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Avai lab le a t www.sc iencedi rec t .com

journa l homepage : www.e lsev ie r . com/ loca te /wat res

Direct quantification of bacterial biomass in influent, effluentand activated sludge of wastewater treatment plants by usingflow cytometry

P. Foladori a,*, L. Bruni b, S. Tamburini a, G. Ziglio a

aDepartment of Civil and Environmental Engineering, University of Trento, via Mesiano, 77, 38050 Trento, ItalybAgenzia per la Depurazione, Provincia Autonoma di Trento, via Pozzo, 6, 38100 Trento, Italy

a r t i c l e i n f o

Article history:

Received 3 October 2009

Received in revised form

25 February 2010

Accepted 26 April 2010

Available online 29 April 2010

Keywords:

Bacterial biovolume

Bacterial biomass

Activated sludge

Wastewater

Flow cytometry

Viability

Activity

* Corresponding author. Fax: þ39 461 882672E-mail addresses: paola.foladori@ing.uni

Tamburini), giuliano.ziglio@ing.unitn.it (G. Z0043-1354/$ e see front matter ª 2010 Elsevdoi:10.1016/j.watres.2010.04.027

a b s t r a c t

A rapid multi-step procedure, potentially amenable to automation, was proposed for

quantifying viable and active bacterial cells, estimating their biovolume using flow

cytometry (FCM) and to calculate their biomass within the main stages of a wastewater

treatment plant: raw wastewater, settled wastewater, activated sludge and effluent.

Fluorescent staining of bacteria using SYBR-Green I þ Propidium Iodide (to discriminate

cell integrity or permeabilisation) and BCECF-AM (to identify enzymatic activity) was

applied to count bacterial cells by FCM. A recently developed specific procedure was

applied to convert Forward Angle Light Scatter measured by FCM into the corresponding

bacterial biovolume. This conversion permits the calculation of the viable and active

bacterial biomass in wastewater, activated sludge and effluent, expressed as Volatile

Suspended Solids (VSS) or particulate Chemical Oxygen Demand (COD). Viable bacterial

biomass represented only a small part of particulate COD in raw wastewater (4.8 � 2.4%),

settled wastewater (10.7 � 3.1%), activated sludge (11.1 � 2.1%) and effluent (3.2 � 2.2%).

Active bacterial biomass counted for a percentage of 30e47% of the viable bacterial

biomass within the stages of the wastewater treatment plant.

ª 2010 Elsevier Ltd. All rights reserved.

1. Introduction thousands of single bacterial cells with mean sizes smaller

The most widely applied treatments of municipal wastewater

are based on biological processes, such as activated sludge, in

which the degradation of pollutants occurs under aerobic or

anoxic conditions mainly by proteobacteria from the alpha,

beta, or gamma subclass (Wagner et al., 1993). Microorgan-

isms, colloids, organic polymers and cations take part in the

complex formation of activated sludge flocs. Flocs are typi-

cally distinguished as small (apparent diameter <150 mm),

medium (150e500 mm) and large (>500 mm) (Jorand et al., 1995;

Jenkins et al., 1993) and are made up of several tens of

.tn.it (P. Foladori), laura.biglio).ier Ltd. All rights reserved

than micrometers (Frølund et al., 1996; Vollertsen et al., 2001;

Foladori et al., 2008).

The most common parameter used for quantifying acti-

vated sludge is the content of suspended solids, expressed as

Total Suspended Solids (TSS) or Volatile Suspended Solids

(VSS). However, VSS do not coincide with the effective

bacterial biomass in activated sludge because VSS also include

endogenous biomass (the residue produced by bacterial death

and lysis) and organic non-biotic particulate matter fed into

the plant with the influent wastewater. The bacterial biomass

in activated sludge (usually called active biomass) is generally

runi@provincia.tn.it (L. Bruni), sabrina.tamburini@ing.unitn.it (S.

.

AIRETIRC TSET NOISIVID LLEC YTIVITCA CILOBATEMENARBMEM

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Fig. 1 e Relationship of physiological states of cells

according to Nebe-von-Caron et al. (2000).

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 0 7e3 8 1 83808

estimated by theoretical calculations based on substratemass

balances using kinetic and stoichiometric parameters (Ekama

et al., 1984; Henze et al., 2000). On the other hand, knowledge

of the actual amount of bacterial biomass in an activated

sludge system is a key parameter to understanding the

processes, kinetics and dynamics of substrate removal.

Bacterial biomass in raw wastewater influent in a waste-

water treatment plant (WWTP) is rarely quantified and its

content is often neglected in mass balances, due to its low dry

mass. Finally, in treated effluents, only the quantification of

faecal indicators like Escherichia coli, total coliforms, strepto-

cocci, etc. is generally considered, and then only to evaluate

disinfection performance.

Several in-depth investigations have been referred to in the

literature on the microbiology of activated sludge and

influent/effluent wastewater (inter alia Gilbride et al., 2006),

but no routine methods are currently available to rapidly

quantify the bacterial biomass in activated sludge and

wastewater. The major barriers relate to:

1) the highly aggregated structure of activated sludge, in

which bacteria are embedded, disturbs microscopic

images, limiting the efficiency of direct cell counting,

2) the difficulty in disaggregating activated sludge flocs due to

the risk of disrupting the cells and losing their viability,

3) the need to convert the number of bacteria into an equiv-

alent mass of bacteria expressed as dry weight,

4) the fact that the quantification of bacteria is often based on

observations under the microscope; this approach is much

too time-consuming, requiring several hours to quantify

and measure bacteria in each sample.

