conditions were obtained by addition of sodium acetate, which is a common, readily biodegradable constituent of wastewater (Raunkjr et al., 1995). Acetate was added in a concentration of at least 100 g COD m3. Initial experiments with addition of acetate up to 7000 g COD m3 yielded identical XB concentrations and did not inhibit the respiration rate or the growth rate in the wastewater batch experiments. Some measurements of OUR rates were}for practical reasons} measured at other temperatures than 208C. In these cases, respiration rates and maximum growth rates were converted to 208C using an Arrhenius relationship with an Arrhenius constant of 1.07 (Hvitved-Jacobsen et al., 1998). OUR was measured in stainless-steel batch reactors with a reactor volume of 2.2 l as described by Vollertsen and HvitvedJacobsen (1999). Cell biomass Direct cell counts on acridine orange (AO) stained samples is a commonly used technique for the determination of total cell numbers (Hobbie et al., 1977). AO is a DNA/ RNA specic stain. Wastewater was xed by adding formaldehyde (2% nal concentration). AO (1 mg ml1 nal concentration) was added to a homogenized and diluted sample, and after 2 min of staining, the solution was ltered through a 0.22 mm MicronClear polycarbonate black lter. CTC (5-cyano-2,3-ditolyl tetrazolium chloride) has been proposed for the determination of actively respiring cells. CTC is readily reduced by electron transport activity into CTCformazan crystals, which are non-soluble and uorescent in metabolically active cells (Rodriguez et al., 1992). DAPI (uorocrom 40 , 60 -diamidino-2-phenylindoldihydrochloride-dilactate)}a commonly used DNA-specic stain for the determination of the total cell number}was used together with CTC to determine the total cell number in the same sample as CTC (Griebe et al., 1996). Initial experiments showed that wastewater should be incubated for 2 h with acetate as substrate. A homogenized and diluted sample was ltrated through a 0.22 mm MicronClear polycarbonate black lter. CTC and DAPI cell counts were carried out simultaneously and on the same sample.

Microbial biomass in wastewater The LIVE/DEAD1 BacLightTM stain (LD) from molecular probes has been published to distinguish between living and dead bacteria on the basis of an intact cell membrane system (Boulos et al., 1999). It contains two dyes: SYTO 9 green and propidium iodide. The DNA-stain SYTO 9 stains all cells, but propidium iodide only penetrates cells with damaged cell membranes. In the cell, SYTO 9 is reduced when propidium iodide is present, resulting in propidium iodide staining those bacteria with damaged cell membranes red (Molecular Probes, 1998). LD cell counts were made on suspensions, determining the ratio between living and dead cells. DAPI cell counts were determined as described above and used to calculate total cell numbers. LD stain was added as specied by the manufacturers protocol, the sample incubated for 15 min. By microautoradiography (MAR), the microbial in situ uptake of dierent radioactive labeled compounds can be studied on a micro-scale (Andreasen and Nielsen, 1997). The microbial uptake of acetate was studied in wastewater samples by incubation for 50 min with 2 mM acetate and 10 mCi [3H]-acetate (Amersham Pharmacia Biotech). Paraformaldehyde was added (4% w/v) for 2 h on ice to avoid further substrate uptake and to x the cells. Subsequently, the samples were washed three times in tap water. Chemography and other artifacts were tested for by additional controls with pasteurized wastewater (808C for 10 min) and controls performed without addition of tracer. After xation, the sample was homogenized thoroughly with a tissue grinder in order to avoid aggregates. The sample was diluted in ltered tap water before ltrating onto a white 0.22 mm polycarbonate lter (Nucleopore). The lters were xed on slides before carefully dipping them in a liquid lm emulsion (LM-1, Amersham Pharmacia Biotech). After an exposure period of 34 weeks, the emulsion was developed by normal standard photographic lm techniques and the distribution of silver grains could be viewed under normal bright-eld microscopy (Nielsen and Nielsen, submitted; Andreasen and Nielsen, 1997). Cell volume determination is necessary to nd cell biomass after total cell counts. Image processing using IP Lab Spectrum (Signal Analytics Corporation, 1994) was performed on AO-stained samples to determine cell volumes. Images were taken with a Photometrics Image Point video camera. Cell volumes (Vcell ) were calculated from measurements of major axis (Lmajor ) and minor axis (Lminor ), using equation (2) (Fuhrman, 1981; Krambeck et al., 1981). Each determination of biomass volume involved measurement of between 50 and 500 individual cells:   p Lminor 2 Vcell L2 minor Lmajor 4 3 All AO, DAPI, CTC and LD cell counts were carried out with a Leitz Laborlux D epiuorescence microscope.
RESULTS

