Annls Limnol.
31 (1) 1995 : 75-80
Détermination of the biodegradable fraction of dissolved and
particulate organic carbon in waters
1
P . Servais
A . Barillier
J. G a m i e r
2
3
Keywords : Dissolved organic carbon, particulate organic carbon, total organic carbon, biodégradation, river water,
vaste water.
A simple procedure is described in order to estimate the biodegradable fraction of dissolved organic carbon (DOC),
particulate organic carbon (POC) and total organic carbon (TOC) in water samples. Methodological improvements of
the method which includes aerobic incubation in batch and carbon measurements are presented. First applications to
natural and waste water samples show the usefulness of the method in the field of water treatment and management
of surface water.
Détermination de la fraction biodégradable du carbone organique dissous et particulate dans les eaux douces
Mots clés : carbone organique dissous, carbone organique particulaire, carbone organique total, biodégradation, eau
de rivière, eau usée.
Une procédure simple est décrite, permettant la détermination dans des échantillons d'eau de la fraction biodégradable du carbone organique dissous (COD), du carbone organique particulaire (COP) et du carbone organique total (COT).
L'article présente des expériences justifiant la procédure retenue. Les premières applications de la méthode montrent
son utilité à la fois dans le domaine du traitement des eaux usées et dans celui de la gestion des eaux de surface.
1. Introduction
Biodegradable organic matter (BOM) can be defined as t h e fraction of organic carbon which can b e
metabolised b y bacteria within a period of a few
h o u r s t o a few weeks. BOM is so the actual signal
t o which growth and activity of heterotrophic
microorganisms respond in natural waters. Its knowledge is therefore required for understanding a n d
modelling bacterial activity in aquatic ecosystems.
A s chemical analysis of organic c o m p o u n d s in natural waters is very complex and cannot give complete
1. Groupe de Microbiologie des Milieux Aquatiques (GMMA),
Université Libre d e Bruxelles, Campus de la Plaine, C P 221, boulevard du Triomphe, B-1050 Bruxelles.
2. C E M A G R E F , Division Qualité des Eaux, 14, avenue de
Saint-Mandé, F-75012 Paris.
3. C N R S , U R A 1367, Laboratoire de Géologie Appliquée, 4 ,
place Jussieu, Tour 26, 5' étage, F-75005 Paris.
information on its biodegradability (Hama & H a n d a
1980, D a w s o n & D u u r s m a 1981), the use of bioassay procedures is thus required in order to determine
the biodegradable fraction of organic matter.
During the last decade, accurate analytical
m e t h o d s have been made available for accurate
determinations of dissolved organic carbon ( D O C )
a n d particulate organic carbon ( P O Q . As there was
a n increasing interest to estimate the biodegradable
dissolved organic c a r b o n ( B D O C ) , especially in the
field of drinking water production and distribution,
several methods have been p r o p o s e d in the literature to estimate B D O C as the difference of D O C
before a n d after an incubation of the water sample
in the presence of bacteria (see the review by H u c k
1990). A m o n g these methods, Servais et al. (1987,
1989) use indigenous suspended bacteria t o inoculate the water sample while fixed bacteria o n t o
sand or inert support are used as inoculum in the
Article available at http://www.limnology-journal.org or http://dx.doi.org/10.1051/limn/1995005
Downloaded from https:/www.cambridge.org/core. IP address: 88.99.165.207, on 14 Jun 2017 at 13:25:49, subject to the Cambridge Core terms of use, available at
https:/www.cambridge.org/core/terms. https://doi.org/10.1051/limn/1995005
76
P. S E R V A I S , A . BARILLIER, J. GARNIER
p r o c e d u r e s p r o p o s e d respectively by Joret & Levi
(1986) a n d Ribas et al. (1991). Block et al. (1992)
h a v e s h o w n t h a t the origin of the inoculum in this
k i n d of bioassay was a low source of variance.
H o w e v e r , these m e t h o d s only concern the dissolved
fraction of organic matter. In a lot of natural aquatic environments a n d in waste water, the concentrat i o n of particulate organic m a t t e r is often as high
as the dissolved fraction or even higher ; in these
c o n d i t i o n s , it is therefore important t o develop
m e t h o d s t o estimate the biodegradable fraction of
b o t h particulate a n d dissolved organic carbon.
