Chemical Engineering Journal 255 (2014) 492–499

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Chemical Engineering Journal

journal homepage: www.elsevier.com/locate/cej

Anaerobic mesophilic co-digestion of sewage sludge and sugar beet pulp

lixiviation in batch reactors: Effect of pH control
Rocío Montañés ⇑, Montserrat Pérez, Rosario Solera
Department of Environmental Technologies, Faculty of Sea and Environmental Sciences, University of Cádiz, 11510 Puerto Real, Cádiz, Spain

h i g h l i g h t s

 Accumulation of volatile fatty acids because of pH value.

 Sugar beet pulp lixiviation (SBPL) improves cumulative net methane generation with initial pH control.
 Different SS/SBPL ratios have been tested in biochemical methane potential tests.
 Initial pH in batch test affects the biodegradability of anaerobic co-digestion.
 A pH out of range for methanogenic microorganism, inhibits their growth.

a r t i c l e i n f o a b s t r a c t

Article history: In this study, biochemical methane potential (BMP) tests were conducted to investigate the effect of pH
Received 10 March 2014 control on the co-digestion of sewage sludge (SS) and sugar beet pulp lixiviation (SBPL) at mesophilic
Received in revised form 10 June 2014 range (35 °C). Microbial concentrations (Eubacteria and methanogenic Archaea) are linked to traditional
Accepted 16 June 2014
parameters, biogas production and total volatile solids (TVS) removal. Also, the relationship between
Available online 27 June 2014
Eubacteria and Archaea has been analysed. Organic matter being equal, higher net methane generation
was reported for the assay with pH adjustment at the beginning of the biochemical methane potential
Keywords:
(BMP) test. This showed that there was inhibition of methane generation as measured by the BMP test
Biochemical methane potential (BMP) test
pH
in the absence of pH adjustment. Evidence of this inhibition was also supported by the methane yield
Anaerobic co-digestion and TVS removal data. Microbial populations in the reactor at the end of both assays were composed
Sewage sludge of Eubacteria and Archaea, with a higher proportion of Eubacteria in all cases. It is noteworthy that in test
Sugar beet pulp lixiviation 1, when inhibition occurred due to a system pH that was not optimal for the activity of methanogenic
Archaea, the analysis showed the largest number of Archaea present. In terms of productivity, it can be
said that methanogenic Archaea were inactive.
Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction operational parameters. Temperature, organic strength, buffering

Anaerobic digestion is a biological process in which a group of
microorganisms biodegrade organic matter (substrate) in the
absence of free molecular oxygen (O2). As a result of this complex
biological process, organic matter is mainly converted into a mix-
ture of methane (CH4) and carbon dioxide (CO2), as well as new
bacterial cells [1]. Throughout the process, complete bioconversion
of organic matter into stable end products is accomplished by a
series of interdependent metabolic reactions in which different
classes of microorganisms take part.
Efficiency of anaerobic digestion depends highly on waste
characteristics, in addition to reactor configurations and other
⇑ Corresponding author. Tel.: +34 956016158.
E-mail address: rocio.montanes@uca.es (R. Montañés).
capacity, solids and nutrient content are considered crucial
waste characteristics affecting anaerobic biodegradation. If the
characteristics of the waste are inappropriate for targeted treat-
ment efficiency, some measurements can be taken to improve its
digestibility.
The biochemical methane potential (BMP) test used to deter-
mine methane potential is a batch procedure carried out over a
period of time that is sufficient for the available carbon in the test
substrate to be converted to biogas. This is normally considered to
be the point at which the biogas production from the test reactors
equals that from control reactors to which no substrate has been
added. In practice, this can be in the range of 5–100 days, depend-
ing on the degradability of the material. The approach has limita-
tions and only provides an approximate basis for estimation of
the biogas yield likely to be achieved in a continuous flow process.