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Wastewater was sampled on 13 occasions for determinations of cell biomass, active cell biomass and model biomass concentrations. On these samples, three biomass growth experiments were carried out to monitor changes in biomass composition and concentration throughout non-limited growth. Bacterial cell volume distribution in wastewater In order to determine the total cell biomass in wastewater, cell volume distributions were measured on AO stained samples and mean cell volumes determined. Distributions of cell volumes were measured on seven dierent wastewater samples (Table 1). Cells with volumes above 1 mm3 were discarded because these determinations most likely arise from erroneous counting of cell agglomerates or eucaryotic cells (Fry, 1988). Between 2 and 10% of the counts gave cell volumes above 1 mm3. In the sampled wastewater, cells generally were single and cell agglomerates were small and infrequent. Cell volumes were not normally distributed, and a relatively large number of cells were smaller than the mean cell volume. An example showing a cell volume distribution illustrates this (Fig. 2). Generally, the median was about 0.22 mm3 and the mean was about 0.29 mm3 (Table 1).

Fig. 2. Distribution of AO cell volumes in wastewater. Mean cell volume 0.321 mm3 (cf. Table 1).

Biomass growth In three experiments performed on wastewater sampled on two dierent occasions in Aalborg, Denmark, the concentration of biomass was followed during several hours of substrate non-limited growth. The growth experiments were conducted at 288C in the batch reactors for OUR measurements. In initial studies, the naturally available, readily biodegradable substrates in wastewater were found to be insucient to induce an increase in the bacterial number large enough to be detected by cell counts. Therefore, acetate was added to enhance the growth. Preliminary studies showed that the batch became nutrient limited after acetate consumptions of 10001500 g m3. To avoid nutrient limitation, approximately 0.6 g (NH4)2SO4 and 0.2 g KH2PO4 were added per gram of acetate.

Table 1. Cell volumes determined on seven dierent AO-stained wastewater samples Mean (mm3) 25% 0.242 0.321 0.211 0.293 0.321 0.302 0.307 Average Std. dev. 0.285 0.042 0.095 0.114 0.074 0.137 0.136 0.135 0.121 0.116 0.024 Percentiles (mm3) 50% 0.180 0.237 0.166 0.235 0.258 0.235 0.235 0.221 0.034 75% 0.315 0.482 0.278 0.421 0.457 0.427 0.461 0.409 0.078

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Transformation between units Biomass in wastewater samples was characterized using the described methods. However, XB was found as a COD concentration and the cell biomass as a cell number. Comparing the dierent methods, cell numbers were converted into cell COD and vice versa, applying a mean cell volume and an estimated COD content per cell volume. The actual cell volume distribution was measured in several cases. In experiments where the cell volume distribution had not been measured, the average cell volume of 0.285 mm3 found in this study was used (Table 1). The conversion factor between cell volume and cell COD was 0.37 g COD (ml cell)1. The factor was calculated from an estimated cell density of 1.05 g ml1, an estimated dry matter content of 25% and an estimated ratio between COD and organic matter of 1.42 g COD (g organic matter)1. Furthermore, the inorganic fraction of the cell dry matter was assumed to be insignicant (Bakken and Olsen, 1983; Fry, 1988; McCarty, 1964).