Early investigations which aimed to recover bacteria from

activated sludge for quantification were based on cultivation

methods (Williams et al., 1971; Banks and Walker, 1977). In

recent decades it has been demonstrated that the culture-

dependentanalysisofmicrobialpopulations inactivatedsludge

producespartial andheavilybiasedresults (Wagneretal., 1993).

To obtain a more accurate view of bacterial populations, the

application of in-situ techniques or direct molecular

approaches is needed. With regard to the recovery of bacteria

from complex matrices such as sludge in order to count them,

sonication has been proposed in several studies, to maintain

cell viability (Falcioni et al., 2006; Foladori et al., 2007) or to

induce cell lysis and bacteria inactivation (Zhang et al., 2007).

The advantages of associating flow cytometry (FCM) with

fluorescent molecular probes for the rapid quantification of

viable, active or dead cells in bacterial suspensions have been

underlined many times throughout the environmental field

(inter alia Porter et al., 1997; Steen, 2000; Vives-Rego et al., 2000;

Bergquist et al., 2009). FCM, capable of counting more than

1000 cells s�1, is a powerful single-cell analysis that can

provide a direct, rapid and precise quantification of free cells

in a suspension. FCM provides a multi-parametric analysis,

recording two or more fluorescence signals and two light

scattering signals simultaneously for each bacterial cell. With

respect to the conventional observation of bacterial suspen-

sions under the epifluorescence microscope (EM), FCM anal-

ysis is faster, gives more reliable results and can analyse

a greater number of samples per day.

The physiological status of cells (integrity, death, activity

and reproductive growth) can be described taking into account

the scheme in Fig. 1 introduced by Nebe-von-Caron et al.

(2000). Membrane integrity demonstrates the protection of

constituents in intact cells and the capability of metabolic

activity/repair and potential reproductive growth. Cells

without an intact membrane are considered as permeabilised

and can be classified as dead cells. As their structures are

freely exposed to the environment they will eventually

decompose (Nebe-von-Caron et al., 2000). The concept of

“viability” is defined on the basis of this scheme assuming that

viable cells are intact cells.

Viable and dead cells are identified simultaneously on the

basis of their membrane integrity applying a double fluores-

cent staining, such as Propidium Iodide (a dye which only

enters permeabilised cells, identified as dead) and SYBR-

Green I (which enters all cells, both intact or permeabilised)

(Ziglio et al., 2002). Metabolic activity is a more restrictive

condition, because it requires cells to demonstrate one of the

following functions: biosynthesis, pump activity, membrane

potential or enzyme activity. Among these, enzymatic activity

can be identified by using fluorogenic substrates such as

BCECF-AM and FDA (Ziglio et al., 2002), FDG (Plovins et al.,

1994) or others, which release, after hydrolysis, fluorescein-

based compounds, retained inside cells which become

fluorescent.

The direct fluorescent staining of bacteria in activated

sludge distinguishes them from other constituents of biolog-

ical flocs such as non-biotic organic and inorganic particles,

which constitute most of the total solid content.

This paper aims to apply a procedure based on FCM for the

rapid and direct quantification of viable, dead or active

bacteria, their biovolume and biomass in raw wastewater,

settled wastewater, activated sludge and effluent. The use of

FCM in conjunction with fluorescent dyes was proposed to

count bacteria in activated sludge by Falcioni et al. (2006), but

the quantification of bacterial biomass in sludge and waste-

water by FCM is new. In particular, in this study FCM was

coupled with the fluorescent staining of cells to identify

viability, death and activity of bacterial cells in flocs, previ-

ously disaggregated by applying mechanical dispersion and

ultrasonication, with the aim of obtaining a single-cell

suspension while maintaining cell integrity. A specific proce-

dure was used for the first time to convert the number of

bacterial cells into an equivalent biomass, expressed as dry

weight, taking into account the bacterial biovolume estimated

from FCM. This FCM method was finally applied to several

samples of raw and pre-settled wastewater, activated sludge

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 0 7e3 8 1 8 3809

and effluents, with the aim of quantifying viable and active

bacterial biomass and to calculate the biomass fraction then

compared to conventional parameters like TSS and COD

(Chemical Oxygen Demand). The use of FCM permits rapid

analyses (a few minutes) and the analysis of dozens of

samples per day.

2. Materials and methods

2.1. Outline of the entire procedure for viable and activebiomass measurement

Fig. 2 shows the whole 10-step procedure. Briefly, samples

underwent a dilution with PBS, pre-treatment to disperse

coarse aggregates (optimal pre-treatment was ultra-

sonication, see Section 3.1), a second dilution and filtration

using 20-mm-filters to separate coarse particles which could

have clogged the nozzle of the flow cytometer. The resulting

suspension was stained with fluorescent dyes (SYBR-Green

I þ Propidium Iodide) or a fluorogenic substrate (BCECF-AM,

for non-specific intracellular esterases), for the assessment of

viable or active biomass respectively. The Forward Angle Light

Scatter (FALS) signal obtained from FCM was then converted

into cellular biovolume. To convert FALS into biovolume,

a calibration curve calculated between silica beads of known

volume (with refractive index similar to bacteria) and corre-

spondent FALS intensity was used, according to the procedure

recently proposed by Foladori et al. (2008) and developed

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Fig. 2 e Outline of the 10-step procedure based on FCM for the q

sludge and effluent.

specifically for bacteria in activated sludge and wastewater.