http://dx.doi.org/10.1016/j.cej.2014.06.074
1385-8947/Ó 2014 Elsevier B.V. All rights reserved.
 R. Montañés et al. / Chemical Engineering Journal 255 (2014) 492–499
This will depend not only on the degradability of the substrate, but
also on factors such as process loading, retention time, tempera-
ture and type of digester.
Co-digestion is one of the options used for the enhancement of
anaerobic degradation of wastes with different characteristics.
Anaerobic co-digestion is the simultaneous biodegradation of dif-
ferent wastes in a reactor to establish positive synergism in the
digestion medium [2]. Merits of co-digestion include: creating a
suitable ratio of required nutrients, diluting potential toxic com-
pounds [3], supplying buffering capacity [4], sharing of equipment,
establishing required moisture content and easing the handling of
wastes [2]. In addition, anaerobic co-digestion is advantageous if
the amount of a single waste generated at a particular site is not
sufficient to make anaerobic digestion cost effective [5]. There
are numerous studies in the literature regarding the anaerobic
co-digestion of various wastes including: food industry wastes
[6,7], animal manure [8,9], municipal solid waste [10,11], waste-
water sludge [1], fish wastes [4] and algal sludge [12]. In most of
these studies, remarkable improvements were observed in both
treatment efficiencies and biogas productions.
Besides the advantages listed above, the co-digestion method-
ology of organic residues of different origin has proved successful
in both thermophilic and mesophilic regimes so that these vari-
ants would be applicable to the co-digestion of said waste. Good
results have been achieved from the co-digestion of sewage
sludge (SS) with fruit and vegetable wastes [13]. Improvements
have also been achieved in the production of biogas slurry mix-
ture or bovine manure and plant debris [14] with mixtures of
bovine and fruit and vegetable waste [15]. To date, no BMP test
has been performed with sewage sludge and sugar beet pulp lix-
iviation (SBPL).
The pH of the system, as well as temperature, has a marked
effect on the rate of growth and in selecting the type of microor-
ganisms predominant in the process. The growth of each type of
microorganism occurs only within a characteristic pH range. The
maximum growth rate occurs at an optimum pH value which
usually coincides with the average value of the interval (7-8)
[16].
The system pH is one of the most important parameters
influencing anaerobic digestion in reactors, as it affects both the
chemical reactions and the activity of microbial consortia in the
sludge [17,18]. The optimum pH for most anaerobic microorgan-
isms is 7–7.5, except for hydrolysing/fermentative bacteria whose
optimum pH is 5–7 [19,20]. Generally, a pH of 8.5 is considered
unfavourable for methanogenic microbes, and pH values lower
than 5 are considered inhibitory.
The different microbial groups involved in the digestion process
have varying sensitivities to environmental changes. The acetogen-
ic bacteria and methanogenic Archaea have more stringent require-
ments (in the thermophilic pH range 7.5–8.5) and greater difficulty
acclimating when a change in environmental conditions occurs
and, therefore, have lower growth rates. As a result of distortions
in the environmental conditions of the medium, acetogenic bacte-
ria are inhibited temporarily. However, they continue to produce
volatile organic acids, which are then converted to acetic acid,
hydrogen (H2) and carbon dioxide. This causes a significant
decrease in pH of the medium and, therefore, inhibits the activity
of the bacteria. This process is known as an acidification reaction
[21,22]. In this regard, a neutral or basic feed, or even direct control
of medium pH should favour degradation activity and, therefore,
the purification process.
There is no conclusive theory to explain the role of the system
pH. Bacterial performance is associated with the concentration of
volatile fatty acids (VFAs), redox potential, hydrogen partial pres-
sure and alkalinity. When the pH of the residue fed to a reactor
decreases, there is an associated decrease in alkalinity.
493
The organic fraction of the sewage sludge and beet pulp lixivi-
ation is converted to methane and carbon dioxide by anaerobic
co-digestion, which takes place by the coordinated action of differ-
ent groups of microorganisms and passes through several interme-
diate stages. The intermediates are the following volatile fatty
acids: acetic acid, propionic acid and butyric acid. The conversion
of acetate to methane by methanogenic bacteria becomes the lim-
iting step in the production of biogas since microorganisms that
are known methanogens grow slowly, resulting in a relatively
small population [23]. Methanogens are typically divided into
two main groups based on their substrate conversion capabilities.
Acetoclastic methanogens are capable of converting acetate into
methane and carbon dioxide. These microorganisms are consid-
ered to play a dominant role in the production of methane since
about 70% of the methane produced in the digester comes from
acetate [23]. The H2-utilising methanogenic bacteria also play a
key role in anaerobic digestion since they are responsible for main-
taining the partial pressure of H2 at a very low level (<10 Pa). This
condition is needed for the operation of the intermediate group,
which is responsible for the conversion of organic acids and alco-
hols to methane [24].
Process imbalances explain the formation and accumulation of
volatile fatty acids (acetic, propionic and butyric acid), hydrogen
and carbon dioxide in gaseous and liquid effluents when their
production by acidogenic bacteria are not balanced with their
degradation.
In this study, anaerobic batch reactors were used to determine
the anaerobic biodegradation and biogas generation potential [25]
of sewage sludge and sugar beet pulp lixiviation. For this purpose,
both substrates were subjected to anaerobic biodegradation in
batch reactors. Since sugar beet pulp is a waste product of sugar
beet processing plants and is known to be suitable for biological
degradation, this study aimed to investigate the potential benefits
of co-digestion with sewage sludge as well as separate digestion of
these wastes. The effect of pH on sewage sludge and sugar beet
pulp lixiviation using the BMP test was examined in this study
for the first time, as it has not previously been reported in the
literature.
Notations
BMP, biochemical methane potential 1-1, 100% SBPL test 1
OLR, organic loading rate 1-2, 75% SBPL–25% SS test 1
COD, chemical oxygen demand 1-3, 50% SBPL–50% SS test 1
H-Ac, acetic acid 1-4, 25% SBPL–75% SS test 1
H-Bu, butyric acid 1-5, 100% SS test 1
H-Pr, propionic acid 2-i, inoculum test 2
TS, total solids 2-1, 100% SBPL test 2
TVS, total volatile solids 2-2, 75% SBPL–25% SS test 2
VFA, volatile fatty acids 2-3, 50% SBPL–50% SS test 2
SS, sewage sludge 2-4, 25% SBPL–75% SS test 2
SBPL, sugar beet pulp lixiviation 2-5, 100% SS test 2
Test 1, assay without pH control Subscripts
Test 2, assay with pH control t, s, total, soluble
1-i, inoculum test 1
2. Materials and methods
2.1. Feedstock
The substrates that were used in batch tests are sugar beet pulp,
from Azucarera Ebro company in Jerez de la Frontera (Cádiz) and
sewage sludge from the municipal wastewater treatment plant of
San Fernando-Cádiz (Spain). Pellets were subjected to biological
pretreatment before the co-digestion process in order to promote
hydrolysis and solubilisation of the organic matter and therefore,
improve anaerobic digestion in the generation of biogas and
possible final residue agronomic valorisation [26].
494 R. Montañés et al. / Chemical Engineering Journal 255 (2014) 492–499