DAPI cell biomasses were closer to XB , 86 and 69% of XB respectively (Fig. 3). An average of 32% of the DAPI cell biomass was found as LD dead cells. This percentage was rather constant with a standard deviation of only 9%. The best correlations between XB and cell biomass were found with AO, DAPI and LD life cell biomass, while poorer correlations were found with LD dead, CTC and MAR cell biomass (Fig. 4). Cell respiration XB was found from simulation of wastewater OUR, i.e. from the mean respiration activity of the biomass in the wastewater. The respiration rates based on AO, DAPI, LD life, CTC and MAR cell biomasses were calculated and are presented in Table 2. The variability of the cell respiration rates was generally low, indicating a rather constant cell respiration rate in the wastewater sampled. However, the variability of respiration rates for CTC cell biomass was signicantly higher than that for other cell biomasses. Biomass growth on acetate as substrate The concept dening XB (Fig. 1) predicts that the biomass concentration increases exponentially whenever readily biodegradable substrate is present in sucient concentrations. The validity of this aspect of the concept was tested in three experiments, where acetate was added to wastewater in the batch reactors in which OUR was measured. The increases in AO cell biomass, in OUR and the decrease in acetate concentrations were followed for 9 h throughout the growth periods. The OUR of the wastewater increased exponentially for the rst 4 h approximately, and OUR increased about 8-fold. After 4 h, the rate of OUR increase declined, and after about 6 h, OUR decreased rapidly (Fig. 5). The explanation for OUR not continuing its exponential increase after the rst 4 h might be growth limitations due to the lack of nutrients other than N, P and K (which were added).

Biomass determination in wastewater Cell biomass and XB in domestic wastewater were determined on 13 samples, 11 of which were collected from dry weather runo and 2 were collected from combined stormwater runo. For all samples AO, DAPI, CTC, LD life and LD dead cell biomasses were determined. Furthermore, XB was calculated from OUR measurements (equation (1)). Cells taking up radioactive acetate (MAR) were determined in 3 of the 11 dry weather runo samples (Fig. 3). The combined stormwater samples were strongly diluted and found to contain about 10% of the amount of biomass found in the dry weather runo samples. The biomass determinations on the 11 dry weather runo samples showed the cell biomass to yield lower values than those predicted by XB . CTC cell biomass yielded 15% of XB , and the 3 MAR cell biomass determinations yielded on average 40% of the corresponding XB . Hence, XB seemed to overestimate the active cell biomass by a factor of 27. AO and

Fig. 3. Average determinations of XB and cell biomasses on all 11 dry weather runo samples and on the 3 samples, where also MAR was determined. The average COD of these samples was 580 gCOD m3.

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Fig. 4. Correlation between XB and cell biomass (AO, DAPI, LD, CTC: 13 samples, MAR: 3 samples). a is the slope of the regression and r is the correlation coecient (r).

Table 2. Mean respiration rates of cell biomass based on AO, DAPI, LD life, CTC (13 samples) and MAR (3 samples). The unit is 1015 g O2 cell1 h1 Mean AO DAPI LD life CTC MAR 33 42 61 404 105 Std. dev. 11 16 22 602 49

looking at absolute increases, both AO and DAPI cell counts increased by the same number during the growth periods. Furthermore, the increase in LD life cells could account for the increases in AO and DAPI-stained cell biomass, whereas the number of dead cell biomass hardly changed throughout the growth experiments. However, the COD mass balance during the growth period was not fullled. That is the sum of DO uptake and XB did not equal the acetate consumed: 4053% of the consumed acetate remained unaccounted for (Table 3).

