Bioresource Technology 74 (2000) 231±239
In¯uence of the content in fats and proteins on the anaerobic
biodegradability of dairy wastewaters
G. Vidal a, A. Carvalho b,1, R. Mendez b,*, J.M. Lema b
b
a
EULA ± Chile Centre, University of Concepci
on, P.O. Box 160-C, Concepci
on, Chile
Department of Chemical Engineering, Instituto de Investigaciones Tecnol
ogicas, University of Santiago de Compostela, Avda. de las Ciencias s/n,
15706 Santiago de Compostela, Spain
Received 4 August 1999; received in revised form 3 January 2000; accepted 5 January 2000
Abstract
The relative amounts of fats, proteins and carbohydrates in wastewaters from dairy industries cause problems during their
anaerobic treatment. The anaerobic biodegradability of two synthetic wastewaters, one rich in fats (chemical oxygen demand (COD)
ratio; Fats/Proteins/Carbohydrates: 1.7/0.57/1) and the other with a low fat content (COD ratio; Fats/Proteins/Carbohydrates: 0.05/
0.54/1) was studied in samples with total COD ranging from 0.4 to 20 g/l. There were no problems of sludge ¯otation and the
maximum biodegradability and methanisation were obtained when operating with wastewaters in the range of 3±5 gCOD/l. The
intermediates of fat degradation (glycerol and long chain fatty acids) seemed not to reach concentrations high enough to a€ect the
process. The anaerobic biodegradation of fat-rich wastes was slower than carbohydrate-rich wastes due to the slower hydrolytic step
of fat degradation which prevented the accumulation of volatile fatty acids (VFAs) and favoured the overall process. Carbohydraterich wastewater degradation produced free ammonia (FA) at concentrations near to inhibitory levels (62.2 mg FA/l), but in this case,
ammonia production facilitated regulation of fall in pH caused by of the accumulation of VFA. Ó 2000 Elsevier Science Ltd. All
rights reserved.
Keywords: Anaerobic biodegradation; Inhibition; Dairy wastewaters; Protein; Ammonia-nitrogen
Nomenclature
AA
%BD
BOD
COD
CODCH4
CODVFA
EGBS
FA
FAA
FVFA
IC
LCFFA
LCFA
*
acetic acid
anaerobic biodegradability percentage
biological oxygen demand
chemical oxygen demand
COD associated with methane
production
COD associated with volatile fatty
acids
expanded granular sludge bed
free ammonia
free acetic acid
free volatile fatty acids
inorganic carbon
long chain free fatty acids
long chain fatty acids
Corresponding author. Tel.: +34-981-563-100; fax: +34-981±595012.
E-mail address: [email protected] (R. MeÂndez).
1
Current address: Department of Chemical Engineering, University
of Aveiro, Aveiro, Portugal.
%M
N
%R
TA
TOC
TSS
UASB
VFA
%VFA
VSS
Percentage of COD methanised
Relationship between FA and TA
%COD removed, based on the ®ltered
substrate COD concentration
total ammonia
total organic carbon
total suspended solids
up¯ow anaerobic sludge blanket
volatile fatty acids
percentage of COD acidi®ed
volatile suspended solids
1. Introduction
The dairy industry produces many di€erent products
such as pasteurised-, condensed-, skimmed- and powdered-milk, yoghurt, butter, di€erent types of desserts
and cheeses and sometimes cheese whey (Carozzi, 1993).
Wastewaters from the dairy industry are generally
produced in an intermittent way, and the ¯ow and
characteristics of wastewaters change from one factory
0960-8524/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 0 - 8 5 2 4 ( 0 0 ) 0 0 0 1 5 - 8
232
G. Vidal et al. / Bioresource Technology 74 (2000) 231±239
to another depending on the kind of systems and the
methods of operation (Rico et al., 1991). The main
contributors to the organic load of these wastewaters are
lactose, fats and proteins (Hansen and Hwang, 1990;

Ozturk
et al., 1993; Perle et al., 1995).
Prevention and reduction of dairy wastewater pollution can be achieved by means of direct recycling and
reutilisation of waste components (e.g., the use of cheese
whey for animal feed; Perle et al., 1995) or by using different wastewater treatments: physical-chemical, aerobic
and/or anaerobic biological treatments (Radick, 1992).
