Bioresource Technology 154 (2014) 305312

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Performance of up-ow anaerobic xed bed reactor of the treatment

of sugar beet pulp lixiviation in a thermophilic range
Roco Montas Alonso , Montserrat Prez Garca, Rosario Solera del Ro
Department of Environmental Technologies, University of Cdiz, Spain

h i g h l i g h t s

 SBPL as substrate; high content of microorganisms in SBPL.

 Improve biogas productivity and the organic matter removal.
 Different OLRs and hydraulic retention times (HRTs) were carried out.
3
 The greatest efciency; 90% COD removal for an OLR of 4.3 kg COD/m d.
 OLR was directly correlated with active microbial biomass.

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

Article history: The acclimatization and performance study of lixiviation of sugar beet pulp are carried out in upow
Received 16 October 2013 anaerobic xed bed reactor in thermophilic range of temperature (55 C). Several hydraulic retention
Received in revised form 11 December 2013 time is conducted (11, 8, 6, 4, 2, and 1.5 days). The performance study showed that Chemical Oxygen
Accepted 14 December 2013
Demand removal efciency is 90% for 6 days-HRT. While COD removal efciency was reduced within
Available online 22 December 2013
the range of 74.3% and 59.4% in others HRT. Organic loading rates greater than 10 kg COD/m3 d in inu-
ent, (2 days-HRT), produces a destabilization of the process due to total acidity accumulation in reactors
Keywords:
although is the HRT with highest methane production.
Anaerobic digestion
Upow anaerobic xed bed reactor (AUF)
The results showed that an increase in OLR was directly correlated with active biomass inside reactor
Methane yield but not with the amount in microbial community. The bacterial concentration inside the reactor is
Sugar beet pulp strongly inuenced by the content of microorganisms in the lixiviation of sugar beet pulp.
Population dynamic 2013 Elsevier Ltd. All rights reserved.

1. Introduction
The anaerobic digestion process provides a very efcient way to
transform organic waste into an innocuous gas (methane) which is
capable of providing energy. However the low settling velocity of
anaerobic digestion in a conventional digester makes solidliquid
separation difcult, which results in a large reactor volume, poor
efuent quality, and unstable anaerobic digestion. The immobiliza-
tion technology which has been developed to increase productivity
in the fermentation industry (Mozaffar et al., 1986) has been
applied to anaerobic treatment in order to overcoming the demer-
its of the conventional process (Scherer et al., 1981). Regardless of
their adhesiveness to biocarriers, almost all of the microorganisms
contributing to anaerobic digestion can be held in the biosystem by
using immobilization technology, which offers anaerobic bacteria
protection from the effects of inhibitory substances (Speece, R.E.,
Corresponding author. Tel.: +34 660 92 12 71.
E-mail addresses: rocio.montanes@uca.es (R. Montas Alonso), montserrat.
perez@uca.es (M. Prez Garca).
1983) and gives a high ratio of SRT (solid retention time) to HRT.
This high ratio is favorable since maximal SRT provides process sta-
bility and minimal sludge production, and minimal HRT minimizes
the reactor volume.
Spain has approved several National Plans covering different
waste categories and contaminated land over the past few years.
The new National Integrated Waste Plan 20082015 (PNIR, Plan
Nacional Integral de Residuos), approved by the Council of Minis-
ters in December 2008, is intended to serve as a guide for the
development of specic policies to improve waste management
by reducing its generation and promoting its correct treatment.
This Plan establishes specic waste reduction, reuse, recycling,
assessment and elimination targets and covers the treatment of
household waste, specic waste, contaminated land and some
non-hazardous agricultural and industrial waste. It also includes
a Biodegradable Waste Discharge Reduction Strategy.
There already exists recent literature about applications and
benets of the anaerobic digestion process to produce renewable
energy from various sources of biomass (Angelidaki et al., 2006).
Furthermore, there also exist several works about continuous