Parallel experiments without acetate addition (blinds) showed that initial concentrations of readily biodegradable substrate present in the wastewater were insignicant compared to the added acetate concentrations. Therefore, the modeled readily biodegradable substrate was equivalent to the added acetate. When AO cell biomass was compared to XB during the growth experiments (Fig. 6), the deviation between the two measures of biomass is obvious. The same deviation is observed when other staining methods were compared with XB (Table 3). CTC, DAPI and LD cell biomasses were determined only at the beginning and the end of the three growth periods. AO cell biomass did not increase detectable during the rst 23 h, while acetate uptake rate and OUR increased exponentially in the same period. As mean cell sizes were the same at the beginning and the end of the growth phase, the cell biomass was therefore not proportional to the biomass activity as measured by acetate and oxygen consumption (Figs 5 and 6). Furthermore, as XB was measured by the biomass activity, cell biomass was not proportional to XB . The methods applied for cell biomass determination showed a consistent picture of the biomass:

DISCUSSION

Of the two methods for the determination of total cell numbers, DAPI generally is considered to be the most correct and AO is considered to overestimate the true number slightly (Cloete and Muyima, 1997). Among the cells counted with DAPI, some could be dead or damaged, some inactive and some active (Karner and Fuhrman, 1997). LD, CTC and MAR should reect these fractions. LD life is expected to reect the living cells}active as well as inactive} and MAR should yield the fraction showing active acetate uptake under the conditions tested. That is MAR counts aerobic, heterotrophic, acetate consuming cells, where DAPI also includes dead, inactive, anaerobic and autotrophic cells. XB }applied for solving engineering problems} and the cell biomass}used in studies of environmental microbiology}showed a good correlation (Figs 3 and 4). It was expected that the best agreement with XB would be with MAR, because MAR measures cells which take up a specic organic substrate, and organic substrate uptake rate is proportional to XB when substrate is not limiting

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Fig. 5. Removal of acetate in wastewater. Measured and simulated OURs and acetate concentrations.

Fig. 6. Addition of acetate to wastewater. Measured AO cell biomass and calculated XB .

Table 3. Increase in biomass, acetate consumed and DO consumed during the growth phase (Figs 5 and 6). Units are g COD m3 and g O2 m3 respectively Model biomass Mean cell size (mm3) AO A B C 743 583 885 0.30 ! 0.34 0.31 ! 0.30 0.31 ! 0.35 38 56 50 DAPI 45 45 42 Cell biomasses LD life 44 43 42 LD dead 0 2 0 CTC 11 39 59 1543 733 1357 683 360 768 Acetate consumed DO consumed

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the growth process. Ideally, CTC should also be in good agreement with XB , because CTC is used to identify actively respiring cells. Therefore, CTC and MAR should yield identical biomasses. However, the CTC cell biomass found was only one third of the MAR cell biomass. The poor agreement between CTC and MAR conrmed results from other studies that CTC is a rather unreliable method of detecting active cells. The reasons for this are, however, not known (Ullrich et al., 1996; Nielsen, unpublished). There may be several reasons why XB overestimated the active biomass. Critical parameters in calculating XB are the heterotrophic yield (YH ) and the maintenance energy requirement rate constant (qm ). These parameters have been chosen on the basis of previous ndings but could have been determined according to Vollertsen and HvitvedJacobsen (1999). YH was 0.65 with a standard deviation of 0.05 and qm was 1.6 d1 with a standard deviation of 0.4 d1 (Vollertsen and Hvitved-Jacobsen, 1999). Dierences in YH and qm will result in the calculation of dierent values for XB (equation (1)). Using the parameter mean values plus or minus the standard deviations as a rough indication of XB determination accuracy, it was found that the average XB varied by only 25%, due to parameter uncertainty. Another explanation of the discrepancy between XB and cell biomass could be the conversion from the unit of cells ml1 to the unit of g COD m3. For this conversion, the parameters cell density, cell organic matter content and COD of the cell organic matter were estimated on the basis of values from the literature. Furthermore, as XB is dened from the total turnover of substrate, XB does not only cover cell biomass, but also extracellular polymers (EPS) formed during the growth of the biomass. The extent of polymer production by the wastewater biomass is not known, but in sewer biolms, EPS has been found to account for 5080% of the total biolm organic matter (Nielsen et al., 1997). However, not all EPS produced are particulate polymers. Also a production of soluble EPS components, often called soluble microbial products (SMP), probably takes place. In biological wastewater treatment systems, this fraction is not unimportant (Barker and Stuckey, 1999), but in sewer systems, the extent of SMP formation is unknown. These uncertainties, and the fact that EPS and SMP are a part of XB , could explain why XB was higher than the cell biomass. The cell volumes found in wastewater (Table 1) were comparable to cell volumes found in other systems. For soil bacteria, Bloem et al. (1995) reported average cell volumes in the range of 0.25 0.41 mm3 and Bjrnsen (1986) reported average cell volumes in the range of 0.190.34 mm3 for freshwater bacterioplankton. Frlund et al. (1996) found average cell volumes in activated sludge of 0.25 mm3. For sewer biolms, Jahn and Nielsen (1998) reported mean cell volumes in the range of 0.130.37 mm3.

