Energy 73 (2014) 866e874

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Energy
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Effects of operational shocks on key microbial populations for biogas

production in UASB (Upflow Anaerobic Sludge Blanket) reactors
~o b, M.I. Capela a, c, L.M. Arroja a, c,
C.S. Couras a, 1, V.L. Louros a, 1, A.M. Grilo b, J.H. Leita
M.H. Nadais a, c, *
a
Environment and Planning Department, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal
b
IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior T
ecnico, Universidade de Lisboa, Av.
Rovisco Pais 1, 1049-001 Lisboa, Portugal
c
CESAM e Centre for Environmental and Marine Studies, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal

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

Article history: This work compares the overall performance and biogas production of continuous and intermittent UASB
Received 22 December 2013 (Upflow Anaerobic Sludge Blanket) reactors treating dairy wastewater and subjected to fat, hydraulic and
Received in revised form temperature shocks. The systems were monitored for methane production, effluent concentration, vol-
10 May 2014
atile fatty acids, and microbial populations of the Eubacteria, Archaea and Syntrophomonadaceae groups.
Accepted 26 June 2014
This last microbial group has been reported in literature as being determinant for the degradation of fatty
Available online 25 July 2014
substrates present in the wastewater and subsequent biogas production. Results show that both
continuous and intermittent systems supported the applied shocks. However, the intermittent systems
Keywords:
Biogas production
exhibited better performance than the continuous systems in biogas production and physical-chemical
UASB reactors parameters. Syntrophomonadaceae microbial group was present in the intermittent systems, but was
Intermittent operation not detected in the biomass from the continuous systems. Hydraulic and temperature shocks, but not the
Operational shocks fat shock, caused severe losses in the relative abundance of the Syntrophomonadaceae group in inter-
Microbial populations mittent systems, leading to undetectable levels during the temperature shock. The severity of the effects
Syntrophomonadaceae of the applied shocks on the key microbial group Syntrophomonadaceae, were classified as:
fats < hydraulic < temperature. Results from a full-scale anaerobic reactor confirm the effect of inter-
mittent operation on the presence of Syntrophomonadaceae and the effect on reactor performance.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction bioenergy is an important measure to improve energy structure,

Interest in the development and utilization of non-petroleum
based renewable sources of energy has been fostered by concerns
on energy security and greenhouse gases emissions [1]. Among
renewable energy resources, bioenergy is one of the fastest
growing energy alternatives, with tremendous potential in many
regions of the world [2e5]. For example, the European strategy for
renewable energy sources identifies bioenergy as the most impor-
tant renewable energy source for the future. The development of
* Corresponding author. Environment and Planning Department, University of
Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal. Tel.: þ351 234 370 349;
fax: þ351 234 370 309.
E-mail addresses: catiacouras@ua.pt (C.S. Couras), vitorialouros@ua.pt
(V.L. Louros), andre.grilo@tecnico.ulisboa.pt (A.M. Grilo), jorgeleitao@tecnico.
~o), icapela@ua.pt (M.I. Capela), arroja@ua.pt (L.M. Arroja),
ulisboa.pt (J.H. Leita
nadais@ua.pt (M.H. Nadais).
1
Both authors contributed equally to the work.
safeguard energy security, protect the environment, and promote a
sustainable development [2]. In this context, the anaerobic treat-
ment of dairy industry wastewater containing high concentrations
of complex organic matter is highly attractive because of high
methane yields and potential for energy production. Besides its
complex chemical nature, dairy wastewater is characterized by a
wide variation in flow, organic matter composition and concen-
tration, due to the cyclic nature of production and cleaning pro-
cesses. In general, wastewater treatment plants include a flow
equalization tank which reduces the flow and load fluctuations.