0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.12.071
306 R. Montas Alonso et al. / Bioresource Technology 154 (2014) 305312
Notation
UAF upow anaerobic xed bed (OR FILTER)
COD chemical oxygen demand (soluble)
HRT hydraulic retention time
OLRr organic load rate removed
anaerobic digestion of sugar beets for production of methane
(Gallert and Winter, 1997; Jarrell and Kalmokoff, 1988).
Most renewable energies, and in particular, agricultural bio-
mass waste, are energy sources scattered around a given country.
The agro-industrial biomass waste generated is concentrated and
its management in the same places from where it originates
requires sufcient quantities of waste for energy recovery to be
technically and economically viable.
Furthermore, production of the beet crop has risen in recent
years, justied by its high energy productivity (3.54.5 t bioetha-
nol/Ha). This crop generates high quantities of waste, such as sugar
beet pulp, which has to be treated. In this regard, the anaerobic
digestion of organic waste is a good choice.
Pretreatment with sugar dried pellets yields a homogeneous
liquid efuent with high organic load, suitable for use in xed
bed reactor.
Section 2 shows the characteristics of the substrate that was
used in terms of the organic matter content. 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 nal residue agronomic valorisation.
Other pretreatments have been studied in order to improve
solubilize the substrate, substrate reduction or for the enhance-
ment of biodegradability (Kavitha et al., 2013; Esakki et al., 2013;
Rani et al., 2011, 2012).
Anaerobic processes operating under thermophilic conditions
(55 C) have attracted a great deal of attention in recent years
due to their apparent advantages, which include high pathogen
destruction, enhanced hydrolysis of complex organic/biological
materials, and foaming reduction (Hartmann and Ahring, 2005).
Anaerobic digestion which is ultimately converted into meth-
ane and carbon dioxide, is carried out by the coordinated action
of various groups of microorganisms and goes through several
intermediate stages. The intermediary products are volatile fatty
acids, acetic, propionic, and butyric acids. Two-thirds or more of
the methane produced in anaerobic bioreactors is derived from
acetate (Zinder, 1993). The conversion of acetate to methane by
methanogenic populations becomes the rate-limiting step in
biogas production, as methanogens are known for their slow
growth, resulting in a relatively small population size (Zinder,
1993). The parameters normally employed in the control of
anaerobic digestion, such as the percentage of COD removal, the
concentration of volatile fatty acids and the amount and composi-
tion of biogas generated in the process, are not always representa-
tive of the composition and physiological state of biomass
contained within the system. From a practical standpoint, given
the importance of methanogens in anaerobic treatment processes,
the ability to monitor methanogens and understand their ecology
is essential to make effective controls of the start-up and operation
of anaerobic bioreactors possible. Consequently, and in order to
acquire more detailed information regarding of this biomass, other
parameters have also been used in the characterization of the
microorganisms responsible for the anaerobic processes. Direct
counting procedures by microscopic methods reach the highest
estimation of members of microorganisms and are occasionally
OLR0 initial organic load rate
TSS total suspended solids
VSS volatile suspended solids
SBPL sugar beet pulp lixiviation
used for indirect calculation of biomass. Epiuorescence micros-
copy with uorometric stainings are widely used for direct count-
ing of microorganisms, since it does not require culturing (Kepner
and Pratt, 1994).
Molecular tools as uorescence in situ hybridization (FISH),
based on sequence comparison of small-subunit (SSU) ribosomal
RNA (rRNA) molecules have made it possible to detect specic
whole cells/organisms in biological samples as anaerobic reactors
(McMahon et al., 2001; Montero et al., 2008, 2009; Zahedi et al.,
2013).
Generally, the treatment capacity of an anaerobic digestion sys-
tem is determined by the amount and activity of microorganisms,
which in turn is inuenced by wastewater composition, system
conguration and operation of the anaerobic reactor. Unlike the
conventional biolm systems in which the growth support media
are xed in space either by gravity or by direct attachment to the
reactor wall, the anaerobic uidized bed system retains the growth
support media in suspension by drag forces exerted by upowing
wastewater. Moreover, the distribution of biomass hold-up (in
the form of a biolm) is relatively uniform because of the com-
pletely mixed conditions maintained and the continuous biolm
sloughing process, which counterbalances the accumulation of bio-
mass due to the growth. This is because of the biomass hold-up,
which can be directly measured in terms of attached volatile solids
using the techniques developed by Mulcahy and Shieh (1987).
A good design, starting and operation of these systems require
knowledge of the mechanisms of absorption and xation of micro-
organisms. It is necessary to establish the effects caused by the ten-
sions generated between the efuent and the particles. The high
complexity of all interactions leads to an unknown complete
mechanisms of the colonization process.
The microbial biolm can be developed on any surface where
microorganisms can grow. This can be considered as the result of
various physical, chemical and biological processes that constitute
the following steps:
Transport and absorption of organic molecules on the surface.
Microbial movement of the cells to the surface.
Surface adhesion of microorganisms and monocell formation.
Development and accumulation of biolm as a consequence of
microbial activity and ora growth.
Detachment of biolm due to hydraulic uid tensions.
When the surface of a solid particle or even reactor walls are in
contact with an organic matter suspended in inuent, a spontane-
ously initial absorption of nutrients and microorganisms occurs.
Physical, chemical and biological characteristics of biolm
depend on certain environmental conditions prevailing the base