Physical-chemical treatments allow the partial removal of the organic load by protein and fat precipitations with di€erent chemical compounds such as
aluminium sulphate, ferric chloride and ferrous sulphide
(K
arpati et al., 1995; Ruston, 1993). However, the reagent cost is high and the removal of soluble chemical
oxygen demand (COD) is poor; therefore, biological
processes are often used.
Several conventional aerobic treatments have been
used extensively in the dairy industry: aerated lagoons,
activated sludge processes (Stephenson, 1989; Shack and
Shandhu, 1989), trickling ®lters (Walsh et al., 1994) and
rotating biological contactors (Radick, 1992). However,
the energy requirements for aeration in these installations are high and problems such as bulking and excessive biomass growth often occur (Timmermans et al.,
1984).
Interest in anaerobic digestion is increasing because
of the well-known advantages for the treatment of high
organic concentration wastewaters (Sayed et al., 1988;
Mendez et al., 1989; Rico et al., 1991; Hawkes et al.,
1995). In particular, the following advantages may be
adduced: no need for aeration equipment, lower excess
of sludge than that produced by aerobic processes, and a
relatively low land demand. (Colleran, 1991; Perle et al.,
1995).
Treatment of dairy wastewaters by means of up¯ow
anaerobic sludge blanket (UASB) reactors (Hansen and
Hwang, 1990; Rico et al., 1991; Hawkes et al., 1995),

hybrid UASB reactors (Ozturk
et al., 1993), expanded
granular sludge bed (EGSB) reactors (Petruy and Lettinga, 1997), as well as others based on anaerobic ®lters
(Mendez et al., 1989; Viraraghavan and Kikkeri, 1990;
Veiga et al., 1994), have been reported in the literature.
These papers show that anaerobic treatment can be effectively used for these e‚uents, in spite of the di€erent
operational problems quoted in the literature, such as
sludge ¯otation or toxicity/inhibition processes.
As was stated above, the characteristics of dairy
wastewaters can be very di€erent depending on the kind
of products produced by the factory. The relative proportions of fats, proteins and carbohydrates can be very
di€erent and they can signi®cantly a€ect the kind and
quantity of the intermediates. Fig. 1 shows some of the
intermediate compounds generated during the anaerobic
Fig. 1. Anaerobic biodegradation of milk compounds: (a) Fats: Triglycerides of di€erent acids: e.g. butyric, capric, lauric and oleic acids;
(b) Protein: e.g., casein, albumin; (c) Carbohydrates: e.g., lactose; (d)
Acids: LCFA: long chain fatty acids. LCFFA: long chain free fatty
acids. VFA: volatile fatty acids. FVFA: free volatile fatty acids. AA:
acetic acid. FAA: free acetic acid.
biodegradation of dairy wastewaters and their interactions; some of these compounds present inhibition/toxicity and biodegradability diculties in anaerobic
processes which will be discussed below.
Sludge ¯otation. Sludge ¯otation and/or the development of sludges with di€erent physical characteristics
and/or poor activity are attributed to the presence of fats
(Perle et al., 1995). Rinzema et al. (1993) reported sludge
¯otation and a total sludge washout in a UASB reactor
with a lipid loading rate exceeding 2±3 gCOD/l d. Alves
et al. (1997) operated two anaerobic ®xed-bed reactors
treating dairy wastewaters at OLR of up to 9 gCOD/lád
and they showed that the presence of higher quantities
of lipids hardly a€ected the overall performance but it
reduced the adhered fraction of biomass, although the
biomass content of both reactors was practically the
same. They also concluded that methanogenic activity
using butyrate as the substrate was enhanced by the
presence of lipids, but no signi®cant response to other
intermediates was detected.
On the other hand, fat adsorption to the surface of
the anaerobic sludge may limit transport of the soluble
substrates to the biomass and consequently cause the
conversion rate in substrates to decrease (Sayed et al.,
1988; Rinzema et al., 1993). Petruy and Lettinga (1997)
showed that 70% of lipids were adsorbed by the granular
sludge, within approximately one day, and thereafter,
the remaining lipids were slowly converted into methane
gas. Sayed et al. (1988) reported similar adsorption
problems in the sludge ¯ocks of protein-rich wastewaters.