Finally the viable and active biomass of bacteria in the sample

were calculated (expressed as VSS or particulate COD).

Statistics to compare differences between groups were

calculated by applying t-test; a difference was considered as

significant when P < 0.05.

2.2. Sampling

Grab samples of raw wastewater, settled wastewater, acti-

vated sludge and effluent were collected from the municipal

wastewater treatment plant (WWTP) of Trento Nord (Italy).

The activated sludge stage operated at a specific organic load

of 0.15 kg BOD5 kg TSS�1 d�1, with a sludge age of about 12

days and a mean VSS/TSS ratio of 0.78. A synthesis of the

conventional parameters used to characterise wastewater,

activated sludge and effluent is indicated in Table 1.

Samples were collected and processed in the lab within

1e2 h of collection. Wastewater, activated sludge and effluent

were diluted 1:20 v/v, 1:400 v/v and 1:1 v/v respectively, using

Phosphate-Buffered-Saline (PBS, 3 g K2HPO4, 1 g KH2PO4 and

8.5 g NaCl L�1; pH ¼ 7.2) in order to reach a concentration of

bacteria of around 106e107 cells/mL, which is optimal for FCM

analysis.

2.3. Chemical analyses

TSS, VSS, total and soluble COD were measured according to

Standard Methods (APHA, 1995). Soluble COD was measured

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uantification of bacterial biomass in wastewater, activated

Table 1 e Parameters characterising organic content ofwastewater, activated sludge and effluent in the WWTP.The mean values and standard deviations are indicated.

Total COD[mg/L]

Soluble COD[mg/L]

VSS[mg/L]

Raw wastewater 721 � 212 124 � 25 404 � 137

Settled

wastewater

330 � 97 112 � 33 147 � 47

Activated sludge 4555 � 200 36 � 5 3053 � 138

Effluent 30 � 6 18 � 2 8 � 4

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 0 7e3 8 1 83810

after filtration on a 0.45-mm-membrane. Particulate COD was

quantified as the difference between total and soluble COD.

2.4. Mechanical dispersion

5-mL sub-samples of diluted activated sludge suspensions

underwent 15 min of mechanical treatment using an IKA-

Ultra-Turrax T8 (IKA Labortechnik, Staufen, Germany),

according to the method proposed by Ziglio et al. (2002).

2.5. Ultrasonication

Ultrasonicationwas applied to 100-mL sub-samples of various

suspensions by using a Branson 250 Digital Ultrasonifier

operating at 20 kHz. The device is equipped with a horn tip

and a temperature probe. The tip (diameter 12.7 mm), was

placed in the centre of the 150-mL glass tubes, containing 100-

mL samples. The main parameters (transferred power, P;

time, t; treated volume, V) were used to calculate the trans-

ferred specific energy as reference parameter, indicated

afterward as Es and expressed in kJ L�1 (Es ¼ P � t/V). Trans-

ferred power instead of applied power has to be used for Escalculation, in order to obtain results comparable with results

from different instruments. In this research, the method

based on calorimetry was used to measure the actual power

input (Mason et al., 1992).

2.6. Fluorescent staining of bacteria

To distinguish viable and dead bacteria, the pre-treated

suspensions were stained with SYBR-Green I (SYBR-I, 1:30

dilution of commercial stock; from Invitrogen, USA;

lex ¼ 495 nm, lem ¼ 525 nm) diluted in dimethyl sulfoxide

(DMSO, Merck, Germany) and Propidium Iodide (PI, stock

solution concentration 1 mg mL�1; Invitrogen, USA;

lex ¼ 536 nm, lem ¼ 617 nm). An amount of 10 mL of both

fluorochromes was added to 1 mL of bacterial suspension

containing about 106e107 cells/mL. Sampleswere incubated in

the dark for 15 min at room temperature. SYBR-I is capable of

staining all cells, whereas the polarity of PI allows it to pene-

trate only cells with permeabilised membranes, characteristic

of dead cells (Ziglio et al., 2002). In dead cells, the simultaneous

staining with SYBR-I and PI activates energy transfer between

the fluorochromes. As a consequence, viable bacteria emit

green fluorescence, while dead bacteria emit red fluorescence.

To identify enzymatically active bacteria, the fluorogenic

substrate BCECF-AM (Invitrogen, USA) was used, which is

hydrolysable by intracellular non-specific esterases in fluo-

rescent molecules (fluorescein with lex ¼ 490 nm,

lem ¼ 535 nm). An amount of 10 mL of BCECF-AM 0.2 mM

solution in DMSO was added to 1 mL of bacteria suspension

containing about 106e107 cells/mL. Incubationwas carried out

at 36 � 1 �C for 30 min in the dark.

2.7. Epifluorescence microscopy

A microscope (Nikon Labophot) at 1000� magnification and

equipped with epifluorescence apparatus was used. Image

analysiswas performed by using a CCD camera (Photometrics)

and an image processing program (Lucia, Nikon).