In this paper we propose a simple procedure based
o n c a r b o n measurements t o estimate the biodegrad a b l e fraction of D O C and P O C in b a t c h experim e n t s . Briefly, the water sample is incubated in
aerobic conditions, D O C and P O C are measured at
t h e beginning of the incubation (initial concentrations) a n d at the end of incubation (final concent r a t i o n s ) when the biodegradable fractions of D O C
a n d P O C have been mineralised by bacteria. T h e
b i o d e g r a d a b l e dissolved organic carbon (BDOC)
a n d biodegradable particulate organic carbon
( B P O C ) are determined as the difference between
initial a n d final concentrations of D O C a n d P O C
- respectively. T h e biodegradable total organic carb o n ( B T O C ) is calculated as the sum of B D O C and
B P O C . T h e paper first presents experiments perform e d in order t o determine the incubation period in
this kind of b a t c h experiments required for bacteria t o consume the biodegradable compunds and so
t o reach stable concentrations in D O C a n d P O C .
D a t a of B D O C determinations using the procedure
described in this p a p e r have been c o m p a r e d t o data
obtained o n the same samples using the B D O C techn i q u e described by Servais et al. (1989).
A s a first application, B T O C estimations were
performed o n raw a n d treated waste water effluents,
d a t a obtained o n river waters are also presented.
2. Materials and methods
2 . 1 . B T O C determination in batch experiments
T h e experimental procedure we proposed to determ i n e in paralell the biodegradable fraction of DOC
( B D O C ) a n d of P O C (BPOC) was as follows : just
after collection, t h e water sample t o be analysed
(2 litres) was first sieved t h r o u g h a nylon membrane
(2)
of 1 m m porosity and than incubated at 20 + / —
0.5°C ; during the incubation the sample was intermittently oxygenated by air bubbling to insure aerobic conditions during biodégradation. T o avoid carbon contamination by the aeration of the batch
system, the air was previously bubbled in t w o successive bathes of sulfochromic mixture and one bath
of distilled water before injection in the batch
system. Subsamples were collected in the batch just
after the beginning of the incubation and at regular
periods ôf time for D O C and P O C determinations.
The batch was incubated for 45 days as we have
shown that such a period was long enough t o reach
stable values of D O C and P O C (see section 3.1 of
the paper) ; the remaining organic carbon at this
moment was considered as the refractory part of the
organic matter. For D O C analysis, two subsamples
of 30 ml were taken out of the batch at each collection time a n d filtered t h r o u g h precombusted
(4 hours at 550° C) fibreglass W h a t m a n n G F / F filters ; D O C measurements were performed using carbon analysers (Dohrman D C 180 or Biorotech 700).
For P O C analysis, three subsamples (4 to 20 ml)
were filtered through a 1 cm diameter precombusted fibreglass filter ( W h a t m a n G F / F ) using a
syringe ; organic matter retained on the filters was
determined using the Bioritech 700 analyser. T h e
accuracy of the D O C and P O C determinations were
about 0.05 m g C / 1 . BDOC and B P O C were calculated as the difference between concentrations, in
D O C a n d P O C respectively, at the beginning of the
batch experiment and the stable values reached at
the end of the incubation that were defined as the
refractory fraction of D O C ( R D O C ) and of P O C
( R P O C ) . Total organic carbon (TOC) in the sample was calculated as the sum of D O C and P O C at
the beginning of the experiment and the biodegradable fraction of T O C (BTOC) as the sum of BDOC
and BPOC.