material for the attachment of the microorganisms. Factors such
as temperature, pH, salts and nutrient levels affect the process.
Differents methanogenic species attached to support in an orga-
nized manner making easier the transfer of substrate (especially
H2, important intermediate metabolite). Other chemical properties
as the composition of the inuent, the rate of biolm development,
cellular interactions are factors to take into account in the evolu-
tive process of colonization of the support.
 R. Montas Alonso et al. / Bioresource Technology 154 (2014) 305312
High organic loading rates and high concentrations of VSS help
to attached microorganisms in the solid surface. The rate of such
accumulation increases also in terms of the ow to the reactor,
because of substrate transfer promotes the formation thereof. How-
ever too high velocities (detachment phenomena cause biomass)
maintain biolm with thickness values (Characklis et al., 1982).
In summary, the formation of biolm is a process which
depends on the characteristics of the inoculum, the feeding compo-
sition, the physiology of bacterias and hydrodynamic characteris-
tics of the reactor, as well as the own support.
The purpose of the study described here was to elucidate the
treatment efciency in an upow anaerobic xed bed reactor
(UAF) that decomposes the lixiviation of sugar beet pulp. The
experimental protocol was designed to examine the effect of
organic loading rate on the efciency of COD removal under differ-
ent hydraulic retention times.
2. Methods
The experimental protocol was designed to examine the effect
of increasing organic loading rate (OLR) on the efciency of upow
anaerobic xed bed reactor (UAF) in the thermophilic anaerobic
treatment of sugar beet pulp lixiviation.
The methods and material used are briey described in this
section. The essay was carried out in duplicate and all the showed
results are the average values of the last data obtained.
2.1. Experimental systems
A schematic diagram of the upow anaerobic xed bed reactor
used in the laboratory study is shown in Fig. 1.
The study was conducted on a laboratory scale over a four-
month period. The reactor was built as a vertical cylindrical tank
(25 cm length and 10 cm internal diameter). The active liquid vol-
ume was 1.7 L, and the empty volume was 2.4 L. The reactor was
lled with 600 randomly distributed media support entities
(16 mm length every one). Reactor temperature was maintained
at 55 C with a heating bath consisting of a pump through which
was achieved by recirculating the hot water through the jacket of
the reactor. Efuent recirculation was used to mix and homogenize
the liquid in the system (recycle rate: 8.3 l/min). Under these con-
ditions, the liquid phase is perfectly mixed. Gas produced in the
reactor was collected in a Tedlar bag.
Once a day the reactors were fed, and an efuent recirculation
was used to mix and homogenize the liquid in the system.
40-L
TEDLAR
BAG
(BIOGAS)
2-L REACTOR
FLOCOR-R
RECYCLE FLOW
HEATING
BATH
(55C)
Fig. 1. Semi-continuous upow anaerobic xed bed reactor (UAF).
307
2.2. Feeding solutions
Pellets were subjected to biological pretreatment before the
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 nal residue agronomic
valorisation. Pretreatment was divided into different steps, as
was describe in Montaes et al. (2013).
The main characteristics of the sugar beet pulp lixiviation are
described. As has been observed, the feeding has an acid pH, the
evolution is shown in Fig. 2. The different physic-chemical charac-
teristics of the lixiviation of sugar beet pulp are the followings; sol-
uble COD, total COD, TOC, TSS, VSS, total acidity concentrations and
alkalinity. COD values and TOC concentrations were 17.75 kg O2/
m3 and 5274 ppm C, respectively. The amount of total suspended
solid and volatile suspended solid in the lixiviation are 1.2 kg
TSS/m3 and 1.1 kg VSS/m3 accordingly. The average value of total
acidity concentration is 3617 g AcH/m3 and the alkalinity is 305 g
CaCO3/m3.
2.3. Media support
Corrugated plastic tubes (non-porous media) were used as sup-
port media for cell immobilization and retention in this research
suitable to use them as stationary packed media in lter reactors.
According to Prez Garca (1995), the support media that deliver
better cleaning efcacy results are plastic support, so that in this
case FLOCOR-R have been used, whose characteristics are reported
in Table 1.
2.4. Chemical and microbial analyses
The volume and composition of biogas were determined daily,
the biogas produced was quantied using a gas ow meter (Ritter
TG1) and a gas suction pump (KNF Laboport). Gas chromatography
was used to analyze the different components of the biogas. The
gases analyzed were: H2, CH4, CO2, O2 and N2 (GC-2010 Shimadzu).
The rst ve components were analyzed 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 under the following operating conditions: Split = 100; con-
stant pressure in the injection port (70 kPa); 2 min at of 40 C;
ramped at 40 C/min until 200 C; 1.5 min at 200 C; detector tem-
perature: 250 C; injector temperature: 200 C. The carrier gas was
helium employing a ow rate of 266.2 ml/min. A commercial mix-
ture of H2, CH4, CO2, O2, N2 and H2S (Abell Linde S.A.) was used to
calibrate the system.
The following analytical determinations were performed to
monitor and control the process in the substrate and the efuent:
TSS, VSS, pH, dissolved COD, alkalinity and VFA. The pH was mea-
sured daily using a Crison 20 Basic pH-meter. TVS, COD and VFA
were analyzed three times a week for a 114 d HRT; daily in the
2 and 1.5 d HRT; These determinations were performed according
to APHA (1995).
Organic matter removal was calculated as the percentage differ-
ence between the VSS of the inuent and the VSS of the efuent
within the substrate TVS. Total acidity was calculated by addition
of the individual fatty acids.
2.5. Analytical determinations to count microbial population
The main steps of uorescence in situ hybridization (FISH) of
whole cells using 16S rRNA-targeted oligonucleotide probes are
cell xation, consequent permeabilization and hybridization with
the desired probe(s).
308 R. Montas Alonso et al. / Bioresource Technology 154 (2014) 305312

