Chapter 3

ACTIVATED SLUDGE BIOFILM

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80
3. ACTIVATED SLUDGE BIOFILM

3.1. INTRODUCTION

The problem pertaining to the biological degradation of aromatic sulfonates have received
considerable attention in recent years as a result of the role of these compounds in water
pollution. Because of the extensive use of detergent products and dye stuffs, it is not surprising
that the presence of sulfonated aromatic compounds has been detected frequently in
wastewater, soil and ground water. Both the general chemical stability of these molecules and
the failure of microbial biota to easily metabolize them are factors in their environmental
persistence.

Among these persistent compounds, p-toluene sulfonate (pTS) has been chosen as model to
study its degradation by microbial flora. Pitter and Chudoba (1990) have measured its
biodegradation in aquatic environment, which was 98.7% of the Chemical Oxygen Demand
(COD). However, the maximum removal rate was only about 8.4 mg g-1 h-1 and
microorganisms needed 4.5 days to be acclimated to this new substrate. Then, pTS was rarely
completely removed by classical wastewater treatment plants.

Recent works have revealed the existence of complex interactions between synthetic
surfactants, bacteria which degrade them and solid surfaces to which both become attached
(Diekmann and Hempel, 1989; Hattendorf and Hempel, 1990; Lee et al., 1995). Furthermore,
it was demonstrated that the presence of sediments collected from riverine sites contaminated
by linear alkylbenzenesulfonates (LAS) accelerated the degradation (Takada et al. 1994). The
same authors have shown that LAS biodegradation by the biofilm on the stream bed is the
predominant LAS removal mechanism in the stream. Continuous exposure of benthic biofilm
on the surfactants can enhanced their ability to degrade the persistent compounds.

Therefore, the purpose of this research was to determine the ability of activated sludges from a
domestic wastewater treatment plant to degrade pTS in a laboratory biofilm reactor under
continuous culture conditions.

81
3.2. MATERIALS & METHODS

3.2.1. Operation of the biofilm reactors

Activated sludge biofilms have been grown in the same three bioreactors than pure biofilm of
C. testosteroni. They have been previously described in Chapter 2. The dilution rate applied
was also D=0.5 h-1.
Air was provided to the feeding reactor instead of pure gaseous oxygen. It was sufficient to
maintain a concentration of 80% of dissolved oxygen in the outflows of the 3 biofilm reactors.
A batch phase has also been performed after each inoculation of fresh activated sludge in order
to allow biomass fixation during approximately two days.

3.2.2. Medium
The growth medium contained 6 mM of pTS; the concentration of the carbon source has not
been changed during the experiment. Composition of the growth medium is described in
Appendix 2.

3.2.3. Microorganisms
Activated sludges were sampled from a domestic wastewater treatment plant (Vidy,
Switzerland). They were directly harvested and washed with potassium phosphate buffer
(40 mM - pH=7) three times successively to avoid addition of exogenous carbon into the pTS
growth medium. Inoculation occurred two hours after the sampling of sludge. Activated
sludges were not grown on pTS medium as enrichment culture prior to inoculation.

3.2.4. Analytical methods

Analytical procedures have been exactly the same as for C. testosteroni biofilm growth (see
Chapter 2).
An isolation and identification of the major bacterial strains of the fixed biomass have been
performed:
- To determine cell numbers, dilution series were made with sterile potassium phosphate buffer
(40 mM) and were plated onto selective pTS agar and on Plating Count Agar (PCA) in order
to count pTS degrading microorganisms and total flora, respectively. Each dilution was
plated in triplicate on both media. Growth was performed at 30°C.
- Isolation of the major strains of the microbial population (more than 108 cfu/mL) was
performed both on PCA medium and on pTS selective agar. Colonies were isolated and
purified by successive plating. Classical microbiological methods were applied to identify all
the bacteria founded including pTS degraders.

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3.3. RESULTS

3.3.1. Growth of mixed population biofilm

Figure 3.1 summarizes the growth of a pTS degrading biofilm under aerobic continuous
culture at D=0.5 h-1.
The various phases of biofilm accumulation can be identified:
- a "lag-phase" during the first week of culture;
- the exponential and linear phase lasted more than two weeks;
- after the 22nd day of culture, the biofilm reached a plateau with 0.29 mg of biomass dry
weight per square centimetre.
At that time, the mixed population biofilm contained more than 50% of protein with a
concentration of 0.15 mg cm-2. Polysaccharide represented a low fraction of the fixed biomass:
0.05 - 0.07 mg cm-2 and did not increase during the biofilm development.
After one month of continuous culture, the mixed population biofilm was rather homogenous.
Its thickness was about 400 to 500 µm (Fig. 3.2). The density was then between 4.5 and
5.5 mg cm-3.
Microbial analysis of the immobilized flora has been performed in order to determine the ratio
of microorganisms able to degrade pTS as unique source of carbon. 2.97 105 and 1.31 107
colonies have grown respectively on pTS agar and Plate Count Agar, respectively. That means
a ratio of pTS degraders to total population of 2.26% (Fig. 3.3).