2.2. Détermination of BDOC following the Servais
et al. (1989) procedure
After a prefiltration through a W H A T M A N
G F / F precombusted filter, a 200 ml water sample
was sterilized by filtration t h r o u g h a 0.2 ^ m p o r e
size m e m b r a n e (Nuclepore m e m b r a n e or cellulose
acetate Sartorius membrane) carefully rinsed first
with distilled water (300 ml) and then with the water
sample (100 ml). A 2 ml inoculum containing indigenous bacteria was added ; the inoculum was in
Downloaded from https:/www.cambridge.org/core. IP address: 88.99.165.207, on 14 Jun 2017 at 13:25:49, subject to the Cambridge Core terms of use, available at
https:/www.cambridge.org/core/terms. https://doi.org/10.1051/limn/1995005
(3)
77
DISSOLVED A N D PARTICULATE ORGANIC CARBON IN WATERS
fact water, from the same environment as the sample, filtered trough a 2 ¿¿m pore size membrane
(Nuclepore membrane). Incubation of the inoculated
sample was performed at 20 + / - 0.5°C in the dark
for 4 weeks on a shaking table. A strong agitation allowed to avoid oxygen depletion during the incubation
of samples with high B D O C . Two 30 ml subsamples
were collected at the beginning of the incubation (just
after the addition of the inoculum) and at the end of
the incubation for D O C deteraünations that were performed using the D o h r m a n DC180 carbon analyser.
T h e B D O C values were calculated as the difference
between initial a n d final D O C .
3. Results and discussion
3.1. Methodological improvement
performed following the here above described procedure with an urban effluent from Brussels. It
contained at the beginning of the experiment
49.4 mgC/1 of P O C and 25 m C / 1 of DOC. A rapid
decrease of D O C was observed during the first day
of incubation when 6.3 mgC/1 were consumed in 24
hours ; this corresponds to a first order rate of
0.3 d-'. The average decrease rate of D O C was
0.08 d for the first week of incubation. The concentration in D O C reached a stable value, at the accury
of the measurement method, between 20 and 30 days
of incubations. For the P O C , the average decrease rate
for the first week of incubation was a little lower than
for D O C (0.65 d-l). Then, P O C decreased m o r e
slowly (decrease rate around 0.01 d" between day 7
and day 30 of incubation). The plateau was reached
between 30 and 45 days of incubations.
1
1
Figure 1 shows a n example of the fluctuations of
D O C , P O C and T O C during a batch experiment
80 ,
Days
Fig. 1. Fluctuations in D O C (A), P O C ( • ) and T O C ( • ) concentrations during a batch experiment performed with a
sample of-urban effluent (Brussels, March 1992).
Fig. 1. Fluctuations des concentrations en C O D (A), C O P ( • ) et C O T ( • ) durant une expérience batch réalisée sur un échantillon d'eau usée urbaine (Bruxelles, mars 1992).
Downloaded from https:/www.cambridge.org/core. IP address: 88.99.165.207, on 14 Jun 2017 at 13:25:49, subject to the Cambridge Core terms of use, available at
https:/www.cambridge.org/core/terms. https://doi.org/10.1051/limn/1995005
78
(4)
P. S E R V A I S , A . BARILLIER, J. G A R N I E R
T h e c o n c e n t r a t i o n s of D O C a n d P O C reached at
t h e e n d of the incubation were respectively 6.4 a n d
2 4 . 6 m g C / 1 . B D O C was thus equal t o 18.6 m g C / 1
(74.4 % of the initial D O C ) a n d B P O C t o
24.8 m g C / 1 (50.2 % of the initial P O C ) . T h e b i o degradable fraction of the T O C was so for this sample 4 3 . 4 m g C / 1 (58 % of the initial T O C ) .
30
Series of similar experiments were carried out with
waste water samples from different treatment plants
a n d with samples collected in rivers. I n all t h e tested samples, constant values of D O C a n d P O C were
r e a c h e d before 45 days of incubations as for t h e
experiment described in figure 1. So for routine meas u r e m e n t s we decide t o fix the incubation period t o
d e t e r m i n e B T O C t o 45 days.