10 days-HRT 8 days-HRT 6 days-HRT 4 days-HRT 2 days-HRT 1,5 days-

9 HRT
8
7
6
5
pH

4
3
2
1
0
0 2 5 7 9 13 15 19 29 34 36 41 43 47 49 51 55 61 63 65 69 71 75 77 79 83 85 89 91 96 98 100 104 106
days
inuent euent

Fig. 2. pH inuent and efuent evolutions.

Table 1
Main characteristics of ller used FLOCOR-R.
Physical properties Corrugated plastic
Density (g/L) 1161.4
Bulk density (g/L) 73.0
Porosity (%) 93
Specic area (m2 m 3
) 450
Height (cm) 1.6
Diameter 1.6
The samples were collected from thermophilic upow anaero-
bic xed bed reactor 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 xed. The exper-
imental procedure was conducted according to Montero et al.
(2009).
The technique used for xation and permeabilization of cells
was based on the one used by Amann et al. (1990a). The following
16S rRNA-targeted oligonucleotide probes were used in this study:
Bacteria-universal probe EUB338 (Amann et al., 1990a,b), Archaea-
universal probe ARC915 (Stahl and Amann, 1991), H2-utilising
methanogens probe MB1174 (specically Methanobacteriaceae).
(Stahl and Amann, 1991). Acetate-utilizing methanogens were cal-
culated as difference of the relative amounts of Archaea and H2-
utilising methanogens.
The samples were examined visually and cells counted using a
an Axio Imager Upright epiuorescence microscope (Zeiss) with a
100 W mercury lamp and an 100 oil objective. According of la-
belled probe, if the uorochrome was 6-FAM, the lter was used
B-2A (DM 510, Excitation 450490 and Barrer 520) and Cy3, the l-
ter was G-2A (DM 580, Excitation 510560 and Barrer 590).
2.6. Performance and reactor operation
The experimental protocol was designed to examine the effect
of organic loading rate on the efciency of COD and VSS removal
of UAF reactor in the treatment of sugar beet pulp lixiviation.
Experiments were performed with the xed bed height in the
reactor controlled, yielding a working volume of 1.7 L with 8.3 L/
h of recirculation rate. Under these conditions tested, the liquid
phase in the reactor was completely mixed.
The main characteristics of the different stages of the operation
are shown in Table 2. The UAF reactor was subjected to a pro-
gramme of steady state operation over a range of HRT between
11 and 1.5 days. The volumetric COD loadings were between 1.1
and 10.7 kg COD/m3 d. Initially, the reactor was fed with lixiviation
of sugar beet pulp to give an organic loading rate of 1.1 kg COD/
m3 d with an HRT of 11 days. After this period of adaptation, the
organic loading rate was maintained constant until three times
the same HRT. The HRT was gradually decreased and remained
constant during each stage until reaching the steady-state condi-
tions. The attainment of the steady state was veried after an ini-
tial period (three times the HRT) by checking whether the constant
efuent characteristic values (COD removal and methane genera-
tion) were the mean of the last measurements in each stage
(Rodrguez-Cano, 2003).
The cellular concentration of Eubacteria, Archaea, H2-utiliz-
ing methanogens, and acetate-utilizing methanogens were
obtained by uorescent in situ hybridization and the data is
shown in Table 2 as well as biomass measured as total volatile
suspended solid and organic loading rate of the feeding at differ-
ent HRTs studied.
The substrate shows a high content in anaerobic microorgan-
ism, mainly those belonging to Eubacteria group that constitute
almost 90% of the total population. The biodegradable substrate
is liable to start the degradation during the pretreatment pro-
cess. The methanogens microorganism are present as a residual
part and are mainly H2-utilizing, whose growth is favored in the
early consequence of biomethanisation as a result of hydrogen
released in the early stages of the process and the acidity of
the medium.
3. Results and discussion
The aim of the present study is to optimize the operating condi-
tions, HRT and OLR of the anaerobic digestion process of sugar beet
pulp lixiviation in a thermophilic upow anaerobic xed bed (UAF)
reactor. The effect of the variations in organic loading has been
studied at laboratory scale measuring COD, VFA, VSS, TSS, microbi-
ological concentration and methane production. Relationships
between OLR, methane generation and total number of microor-
ganisms were also considered.
The pH remained within the range 7.58.5 throughout the pro-
cess, thus demonstrating the stability of this system until 2 days
HRT is reached. Acclimatization of sugar beet pulp lixiviation is
done with the organic loading rate of 1.1 kg O2/m3 d which
corresponds to 11 days of HRT and allows the anaerobic bacterias
present in the feeding could entirely acclimatize to the new envi-
ronmental conditions in the reactor. The pH of the reactor decrease
under 7.5 when 2 days HRT is achieved as show in Fig. 2. This is
due to fatty acids, in the feeding, are not transformed into organic
acids which mainly consist of acetic acid with hydrogen and
carbon dioxide.
 R. Montas Alonso et al. / Bioresource Technology 154 (2014) 305312 309