Casein biodegradability. Casein is the main protein in
milk (80% of total proteins). In general, proteins are
hydrolysed by proteases into polypeptides and amino
acids; in anaerobic conditions this hydrolysis is slower
than the hydrolysis of carbohydrates (Pavlostathis and
Giraldo-Gomez, 1991). The rate of casein hydrolysis
depends on the acclimation of the biomass. Some
G. Vidal et al. / Bioresource Technology 74 (2000) 231±239
experiments showed that gas production, in the case of
casein (3.7 gCOD/l d) fed to non-acclimated sludge (4.5
gVSS/l), was low (0.25 ml CH4 /h) with a lag phase of
approximately 50 h compared with gas production in
the case of casein fed to acclimated sludge (0.79 ml CH4 /
h). However, casein was found not to inhibit anaerobic
biodegradability in the range 0±3 g/l (Perle et al., 1995).
Inhibitory and toxic e€ects of intermediate products.
The biodegradation of soluble carbohydrates (such as
lactose) is generally faster and almost total in anaerobic
conditions (Pavlostathis and Giraldo-Gomez, 1991).
The degradation of lactose leads to the formation of
products such as propionate, ethanol or acetate. The
accumulation of these intermediate products, especially
in the undissociated form, leads to the inhibition of
several microbial species with the consequent decrease in
methane production (Aguilar et al., 1995).
The biodegradation of lipids is dicult because of
their low bioavailability (Petruy and Lettinga, 1997).
Fats in dairy wastewaters produce glycerol and LCFA
during the hydrolytic step (Fig. 1). Glycerol was found
to be a non-inhibitory compound (Perle et al., 1995).
However, LCFA (saturated fatty acids with 12±14 carbon atoms and unsaturated fatty acids with 18 carbon
atoms) are reported to be inhibitors of various microorganisms (Rinzema, 1988), particularly of methanogenic bacteria (Koster, 1987); they also decrease the
concentration of adenosine triphosphate (ATP) (Perle
et al., 1995; Hanaki et al., 1981). The inhibitory e€ect
increases with the number of double bonds and cis-isomers which are abundant in natural lipids (Rinzema,
1988). As in the case of volatile fatty acid (VFA), the
toxicity of LCFA seems to be related with the unionised
form of these acids, namely long chain free fatty acids
(LCFFA).
Studies carried out by Hanaki et al. (1981), show that
LCFA a€ect the amount of hydrogen produced by
acetogenic bacteria which are responsible for the b-oxidation of LCFA. Inhibition of acetogens and acetotrophic methanogens causes a pronounced lag phase in
batch experiments, whereas inhibition of hydrogenotrophic methanogens merely causes a decrease in the
hydrogen conversion rate (Rinzema et al., 1994).
The main products of the biodegradation of proteins
in anaerobic conditions are ammonia and di€erent aminoacid compounds. However, the ammonia produced
may be toxic for methanogenic bacteria (Parkin et al.,
1983; Koster and Lettinga, 1988; Soubes et al., 1994).
McCarthy (1964) showed that when the ammonia±nitrogen content ranged between 50 and 200 mg/l it
stimulated the methanogenic bacteria, but higher concentrations may be toxic, in particular to this kind of
bacteria. According to Omil et al. (1995) concentrations
of free ammonia (FA) from 25 to 140 mg N ) FA/l inhibit mesophilic treatment. The pH is important to determine the speciation between ionised and unionised
233
ammonia (Parkin et al., 1983), because the unionised
form is especially toxic (Anderson et al., 1982) therefore
the higher the pH, the more noxious the e€ects.
The objectives of this work were to evaluate the in¯uence of absolute and relative concentrations of carbohydrates, fats and proteins on the anaerobic
biodegradability of dairy wastewaters.
2. Methods
2.1. Wastewater composition
Two media with di€erent contents in proteins, carbohydrates and fats were used to simulate wastewaters:
W-type wastewaters, obtained by dissolving full creammilk powder, and S-type wastewaters, obtained from
skimmed-milk powder. Table 1 shows the composition
of the powdered-milks used to prepare both substrates.