Nevertheless, in real scale dairy plants, variations in flow, organic
load and fat concentrations can reach an order of 100% compared to
the average values [6]. These fluctuations are important since the
major drawbacks of anaerobic technology are unstable operation,
poor resistance to high load variations, and transient operational
conditions [7,8]. In dairy wastewater, the most problematic con-
stituents for anaerobic treatment are fats and LCFA (Long Chain

http://dx.doi.org/10.1016/j.energy.2014.06.098
0360-5442/© 2014 Elsevier Ltd. All rights reserved.
 C.S. Couras et al. / Energy 73 (2014) 866e874
Fatty Acids) that result from fat hydrolysis [9e11]. Several in-
vestigations have been published in recent years concerning the
anaerobic degradation of fats and LCFA and their effects on the
overall process performance [12,13]. Fats are degraded via meta-
bolic pathways distinct from those of proteins and hydrocarbons. In
a first step, neutral fats are hydrolyzed (lipolysed) into free LCFA
and glycerol, a process catalyzed by extracellular lipases. The free
LCFA are converted to acetate and H2 by syntrophic acetogenic
bacteria through the b-oxidation process. Microorganisms of the
Syntrophomonadaceae group have been found to play an impor-
tant role in this step of LCFA anaerobic degradation [14e16], and are
therefore considered as a key microbial group for the anaerobic
degradation of fat-containing wastewaters (viz dairy wastewater)
and subsequent biogas production. Since lipids and LCFA have high
methane potential, the Syntrophomonadaceae group is also a key
microbial group for the maximization of biogas production. The last
step of the anaerobic process is the production of methane by
methanogens (Archaea microorganisms). The b-oxidation step has
been reported as the limiting step in the anaerobic degradation of
fats [11]. LCFA exert an inhibitory effect on the b-oxidation process,
which was initially considered as permanent [9], but was later
demonstrated to be reversible after a lag phase [17]. Recent studies
emphasize the importance of a well-balanced microbial commu-
nity for the stability and good performance of anaerobic reactors
[8,18e20], and for optimized biogas production. However, transient
operational conditions are known to alter this necessary equilib-
rium [21]. It has been reported that anaerobic populations suffered
shifts caused by overloading [8,22] or by temperature changes [23].
Dairy wastewaters and other effluents, such as food processing
effluents, are subject to sharp fluctuations in composition which
may affect anaerobic systems performance. Significant decreases in
the overall efficiency were observed by Schmidt and Ahring [24]
when UASB (Upflow Anaerobic Sludge Blanket) reactors feed
composition was changed. Variations in the carbon source present
in the wastewater were reported to cause gradual changes in the
physical structures, bacterial distribution and settling characteris-
tics of the sludge in UASB reactors [25]. Research by Fukuzaki et al.
[26] also demonstrated that variations of the carbon source present
in the wastewater caused changes on the physical structure,
chemical contents (extracellular polymeric substrates) and bacte-
rial distribution. Specifically in what concerns sudden overloading
of fatty substrates, serious drops of methanogenic activity and
biogas production have been reported as a result of inhibition,
accompanied by deterioration of sludge quality [27]. Flotation
frequently results, due to the adsorption of LCFA at the sludge
surface [27,28]. Another effect is the disintegration of sludge ag-
gregates, which can occur when lipids are present, due to the
surfactant effect of LCFA. The hydrophobic acetogens that can
degrade LCFA are severely affected by this surfactant disaggregating
effect [29].
The response of anaerobic reactors to hydraulic shocks has
been reported in the literature as resulting in the drop of removal
efficiency [27], disaggregation and washout of filamentous or-
ganisms [29,30], and VFA (volatile fatty acids) accumulation and
inhibition of methanogenic bacteria with consequent drop in
biogas yield [31]. In this type of operational shock, an improve-
ment effect has also been observed in the COD (chemical oxygen
demand) concentration of the treated effluent, attributed to
changes in the structure of microbial populations inside the
reactor [32], or to changes in the Monod half saturation constant
[7]. Some authors suggest that the substrates diffusion rate in-
crease with higher substrate concentration (Fick's law) and
decrease with a higher flow velocity [33], whilst other authors [34]
found that the external mass transfer resistance can be decreased
by increasing the flow velocity. Brito and Melo [35] reported that
867
under conditions of turbulent liquid flow, and thus higher shear
stress, the flow velocity had a pronounced effect on the biofilm
thickness and compactness, leading to different mass transfer
coefficients. If the bulk liquid suffers a shift in velocity, there is an
increase in internal mass transfer coefficient. In what concerns
biomass washout caused by hydraulic overload it was reported
[36] that the microorganisms responsible for the degradation of
LCFA were the most susceptible to washout at low HRT (hydraulic
retention time).