Table 2
Main characteristics of the feeding.

HRT (days) OLR (kg COD/m3 d) VSS (kg VSS/m3) Eubacteria (%) Archaea (%) H2-utilizing methanogens (%) Acetate-utilizing methanogens (%)
11 1.1 0.654 58 42 97 3
8 2.7 1.568 82 18 72 28
6 4.3 1.093 89 11 72 28
4 4.5 1 74 26 36 64
2 10 1.63 81 19 78 22
1.5 10.7 0.87 81 19 80 20

The acidity registered in the efuent is related to the nature of Table 4

sugar beet pulp lixiviation. Nevertheless, the measured values are Average biogas production.

not very high until 2 days HRT is reached, again indicating the HRT OLRremoval kg Methane yield
stability of the process (Desh et al., 1994). (days) CODremoval/m3 d
m3 CH4/ m3 CH4/kg m3 CH4/kg %
The alkalinity of the efuent remains relatively constant m3digester d CODremoval VSSremoval CH4
throughout the assay (all HRT tested). However, the system has a 11 0.65 0.15 0.26 3.4 68.6
high buffer capacity and the acidity/alkalinity ratio remained below 8 1.67 0.67 0.43 4.4 63.4
0.1 until 2 days HRT is reached in the assay (Desh et al., 1994). 6 2.95 1.40 0.47 8.8 67.5
Table 3 summarized the steady state performance of UAF reac- 4 3.47 1.48 0.43 6.8 65.7
2 6.26 3.20 0.51 5.8 63.0
tor at six different HRT of 11, 8, 6, 4, 2, and 1.5 days. Hence the COD
1.5 6.38 3.60 0.51 9.4 59.5
removal efciency was in the range of 59.4% until 90%. The higher
COD removal efciency for the UAF is 90% for 6 days of HRT. The
COD removal efciency becomes poorer at shorter HRT.
production was reached with a value of 0.51 m3 CH4/kg CODremoval,
with a methane production of 3.2 and 3.6 m3/m3 d, respectively.
2.8. Biogas production The volumetric methane production activity (as m3/m3 d) could
be expressed as a linear function of removal organic loading rate
The rate of CH4 was increasing due to the increasing organic (OLRremoval, as gCODremoval/m3 d), by the linear equation
loading rate or decreasing HRT. At 11 days-HRT the system has a y = 0.515x with a regression coefcient of 0.9706. It regression rep-
bad behaviour because of the low methane rate, but when the sys- resents methane production assuming that the incoming COD is
tem reached 6 days-HRT the high rate of methane makes the sys- transformed into methane.
tem a good option to degradate the sugar beet pulp lixiviation. The specic methane production value obtained in the present
At the end of each HRT studied, a stabilization of biogas production study (0.430.51 m3 CH4/kg CODremoval) could explain that a high
was observed with a percentage of CH4 at least of 60%. concentration of metabolized substrate have been converted to
Table 4 shows data about yield of biogas at each stage of the methane. The unconverted substrate to biogas could be explained
reactor. In the rst HRT, the OLR was of 1.1 kg COD/m3 d and the by the presence of other agents that would consume COD without
production of CH4 was 0.15 m3/m3 d, with a corresponding specic producing methane and by the synthesis cell (Metcalf and Eddy,