O n a series of eleven samples of natural a n d waste
w a t e r s , B D O C was estimated using t w o different
p r o c e d u r e s . O n o n e hand, they were estimated using
t h e p r o c e d u r e p r o p o s e d by Servais et a l . (1989) in
w h i c h t h e sample is filtered a n d reinoculated with
i n d i g e n o u s suspended bacteria before t h e beginning
of t h e i n c u b a t i o n a n d , o n the other h a n d , using t h e
p r o c e d u r e p r o p o s e d in this p a p e r for estimating in
parallel B D O C a n d B P O C . M a j o r differences between t h e s e t w o techniques lies, first, i n the presence
or the absence of the particulate fraction of the organic m a t t e r d u r i n g t h e course of t h e i n c u b a t i o n ,
secondly in t h e incubation period, 45 d a y s versus
30 d a y s a n d , lastly, in the fact t h a t D O C is considered as the fraction passing t h r o u g h a 0 . 7 ¿¿m p o r e
size m e m b r a n e ( W h a t m a n G F / F filter), in o n e
m e t h o d , a n d t h r o u g h a 0.2 ^ m pore size m e m b r a n e ,
in t h e other o n e . I n figue 2, the B D O C values determ i n e d using o n e m e t h o d were plotted agains the
B D O C d a t a determined using the other m e t h o d s . A
very significant correlation was observed (r = 0.99,
n = 11). T h e slope of the correlation straight line
(1.03), very close t o 1, indicated that there was n o
significant difference between b o t h estimates of
B D O C . I n o t h e r w o r d s , this means that :
BDOC
mgC/1
Fig. 2. B D O C values measured using the procedure proposed in
this paper (y axis) plotted against B D O C values estimated o n
the same samples of waste waters and river waters using the
Servais et al. (1989) method (x axis).
Correlation straight line : y = 1.03 x
( r = 0.99, n = 11)
Fig. 2. Valeurs de C O D B estimées selon la procédure proposée
dans cet article (axe Y), portées en fonction des valeurs de
C O D B estimées sur les mêmes échantillons d'eaux usées et
de rivières par la méthode proposée par Servais et al. (1989)
(axe x).
Droite de régression : y = 1.03 x
(r = 0.99, n = 11)
• The 30 day incubation period is long enough
to c o n s u m e the entire biodegradable fraction of
D O C as already pointed out by Servais et al. (1989)
and in the previous section of this paper.
• T h e fraction of organic c a r b o n in the size range
between 0.7 and 0.2 ^ m is insignificant. This results
is in concordance with the d a t a of Barillier (1992)
showing t h a t a r o u n d 1 % only of the t o t a l organic
carbon was in this size range for various waste
waters a n d river waters.
3.2. BTOC determination in different water samples
• T h e presence of the particulate organic m a t t e r
d u r i n g t h e incubation does not seem t o influence the
B D O C d a t a , i.e. P O C does n o t act as a cosubstrate
for further d e g r a d a t i o n of D O C (Barillier & Garnier, 1993). M o r e o v e r , it is i m p o r t a n t t o n o t e t h a t ,
if a fraction of P O C is solubilized during t h e incub a t i o n , it as also mineralised before t h e e n d of
experiment.
I n addition to the measurement performed o n the
Brussels effluents, B T O C was also determined o n
raw and treated water of the Achères treatment plant
collected in J u n e 1992 (Table 1). This plant located
in t h e Parisian suburbs is the most i m p o r t a n t treatment plant in E u r o p e ; it receives the waste water
of a b o u t 8.000.000 equivalent inhabitants a n d
Downloaded from https:/www.cambridge.org/core. IP address: 88.99.165.207, on 14 Jun 2017 at 13:25:49, subject to the Cambridge Core terms of use, available at
https:/www.cambridge.org/core/terms. https://doi.org/10.1051/limn/1995005
(5)
79
DISSOLVED A N D PARTICULATE ORGANIC CARBON IN WATERS
Table 1. Concentrations in the different fractions of organic carbon expressed in mgC/1 (dissolved D , particulate P and total T) in
the tested wastewaters and river water samples (B for biodegradable and R for refractory fractions).
Tableau 1. Concentrations des différentes fractions du carbone organique exprimées en mgC/1 ( D pour dissoute, P pour p a r t i c u l a t e
et T pour total) dans les échantillons d'eaux usées et de rivières analysées (B pour biodégradable et R pour réfractaire).