The relative contributions of proteins/ sugars/fats expressed in terms of %COD were 17.4/30.5/52.1 for
W-type wastewaters and 34.2/62.8/3.0 for S-type wastewaters, respectively.
2.2. Anaerobic biodegradability batch assays
Anaerobic biodegradability batch assays were performed in closed glass ¯asks with a total volume of 500
ml. A plastic tube connected the vial to a 500-ml inverted ¯ask containing an alkaline solution (2.5%
NaOH) which allowed methane production to be measured by the displacement of the liquid. Anaerobic
sludge from a UASB treating sugar-production wastewaters (2.04 gVSS/l; speci®c methanogenic activity 0.3
gCOD/gVSS d), distilled water and a known amount of
COD supplied by full cream- or skimmed-milk were
transferred to the ¯asks containing 50 ml of a nutrient
stock solution and 5 ml of a reducing medium containing 100 mg Na2 S 9H2 O/l. The COD after dilution
to the ®nal volume ranged from 0 to 20 gCOD/l and the
pH was adjusted to 7:00 0:05. A blank assay without
substrate was also carried out to evaluate the biodegradability of the sludge. The standard nutrient stock
solution was constituted by macronutrients (N, P and S)
and trace elements (Table 2) as outlined previously
Table 1
Composition of full cream and skimmed-milk per 100 g of milk
powder
Parameter
Full cream-milk
Skimmed-milk
Proteins (%)
Sugars (%)
Fats (%)
Salts (%)
COD (g/100 g powder)
25.4
38.9
26.0
9.7
159
33.9
52.1
0.9
13.1
115
234
G. Vidal et al. / Bioresource Technology 74 (2000) 231±239
2.5. Determination of FA
Table 2
Standard stock nutrients solution
a
Compound
Concentration
(g/l)
Compound
Concentration (g/l)
NH4 Cl
KH2 PO4
MgSO4 7H2 O
2.8
2.0
0.1
CaCl2
Micronutrienta
NaHCO3
0.076
10
4
Sierra-Alvarez and Lettinga (1991).
(Sierra-Alvarez and Lettinga, 1991). Before closing, the
bottles were ¯ushed with a 70% nitrogen and 30% carbon dioxide mixture to remove air in the ¯ask head prior
to incubation; the temperature was maintained at
36 1 C (Soto et al., 1993). Methane production, TOC,
COD and VFA concentrations were determined during
the assay. Protein, sugar and ammonia concentrations
were measured at the beginning and at the end.
Ammonia generated during anaerobic breakdown of
proteins will be present in the liquid in two forms: ionic
(NH‡
4 ) and FA. It has been suggested that FA is the
active component responsible for ammonia inhibition
and it is possible to calculate FA concentration from the
total ammonia concentration in the liquid (TA) and the
fraction of FA (N), using the equation (Omil et al.,
1995):
Nˆ
TA
1
ˆ
;
FA 1 ‡ kb 10ÿpH =kw
where kb and kw are the dissociation constants for ammonia and water, respectively (1:855 10ÿ5 and 2:355
10ÿ14 mol/l at 37°C).
2.3. Analytical methods
3. Results and discussion
Protein was determined by spectrophotometry (Lowry et al., 1951), while total sugars were determined
using Miller (1959). VFA were analysed by gas chromatography using a Hewlett Packard 5890 equipped
with a 3 m ´ 2 mm glass column packed with Chromosorb W-AW (100±120 mesh) coated with 25% NPGA
and 2% H3 PO4 . TA was determined by an anion-selective electrode. COD, Sugar, total suspended solids (TSS)
and volatile suspended solids (VSS) were measured as
described in Standard Methods (APHA, 1985). Total
organic carbon (TOC) and inorganic carbon (IC) were
determined using a Shimadzu TOC-5000 system equipped with an infrared detector, and methane gas was
analysed using a Hewlett Packard 5890 Series II gas
chromatograph equipped with a 2 m 2 mm steel column.