The respiration rates found for wastewater biomass (Table 2) corresponded well with studies where mono cultures have been grown at high growth rates. Values reported are e.g. 0.7 g O2 (g cell dry weight)1 h1 for Klebsiella aerogenes grown in a chemostat (Tempest and Neijssel, 1984) and 0.55 g O2 (g cell dry weight)1 h1 for Klebsiella pneumoniae also grown in a chemostat (Tempest and Neijssel, 1992). Pseudomonas aeruginosa grown in a biolm was reported with 2.2 g O2 (g cell dry weight)1 h1 (Turakhia and Characklis, 1989). Assuming mean cell sizes and dry matter contents as in this study and furthermore assuming that the measured cell dry weight was cell biomass only, the corresponding single-cell respiration rates are 52 1015, 41 1015 and 165 1015 g O2 cell1 h1, respectively. However, as extracellular matter probably was present in the cultures, these values underestimate the real cell respiration rates. In this study cell respiration rates (MAR) were found to 105 g O2 cell1 h1 (Table 2). Cell activity In Table 4, it is assumed that all cells are equally active. As the relative increase in cell number was smaller than the relative increase in oxygen uptake, it follows that the specic cell respiration rate increased substantially, regardless of the staining method applied. This was not due to a change in cell sizes, indicating that the cells found in the wastewater had achieved their maximum sizes already. Furthermore, their oxygen and acetate uptake activity did not show a lag phase prior to the exponential increase in activity, indicating that the cells were actively growing. Therefore, it seems unrealistic that the specic biomass respiration rates should increase as dramatically as shown in Table 4. For the same reasons it also seems unreasonable to assume that a lot of cells should change from an almost inactive state to a highly active state during the experiments. The methods applied for cell biomass determination cannot quantify the actual activity on single cell basis, and it seems realistic to assume that not all the cells were equally active. Some cells are known to be inactive or dead, and some are probably rather slow growing (Fig. 7). A rough estimation of the fraction of cells which is fast growing can be made on the basis of the growth experiments performed. For this purpose, it is necessary to assume that the substrate

Table 4. Absolute increase in the specic cell respiration rate from the start of a growth experiment until the end of the growth phase (Figs 5 and 6). All cells counted are assumed to be active. The unit is 1015 g O2 cell1 h1 AO A B C 38 ! 334 37 ! 204 37 ! 314 DAPI 50 ! 353 57 ! 653 57 ! 407 LD life 48 ! 367 55 ! 240 55 ! 357 CTC 139 ! 1200 193 ! 415 193 ! 416

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Fig. 7. Growth of wastewater biomass.

uptake of the slow growing cells is negligible, that the fast growing cells are all growing at identical rates, and that they have a constant oxygen uptake rate per cell. In this case equation (3) is valid, i.e. the entire oxygen uptake at both time t0 and time t1 (Fig. 7) is due to the fast growing cells, and the OUR is proportional to this biomass. OURt0 DAPIt0 DAPIinactive OURt1 DAPIt1 DAPIinactive 3