2.8. Flow cytometer

FCM analyses were performed with an Apogee-A40 flow

cytometer (Apogee Flow Systems, UK) equipped with an Ar

laser (488 nm) and a Bryte-HS flow cytometer (BioRad,

Hercules, USA) equipped with a short-arc Xenon lamp. The

A40 demonstrated high precision in counting and sizing small

particles, such as small bacteria, because of its better light

scatter performance. In both flow cytometers, collected

signals were FALS, green and red fluorescence. The fluores-

cent signals depend on cell staining, while the FALS signal is

related to cell size (Foladori et al., 2008). Data acquisition was

gated on the green fluorescent distribution, in order to acquire

the FALS signal of viable or active bacteria only, and to elim-

inate scattering signals produced by non-bacterial particles,

non-viable or inactive bacteria and other debris. Green and red

fluorescences were collected with logarithmic gain while the

FALS signal was collected with linear gain.

Fluorescent polystyrene spheres were used to verify

steady-state optical properties of the instrument during

operation. Non-fluorescent silica microspheres (MicroParti-

cles GmbH, Germany) of different diameters were used to

assess the calibration curve of FALS intensity used in bacteria

sizing. Six sizes of silica microsphere with diameters between

0.5 and 1 mm were chosen.

3. Results and discussion

In this section the results obtained in the application of FCM

procedure for bacteria identification and quantification in

wastewater, activated sludge and effluent and for calculating

bacterial biomass are explained and discussed step by step,

according to the 10-step procedure outlined in Fig. 2.

3.1. Pre-treatment of wastewater and activated sludgeby ultrasonication (step 2 in Fig. 2)

Activated sludge flocs have different shapes, different

porosity, irregular boundaries and sizes, ranging widely from

a few microns (small clumps of microbial cells) to several

hundred microns. An example of floc structure observed

under the epifluorescence microscope after staining with

SYBR-I and PI is shown in Fig. 4A.

Both mechanical treatment (according to Ziglio et al., 2002)

and ultrasonication (according to Foladori et al., 2007) were

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 0 7e3 8 1 8 3811

applied and compared as pre-treatments to disaggregate acti-

vated sludge flocs, with the aim of obtaining a suspension

made up mostly of free single cells, suitable for FCM analysis.

Mechanical dispersion is caused by shearing forces as

a consequence of the high speed of the rotor

(15.000e20.000 rpm), while ultrasonication causes floc disper-

sion through the action of acoustic waves. Using different

types of mechanical blenders or dispersers it is not possible to

apply a standardised intensity of the shearing forces; while

using ultrasonication, an absolute transferred specific energy,

Es, can be used, which is easily calculated for each sonicator.

Fig. 3 indicates the concentration of free bacteria after the

application of mechanical dispersion and ultrasonication.

Viable and dead cells are discriminated on the basis of cell

membrane integrity, coupling SYBR-I þ PI and are indicated in

Fig. 3A and B respectively. An image of the free bacteria

released in the bulk liquid after ultrasonication is shown in

Fig. 4B.

By increasing the mechanical treatment times and

increasing Es in ultrasonication, a progressive increase in the

concentration of free bacteria in the bulk liquid was observed

until a plateau was reached. The value of this plateau, which

identifies the maximum number of disaggregated cells,

changed significantly for ultrasonication compared to

mechanical treatment. Adopting mechanical treatment (for

times over 15 min), the maximum number of free viable cells

was in the range 0.95e1.06 � 1012 cells/L (grey area at the

bottom of Fig. 3A), while using ultrasonication at Es higher

than 80 kJ L�1 the maximum range of viable cells was

2.9e3.6� 1012 cells/L (grey area at the top of Fig. 3A) whichwas

3.2 times higher than mechanical treatment. The maximum

number of free dead cells was in the range

0.19e0.32� 1012 cells/L for mechanical treatment (grey area at

the bottom of Fig. 3B), and 0.89e1.1 � 1012 cells/L for ultra-

sonication (grey area at the top of Fig. 3B), 3.9 times higher.

The pre-treatment based on ultrasonication was more

effective than mechanical treatment, with some caveats. Due

to thecomplexaggregationofbacteria inactivatedsludgeflocs,

the exact number of cells present in untreated activated sludge

is not known and cannot be calculated exactly. Bacterial pre-

treatment is therefore needed, making it impossible to decide

which method is better able to measure the exact number of

0.E+00

1.E+12

2.E+12

3.E+12

4.E+12

0 20 40 60 80 100 120 140 160

Specific energy of ultrasonication, Es (kJ L-1)

conc

entra

tion

of v

iabl

e ba

cter

ia (c

ells

L-1

)

0 5 10 15 20 25 30time of mechanical treatment (min)

ultrasonicationmechanical treatment

A

Fig. 3 e The concentration of free bacteria released in the bulk li

treatment (as a function of time) and ultrasonication (as a funct

cells. We can only prefer the method which is able to release

the highest number of free single cells in the bulk liquid

without losses in bacterial viability. In fact, possible damage to

part of the bacteria during the disaggregation step cannot be

fully excluded. Theoretically, some problemsmay occur in the

disaggregation of particle-clusters or in the division of fila-

mentous bacteria. In order to investigate eventual losses in

bacterial viability after ultrasonication and mechanical treat-

ment, the ratio of dead/viable free cells was evaluated, and it

was found that it doesnot changesignificantly for increasingEsduring sonication, or for mechanical treatment times below

20 min (Fig. 5). The values of this ratio are highly variable for

low Es (under 80 kJ L�1) or short mechanical treatment times,

due to the incomplete disaggregation of the flocs. At

Es > 80 kJ L�1 the ratio remained quite constant, assuming

values in the range 0.16e0.26. Therefore, considering the

higher number of viable cells recovered by ultrasonication

without significant changes in the dead/viable ratio, we

preferred to use ultrasonication as the pre-treatment step.