Table 1 Prior to incubation, all of the 24 reactors were purged with

Inocula characteristics. 100% N2 for 3–4 min in order to maintain anaerobic conditions
Mesophilic inocula with proper pH. Then the reactors were sealed with natural rubber
pH 7.4 stoppers and plastic screw-caps. Prepared reactors were incubated
CODt (kg/m3) 21.3 in a temperature controlled bath at 35 °C. Manual mixing was
CODs (kg/m3) 1.2 applied three times a day during the test.
ST (kg/m3) 14.50 During digestion period, biogas productions and biogas compo-
SV (kg/m3) 8.58
ST (%) 1.45
sitions were determined daily. After the digestion period was
SV (%) 0.86 ended, all reactors were subjected to pH, TS, TVS, VFA, alkalinity
Alkalinity (mg CaCO3/l) 2480 and COD total and soluble determinations, in order to analyse
VFA t (mg H-Ac/l) 45.5 the treatment efficiencies, as well as the microbiology analyses.
H-Ac (mg/l) 45.5
The process control parameters were: the degradative capacity
H-Pr (mg/l) 0.0
H-Bu (mg/l) 0.0 of the system, measured as a percentage TVS removal and carbon
Total microorganism (cell/ml) 6.5
108 oxygen demand (COD) and as well as the productivity of biogas,
% Eubacteria 59.4 especially quantifying the cumulative net methane generation.
% Archaea 40.6 Initially, mesophilic inoculas were characterised regarding the
activity of the microorganisms presented. At the end of the tests,
the same parameters were analysed. Volume and composition of
2.2. Inoculum biogas were measured daily.

Primary Sludge from the WWTP of San Fernando-Cádiz was
used as inoculum in both tests.
The ultimate methane yields as well as the methane production
rates are dependent on the specific substrates and inoculum. Large
inoculations volumes ensure high microbial activity, low risk for
overloading and low risk of inhibition [27]. In this study, the anaer-
obic seed was obtained from the municipal wastewater treatment
plant of San Fernando-Cádiz (Spain). The seed culture was the
effluent of a completely mixed anaerobic sludge digester having
an SRT of 30 days and operating at mesophilic range.
Mesophilic inocula with 1.45% of TS were added to the assays
without and with pH control, until the desired conditions were
achieved. Table 1 shows the pH, TS, TVS, CODt, CODs, Alkalinity,
VFA and microbial characterisation of inocula used.
2.3. Experimental set-up and procedures
Separate and co-digestion of sewage sludge and sugar beet pulp
lixiviation were studied in 250 ml serum bottles with effective vol-
ume of 130 ml.
The digesters were initially loaded with a mixture of inoculum
and substrate, resulting in a final concentration of 40% w/w of inoc-
ulum, which is considered optimum for biogas production and sub-
strates acclimatize, establishing a TVS concentration of 8.58 kg/m3
for test without and with pH control, respectively. Then the wastes
were added to the reactors at different amounts to give SS/SBPL
ratios in the range of 0.25, 0.5 and 0.75 (Table 2). Control reactors,
containing only anaerobic inoculums, were also incubated to
determine the background gas production. All reactors were run
in duplicates and presented data composed of the averaged values.
2.4. Analytical methods
Methods of analysis performed in this study can be grouped
into two categories: physical and chemical parameters of the
process control of degradability and methods for quantification
of the microbial population in the reactors.
The determination of pH, total and volatile solids, total and sol-
uble chemical oxygen demand and alkalinity will be established
according to the standard method [28]. Volatile fatty acids and
the composition of the biogas are determined by gas chromatogra-
phy. Gas chromatography was used to analyse the different com-
ponents of the biogas. The gases analysed were: H2, CH4, CO2, O2
and N2 (GC-2010 Shimadzu). The first five components were ana-
lysed by means of a thermal conductivity detector (TCD) using a
Supelco Carboxen 1010 Plot column. Samples were taken using a
1 ml Dynatech Gastight gas syringe.
Organic matter removal was calculated as the percentage differ-
ence between the TVS of the initial and final substrates in the
assays. Total acidity was calculated by addition of the individual
fatty acids.
In order to compute methane generations, ideal gas balance
evaluations were carried out for each reactor, calculations were
performed daily. During calculations, methane, generated in con-
trol reactor was subtracted from that of reactor of interest, to be
able to determine the net methane generation, resulted from the
stabilisation of the waste in the corresponding reactor [29].
2.5. Microbial analyses
The main steps of fluorescence in situ hybridisation (FISH) of
whole cells using 16S rRNA-targeted oligonucleotide probes are

Table 2
Initial characteristics from substrates in bottle serum.