Operational temperature is a major factor on the performance of
anaerobic reactors [37,38]. Thermophilic operation has been
pointed out as presenting some advantages over mesophilic oper-
ation, namely in terms of substrate degradation rates and biogas
production [23,39]. However, mesophilic reactors present a higher
operational stability [38,40]. Significant methanogenic biomass
washout was reported by Khemkhao et al. [23] due to mesophilic to
thermophilic transition of the operational temperature of a UASB
reactor treating palm oil mill effluent. Other effects resulting from
the rise of operational temperature include increased biogas pro-
duction and lower methane content of the biogas. When the pro-
cess is exposed to a sudden alteration of temperature, the process
conditions may be unbalanced because of different responses of the
various metabolic groups [41]. Van Lier et al. [42] found that
exposure of a UASB reactor to temperatures above 45  C resulted in
serious drop in the activity of mesophilic granular sludge due to
high bacterial decay. Immediately after the temperature shock, a
raise in biogas production was observed, followed by a sharp
decrease. The microorganisms that oxidize propionate are the most
susceptible to temperature raise, and it was also reported that
methanogens are more susceptible to temperature shocks than
acidogens. It is also known that temperature variations can affect
the sludge retention capacity, since temperature affects viscosity,
and consequently, the hydraulic shear forces exerted on the sludge
particles [43].
UASB (Upflow Anaerobic Sludge Blanket) reactors are the most
widely used anaerobic technology for the treatment of industrial
wastewater [44,45]. The performance of these systems may be
largely enhanced by a new form of operation named intermittent
operation, in which feed and feedless periods are combined to raise
biogas production [46]. The beneficial effect of intermittent oper-
ation is ascribed to a forced adaptation of the anaerobic biomass to
substrates resistant to degradation (fats and LCFA) which occurs
during the feedless periods [47]. Studies on the microbial pop-
ulations striving in continuous and intermittent UASB reactors
treating dairy wastewater have shown a significant raise in Syn-
trophomonadaceae relative abundance in intermittent reactors as
compared to continuous reactors [47]. To date, no studies have been
published comparing the performance of continuous and inter-
mittent UASB reactors submitted to operational shocks and their
effects on the Syntrophomonadaceae key microbial group.
2. Materials and methods
This work compares the performance of intermittent and
continuous UASB reactors treating dairy wastewater at a load of
12 g COD/L/day and subject to operational shocks. Two replicate
laboratory-scale UASB reactors (working volume of 6 L) were
operated in a continuous mode and two were operated in an
intermittent mode. The intermittent cycle consisted of 48 h feed
followed by 48 h feedless [46]. The feed concentration for the
intermittent reactors was always double the feed concentration
used for the continuous reactors, so that in a 96 h period the total
COD mass admitted to each reactor was the same. The feed was
composed of diluted semi-skimmed milk or whole milk, supple-
mented with nutrients and alkalinity. The reactors were seeded
868 C.S. Couras et al. / Energy 73 (2014) 866e874
with approximately 4 L of flocculent sludge adapted to dairy
wastewater from an industrial wastewater treatment plant. The
inoculation sludge had a SMA (specific methanogenic activity) of
7.1 mL CH4/g VSS/day measured with sodium acetate at (35 ± 1)  C.
Throughout the entire experiment, effluent from the reactors was
collected in 24 h composite samples. All physical-chemical de-
terminations were made according to Standard Methods [48]. The
produced biogas was measured by wet gas meters (Schlumberger).