methane production of 0.26 m3 CH4/kg CODremoval (cubic meter of 1991).
methane per kilogram Chemical Oxygen Demand removal). This
value indicates that the performance of the reactor is acceptable 2.9. Microbial population dynamics
as can be seen in other work (Nebot, 1992). Nebot indicated a value
of 0.20 m3 CH4/kg CODremoval as reference for a similar OLR of Anaerobic digester efuent (sugar beet pulp lixiviation) from a
1.5 k g COD m3 d) and a portion of the metabolized substrate is UAF reactor in mesophilic range was used as inoculum. The evolu-
used in methane production. At 8 and 4 HRT the specic methane tion of the microbial population was studied in the stabilization
production was of 0.43 m3 CH4/kg CODremoval, with a correspond- periods in thermophilic anaerobic digestion in all HRT tested.
ing methane production of 0.67 m3/m3 d and 1.48 m3/m3 d, respec- Results of the relative percentages of the main microbial groups
tively. There is an optimum HRT in terms of % COD removal are shown in Table 5.
(6 days-HRT), although the value of specic methane production Microbial population suspended in the reactor, and therefore
is not the highest with a value of 1.40 m3 CH4/m3 d and nally, leaves the system daily with the efuent, is constituted by Eubac-
for lows HRT of 2 and 1.5 days, a notable increase of specic methane teria and Archaea with similar proportions from 11 to 4 days of

Table 3
Efuents properties.

Parameter Average concentration for HRT (day) of

11 8 6 4 2 1.5
pH 8.14 7.89 7.76 7.64 7.38 7.35
Efuent COD (kg O2/m3) 3.1 3.3 2.5 2.4 6.5 6.5
COD removal efciency (%) 74.3 83.2 90.0 74.3 69.7 59.4
Efuent VSS (kg/m3) 0.173 0.243 0.176 0.148 0.478 0.379
VSS removal (%) 73.6 84.5 73.9 85.2 70.7 56.4
Alkalinity (kg CaCO3/m3) 3605 2592 2720 2683 2475 3590
Total acidity (mg AcH/L) 164 351 30 43 2512 2148
Total acidity removal (%) 96.9 82.0 98.9 97.9 0 0
Total acidity/alkalinity 0.08 0.04 0.02 0.03 0.91 0.87
310 R. Montas Alonso et al. / Bioresource Technology 154 (2014) 305312

Table 5
Evolution of Eubacteria, Archaea, H2-utilising methanogens and acetate-utilising methanogens in the efuent of UAF reactor.

Efuent
HRT = 11 d HRT = 8 d HRT = 6 d HRT = 4 d HRT = 2 d HRT = 1.5 d
Eubacteria (cell/ml) 7.4  107 1.1  108 7.4  107 7.04  107 7.5  108 3.4  108
Archaea (cell/ml) 8.8  107 7.3  107 9.3  107 5.9  107 3.4 107 4.2  107
H2-utilising methanogens (cell/ml) 5.1  107 6.2  107 3.1 107 6  107 2.3  107 6.2  107
Acetate-utilising methanogens (cell/ml) 3.8  107 1.1  107 6.2  107 0 1.1  107 0
Total microorganism 1.6  108 1.8  108 1.7  108 1.3  108 7.8  108 3.8  108
% Eubacteria 45.66 60.06 44.35 54.25 95.64 89.07
% Archaea 54.34 39.94 55.65 45.75 4.36 10.93
% H2-utilising methanogens 57.51 85.27 33.33 100 68.87 100
% Acetate-utilising methanogensa 42.49 14.73 66.67 31.13
a
Acetate-utilising methanogens has been calculated in relation with Archaea.