In our investigation we selected the following conditions

for an optimised pre-treatment with ultrasonication:

- Es around 80 kJ L�1, for activated sludge,

- Es around 25 kJ L�1, for raw and settled wastewater (data not

shown).

The difference in these Es levels is due to the fact that

bacterial aggregates in wastewater and activated sludge react

differently to ultrasound (Foladori et al., 2007).

Biggs and Lant (2000) indicated ultrasonication at Es of

90 kJ L�1 (100 mL of sludge sonicated for 3 min at 50 W) as the

optimal treatment for dispersing activated sludge without

extensive cell destruction.

We checked that ultrasonication was not necessary for

effluents, because the minimal presence of aggregated

bacteria can be dispersed by simply mixing (Vortex) the

diluted suspensions.

3.2. Filtration with 20-mm-filters (step 4 in Fig. 2)

Before FCM analysis, the various suspensions undergo a 20-

mm filtration, in order to recover free bacteria from the bulk

0.0E+00

5.0E+11

1.0E+12

1.5E+12

0 20 40 60 80 100 120 140 160

Specific energy of ultrasonication, Es (kJ L-1)

conc

entra

tion

of d

ead

bact

eria

(cel

ls L

-1)

0 5 10 15 20 25 30time of mechanical treatment (min)

ultrasonicationmechanical treatment

B

quid after disaggregation of activated sludge by mechanical

ion of Es): (A) viable bacteria, (B) dead bacteria.

Fig. 4 e (A) An example of activated sludge flocs; (B) an example of bacteria suspension after ultrasonication and appropriate

dilution.

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 0 7e3 8 1 83812

liquid and any separate aggregated coarse flocs that could clog

the flow cytometer. The aggregates remaining on the

membrane filter amounted to less than 3% of the initial flocs

(quantified using epifluorescence microscopy with a digital

image acquisition system). Small aggregated clusters, made

up of more than one bacterium, passed through the filter and

were counted by FCM as single particles. This drawback was

partially overcome because the actual biovolume of each

particle (see Section 3.4) was taken into account in the

calculation of bacterial biomass.

3.3. Number of viable, dead and active bacteria (steps5e7 in Fig. 2)

The bacteria suspensions stained with SYBR-I þ PI undergo

FCM analysis tomeasure simultaneously the number of viable

and dead bacteria. The use of the two dyes, SYBR-I þ PI, and

the setting of two thresholds on red and green fluorescences

generates four areas in the cytograms in Fig. 6 (2nd column).

Depending on the intensity of the green and red fluorescences

0.0

2.0

4.0

6.0

8.0

0.1

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ratio

dea

d/vi

able

free

cel

ls

035202510150)nim( tnemtaert lacinahcem fo emit

Fig. 5 e Ratio of dead/viable free cells released in the bulk

liquid after disaggregation of activated sludge by

ultrasonication and mechanical treatment.

emitted, the following populations are distinguished: dead

bacteria (red), viable bacteria (green), small aggregates (both

red and green) and instrument background (neither red nor

green). Instrument background includes non-biotic particles

without fluorescence at either wavelength. The fluorescent

staining allowed bacteria from other organic matter and

debris to be distinguished. Raw wastewater contains some

small aggregates (22% of events), formed by clusters of viable

and dead cells clumped together, appearing fluorescent both

in red and green. After disaggregation, amoderate presence of

small aggregates remains, reduced to 13% in activated sludge

by the optimised ultrasonication step. In effluents, small

aggregates represent only 7.6% of events.

Total cells are considered as the sum of viable and dead

cells and small aggregates. These small aggregates contain

more than one cell, which affects the estimation of the total

number of cells. However, final calculation of bacterial

biomass is more accurate, because it is calculated considering

the biovolume of each particle (i.e. its size, as described in

Section 3.4).

The FCM procedure achieves statistically representative

analytical results due to the high number of bacteria that can

be quantified by means of this technique.

Five independent replicates of the complete procedure

(pre-treatment þ FCM) applied to a sample of activated sludge

give coefficients of variation for viable and dead cells of 8.7%

and 15.3% respectively.

Enzymatically active bacteria in wastewater, activated

sludge and effluents were measured after staining with the

fluorogenic substrate BCECF-AM, obtaining the cytograms

shown in Fig. 6 (3rd column), where FALS vs. green fluores-

cence are plotted. Due to fluorescein production, cells with

enzymatic activity are identified on the basis of a green fluo-

rescent signal higher than the threshold, thus excluding

instrument background and inactive cells.