1-1 1-2 1-3 1-4 1-5 2-1 2-2 2-3 2-4 2-5
pH 4.8 5 5.1 5.5 6 7.3 7.4 7.3 7.5 7.8
CODt (kg/m3) 31.6 32.1 31.3 35.3 33.9 29.1 40.3 47.2 63.2 72.1
CODs (kg/m3) 13.4 12.2 9.9 8.3 6.5 12 10.3 7.8 5.9 1.5
ST (kg/m3) 14.9 17.5 18.4 19.1 20.5 17.3 22.6 25.6 31.2 34.5
SV (kg/m3) 12 12.8 13.5 13.6 14 10.7 15.3 18.5 23.4 26.3
ST (%) 1.6 1.7 1.8 1.9 2 1.7 2.3 2.6 3.1 3.4
SV (%) 1.2 1.2 1.3 1.3 1.5 1.1 1.5 1.8 2.3 2.6
Alkalinity (kg CaCO3/m3) 1.4 1.5 1.5 1.7 2 2.2 3.9 3.4 3 2.5
VFA t (mg H-Ac/l) 5441 3921 3435 3521 3743 0 20.2 8.5 0 17
H-Ac (mg/l) 1734 936 1104 1153 1154 0 0 0 0 0
H-Pr (mg/l) 727 758 780 957 1246 0 0 0 0 0
H-Bu (mg/l) 1671 1155 693 531 347 0 0 0 0 0
 R. Montañés et al. / Chemical Engineering Journal 255 (2014) 492–499 495

cell fixation, consequent permeabilisation and hybridisation with
the desired probe(s).
The samples were collected from batch reactor at the end of the
assays into sterile universal bottles. Absolute ethanol was added to
the bottles in a volume ratio of 1 sample: 1 ethanol. The samples
were stored at 20 °C until they were fixed. The experimental pro-
cedure was conducted according Montero et al. [30].
The technique used for fixation and permeabilisation of cells
was based on the one used by Amann et al. [31]. The following
16S rRNA-targeted oligonucleotide probes were used in this study:
Bacteria-universal probe EUB338 [31,32], Archaea-universal probe
ARC915 [34], H2-utilising methanogens probe MB1174 (specifi-
cally Methanobacteriaceae) [33].
The cellular concentration and percentages of Eubacteria,
Archaea, H2-utilising methanogens were obtained by FISH. The
total population was calculated as the sum of the relative amounts
of Eubacteria and Archaea, because the main anaerobic groups in
the anaerobic reactors are contained within these two domains
[33]. Acetate utilising methanogens were calculated as the
difference in the relative amounts of Archaea and a H2-utilising
methanogens. The main steps of FISH of whole cells using 16S
rRNA-targeted oligonucleotide probes are cell fixation, consequent
permeabilisation, and hybridisation with the desired probe(s).
The samples were examined visually and cells counted using an
Axio Imager Upright epifluorescence microscope (Zeiss) with a
100 W mercury lamp and an 100 oil objective. According of
labelled probe, if the fluorochrome was 6-FAM, the filter was used
B-2A (DM 510, Excitation 450–490 and Barrer 520) and Cy3, the
filter was G-2A (DM 580, Excitation 510–560 and Barrer 590).
3. Results and discussion
3.1. Evolution of biogas generation
Reactors were operated until no significant biogas production
was detected. Fig. 1 illustrates the cumulative net methane
generation in all reactors. Cumulative biogas production is
depicted for 20 and 35 days of operation in test 1 and 2,
respectively.
In anaerobic treatment systems, waste stabilisation is achieved
by methane production [34]. Therefore, the rate of methane produc-
tion directly reflects the rate of process stabilisation, which is crucial
information for the design and operation of anaerobic treatment
systems. Thus, determining the rate-limiting step, as well as analys-
ing the overall biodegradation rate, is of fundamental importance.
As observed in biogas production, higher net methane genera-
tion was reported for assay 2 when NaOH was added to adjust
the pH at the beginning of the BMP test. This explained the inhibi-
tion in the first test compared to the second test. This effect was
also supported by methane yield records and TVS removal data
(Table 3).
Organic matter being equal, the reactors with the same sub-
strate composition (SS/SBPL ratio) in both assays can be evaluated
in terms of treatment efficiencies in order to compare relative bio-
degradability of SS/SBPL ratios with and without pH adjustment.
The highest values of methane yield (544.4 ml/g TVS added
[TVSadd]) and TVS reduction (63.5%) were observed in reactor 2-
1, which was fed only with sugar beet pulp lixiviation. In fact, it
is widely accepted that sugar beet pulp lixiviation is highly biode-
gradable because it is made up of soluble carbohydrates, mainly
sucrose [35,36]. Lower methane yield, chemical oxygen demand
(COD) removal and TVS reduction were observed in test 1. These
observations are a direct result of inhibition in the assay due to a
system pH that is out of the methanogenic range and the high val-
ues of VFAs in the initial substrates.
Thus to SS/SBPL ratio, initial pH was influential on the
treatment performance of the reactors (Table 4). Still, in all
SS/SBPL ratios tested with pH adjustment, treatment efficiencies
(48–61.5% total COD removal and 47–63.5% TVS reduction) are
indications of high biodegradability for both substrates.
Fig. 1a and b shows that, in test 1, only reactors with a high per-
centage (75% and 100%) of sewage sludge in the feed generated

Fig. 1. Cumulative net methane generations: (a) test 1; (b) test 2.

Table 3
Final characteristics from substrates in bottle serum.