Figure 3.2. Activated sludge biofilm after 28 days of continuous culture at D=0.5 h-1 with
6 mM of pTS.

83
 0,35

0,30

0,25

0,20
Fixed biomass [mgdw/cm2]

Polysaccharide [mg/cm2]
Protein [mg/cm2] 0,15

0,10

0,05

0,00
0 10 20 30 40
Time [d]

Figure 3.1. Growth of mixed population biofilm under continuous culture at D=0.5 h-1 with
pTS as unique source of carbon: (n), biofilm dry weight; (m), protein and (s),
polysaccharide.

107

106

105

104

103
pTS degraders [cfu/cm2]
Total flora [cfu/cm2]

102

101

100
0 5 10 15 20 25 30
Time [d]

Figure 3.2. Mixed biofilm composition: ( ), total flora and (¦), pTS degraders.

84
3.3.2. pTS degradation
The inlet concentration of pTS was 6 mM. An average concentration of 4.03 mM ±0.4 was
measured in the effluents of the bioreactors only two days after the start of the continuous
feeding (Fig. 3.4). This was the lowest concentration obtained during the month of biofilm
growth. Indeed, pTS average concentration was about 4.45 mM and even 5.5 mM at day 23.
Moreover, differences in pTS degradation between the three reactors have been important and
standard variations were always about ±0.5, i.e. ±100 mg L-1.

It can be noticed that sulphate did not follow the same behaviour as the aromatic sulfonate
compound (Fig. 3.4). The ion concentration remained stable at 130 mg L-1 during the four first
days. Then, it raised suddenly to 360 mg L-1 at day 8. However, this phenomenon was
temporary and a stable concentration oscillating between 250 to 275 mg L-1 was measured till
the end of experiment.
Concerning dissolved organic carbon, Figure 3.4 shows that strong perturbations have
occurred in the culture. Important variations have also appeared between the bioreactors. It
has to be said that inoculated activated sludge were sampled from a domestic wastewater
treatment plant. It contained fibbers, particles and other scraps more or less adsorbed into the
sludge. Dissolved organic carbon concentrations can be partly due to the lysis or detachment of
these sludge components. Suspended dry matter had the same uncontroled behaviour as
organic carbon and biofilm detachment played also a role in this phenomenon. Table 3.1
summarizes the degradation efficiencies of the mixed biofilm and C. testosteroni for
comparison.

Table 3.1. Characteristic degradation rates at the end of growth of Comamonas biofilm and
mixed population biofilm with 6 mM of pTS in the inlet flow. Each value represents
the average of the three reactors. Details of the calculation are described in
Appendix 6.

C. Mixed
testosteroni biofilm
Biodegradation %=(S/Si)*100 97.28 25.8 %
pTS removal rate rS=D (Si-S) 2.92 0.55 µmolpTS.cm-3.h-1
Surface removal rate rSA=F (Si-S) A-1 1.96 0.38 µmolpTS.cm-2.h-1
Growth rate µ=V [dX/dt+D XL]* 0.012 0.076 h-1
[XL V+XF A]-1
µmolpTS.mgX.h-1
-1
Specific consumption qS=[D V (Si-S)]*[XL V+XF A] 3.67 1.01
rate
µmolpTS.mgProt.h-1
-1
Proteic consumption qSprot=[D V (Si-S)]*[Prot A] 12.40 3.83
rate
Yield coefficient YX/S=µ.[XL V+XF A]*[DV (Si-S) 2.93 192.77 mgX.mmolpTS-1
-1
-V dS/dt]

85
 6,5 400

6,0 350

5,5 300

5,0 250

4,5 200

4,0 150

3,5 100
pTS [mmol / L]

Sul f ate [mg/L]

3,0 50

2,5 0
0 10 20 30 40 0 10 20 30 40
Time [d] T i me [d]

1000 0,35

900 0,30

800 0,25

0,20
700

0,15
600
Suspended matter [mg/L]

0,10
DOC [mg/L]

500

400 0,05

0,00
300

-0,05
200
0 10 20 30 40 0 10 20 30 40
Time [d] T i me [d]

Figure 3.3. Evolution of the xenobiotic removal by mixed population biofilm: (n) pTS, (s),
sulphate, (u), dissolved organic carbon and (l), suspended dry matter. Each curve
is the average of the three reactors values.