Using equation (3) on data from the growth experiments, an average of 16% (standard deviation 5%) of the total DAPI cell biomass is found to be fast growing cells. This simplication of the real situation shows that XB overestimated the (highly) active biomass by roughly a factor 10, as the DAPI count on average was 69% of XB (Fig. 3). Errors in the assumed conversion factors, model parameter uncertainties and formation of extracellular matter might explain the resulting error in the COD mass balance. Another consequence is that the cell respiration rate for the fast growing cells becomes about 260 1015 g O2 cell1 h1, i.e. somewhat higher than the reported cell respiration rates for pure cultures. The model concept The fact that there is an obvious conict between XB and the active cell biomass leads to the question of whether and how the model concept should be modied to accommodate this new understanding. Active and inactive cell biomass and production of EPS and SMP could be taken into account in the model. However, the model was developed as an engineering tool for characterization of wastewater COD and for simulation of in-sewer transformations of organic matter. It is a tool for design and evaluation of sewer systems. For these purposes, the concept must be rather simple and simple experimental techniques must be applied to determine model parameters and to calibrate the model.

Electron donor and electron acceptor uptake have been observed to behave as predicted by the model when applied under conditions normally observed in sewers. Furthermore, Vollertsen and Hvitved-Jacobsen (1999) could determine model parameters and validate the model concept based on long-term OUR measurements and acetate additions. When applied for simulation of in-sewer transformation of organic matter, this concept could be validated under eld conditions (Hvitved-Jacobsen and Vollertsen, unpublished). Moreover, the concept has been calibrated and implemented as a design tool under various conditions (Stemplewski et al., 1999; Tanaka et al., 1998). The critical term is the denition of XB . It is shown in this study that XB }even though it is based on biomass respiration activity}is not equivalent to active cell biomass as used in the microbiological terminology. Instead XB should be considered a measure of biomass activity and substrate ows equivalent to a certain COD fraction and not a genuine concentration of bacteria. This equivalent COD concentration is in reasonable agreement with the COD that the total cell biomass represents. From a model point of view, this is important}and in many cases sucient}as it allows the establishment of a fundamental environmental engineering requirement: the COD mass balance. In environmental engineering, information about the overall biomass/substrate transformations and wastewater quality changes is the objective. The understanding of XB as a measure of biomass activity representing an equivalent COD concentration is considered a realistic and sucient description of the involved biomass for engineering purposes, e.g. sewer design and interactions between sewer, treatment plant and receiving water.

CONCLUSION

The conceptual understanding of XB in wastewater based on OUR measurements and applied for characterization of in-sewer wastewater transformation processes correlated well with cell biomass determined by microbial enumeration methods. Total DAPI cell biomass was on average determined to be 69% of the biomass predicted by the model. Observed exponential increases with time in uptake rates of oxygen and acetate in wastewater were not reected by an exponential increase in cell biomass. The results indicated that only a smaller fraction of the total cell biomass was highly active. As this phenomenon is not included in the present model concept, it is important to distinguish clearly between the terms model biomass and cell biomass. Despite the limitations, the concept dening XB has been successfully validated and implemented in

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other studies, including eld experiments. For engineering applications, model components and model parameters which cannot be identied by simple experimental techniques should not be introduced. Therefore, it is suggested that the rather simple model of the substrate ows in wastewater is maintained and XB seen as a measure of biomass activity representing an equivalent COD concentration. A basic engineering requirement, the mass balance, is thereby maintained.