HRT. Stable anaerobic reactors contain a Eubacteria population
much larger than of Archaea (Zahedi et al., 2013) therefore there
is a greater degree of detachment of methanogenic microorganism.
However, when the reactor is destabilized the population that lar-
gely detaches the support and leaves the reactor is Eubacteria (2
and 1.5 days-THR). Is noteworthy the uctuations of subpopula-
tions (H2-utilizing methanogenic and acetate-utilising methano-
gens) were suffering.
The relationship between Archaea and Eubacteria change for
each HRT. A specic analysis of each HRT has been done.
The rst four HRTs (11, 8, 6 and 4) have the same relation
between the number of Archaea efuent and inuent except HRT
for 4 days. These are times with high COD removal rates and good
biogas yields in terms of m3/kg CODremoval except for the rst
hydraulic retention time as shown in Table 4.
The analysis of the last two HRT where the number of bacteria in
the inuent and efuent is less as well as the percentage of organic
matter removal in terms of COD, however the production of biogas
is signicantly higher in those HRT. Drastic reduction of total metha-
nogens may be related with the increase in acidity as acetic accumu-
lates if not consumed by the acetate-utilizing methanogenic.
The biolm formed in the support has been analyzed by the
microorganisms accumulation as the relation between Archaea
and VSSout was studied.
According to Fig. 3 a high amount of microorganism in the efu-
ents is directly related with higher levels of organic matter in the
efuent in terms of VSS. At 2 and 1.5 days-HRT the values of VSS
in terms of kg VSS/m3 increased due to a possible detachment of
the biolm by high organic load and zero total acidity removal.
Fig. 4 shows the evolution of COD over the essay. It is remark-
able the high removal capacity of COD although there are OLR uc-
tuations in the feeding to the system, except for high ow inlet (at
2 and 1.5 days-HRT), where the reactor has no ability to assimilate
OLR and an accumulation of VFA is produced.
Although the determination of the number of microorganisms
is important in many microbial ecology studies (Hanning et al.,
2007), these papers do not assess the activities associated with
the total population. Under some conditions, microbial number

HTR=11 d HTR=8 d HTR=6 d HTR=4 d HTR=2 d HTR=1.5 d

0.7000

0.6000

0.5000

0.4000
kg/m3

VSSout
0.3000

0.2000

0.1000

0.0000
0 1 6 13 15 16 20 34 35 36 41 43 44 48 50 64 70 79 86 91 98 100 105 106
days

9.00E+08
8.00E+08
7.00E+08
6.00E+08
5.00E+08
cell/ml

4.00E+08
3.00E+08
2.00E+08
1.00E+08
0.00E+00
11 8 6 4 2 1.5
Total microorganism

Fig. 3. Relation between VSSout and total microorganism in efuent.

 R. Montas Alonso et al. / Bioresource Technology 154 (2014) 305312 311

HTR=11 d HTR=8 d HTR=6 d HTR=4 d HTR=2 d HTR=1.5 d

4.00E+04 100
3.50E+04 90
80
3.00E+04
70
2.50E+04
kg O2/m3

60

%
2.00E+04 50
1.50E+04 40
30
1.00E+04
20
5.00E+03 10
0.00E+00 0
0 6 13 16 34 36 43 48 51 56 63 65 70 79 84 86 90 97 99 103 105
days
CODo CODremoval %CODremoval

Fig. 4. Proles of inuent COD and CODremoval (kg O2/m3 d) against % COD removal.

4 1.E+08
(a) 9.E+07
(b) 0.6 1.E+08
9.E+07
3.5
8.E+07 0.5 8.E+07

m3 CH4/kg CODremoval
3
m3 CH4/m3 digester d

7.E+07 7.E+07
0.4
2.5 6.E+07 6.E+07
cell/ml

cell/ml
2 5.E+07 0.3 5.E+07
1.5 4.E+07 4.E+07
3.E+07 0.2 3.E+07
1
2.E+07 2.E+07
0.1
0.5 1.E+07 1.E+07
0 0.E+00 0 0.E+00
111 82 63 4 25 1.5
6 111 82 63 4 25 1.5
6
HRT HRT
m3 CH4/m3 digester d cell/ml 107 m3 CH4/kg CODremoval cell/ml 107