86
3.4. DISCUSSION

Activated sludge in laboratory fixed bed reactor in continuous culture has shown its ability to
degrade pTS as unique source of carbon. Nevertheless, the biomass needed almost one month
to reach a stable biofilm composition and dry weight. On the contrary, maximum pTS removal
was obtained only 2 days after the start of continuous feeding. At that time, the biofilm was in
the lag phase and only 30 µg of biomass had been immobilized per square centimeter. This
implies a delay between biofilm development and substrate consumption. Archangeli (1994)
has found the same delay when aerobic degradation of toluene by mixed population biofilm in a
Biodrum (RAR). The degradation of toluene was reached within 20-30 hours while biofilm
growth needed more than forty days. Both of these results do not agree with the theoretical
behaviour of biofilm described by Belkhadir (1986) and Nguyen (1989) for whom the
exponential growth phase is concomitant with a significant decrease of the substrate
concentration across the reactor. However, their results were obtained with synthetic
wastewaters or glucose as substrate. In the case of xenobiotics removal, the selective pressure
modifies the microbial population. A trophic structure can be elaborated on the first flora of
pTS degraders organisms. The establishment of this second population growing on
intermediate metabolites or cell lysis can provoke a delayed growth of the biofilm.

Mixed population biofilm was able to degrade only 20-25% of the xenobiotic compound.
Although, the biofilm contained 0.155 mg cm-2 of protein, which represents more than 50% of
the biofilm dry weight. Toluene degrading biofilm grown by Archangeli (1994) contained only
0.03-0.04 mg cm-2 of protein, i.e. 5% of the total dry weight. This biofilm was able to
mineralize 65% of the toluene. Moreover, the toluene surface loading was 2.5 times higher at
2430 mg m-2 d-1 than pTS surface loading at 1000 mg m-2 d-1. The thickness of the toluene
biofilm was also higher than pTS biofilm, 800 µm and 450 µm, respectively; which implies
higher problems of diffusion. Besides toluene solubility in water is known to be specially low in
contrast to pTS.
Mörsen and Rehm (1990) have reached a phenol removal rate of 6.4 g L-1d-1by defined mixed
culture immobilized on glass. pTS removal rate was half of this value at 3.51 g L-1d-1
A fixed culture of an Alcaligenes sp. was able to degrade 1.16 mmol L-1 h-1 of chlorophenol;
but 0.775 mmol L-1 h-1 have been degraded in our experiment (Menke and Rehm, 1992).
All these results confirmed the recalcitrance of pTS compared to other mono aromatic
compounds. However, when comparing pTS removal with other sulfonated aromatic
substances, its persistence seems mainly due to the non-adaptation of microorganisms toward
such sulfonated molecules.
Kolbener et al. (1994) has studied the degradation of 3-aminobenzenesulfonate and 3-
nitrobenzenesulfonate by six different activated sludges. The authors pointed out the difference
between activated sludge originated from domestic wastewater treatment plant and those from
industrial wastewater plant. None of the domestic sludges were able to degrade the sulfonated
compounds but the addition of industrial sludges into domestic ones improved the xenobiotics
removal. Takada et al; (1994) have performed continuous degradation of a mixture of linear
alkylbenzenesulfonates commonly found in a Japanese river by immobilized benthic cells. They
have obtained a surface removal rate of 3.75 mg m-2 h-1, when pTS surface removal rate
reached in our experiment with activated sludge was about 1.03 g m-2 h-1.
When using a defined co-culture of 7 selected bacterial strains, it has been possible to consume
138 mg of carbon h-1 L-1 with several aromatic sulfonates including pTS as substrates

87
(Thurnheer et al., 1986). This higher consumption of carbon than in the present study:
maximum 65.1 mg of carbon h-1 L-1 have been degraded by non specialized activated sludge
biofilm; reflects the importance of the choice of bacteria.

Obviously, efficiencies of the mixed population biofilm appeared to be negligible in comparison

with C. testosteroni pure biofilm. As it was shown in Chapter 2, the specialized strain was able
to degrade 98% of the 6 mM of pTS in the same culture conditions as for activated sludge
(D=0.5 h-1) and a stable single strain biofilm has been established in two weeks (Table 3.1).