REFERENCES

Andreasen K. and Nielsen P. H. (1997) Application of microautoradiography to the study of substrate uptake by lamentous microorganisms in activated sludge. Appl. Environ. Microbiol. 63(9), 36623668. APHA (1995) Standard Methods for the Examination of Water and Wastewater, 19th ed. APHA, AWWA, WEF Washington. Bakken L. R. and Olsen R. A. (1983) Buoyant densities and dry-matter contents of microorganisms: Conversion of a measured biovolume into biomass. Appl. Environ. Microbiol. 45(4), 11881195. Barker D. J. and Stuckey D. C. (1999) A review of soluble microbial products (SMP) in wastewater treatment systems. Water Res. 33(14), 30633082. Bjerre H. L., Hvitved-Jacobsen T., Teichgra ber B. and Heesen D.te (1995) Experimental procedures characterizing transformations of wastewater organic matter in the Emscher River, Germany. Water Sci. Technol. 31(7), 201212. Bjerre H. L., Hvitved-Jacobsen T., Teichgra ber B. and Schlegel S. (1998) Modeling of aerobic wastewater transformations under sewer conditions in the Emscher River, Germany. Water Environ. Res. 70(6), 11511160. Bjrnsen P. K. (1986) Automatic determination of bacterioplankton biomass by image analysis. Appl. Environ. Microbiol. 54(6), 11991204. Bloem J., Veninga M. and Shepherd J. (1995) Fully automatic determination of soil bacterium numbers, cell volumes, and frequencies of dividing cells by confocal laser scanning microsopy and image analysis. Appl. Environ. Microbiol. 61(3), 926936. vost M., Barbeau B., Coallier J. and Boulos L., Pre Desjardins R. (1999) LIVE/DEAD1 BacLightTM : application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J. Microbiol. Methods 37(1), 7786. Carman K. R. (1990) Radioactive labeling of a natural assemblage of marine sedimentary bacteria and microalgae for the trophic studies: an autoradiographic study. Microbial Ecol. 19(3), 279290. Christon J. H. (ed.) (1997) Manual of Environmental Microbiology, p 894. ASM Press Washington, DC. Cloete T. E. and Muyima N. Y. O. (1997) Microbial community analysis: the key to the design of biological wastewater treatment systems. IAWQ Scientic and Technical Report No. 5. Frlund B., Palmgren R., Keiding K. and Nielsen P.H. (1996) Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Res. 30(8), 17491758. Fry J. C. (1988) Determination of biomass. In Methods in Aquatic Bacteriology, ed. B. Austin, pp. 2772. Wiley, New york. Fuhrman J. A. (1981) Inuence of method on the apparent size distribution of bacterioplankton cells: epiuoriscence microscopy compared to scanning electron microscopy. Mar. Ecol. Progr. Ser. 5(1), 103106.