3.4. Cell biovolume and cell biomass (steps 8e9 in Fig. 2)

Since FCM is a multi-parametric analysis, it was possible to

acquire separately the FALS signal produced only by viable

cells, by dead cells or by small aggregates. The FALS signal

acquired by FCM (expressed in an arbitrary scale) e which

increases with particle size with a non-linear function and is

Type of sample

Cytograms of bacteria stained with SYBR-I + PI to measure viable and

dead bacteria and small aggregates

Cytograms of bacteria stained with BCECF-AM to measure active

bacteria

Rawwastewater

dead bacteria

viablebacteria

small aggregates

instrument background

Green fluorescence (SYBR-I)

)IP( ecnecseroulf deR

active bacteria

SLAF

Green fluorescence (BCECF-AM)

Settledwastewater

dead bacteria

viablebacteria

small aggregates

Green fluorescence (SYBR-I)

)IP( ecnecseroulf deR

instrument background

SLAFGreen fluorescence (BCECF-AM)

active bacteria

Activatedsludge

dead bacteria

viablebacteria

small aggregates

Green fluorescence (SYBR-I)

)IP( ecnecseroulf deR

instrument background

SLAF

Green fluorescence (BCECF-AM)

active bacteria

Effluent

dead bacteria

viablebacteria

small aggregates

Green fluorescence (SYBR-I)

)IP( ecnecseroulf deR

instrument background

Green fluorescence (BCECF-AM)

SLAF

active bacteria

Fig. 6 e Example of FCM cytograms to measure: viable bacteria, dead bacteria and small aggregates on the basis of staining

with SYBR-I D PI (2nd column); active cells on the basis on staining with BCECF-AM (3rd column).

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 0 7e3 8 1 8 3813

widely used to estimate cell volume e was converted into cell

biovolume (expressed as mm3) according to the procedure

proposed by Foladori et al. (2008). As an example, viable

bacteria in activated sludge have frequency distributions of

FALS intensity and their corresponding biovolumes shown in

Fig. 7A and B respectively.

The biovolume (V) of each bacteria or small aggregate was

converted into the corresponding biomass (M, expressed as

VSS or COD), assuming the carbon content per unit of cell

volume (Cs) as 310 fg C mm�3 (Fry, 1990; Andreottola et al.,

2002). Considering the empirical formula of bacterial compo-

sition commonly used in activated sludge studies (C7H10O3N

or C5H7NO2), the carbon content accounts for 53.8% and 53.1%

of cell dry weight (expressed as VSS), respectively.

Therefore, the conversion to an equivalent mass of VSS is:

M½mg VSS� ¼ V � Cs � 10�12

0:53

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 50 100 150 200 250

FALS (arbitrary scale)

)%(

noitubirtsidycneuqerF

A

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0 0.1 0.2 0.3 0.4 0.5 0.6

biovolume ( m3))

%(noitubirtsid

ycneuqerF

B

0.0

1.0

2.0

3.0

4.0

5.0

6.0

0.00 0.10 0.20 0.30 0.40

bacterial biomass, M (mg COD x 10-9)

)%(

noitubirtsidycneuqerF

C

µ

Fig. 7 e Frequency distribution of: (A) FALS intensity acquired by FCM, (B) bacterial biovolume calculated from FALS

intensity, (C) bacterial biomass expressed as COD calculated converting the biovolume into biomass (data refers to viable

cells in activated sludge).

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 0 7e3 8 1 83814

The VSS value of each cell can be converted into particulate

COD multiplying by the conversion factor fcv 1.48 mg COD/mg

VSS:

M½mgCOD� ¼ M½mgVSS� � fcv ¼�V � Cs � 10�12

0:53

�� 1:48

An example of the frequency distribution of bacterial

biomass expressed as COD is indicated in Fig. 7C. The

frequency distributions in Fig. 7B and C are similar, because

they differ only in their constant factor.

The same procedure was applied to calculate the bio-

volume and biomass of enzymatically active bacteria.

The biovolume of viable bacteria in activated sludge,

shown in Fig. 7B, ranged up to 0.4 mm3 with a mean value of

0.16 mm3 (corresponding to an equivalent sphere diameter of

0.67 mm). Small aggregates in activated sludge have a mean

0

1

2

3

4

5

6

3 . 0 2 . 0 1 . 0 0

mu l o v o i b

Freq

uenc

y di

strib

utio

n (%

)

Red

fluo

resc

ence

(PI)

Fig. 8 e Comparison of frequency distribution of biovolume calc

(grey line).

biovolume of 0.43 mm3 and therefore have amean size 2.6-fold

that of viable cells, as can be observed in the biovolume

distributions compared in Fig. 8. Small aggregates contribute

up to 13% of the total number of particles counted by FCM, but

mostly contribute in terms of biovolume and biomass.

3.5. Biomass of viable and active bacteria as VSS orparticulate COD (step 10 in Fig. 2)

Finally, the entire viable bacterial biomass, expressed as mg

COD/L (or VSS/L), is calculated by adding up the values ofM for

all the viable bacteria and half the number of small aggregates

present in 1 L of original sample. Since these small aggregates

are mainly made up of 2e3 bacteria, either viable or dead, we

decided to consider approximately that 50% of the mass of

small aggregates contributes to viable bacterial biomass.

. 0 6 . 0 5 . 0 4 . 0 7

( e µm 3 )

a i r e t c a b e l b a i v s e t a g e r g g a l l a m s

l l a m s s e t a g e r g g a

) I - R B Y S ( e c n e c s e r o u l f n e e r G

e l b a i v a i r e t c a b

ulated for viable bacteria (black line) and small aggregates

0%

20%

40%

60%

80%

100%

rawwastewater

settledwastewater

activatedsludge

treatedeffluent

perc

enta

ge o

f tot

al c

ells

(%)

viable bacteriadead bacteria

Fig. 10 e Percentage of viable and dead cells in wastewater,

activated sludge and effluent (error bars indicate min, max

values).

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 0 7e3 8 1 8 3815

The same procedure was applied to calculate the entire

biomass of enzymatically active bacteria present in 1 L of

original sample.