1-i/2-i 1-1 1-2 1-3 1-4 1-5 2-1 2-2 2-3 2-4 2-5
pH final 7.5 4.7 5.4 7.4 7.1 7.1 7.7 7.7 7.6 7.6 7.6
CODs (kg/m3) 3.8 15.5 14.3 12.4 9.7 8 13.3 12.3 10.7 8.4 6.9
% CODt removal 8.3 0 0 0 0 0 47.8 49.9 56.1 59 61.5
TVS (kg/m3) 6.7 11.9 10.2 10.8 10.5 10.9 3.9 6.4 9.6 12.3 11.6
% TVS removal 22.1 3.1 20.6 20 65.3 27 63.5 57.8 48 47.3 56
Final VFA (mg Ac/l) 21.6 5417 6103 5117 2975 2459 0 20.2 8.5 0 17
Alkalinity (mg CaCO3/l) 4737 2997 1727 2682 2915 2895 1185 2322 2987 2880 2917
ml CH4/g VS added – 0 0 0 5.6 34.8 544.4 520.8 403.4 358.8 255
% CH4 62.9 10.2 9 42.2 60.9 56.2 67 63 61.6 63.3 57.5
% of biogas produced in first 10 days 61.2 – – 100 71.3 3 43.2 47.2 48.4 47 47.2
496 R. Montañés et al. / Chemical Engineering Journal 255 (2014) 492–499

Table 4
Concentrations and percentages of Eubacteria, Archaea, H2-utilising methanogens and acetate-utilising methanogens in test 1 and in test 2.

Test 1
1-i 1-1 1-2 1-3 1-4 1-5
Total microorganism (cell/ml) 1.1
109 7.3
108 6.3
108 9.5
108 6.9
108 5.1
108
% Eubacteria 52.4 68.1 74.8 82.4 74.5 61.5
% Archaea 47.6 31.9 25.2 17.6 25.5 38.5
% H2-utilising methanogens 71.2 100 95.2 72.1 62.7 86.8
% Acetate-utilising methanogens1 28.8 0 4.8 27.9 37.3 13.2
Test 2
2-i 2-1 2-2 2-3 2-4 2-5
Total microorganism (cell/ml) 1.2
108 7.3
107 6.5 107 1.3 108 1.1 108 1.1 108
% Eubacteria 52.6 76.1 61.5 64.3 60.3 53
% Archaea 47.4 23.9 38.5 35.7 39.7 47
% H2-utilising methanogens 100 100 100 100 100 100
% Acetate-utilising methanogensa 0.0 0.0 0.0 0.0 0..0 0..0
a
Acetate-utilising methanogens has been calculated in relation with Archaea.

biogas. The rest of the reactors had no biogas production or
removal of organic matter due to low pH values and high
quantities of VFAs.
Table 4 shows the parameters measured at the end of the
biodegradability test, in which very low methane productions were
observed in all reactors assayed in test 1 for the different SS/SBPL
ratios tested. The cause of these results in test 1 was inhibition
of the process as a result of inappropriately low pH in the digester
during the degradation test. The development of methanogens in
anaerobic digestion requires higher pH values. In addition, the
substrates analysed in test 1 had a high quantity of VFAs, which
inhibits methane generation.

Fig. 2. Comparative cumulative net methane generations in test 1 and test 2 at different SS/SBPL ratios.
 R. Montañés et al. / Chemical Engineering Journal 255 (2014) 492–499
The following figures compare cumulative net methane
generation for all substrates tested in both assays and show how
pH adjustment affects methane generation and prevents system
inhibition.
From the graphs above it is noteworthy that the inocula used
was able to generate the biggest quantity of cumulative biogas
compared with the rest of the reactors in test 1, and the lowest
compared with reactors in test 2. In the rest of the reactors tested,
those in assay 2 have higher biogas production than reactors in
assay 1, as shown in Fig. 2.
3.2. Alkalinity and volatile fatty acids
The monitoring of volatile fatty acids, also known as short
chain fatty acids, is widely applied as a stress indicator in anaer-
obic digestion processes. Allowing their accumulation leads to
drops in pH and even subsequent reactor failures [37–39] as
shows Fig. 3.
This pH reduction is normally counteracted by the activity of
the methanogens, which produce alkalinity in the form of carbon
dioxide, ammonia and bicarbonate. The system pH is controlled
by the CO2 concentration in the gas phase and the HCO3 alkalinity
of the liquid phase. If the CO2 concentration in the gas phase
remains constant, the addition of HCO3 alkalinity will increase
the digester pH [40]. Fig. 4 shows the relationship between total
acidity and alkalinity. It is clear that the digesters in test 2 were
operating with good buffering capacity, indicated by the low or
null amounts of VFAs. In test 2 the reduction in VFA levels and
alkalinity over time did not affect methanogenic activity as the
methane concentration in the gas did not drop. This demonstrated
that the acetogens and methanogens were able to cope with the
Fig. 3. (a) Individual volatile acid (as mg/l) levels in test 1; (b) individual volatile acid (as mg/l) levels in test 2.
Fig. 4. (a) Ratio total acidity/alkalinity in test 1; (b) ratio total acidity/alkalinity in test 2.
497
fluctuations in the VFAs and alkalinity in the digester, indicating
that the conditions were stable and the possibility of methanogen
inhibition was low. Nevertheless, in test 1 reactors the production
and composition of biogas shows that VFAs affected the anaerobic
digestion process.
Total acidity values in test 1 were very high at the beginning
and end of the process due to methanogen inhibition in the assay.
High concentrations of volatile fatty acids were observed fre-
quently in the effluent from anaerobic digesters as a result of
organic causes, such as overloading of the digester, entrance of
toxic compounds and changes in the temperature or pH. Low pH
stimulates acidogenic activity (VFA production) and inhibits meth-
anogenic activity (VFA consumption). This could explain the obser-
vation of VFAs at the end of the assays.
Acetate has been described as the least toxic fatty acid [41],
while an increase in propionate concentration has been shown to
be associated with system failure [42]. Propionate is even more
inhibitory than butyrate.
At the end of the test 2 assay, the level of VFAs decreased con-
siderably. This was a consequence of the total biodegradability that
occurred in all test 2 reactors.
During the entire operating time of the digester, alkalinity
remained constant at around 3000 mg CaCO3/l, except for reactors
where inhibition occurred because of low pH values. As shown in
Fig. 4, the total acidity/alkalinity ratio was very low in test 2 reac-
tors and high in test 1 reactors, except for reactor 1-i (reactor with
inocula). The pH balance is essential for proper operation and opti-
mal degradation of VFAs. A ratio of total acidity/alkalinity between
0.0 and 0.1 is desirable and results in a strong system. Values
between 0.1 and 0.4 indicate favourable operating conditions
without the risk of acidification [43,44].
498 R. Montañés et al. / Chemical Engineering Journal 255 (2014) 492–499