Griebe T., Schaule G., Secker J. and Flemming H. C. (1996) Bestimmung der stowechselaktiven Bakterien im Be kologie der Abwasserorganismen, eds lebtschlam. In: O H. Lemmer, T. Griebe and H.-C. Flemming. Springer, Germany. Hobbie J., Daley R. J. and Jasper S. (1977) Use of Nucleopore lters for counting bacteria by epiourescence microscopy. Appl. Environ. Microbiol. 33, 12251228. Hvitved-Jacobsen T. and Vollertsen J. Contact: T. HvitvedJacobsen, Environmental Engineering Laboratory, Aalborg University, Sohngaardsholmsvej 57, 9000 Aalborg, Denmark, (unpublished). Hvitved-Jacobsen T., Vollertsen J. and Nielsen P. H. (1998) A process and model concept for microbial wastewater transformations in gravity sewers. Water Sci. Technol. 37(1), 233241. Jahn A. and Nielsen P. H. (1998) Cell biomass and exopolymer composition in sewer biolms. Water Sci. Technol. 37(1), 1724. Kappeler J. and Gujer W. (1992) Estimation of kinetic parameters of heterotrophic biomass under aerobic conditions and characterisation of wastewater for activated sludge modelling. Water Sci. Technol. 25(6), 125139. Karner M. and Fuhrman T. (1997) Determination of active bacterioplankton: a comparison of universal 16S rRNA probes, autoradiography, and nucleoid staining. Appl. Environ. Microbiol. 63(4), 12081213. Krambeck C., Krambeck H. -J. and Overbeck J. (1981) Micro-computer-assisted biomass determination of plankton bacteria on scanning electron micrographs. Appl. Environ. Microbiol. 42(1), 142149. McCarty P. L. (1964) Thermodynamics of biological synthesis and growth. Second International Conference on Water Pollution Research, pp. 169199. Molecular Probes (1998) LIVE/DEAD1 BacLightTM Bacterial Viability Kits. Product Information Sheet, Molecular Probes 07007 05/15/98. Nielsen P. H. Contact: P. H. Nielsen, Environmental Engineering Laboratory, Aalborg University, Sohngaardsholmsvej 57, 9000 Aalborg, Denmark, (unpublished). Nielsen P. H., Jahn A. and Palmgren R. (1997) Conceptual model for production and composition of exopolymers in biolms. Water Sci. Technol. 36(1), 1119. Nielsen J. L. and Nielsen P. H. Enumeration of acetateconsuming bacteria under oxygen- and nitrate respiring conditions in activated sludge by application of microautoradiography. Water Res. (submitted for publication). Raunkjr K., Hvitved-Jacobsen T. and Nielsen P.H. (1995) Transformation of organic matter in a gravity sewer. Water Environ. Res. 67(2), 181188. Rodriguez G. G., Phipps D., Ishiguro K. and Ridgway H. F. (1992) Use of a uorescent redox probe for direct visualization of actively respiring bacteria. Appl. Environ. Microbiol. 58(6), 18011808. Signal Analytics Corporation (1994) IP Lab Spectrum version 2.5.5, Users Guide. Signal Analytics Corporation, 19891994. Stemplewski J., Schlegel S., Stein A., Geisler W., Schmelz K. -G., Hvitved-Jacobsen T. and Vollertsen J. (1999) Restructuring the Emscher system. Proceedings of the 11th EWPCA (the European Water Pollution Control Association) Symposium: Sewerage Systems}Cost and Sustainable Solutions, Munich, Germany, May 46, 1999, p. 14 Tanaka N., Hvitved-Jacobsen T., Ochi T. and Sato N. (1998) Aerobic/anaerobic microbial wastewater transformations and reaeration in an air-injected pressure sewer. Proceedings for the 71st Annual Water Environment Federation Conference & Exposition, WEFTEC 98, Orlando, FL, USA. Vol. 2, 37 October 1998, pp. 853864.

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Jes Vollertsen et al. zolium chloride. Appl. Environ. Microbiol. 62(12), 45874593. Vollertsen J., Almeida M. C. and Hvitved-Jacobsen T. (1999) Eects of temperature and dissolved oxygen on hydrolysis of sewer solids. Water Res. 33(14), 31193126. Vollertsen J. and Hvitved-Jacobsen T. (1998) Aerobic microbial transformations of resuspended sediments in combined sewers-a conceptual model. Water Sci. Technol. 37(1), 6976. Vollertsen J. and Hvitved-Jacobsen T. (1999) Stoichiometric and kinetic model parameters for microbial transformations of suspended solids in combined sewer systems. Water Res. 33(14), 31273141.

Tempest D. W. and Neijssel O. M. (1984) The status of YATP and maintenance energy as biological interpretable phenomena. Annu. Rev. Microbiol. 38, 459486. Tempest D. W. and Neijssel O. M. (1992) Physiological and energetic aspects of bacterial metabolite overproduction. FEMS Microbiol. Lett. 100(13), 169176. Turakhia M. H. and Characklis W. G. (1989) Activity of Pseudomonas aeruginosa in biolms: eect of calcium. Biotechnol. Bioeng. 33(4), 406414. Ullrich S., Karrasch B., Hoppe H. -G., Jeskulke K. and Mehrens M. (1996) Toxic eects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl tetra-