Protozoa and metazoa populations in activated sludge

contribute to the total amount of active fraction. In our

procedure, protozoa and metazoa are partly destroyed during

pre-treatment, as confirmed by direct microscope observa-

tions, while larger organisms may be retained during 20-mm

filtration. Therefore, the proposed FCM method is only suit-

able for the quantification of bacterial biomass. Protozoa in

activated sludge samples are about 106 organisms/L, which is

6 orders of magnitude less than the total number of bacteria,

but they may contribute significantly to the overall active

biomass, due to their large size compared to bacteria.

3.6. Quantification of viable (and active) bacterialbiomass in wastewater, activated sludge and effluent

The flow-sheet of theWWTP studied is shown in Fig. 9, where

sampling points are indicated. In this WWTP e as in many

WWTPs equipped with primary and secondary settlement e

part of the excess secondary sludge is re-circulated into the

primary settler, and themixed sludge (primaryþ secondary) is

extracted from the bottom. However, we consider that the

quality of settled wastewater may only be slightly influenced

by this sludge recirculation, due to the low flow rate of re-

circulated sludge. Grab samples were considered in this

2-month monitoring period, without taking into account

hydraulic retention time.

Data regarding conventional parameters (COD, VSS) and

active or viable bacteria, are summarised in the table included

in Fig. 9. CVs calculated for the concentrations of viable and

surnatants fromsludge handling units mixed

sludge

primary settlerdegritting

sieving

sewerage

(2.2 ± 0.3(4.4 ± 0.9) x1010concentration of small aggregates(#/L)

(1.5 ± 0.0(2.0 ± 0.4) x1011concentration of total cells (#/L)

(4.3 ± 1.0(6.9 ± 2.4) x1010concentration of dead cells (#/L)

10.7% 4.8% ± 2.4% viable biomass/COD

6.5 ±11.7 ± 2.7active biomass (mgCOD/L)

(2.8 ± 0.4(4.9 ± 1.2) x1010concentration of active cells (#/L)

30% 47% ± 4% active/viable biomass

21.8 ±25.2 ± 6.1viable biomass (mgCOD/L)

(8.4 ± 1.1(8.4 ± 2.3) x1010concentration of viable cells (#/L)

147 ±404 ± 137VSS (mg/L)

218 ±597 ± 202particulate COD (mg/L)

112 ±124 ± 25soluble COD (mg/L)

330 ±721 ± 212total COD (mg/L)

SETT

WASTEW

RAW

WASTEWATERPARAMETER

Fig. 9 e Layout of the WWTPs and monitoring points for the qu

sludge and effluent. Parameter concentrations are indicated as

dead cells in activated sludge samples were 17.4% and 28.5%

respectively. These values are approximately twice the CV

values calculated on the five independently replicated sample

analyses (see 3.3), indicating that activated sludge quality has

undergone some variations during the 2-month period.

The primary settler removes 54% of total COD and a small

fraction (9.7%) of soluble COD, as expected. Notwithstanding

this high COD removal percentage, the concentration of total

bacteria decreased by 25%, from 2.0 � 1011 cells/L in raw

wastewater to 1.5 � 1011 in settled wastewater, probably

return flow

activatedsludge

secondarysettler

ricirculation of excess secondary sludge

discharge

disinfection

(1.6 ± 0.7) x108(4.5 ± 1.1) x1011) x1010

(2.2 ± 1.1) x109(3.3 ± 0.6) x10127) x1011

(3.6 ± 1.8) x108(5.6 ± 1.6) x1011) x1010

3.2% ± 2.211.1% ± 2.1± 3.1

0.12 ± 0.05224 ± 201.3

(7.1 ± 3.0) x108(1.1 ± 0.11) x10126) x1010

45% ± 5

500 ± 84

(2.3 ± 0.4) x1012

3053 ± 138

4519 ± 204

36 ± 5

4555 ± 200

ACTIVATED

SLUDGE

45% ± 10± 5

0.30 ± 0.153.2

(1.7 ± 0.9) x109) x1010

8 ± 447

11 ± 669

18 ± 233

30 ± 697

TREATED

EFFLUENT

LED

ATER

antification of bacterial biomass in wastewater, activated

mean value ± st.dev.

0.00

0.05

0.10

0.15

0.20

0.25

0.30

rawwastewater

settledwastewater

activatedsludge

treatedeffluent

mea

n ba

cter

ia b

iovo

lum

e ( µ

m3 /c

ell)

Fig. 11 e Biovolume of viable bacteria (error bars indicate

min, max values).

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 0 7e3 8 1 83816

becausemost bacteria are free in suspension and therefore do

not settle. The concentration of total bacteria in activated

sludge is expected to be higher (3.3 � 1012 cells/L), considering

VSS concentrations in the biological reactor (around 3 g

VSS/L). In the effluent the bacteria concentration dropped by 3

orders of magnitude (2.2 � 109 cells/L), in agreement with the

low particulate COD concentration (around 11 mg COD/L).

With regard to viable and dead cells, the percentages of

dead cells were higher in raw wastewater when compared to

activated sludge (P ¼ 0.0007, Fig. 10), probably as a conse-

quence of the septic conditions which developed in the sewer

(several km) and in the sieving and de-gritting stages. The

percentage of dead cells in effluents and activated sludge was

similar (P ¼ 0.35, Fig. 10). The retention time in the secondary

settler (up to 5e6 h) does not influence viability and death

significantly.