3.3. Microbial population dynamics

The microorganism concentrations in the effluent at the end of

the BMP tests were studied. The amounts and relative percentages
of the main microbial groups are shown in Table 4.
Microbial populations in the reactor at the end of both assays
consist of Eubacteria and Archaea, with higher proportions of
Eubacteria in all cases. Stable anaerobic reactors contain a
Eubacteria population much larger than that of Archaea [45]. It is
noteworthy that in most cases studied, the H2-utilising methano-
genic subpopulations are dominant over the acetate-utilising
methanogens.
In test 1, inhibition occurred in reactors 1, 2, 3, 4 and 5 due to
system pH values that were not optimum for the activity of meth-
anogenic Archaea. The analysis showed the largest number of
Archaea present, however, in terms of productivity it can be said
that methanogenic Archaea were inactive.
All the reactors in test 2 showed the same behaviour, and the
relationships between all microbial populations were similar. For
this reason, all substrates tested showed biodegradability.
To evaluate the biochemical activity on initial OLR (in terms of g
TVSadd), it has been considered the parameter to measure metha-
nogenic activity. The relation between those parameters was cal-
culated as the ratio of CH4 volume generated to the number of
Archaea inside the reactor by FISH staining [30]. Fig. 5a and b com-
pares productivity and microbial activity, respectively, in tests 1
and 2. The productivity values of reactors in test 1 showed null
to low productivity in terms of ml CH4/g TVSadd, as well as null to
low microbial activity, due to the inhibition in the system. In
Fig. 5b inhibition is observed in test 1 as seen by the results
obtained in terms of ml CH4/cell. This is because the Archaea
population present in the reactor did not generate methane due
to bacterial inactivity. However, Fig. 5b shows good results for test
2 regarding microbial activity. A more detailed analysis is reflected
in Fig. 5d, which shows a linear relationship between the initial
organic loading, in terms of g TVSadd, and the total Archaea popula-
tion at the end of the assay in batch reactors, except for inoculum
reactor 2-i.
According to Fig. 5d, a high amount of microorganisms in the Fig. 5. (a, b) Analysis of productivity and microbial activity respectively in all
effluent is directly related to higher levels of organic matter in reactors tested. (c) Relation between total Archaea population and initial organic
the initial substrates as defined by TVS. loading rate in terms of g TVSadd in test 2.
Fig. 6a and b shows the relationship between the amount of
Archaea in the effluent and productivity obtained in all SS/SBPL
ratios studied in test 1 and test 2, respectively. due to the increased amount of volatile solids in the initial sub-
Compared to test 2 (Fig. 6b), test 1 (Fig. 6a) showed no or low strates. Comparing Fig. 5d with Fig. 6b shows that the results of
values of productivity in terms of ml CH4/g TVSadd in reactors productivity in terms of ml CH4/g TVSadd are not proportional to
tested, except for inoculum reactor 1-i, although the Archaea the initial organic loading for the reason mentioned above.
population is higher in this test. Previous studies demonstrated links between digester operat-
Fig. 6b shows that the size of the Archaea population has an ing conditions, physical and chemical performance parameters
indirect relationship with productivity in terms of ml CH4/g TVSadd, and microbial population dynamics [30]. As discussed, the results