(c) 12 1.E+08
(d) 100 1.E+08
9.E+07 90 9.E+07
10 8.E+07 80 8.E+07
8 7.E+07 70 7.E+07
kg COD/m3 d

6.E+07 60 6.E+07
cell/ml

cell/ml
6 5.E+07 50 5.E+07
%

4.E+07 40 4.E+07
4 3.E+07 30 3.E+07
2 2.E+07 20 2.E+07
1.E+07 10 1.E+07
0 0 E+00 0 0.E+00
111 82 63 4 25 1.5
6 111 82 63 4 25 1.5
6
HRT HRT
OLR (kg COD/m3 d) cell/ml 107 %CODremoval cell/ml 107

Fig. 5. Relations between physicochemical parameters and microbial concentrations in efuent. Archaea with volume of methane generated, methane yield, organic loading
rate and organic loading rate removal (ad), respectively.

and activity show proportional correlations, whereas this is not the to the OLR than the microbial concentrations, as has been dis-
case under many realistic circumstances. cussed. A correlation between OLR and methane yield throughout
Previous studies had demonstrated links between digester the entire study period has been proved. This study shows that the
operating conditions, physical and chemical performance parame- increase in active biomass inside the reactor is directly propor-
ters and microbial population dynamics (Montero et al., 2008). tional to the OLR (or inversely proportional to the HRT).
It is important to have into account that the microorganisms in The results had clearly indicated that the relative abundance of
the efuent are not representative of the microorganisms present microorganisms in the efuent was directly correlated with OLR,
inside the reactor in the process of anaerobic digestion because suspended solid volatile in the efuent and methane production
of the biolm formation. The microorganisms in the efuent are in UAF reactor treating sugar beet pulp lixiviation until 4 days-
representative of the microorganisms detached from the biolm. HRT.
The microbiology of the efuent of UAF reactors for anaerobic deg- Determine of microorganisms in inuent and efuent only
radation has not been analyzed prior to this study and only the reected the detached microorganisms from the biolm in the sup-
support with microorganisms attached has been studied previ- port, and not the amount of microorganisms in the biolm in the
ously (Gannoun et al., 2013). support.
The results obtained in this study seem to show that the activity Fig. 5 represents the amount of Archaea in the efuent and phy-
of anaerobic microorganisms in the reactor could be more related sic-chemical results obtained in all HRT. The ratio between the OLR
312 R. Montas Alonso et al. / Bioresource Technology 154 (2014) 305312
and the number of Archaea in the efuent (Fig. 5c) is quasi-inver-
sely proportional, that means the majority microorganism
attached to biolm are methanogens bacteria. Fig. 5a and b repre-
sent biogas yields, in terms of production of methane expressed as
m3 CH4/kg CODremoval d and m3/m3digester, for each HRT. The results
show an increasing biogas yield, although a reduction of Archaea in
efuents due to most of microorganisms detached from the biolm
are Eubacteria. Previous investigations indicate that the nature of
the feeding inuences the activity of the methanogenic
microorganisms and, therefore, the generation of biogas
(Rodrguez-Cano, 2003). A survey of the literature in this eld
shows that several recalcitrant wastewaters can be anaerobically
digested and generate only small amounts of biogas (Hansen
et al., 1998). However, the volume of biogas can be increased by
the addition of a co-substrate (van Lier et al., 2001; Rodrguez-
Cano, 2003) and by providing sufcient time for the adaptation
of the biomass (Zeeman et al., 1985).
Fig. 5d, shows the high levels of COD removal with a decline in 2
and 1.5 days-HRT because the detached of microorganisms from
the biolm in the support.
4. Conclusions
Laboratory results conrm that UAF reactor provides a good
reduction in COD when operating at low HRT and high OLR in
anaerobic thermophilic digestion of SBPL. The greatest efciency
in terms of substrate removal was 90% for an OLR of 4.3 kg COD/
m3 d and at 6 days-HRT.
The results have shown that an increase in OLR was directly
correlated with methane yield but not with the amount in micro-
bial community because of the biolm.
Although the generation of biogas could be higher, the UAF
reactor provides good chemical reduction and is a stable process
with change in OLR.
Acknowledgements
The authors wish to express their gratitude to Junta de And-
aluca, especically to Proyecto de Excelencia with reference P09-
TEP-5275, called Codigestin anaerobia de lodos de depuradora
y residuos de cultivos vegetales energticos. Estrategias para mej-
orar produccin de biogas y la valorizacin agronmica del residuo
nal.
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