Biodegradability assays
2.4. Performance parameters calculation
The percentage of COD methanised (%M) and the
anaerobic biodegradability percentage (%BD) were calculated by the equations:
CODCH4
100
…1†
%M ˆ
CODinitial
%BD ˆ %R ‡ %VFA
CODinitial ÿ CODfinal
100
ˆ
CODinitial
CODVFAfinal
100
‡
CODinitial
…2†
where %BD includes the fraction of COD removed and
the fraction of COD acidi®ed contained in the vial at the
conclusion of the experiment. This acidi®ed COD fraction is easily removed in a continuous reactor.
…3†
To evaluate the e€ects of relative concentrations of
fats, carbohydrates and proteins during substrate degradation and methane production, two sets of duplicate assays with COD between 0.68±16.6 gCOD/l and
0.39±19.6 gCOD/l, for W wastewaters (Assays W1±W6)
and S wastewaters (Assays S1±S6), respectively, were
carried out. Table 3 shows the initial and ®nal values of
the most relevant parameters. A direct relation between
the initial amount of proteins and the ®nal ammonia
concentration and pH of the assay can be observed
therein. A larger concentration of residual sugar was
present in W assays, whereas sugar was almost entirely
degraded in S assays.
Figs. 2(a) and (b) show the degradation of the
wastewaters with higher COD concentrations. By comparing both graphs, di€erent anaerobic degradation kinetics can be observed for W and S wastewaters.
Methane production curves of the W wastewaters
showed two exponential increases during the operation.
The ®rst one corresponded to the most easily biodegradable substrates, mainly sugars and some proteins
(Pavlostathis and Giraldo-Gomez, 1991), whereas the
second one corresponded to fat degradation. The
complete degradation of the wastewaters clearly depends on the hydrolysis rate of each di€erent compound. For instance, casein is easily hydrolysed in the
presence of an acclimated biomass (Perle et al., 1995),
but its lag phase is longer than that of carbohydrates in
unacclimated biomasses (Pavlostathis and GiraldoGomez, 1991). In the case of fats, Rinzema et al. (1993)
reported that the overall conversion rate is limited either by the conversion of the LCFA, or by the physical
processes of dissolution and mass transfer of these
acids.
G. Vidal et al. / Bioresource Technology 74 (2000) 231±239
235
Table 3
Initial and ®nal conditions of the anaerobic biodegradability assays
Assay
Initial conditions (mg/l)
COD
Blank
W1
W2
W3
W4
W5
W6
S1
S2
S3
S4
S5
S6
689
1377
3444
5548
11,101
16,639
396
791
1978
6549
13,114
19,668
Final conditions (mg/l, except pH)
Fat
Protein
Sugar
pH
COD
Protein
Sugar
Total ammonia
±
±
±
6.86
6.61
6.60
6.69
6.85
6.94
7.37
6.63
6.69
6.66
6.87
7.48
7.64
±
±
32
53
42
48
135
261
43
4
61
112
±
±
±
14
8
18
28
56
85
16
8
0
0
0
0
91
±
±
±
120
159
384
±
±
±
102
488
1196
121
239
598
964
1928
2890
4
8
20
66
131
197
120
240
600
965
1932
2895
135
24
676
2240
4485
6727
175
350
875
1410
2822
4229
207
414
1035
3427
6863
10,293
Methanisation (%M) and biodegradation (%BD)
percentages for W and S wastewaters are shown in Figs.
3(a) and (b). At lower concentrations (1±5 gCOD/l) it
can be seen that the biodegradability and the methanisation were greater in the case of W wastewater. At
higher concentrations (5±20 gCOD/l) the biodegradability of W wastewaters ranged between 98% and 99%,
whereas for S wastewaters it diminished from 97.5% to
86%. Moreover, Fig. 3(b) shows that the optimum COD
for the methanisation of S wastewaters was around 5
gCOD/l.
It can be seen that there was an apparently recalcitrant fraction of both wastewaters to be biodegraded
anaerobically. Transport limitations of the soluble substrates to the biomass, due to the development of a ®lm
of fats (Rinzema et al., 1993; Sayed et al., 1988) or by
inhibition processes, due to the presence of high concentrations of intermediate compounds, may explain
this fact.