Fig. 6. Relations between physicochemical parameters and microbial concentrations. (a, b) Compare Archaea population with methane yield in terms of ml CH4/g TVSadd in
test 1 and 2 respectively.
 R. Montañés et al. / Chemical Engineering Journal 255 (2014) 492–499
obtained in this study show that the initial organic loading in the
reactors could be more related to microbial concentrations than
to the activity of anaerobic microorganisms.
4. Conclusions
Recent studies have indicated that in addition to sewage sludge,
sugar beet pulp lixiviation has huge potential to be used as a
renewable energy source by using anaerobic co-digestion. Based
on the experimental results, the following conclusions were made:
In tests without pH adjustment, inhibition occurred in anaero-
bic digestion, except in reactor 1 with inocula, because the opti-
mum pH of the methanogens was not reached and there was an
accumulation of volatile fatty acids. In the second test, after an ini-
tial pH adjustment total biodegradability of the substrates tested
occurred. In reactors 2-2, 2-3 and 2-4, where the substrates were
sewage sludge and sugar beet pulp lixiviation in different concen-
trations, more methane was produced, which was an indication of
high biodegradability for both wastes.
Owing to the above mentioned points, the anaerobic co-
digestion of SS and SBPL is a promising application, since addition
of SBPL significantly increases the rate of biomethanation of SS
when an optimum pH is reached.
Acknowledgments
The authors wish to express their gratitude to Junta de And-
alucía, especifically to Proyecto de Excelencia with reference P09-
TEP-5275, financed by FEDER funds, called ‘‘Codigestión anaerobia
de lodos de depuradora y residuos de cultivos vegetales energéti-
cos. Estrategias para mejorar producción de biogas y la valorización
agronómica del residuo final’’.
References
[1] R.T. Romano, R. Zhang, Co-digestion of onion juice and wastewater sludge
using anaerobic mixed biofilm reactor, Bioresour. Technol. 99 (2008) 631–637.
[2] J. Mata-Alvarez, S. Macé, P. Llabrés, Anaerobic digestion of organic solid wastes.
An overview of research achievements and perspectives, Bioresour. Technol.
74 (2000) 3–16.
[3] P. Sosnowski, A. Wieczorek, S. Ledakowicz, Anaerobic co-digestion of sewage
sludge and organic fraction of municipal solid wastes, Adv. Environ. Res. 7
(2003) 609–616.
[4] A. Mshandete, A. Kivaisi, M. Rubindamayugi, B. Mattiasson, Anaerobic batch
codigestion of sisal pulp and fish wastes, Bioresour. Technol. 95 (2004) 19–24.
[5] W. Parawira, M. Murto, R. Zvauya, B. Mattiasson, Anaerobic batch digestion of
solid potato waste alone and in combination with sugar beet leaves, Renew.
Energy 29 (2004) 1811–1823.
[6] G. Carucci, F. Carrasco, K. Trifoni, M. Majone, M. Beccari, Anaerobic digestion of
food industry wastes: effect of codigestion on methane yield, J. Environ. Eng.
131 (2005) 1037–1345.
[7] M. Murto, L. Björnsson, B. Mattiasson, Impact of food industrial waste on
anaerobic codigestion of sewage sludge and pig manure, J. Environ. Manage. 70
(2004) 101–107.
[8] G. Gungor-Demirci, G.N. Demirer, Effect of initial COD concentration, nutrient
addition, temperature and microbial acclimation on anaerobic treatability of
broiler and cattle manure, Bioresour. Technol. 93 (2004) 109–117.
[9] K. Umetsu, S. Yamazaki, T. Kishimoto, J. Takahashi, Y. Shibata, C. Zhang, et al.,
Anaerobic co-digestion of dairy manure and sugar beets, Int. Congr. Ser. 1293
(2006) 307–310.
[10] H. Hartmann, B.K. Ahring, Anaerobic digestion of the organic fraction of
municipal solid waste: influence of co-digestion with manure, Water Res. 39
(2005) 1543–1552.
[11] G.D. Zupancic, N. Uranjek-Zevart, M. Ros, Full-scale anaerobic co-digestion of
organic waste and municipal sludge, Biomass Bioenergy 32 (2008) 162–167.
[12] H. Yen, D.E. Brune, Anaerobic co-digestion of algal sludge and waste paper to
produce methane, Bioresour. Technol. 98 (2007) 130–134.
[13] B. Molinuevo, X. Gómez, A. Marán, M.C. García-González, Anaerobic co-
digestion of livestock and vegetable processing wastes: Fibre degradation and
digestate stability, Waste Manage. 33 (2013) 1332–1338.
[14] R.M. Dinsdale, G.C. Premier, F.R. Hawkes, D.L. Hawkes, Two-stage anaerobic
co-digestion of waste activated sludge and fruit/vegetable waste using
inclined tubular digesters, Bioresour. Technol. 72 (2000) 159–168.
499
[15] G.H. Dar, S.M. Tandon, Biogas production from pretreated wheat straw, lantan
residue, apple and peach leaf litter with cattle dung, Biol. Wastes 21 (1987)
75–83.
[16] F.J. Callaghan, D.A.J. Wase, k. Thayanithy, C.F. Forster, Co-digestion of waste
organic solids: batch studies, Bioresour. Technol. 37 (1999) 117–122.
[17] R. Gaudy, J.P. Guerin, Population dynamics of Tisbe holothuriae (Copepoda;
Harpacticoida) in exploited mass cultures, Netherlands J. Sea Res. 16 (1982)
208–216.