1992
DOC
BDOC
RDOC
POC
BPOC
RPOC
TOC
BTOC
RTOC
Waste water samples
Achères-raw water
June
33.00
24.35
8.65
43.00
26.40
16.60
76.00
50.75
25.25
Achères-treated water
June
26.45
16.15
10.30
21.25
13.05
8.20
47.70
29.20
18.50
Brussels-raw water
March
25.00
18.60
6.40
49.40
24.80
24.60
74.40
43.40
31.00
River water samples
Seine river (La Frette)
June
4.40
1.20
3.20
2.15
0.55
1.60
6.55
1.75
4.30
Seine river (Bac)
June
8.45
4.35
4.10
5.15
2.45
2.70
13.60
6.80
6.80
Meuse river (Liège)
May
5.10
2.00
3.10
4.10
1.25
2.85
9.20
3.25
5.95
treatment includes classical pretreatment and décantation followed b y a n activated sludge stage. T h e
treated effluents of this plant are discharges into the
river Seine a n d constitute t h e m a i n source of organic m a t t e r t o the river d o w n s t r e a m Paris ( G a m i e r
et al. 1992, Servais & G a m i e r 1993).
In the raw waters, D O C a n d P O C equalled respectively 33 m g C / 1 a n d 43 m g C / 1 (Table 1) a n d t h e
biodegradable part represented 74 % for D O C 61 %
for P O C (67 % for t h e total organic carbon). These
values are c o m p a r a b l e with those obtained o n the
raw waters of Brussels.
Regarding the treated water, D O C and P O C were
26.45 a n d 21.25 m g C / 1 corresponding t o a removal
by t r e a t m e n t of 6.55 m g C / 1 of D O C (20 % ) a n d
21.75 m g C / 1 of P O C (51 % ) . T h e biodegradable
fractions of the treated waters represented 61 % for
D O C a n d 61 % also for P O C .
T h e removal of D O C during t r e a t m e n t was
approximately similar to the removal of B D O C (6.55
against 8.2 mgC/1) showing t h a t the treatment
removes the dissolved fraction through a process of
biodégradation r a t h e r t h a n t h r o u g h a physicochemical process. T h e mechanism of removal is different for P O C . T h e r e m o v a l of P O C during
treatment was m u c h higher t h a n the r e m o v a l of
B P O C (21.75 against 13.35 mgC/1), but the percentage of P O C and B P O C removal a r e very similar,
indicating t h a t t h e refractory a n d t h e biodegradable fraction of P O C was proportionally r e m o v e d
with same efficiency. T h a t m e a n s that the i m p a c t
of biodégradation of particular organic m a t t e r in
activated sludge was low with respect to the décantation that was the major process in removing P O C .
B T O C was also estimated o n three river waters.
River Seine was sampled just u p s t r e a m (LaFrette)
a n d d o w n s t r e a m (Bac) the effluent outfall from the
Achères treatment plant ; a sample was also collected in the River Meuse (Belgium) in a n organically
polluted area near Liège (Table 1). In t h e River
Seine, t h e biodegradable fraction of T O C , increased from 27 % u p s t r e a m to 50 °7o d o w n s t r e a m the
effluent outfall. In the River Meuse sample, a n intermediate value of 35 % of biodegradable T O C was
found ; B T O C was composed of 62 °7o of dissolved
material a n d 38 % of particulate o n e .
Regarding all the results, it appears that the importance of the B T O C fraction is associated with the
degree of domestic pollution. Quantifying the biodegradable fraction of organic matter gives insights
o n the bacterial activity that c a n be supported.
Downloaded from https:/www.cambridge.org/core. IP address: 88.99.165.207, on 14 Jun 2017 at 13:25:49, subject to the Cambridge Core terms of use, available at
https:/www.cambridge.org/core/terms. https://doi.org/10.1051/limn/1995005
80
P . S E R V A I S , A . BARILLIER, J. G A R N I E R
(6)
4. Conclusion
References
T h e p r o p o s e d m e t h o d t o determine simultan e o u s l y t h e b i o d e g r a d a b l e fraction of T O C , D O C ,
P O C is very easy t o apply from a l a b o r a t o r y point
of view. In routine, it only requires the measurement
of D O C a n d P O C a t the beginning a n d at the end
of the experiment. During the incubation period that
is p e r f o r m e d at l a b o r a t o r y t e m p e r a t u r e , the only
t h i n g t o d o is t o control the oxygenation of the
sample.
Barillier A . 1992. — Caractérisation et dynamique de la matière
organique d'un milieu fluvial anthropisé, la Seine. Thèse. Université de Paris VI : 104 p .