Table 3.2. C. testosteroni and mixed population biofilms composition.

Dry weight Protein Polysaccharide

[mg cm-2] [mg cm-2] [mg cm-2]
Activated sludge biofilm 0.292 0.155 0.05-0.07
C. testosteroni biofilm 0.550 0.134 0.379

As shown in Table 3.2, C. testosteroni biofilm contained much more polysaccharide than
mixed population biofilm, and this was related also with biofilms dry weights. Such a difference
can be explained both by the typical structure of C. testosteroni biofilm but also because the
mixed biofilm was not completely formed. When continuous culture of activated sludge biofilm
had been grown during three months, accumulation of polysaccharides occurred while protein
content remained constant (see Chapter 6).
It is interesting, however, to notice that protein amounts are quite equivalent in both pure and
mixed biofilms. As proteins are mainly due to cells, this confirms that, for a same quantity of
biomass adapted or non specialized cultures can degrade persistent compounds at complete
different rates. The importance of the specialization of microorganisms also appears when
analyzing proteic pTS removal rates: 12.40 mM cm-3 h-1 gProt-1 and 3.83 mM cm-3 h-1 gProt-1 for
C. testosteroni and mixed biofilms, respectively.
Moreover, the difference between the two biodegradation rates 25 against 98% in favour to
the specialized strain can be also found between the two pTS removal rates: 0.55 µmol cm-3 h-1
for mixed biofilm which represents 19% of 2.92 µmol cm-3 h-1 for specialized biofilm. It is
interesting to notice the growth rates of the two biofilms. Even if the amount of immobilized
C. testosteroni was twice the amount of mixed biofilm dry weight, the growth rate of the
specialized strain was six times lower than the fixed activated sludge. This phenomenon is
specially due to suspended biomass, negligible in the case of the pure strain but important for
mixed flora: 14.5 mg L-1 and 190 mg L-1, respectively. Differences between the suspended
biomass and the pTS removal abilities explained as well the variations of growth yield
coefficients (Table 3.1).

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3.5. CONCLUSION

Maximal pTS removal rate by activated sludge biofilm in continuous culture was reached after
only two days of growth. Mixed biomass has been able to degrade 3.5 g of pTS L-1 d-1 after
one month of growth. Sulphate produced by the reaction showed that the sulfonate group was
effectively removed from the aromatic ring. This constitutes an advantage because sulfonate
mainly contributes to the recalcitrance of the compound. However, it is difficult to predict if
pTS removal by mixed population biofilms would occur in presence of more degradable
substrates.
Comparison between mixed population biofilm and single strain specialized biofilm, in the same
culture conditions, demonstrates the particular adaptation of Comamonas testosteroni toward
pTS degradation when immobilized.

89
3.6. REFERENCES

Arcangeli J.-P. (1994)

Biological degradation of aromatic hydrocarbons in biofilm systems. Ph. D. Thesis,
Technical University of Denmark, Lyngby, DK.
Belkhadir R., B. Capdeville and H. Roques (1986)
Fundamental descriptive study and modelization of biological film growth - I. Fundamental
descriptive study of biological film growth, Water Research, 22: 59-69.
Diekmann R. and D. C. Hempel (1989)
Degradation of aromatic sulfonic acids - Biofilm formation and kinetics of bacterial mixed
culture. Dechema Biotechnology Conferences 3; VCH Verlagsgesellschaft; pp: 841-844.
Hattendorf C. and D. C. Hempel (1990)
Simultaneous degradation of aromatic sulfonic acids by specialized mixed culture. Dechema
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Kölbener P., U. Baumann, A. M. Cook and T. Leisinger (1994)
3-nitrobenzenesulfonic acid and 3-aminobenzenesulfonic acid in a laboratory trickling filter:
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Degradation of mixtures of monochlorophenols and phenol as substrates for free and
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Mörsen A. and H.-J. Rehm (1990)
Degradation of phenol by defined mixed culture immobilized by adsorption on activated
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Nguyen K. M. (1989)
Description et modélisation de films biologiques aérobies. Doctoral thesis Nr. 96, INSA
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Pitter P. and J. Chudoba (1990)
Biodegradability of organic substances in the aquatic environment. CRC Press, Boca Raton,
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Takada H., K. Mutoh, N. Tomita, T. Miadzu and N. Ogura (1994)
Rapid removal of linear alkylbenzene sulfonates (LAS) by attached biofilm in an urban
shallow stream. Water Research, 28(9): 1953-1960.
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90