The biovolume of viable bacteria in activated sludge

(0.23 mm3/cell on average) was lower than for viable bacteria in

raw and settled wastewater (0.28 and 0.27 mm3/cell on average

respectively) (Fig. 11). This difference (P < 0.05) may be due to

Fig. 12 e Viable and active bacterial biomass in activated sludg

values).

the different populations present in wastewater and activated

sludge, different stress conditions at longer sludge age (12 d for

activated sludge) and the predation pressure in activated

sludge. Predatory activity may in fact promote a shift in

bacterial size towards very small cells or the formation of large

grazing-resistant aggregates as observed in planktonic bacte-

rial communities by Jurgens et al. (1999) andCorno et al. (2008).

The biovolume of viable bacteria in the effluent was the

smallest (0.20 mm3/cell on average) indicating that the most

difficult cells to settle are the smallest ones.

Similar results were found by Vollertsen et al. (2001), which

referred to mean and median values of 0.22 mm3/cell and

0.29 mm3/cell, respectively, in raw wastewater, by sizing

bacteria under the microscope. In activated sludge samples,

Frølund et al. (1996) observed a mean biovolume of 0.25 mm3/

cell.

Data regarding viable and active bacterial biomass,

expressed as concentrations (mg COD/L) and percentages of

particulate COD, are shown in Fig. 12. Viable biomass was

higher than active biomass, as expected, in wastewater and

activated sludge, while in the effluent the difference was not

significant. Considering the viable biomass expressed as

a concentration, the values were similar in raw and settled

wastewater (25.2 mg COD/L and 21.8 mg COD/L, respectively),

while the viable biomass expressed as percentages of partic-

ulate COD were 4.8% and 10.7% respectively (P < 0.01). This

fact suggests that the primary settler causes sedimentation of

organic and inorganic particulate, while the bacteria which

are mainly free in the bulk liquid settle only partially. Other

authors have found values ranging from 12 to 17% in primary

settled wastewater (Sperandio et al., 2001) to 2e7% (Wentzel

et al., 1998); 7e15% (Kappeler and Gujer, 1992) and 2e13%

(6.5% on average, Sperandio et al., 2001) in raw wastewater.

In activated sludge the mass of viable bacteria was 500 mg

COD/L on average, corresponding to 11.1 � 2.1% of particulate

COD on average (Fig. 12). Considering the whole bacterial

biomass (viable þ dead) the fraction is 14.5 � 2.8% of partic-

ulate COD in activated sludge. This value may appear lower

than expected, but it is in agreement with other findings

e, wastewater and effluent (error bars indicate min, max

wat e r r e s e a r c h 4 4 ( 2 0 1 0 ) 3 8 0 7e3 8 1 8 3817

referred to in the literature. Munch and Pollard (1997) found

a fraction of bacterial biomass of 9e14% in the activated

sludge of an SBR plant, after cellular staining with acridine

orange, a dye which stains all cells, both viable and dead.

Frølund et al. (1996) indicated a value of active fraction in the

range 10e15% for activated sludge using staining with DAPI

and acridine orange for the identification of all cells.

The average ratio of active/viable biomass was 47% and

30% in raw and settled wastewater respectively. In activated

sludge this ratio was 45% and it was also maintained in

effluent.

For our preliminary investigation on samples taken from

the same WWTP we can observe that the changes in the

bacterial biomass were minimal over a period of 2 months.

An automated procedure based on the outline described (in

Fig. 2) was developed to rapidly assess the bacterial biomass in

an activated sludge or wastewater sample and the whole

procedure only requires about 30 min to be completed. Thus

time is available for the analysis of several samples in

a working day.

4. Conclusions

Fuller knowledge of the actual bacterial biomass e generally

estimated by theoretical calculations e is a key parameter in

WWTP design and management. Although FCM in conjunc-

tion with fluorescent dyes has been widely used to count

bacterial cells, the proposed rapid multi-step procedure leads

to the final calculation of bacterial biomass through the

conversion of the number of bacterial cells into an equivalent

biomass, expressed as dry weight, which takes into account

the bacterial biovolume, again estimated by FCM. The main

experimental results obtained in the WWTPs were:

- the concentration of viable bacteria decreased from an

average concentration of 8.4� 1010 cells/L in raw and settled

wastewater to 1.7 � 109 cells/L in the effluent, while the

highest concentration (2.3 � 1012 cells/L) was in the acti-

vated sludge stage, as expected;

- the biovolume of viable bacteria decreased from 0.27 to

0.28 mm3/cell in raw and settled wastewater to 0.23 mm3/cell

in activated sludge and to 0.20 mm3/cell in effluent, due to

longer sludge age and predation pressure in activated sludge

and the sedimentation effect in the final settler;

- viable bacterial biomass represented only a small part of

particulate COD in wastewater (4.8 � 2.4%) and in activated

sludge (11.1 � 2.1%), in agreement with other findings

referred in the literature;

- active bacterial biomass only represented a fraction of the

viable biomass: the average active/viable biomass ratio in

raw and settled wastewater was in the range 30e47%, and

was 45% in activated sludge.

One great advantage of FCM is to permit rapid analyses

(a fewminutes) of dozens of samples per day. The applied FCM

approach provides a useful tool for understanding the bacteria

dynamics of activated sludge (growth, decay, etc.) and may

help to obtain additional information for modelling biological

processes.

Acknowledgements

The authors thank the anonymous reviewers and H. Shapiro

for their excellent revision of the manuscript.

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