The maximum methane production rate for each assay in terms of COD is shown in Fig. 4. S wastewaters
78
51
91
100
130
175
100
73
176
132
400
3011
had a high degradation rate at low COD, although inhibition occurred at concentrations higher than 6
gCOD/l (2.0 g protein/l). The fast hydrolysis of S
wastewaters soluble substrates produced inhibition of
the acetoclastic methanogenic bacteria, because of the
presence of high concentrations of VFA. On the other
hand, W wastewaters exhibited a linear increase in activity as the COD increased, therefore, in this case, the
methanogenic step was not the limiting stage for the
anaerobic process. In fact, the biodegradability percentages for these wastewaters ranged between 95% and
100% even at higher COD concentrations.
The di€erent behaviour of both types of wastewaters
may be explained by the di€erent proportions of fats
and proteins in W and S substrates.
Fats and intermediate compounds e€ects. The degradation pro®les of W wastewaters suggest that the intermediates of fat degradation (glycerol and LCFFA)
did not reach concentrations high enough to a€ect the
di€erent trophic groups involved in the overall process.
Perle et al. (1995) have shown that oleic acid (chosen as
Fig. 2. Methane production from the di€erent e‚uents (gCOD/l): (a) W assays (H blank, 5.5 gCOD/l (W4), s 11.1 gCOD/l (W5), 16.6 gCOD/l
(W6)); (b) S assays (H blank, 6.5 gCOD/l (S4), s 13.1 gCOD/l (S5), 19.6 gCOD/l (S6)).
236
G. Vidal et al. / Bioresource Technology 74 (2000) 231±239
Fig. 3. Methanisation () and biodegradability (n) percentages in assays W (a) and S (b).
Fig. 4. Maximum methane production rate. Assays W (s) and S (d).
a representative LCFA in milk fat) can begin to cause
inhibition at 1.5 g/l and in our case, the concentration of
LCFA in the medium for assays W5 and W6 might be
0.9 and 1.5 g/l, respectively (values calculated by con-
sidering an accumulation in the form of LCFA of 50%
of milk fats). However, the biodegradability of S
wastewaters at higher concentrations (assays S5±S6)
clearly decreased. It is obvious that this was not due to
the presence of fats and it may only be explained by the
higher relative quantities of carbohydrates (6.9±10.3 g/l)
and proteins (4.5±6.7 g/l) in S wastewaters.
E€ects of carbohydrates (VFA). At low COD concentrations (1±5 gCOD/l), both wastewaters presented
no signi®cant VFA accumulation in the liquid phase.
However, at higher concentrations (5±20 gCOD/l), VFA
accumulation took place during a period of time, butyric
acid being the greatest contributor to the total concentration of fatty acids, especially in the case of S wastewaters. The evolution of VFA for the most highly
concentrated wastewaters is shown in Figs. 5(a) (Assay
W6) and (b) (Assay S6).
Methanisation of W wastewaters was probably controlled by the presence of large amounts of fats (0.1±2.8
g/l), because their hydrolysis rate was slow (Pavlostathis
and Giraldo-Gomez, 1991), which facilitated an equilibrated methanogenic step and avoided temporary VFA
accumulations. In this case (Fig. 5(a)), the butyric concentration of W6 was high (0.8±1 gCOD/l) between 17
and 43 h of the biodegradation assays, whereas the
Fig. 5. Evolution of VFA content in assays W6 (a) and S6 (b) (H total VFA, acetic acid, r propionic acid, n butyric acid).
G. Vidal et al. / Bioresource Technology 74 (2000) 231±239
concentrations of acetic and propionic acids were about
0.4 and 0.2 gCOD/l, respectively. The accumulations of
butyric acid may be explained by its maximum degradation rate, which according to Aguilar et al. (1995) is
23.8 mg/láh. On the contrary, S wastewaters were so
easily hydrolysed and acidi®ed, that signi®cant temporary accumulations (between 10 and 150 h, approximately) occurred (Fig. 5(b)), and probably inhibited
methanogenic bacteria and decreased the %M at higher
concentrations. This concurs with previous results (Aguilar et al., 1995).