Barillier A . & Garnier J. 1993. — Influence o f temperature and
substrate concentration o n bacterial growth yield in Seine
River water batch cultures, Appl. Environ. Microbiol.,
59 :
1678-1682.
Block J . C . , Mathieu L . , Servais P . , Fontvielle D . & Werner P.
1992. — Indigenous bacterial inocula for measuring the biodegradable dissolved organic carbon (BDOC) in waters. Wat.
Res., 26 : 481-486.
Dawson R. & Duursma E.K. 1981. — Stade of the art. In -.Marine
Organic Chemistry, Duursma E.K. & Dawson R., ed. Elsevier, Amsterdam : 497-512.
Garnier J., Servais P . & Billen G. 1992. — Dynamics of bacterioplankton in the river Seine (France) ; impact o f Parisian
effluents. Can. J. Microbiol.,
38 : 56-64.
Hama T . & Handa N . 1980. — Molecular weight distribution
a n d characterization of dissolved organic matter from lake
waters. Arch. Hydrobiol.,
90 : 106-120.
Huck P . 1990. — Measurement of Biodegradable Organic Matter and Bacterial Growth Potential in Drinking Water, Journal
A WWA, 82 : 78.
Joret J . C . & Levy Y . 1986. — Méthode rapide d'évaluation du
carbone éliminable des eaux par voies biologiques. Trib. Cebedeau. 510 : 39 : 3-9.
Ribas F., Frías J. & Lucena F. 1991. — A new dynamic method
for the rapid determination of the biodegradable dissolved
organic carbon in drinking water. J. of Appl. Bacteriol., 71 :
371-376.
Servais P . , Billen G. & Hascoët M . C . 1987. — Determination
of the biodegradable fraction of dissolved organic matter in
waters, Wat. Res., 21 : 445-450.
Servais P . , Anzil A . & Ventresque C. 1989. — A simple method
for the determination of biodegradable dissolved organic carb o n in water. Appl, Environ. Microbiol.,
55 : 2732-2734.
Servais P. & Garnier J. 1993. — Contribution o f Heterotrophic
Bacterial Production to the Carbon Budget of the River Seine
(France). Microbial Ecology, 25 : 19-33.
T h i s m e t h o d c a n b e considered as a n alternative
t o t h e classical m e t h o d of the biological oxygen
d e m a n d ( B O D ) used for estimating biodegradable
m a t t e r in waste water samples. As m a i n differences
between b o t h m e t h o d s , we have to mention the next
p o i n t s . T h e d a t a o b t a i n e d with the m e t h o d p r o p o sed in this p a p e r are expressed in carbon unit rather
t h a n in oxygen unit. This avoids to take into account
in the m e a s u r e m e n t other processes, t h a n biodégrad a t i o n , which as nitrification also consume oxygen.
M o r e o v e r in o u r m e t h o d , dissolved a n d particulate
fractions of t h e b i o d e g r a d a b l e matter are determin e d . T h i s is a n i m p o r t a n t point in the view of wastewater t r e a t m e n t processes design and m a n a g e m e n t
as t h e b e h a v i o u r of dissolved a n d particulate fractions is different during treatment and also after discharge of the effluent in natural aquatic ecosystems.
Ecological implications are of major i m p o r t a n c e as
t h e w a t e r quality of rivers (oxygenation) d o w n s t r e a m effluents outfall will depend o n the fraction
o f b i o d e g r a d a b l e organic m a t t e r available for the
heterotrophic bacteria. Acknowledgements
T h e study has been partly carried out during the course o f the
P I R E N - S e i n e program (Centre National de la Recherche Scientifique - C N R S - France) and partly during the course of the study :
« E t u d e écologique d'un système fluvial perturbé (la basse
Meuse) » supported b y the « Fonds de la Recherche F o n d a m e n tal Collective » (Belgium). T h e authors are indebted to A . Anzil
for her excellent technical assistance.
Downloaded from https:/www.cambridge.org/core. IP address: 88.99.165.207, on 14 Jun 2017 at 13:25:49, subject to the Cambridge Core terms of use, available at
https:/www.cambridge.org/core/terms. https://doi.org/10.1051/limn/1995005