[18] U. Marchaim, Biogas Processes for Sustainable Development, FAO, 1992. p.
238.
[19] P.F. Pind, I. Angelidaki, B.K. Ahring, K. Stamatelatou, G. Lyberatos, Monitoring
and control of anaerobic reactors, in: Biomethanation, Springer-Verlag, Berlin,
2003, pp. 135–182.
[20] A. Princic, I. Mahne, F. Megusar, E.A. Paul, J.M. Tiedje, Effects of pH and oxygen
and ammonium concentrations on the community structure of nitrifying
bacteria from wastewater, Appl. Environ. Microbiol. 64 (1998) 3584–3590.
[21] R.D. Tyagi, Wastewater Treatment by Immobilized Cell, CRC Press, Boca Ratón,
1990, pp. 334–399.
[22] M. Soto, R. Mendéz, J.M. Lema, Sodium inhibition and sulphate reduction in the
anaerobic treatment of mussel processing wastewaters, J. Chem. Technol.
Biotechnol. 58 (1993) 1–7.
[23] S.H. Zinder, Physiological ecology of methanogens, in: J.G. Ferry (Ed.),
Methanogenesis: Ecology, Physiology, Biochemistry and Genetic, Chapman &
Hall, New York, 1993, pp. 128–206.
[24] A. Pauss, R. Samson, S. Guiot, C. Beauchemin, Continuous measurement of
dissolved H2 in an anaerobic reactor using a new hydrogen/air fuel cell
detector, Biotechnol. Bioeng. 35 (1990) (1990) 492–501.
[25] W.F. Owen, D.C. Stuckey, J.B. Healy Jr, L.Y. Young, P.L. McCarty, Bioassay for
monitoring biochemical methane potential and anaerobic toxicity, Water Res.
1 (1979) 485–492.
[26] R. Montañés, M. Pérez, R. Solera, Mesophilic anaerobic co-digestion of sewage
sludge and a lixiviation of sugar beet pulp: optimisation of the semicontinuous
process, Bioresour. Technol. 142 (2013) 655–662.
[27] I. Angelidaki, W. Sanders, Assessment of the anaerobic biodegradability of
macropollutants, Rev. Environ. Sci. Biotechnol. 3 (2004) 117.
[28] APHA, AWWA, WEF. Standard Methods for the Examination of Water and
Wastewater, 19th ed. Washington, DC, New York, USA. 1995.
[29] E. Alkaya, G.N. Demirer, Anaerobic mesophilic co-digestion of sugar-beet
processing wastewater and beet-pulp in batch reactors, Renew. Energy 36
(2011) 971–975.
[30] B. Montero, J.L. García-Morales, D. Sales, R. Solera, Analysis of methanogenic
activity in a thermophilic-dry anaerobic reactor: use of fluorescent in situ
hybridization, Waste Manage. (Oxford) 29 (2009) 1144–1151.
[31] R.I. Amann, B.J. Binder, R.J. Olson, S.W. Chisholm, R. Devereux, D.A. Stahl,
Combination of 16S rRNA-targeted oligonucleotide probes with flow
cytometry for analyzing mixed microbial populations, Appl. Environ.
Microbiol. 56 (1990) 1919–1925.
[32] R.I. Amann, L. Krumholz, D.A. Stahl, Fluorescent-oligonucleotide probing of
whole cells for determinative phylogenetic and environmental studies in
microbiology, J. Bacteriol. 172 (1990) 762–770.
[33] D.A. Stahl, R. Amann, Development and application of nucleic acid probes, in:
E. Stackebrandt, M. Goodfellow (Eds.), Nucleic Acid Techniques in Bacterial
Systematics, John Wiley & Sons, Chichester, U.K., 1991, pp. 205–248.
[34] R.R. Speece, Anaerobic Biotechnology for Industrial Wastewaters, Arachae
Press, Nashville, USA, 1996.
[35] J. Iza, J.I. Palencia, F. Fdz-Polanco, Waste water management in a sugar beet
factory: a case study of comparison between anaerobic technologies, Water
Sci. Technol. 22 (9) (1990) 123–130.
[36] M. Shore, N.W. Broughton, N. Bumstead, Anaerobic treatment of waste waters
in the beet sugar industry, Water Pollut. Control 83 (4) (1984) 499–506.
[37] B.K. Ahring, M. Sandberg, I. Angelidaki, Volatile fatty acids as indicators of
process imbalance in anaerobic digestors, Appl. Microbiol. Biotechnol. 43 (3)
(1995) 559–565.
[38] W. Gujer, A.J.B. Zehnder, Conversion processes in anaerobic digestion, Water
Sci. Technol. 15 (1983) 127–167.
[39] P.L. McCarty, Anaerobic waste treatment fundamentals. Part two:
environmental requirements and control, Public Works 95 (1964) 123–126.
[40] I.S. Turovskiy, P.K. Mathai, Wastewater Sludge Processing, Wiley Interscience.
John Wiley & Sons, New York, 2006.
[41] E.L. Iannotti, J.R. Fischer, D.M. Sievers, Medium for the enumeration and
isolation of bacteria from a swine waste digester, Appl. Environ. Microbiol. 36
(1978) 555–566.
[42] P.N. Hobson, B.G. Shaw, Inhibition of methane production by
Methanobacterium formicicum, Water Res. 10 (10) (1976) 849–852.
[43] D.W. de Haas, N. Adam, Use of a simple titration procedure to determine
H2CO⁄3 alkalinity and volatile fatty acids for process control in waste-water
treatment, Water SA 21 (1995) 307–317.
[44] E. Sánchez, R. Borja, L. Travieso, A. Martin, M.F. Colmenarejo, Effect of organic
loading rate on the stability, operational parameters and performance of a
secondary upflow anaerobic sludge bed reactor treating piggery waste,
Bioresour. Technol. 96 (2005) (2005) 335–344.
[45] S. Zahedi, D. Sales, L.I. Romero, R. Solera, Optimisation of single-phase dry
thermophilic anaerobic digestion under high organic loading rates of
industrial municipal solid waste: population dynamics, Bioresour. Technol.
146 (2013) 109–117.