Protein and FA compounds. In order for milk protein
to a€ect the degradation process, Perle et al. (1995) have
indicated that the previous adaptation of the sludge to
the presence of casein is necessary. In spite of the fact
that the seed sludge had no previous contact with dairy
e‚uents, none of the assays presented initial lag phases.
It is reasonable to assume that the problem is not one of
adaptation to casein but rather one related to ammonia
production.
Fig. 6 shows FA concentrations, which were computed using Eq. (3), to study the possible inhibition by
ammonia. According to this Figure, the ®nal concentrations of FA in W assays were not sucient to inhibit
methanogenic bacteria, a fact con®rmed by previous
studies (Soto et al., 1991; Omil et al., 1995). The inhibition range for mesophilic bacteria is between 30 and
170 mg FA/l. During these assays the highest concentration produced was 5.4 mg FA/l in the reactor of W6
assay. However, for S6 assay, the FA concentration was
62.2 mg/l, thus indicating a possible partial inhibition of
the methanogens.
According to Angelidaki et al. (1993), when ammonia
concentration increases, its toxic e€ect on methanogens
causes an accumulation of VFA which decreases pH and
in turn reduces the concentration of FA. This mechanism of inhibition relief tends to stabilise the process at a
certain VFA concentration and pH level. Besides, the
inhibition of methanogenic bacteria may explain why
237
Fig. 7. Evolution of inorganic carbon (r) Blank, W6 (s) and S6 (d).
the %BD decreased as the protein (or COD) concentration increased in the di€erent assays (see Fig. 3(b)). It
coincided with the kinetics of IC concentration in W6
and S6 assays shown in Fig. 7. The IC concentration in
S6 assay was negligible during the ®rst 50 h, whilst the
pH in the medium decreased, because of VFA accumulation (Fig. 5(b)). However, the ammonia concentration in the system (6.7 g protein/l) may have regulated
the pH, which contributed to increase in IC concentration.
Flotation problems. Assays of the wastewaters with
higher COD presented problems of sludge ¯otation
during anaerobic biodegradation, which were more intense in the ¯asks treating S wastewaters. According to
Perle et al. (1995) and Rinzema et al. (1993) sludge ¯otation might have been explained by the presence of fats
in W wastewaters, because as fat concentration increases, its solubility in water decreases and the surface
activity increases (Bell, 1973). These mechanisms do not
explain ¯otation of S wastewaters. It has been suggested
that carbohydrate-rich substrates favour the development of exocellular polymers, modifying the surface/
volume ratio of aggregates (Breure and Van Andel,
1988). This fact and the fast production of biogas, due
to the easy degradation of milk carbohydrates, may
explain ¯otation in the highly concentrated S wastewaters.
4. Conclusions
Fig. 6. Concentrations of free ammonia at the end of the assays W (s)
and S (d).
From the results obtained, several conclusions may
be drawn and the following recommendations for the
anaerobic treatment of this kind of e‚uent made:
Operation of the reactor at COD concentrations between 3 and 5 kgCOD/m3 is recommended, because
these conditions ensure the highest levels of biodegradability and methanisation of both wastewaters and
eliminate ¯otation problems.
238
G. Vidal et al. / Bioresource Technology 74 (2000) 231±239
The anaerobic biodegradation rate of fat-rich wastewaters (W-type) is slower than that of fat-poor wastewaters (S-type), due to the slower rate of the fat
hydrolysis step. However, this fact avoids the accumulation of VFA and the overall process is favoured,
therefore the presence of fats in the wastewater prevents
the periodic production of high concentrations of VFA,
which may adversely a€ect the process.
The intermediates of fat degradation (glycerol and
LCFA) seem not to reach concentrations high enough to
a€ect the anaerobic process.
Ammonia production is signi®cant in carbohydraterich wastewaters (S-type) when the COD is high. This
fact has two antagonistic e€ects: FA causes a partial
inhibition of the process but it also controls the pH and
therefore inhibition by VFA accumulation is avoided
and the overall process improved.
Acknowledgements
This work was partially ®nanced by CICYT, project
number AMB98-0658, Agencia de Cooperaci
on Iberoamericama Project ICI-USC/UFRO-97 and Xunta de
Galicia (Project PGIDT99MA010E). We are also
grateful to Mar Orge for her technical assistance.
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