Methane content in biogas was monitored using a Shimadzu GC e
9a gas chromatograph equipped with a Supelco Molecular Sieve 5 A
column and a Thermal Conductivity Detector (T ¼ 100  C). Injection
temperature was 45  C and Helium was used as carrier gas
(P ¼ 4.4 kg/cm2). Volatile fatty acids determination was carried out
in a Chrompack CP 9001 gas chromatograph equipped with a
Chrompack CP e sil5 e CB column and a Flame Ionization Detector
(T ¼ 300  C). The injection temperature was 270  C and Helium was
used as carrier gas with a volumetric flow of 8 mL/min. Biomass
samples for FISH (Fluorescence In Situ Hybridization) analyses
were collected immediately before each applied shock, at the end of
the shock period, and 20 days after the end of the shock. After
collection, biomass samples were immediately frozen at 20  C. For
(FISH) experiments, samples were allowed to reach room temper-
ature and were vortexed for 5 min to disrupt granules and other
aggregates, and subsequently fixed with formaldehyde (4% v/v).
Fixed samples were washed 3 times with PBS (phosphate buffered
saline) and stored at 20  C in a mixture containing 50% PBS and
50% ethanol. Samples were spotted onto the surface of glass slides
(2 spots per slide) and allowed to dry for 30 min at 42  C. Samples
were further dehydrated by 3 incubations of 3 min each with
increasing concentrations of ethanol (50, 80 and 100% v/v) and
finally dried at room temperature. Each spot was covered with 9 mL
of hybridization buffer (0.9 M NaCl, 20 mM Tris buffer, 10 mM EDTA
(ethylenediaminetetraacetic acid), 0.01% SDS, pH 7.2) and 1 mL of
probe working solution (50 mg/mL) of each probe. Probes used
were EUB338 (50 -GCGCCTCCCGTAGGAGT-30 , [49]) specific for
Eubacteria, Arc915 (50 - GTGCTCCCCCGCCAATTCCT-30 , [50]) specific
for Archaea, and SYNM700 (50 -ACTGGTN5TTCCTCCTGATATCTA-30 ,
[51]) specific for Syntrophomonadaceae. These probes were chosen
to assess the microbial population composition, namely the total
bacterial population including acidogenic bacteria (EUB338), the
Archaea population which includes the methanogens (Arc915), and
the fatty acid degraders belonging to the Syntrophomonadaceae
(SYNM700). Probes labeled with FITC (EUB338), CY3 (ARC915), or
CY5 (SYNM700) were acquired from Eurofins MWG Operon (Ger-
many). Hybridization was performed in a humidified chamber for
3 h at 45  C. After hybridization, slides were subsequently washed
with washing buffer (180 mM NaCl, 20 mM Tris, 5 mM EDTA, 0.01%
SDS, pH 7.2) for 30 min at 48  C. Slides were further incubated with
10 mL of DAPI (2 mg/mL) in PBS at room temperature for 15 min,
followed by 3 washes with distilled water at room temperature. The
slides were analyzed with an AXIOPLAN microscope equipped with
an external UV light source and filters adequate for the fluorescent
dyes under use. Images were captured by a CCD camera (Ropers
Scientific) and processed with the Metamorph software. The Image
J software was used to quantify the fluorescence intensity obtained
with each probe. Results are the mean values obtained for at least 3
slides, each spotted at least three times.
The reactors were operated in baseline conditions (HRT ¼ 12 h;
OLR (organic loading rate) ¼ 12 g COD/L/d) for several months
before the beginning of operational shock experiments. Three types
of operational shocks were applied, consisting in the raise of feed
fat content, feed volumetric flow, or operational temperature.
The percentage of COD removal was calculated with the
following formula:
CODTfeed  CODSeffluent
Removalð%Þ ¼  100 (1)
CODTfeed
The percentage methanisation was calculated with the
following formula:
COD  CH4
Methanisationð%Þ ¼  100 (2)
CODR
Results for a full-scale anaerobic reactor from a milk processing
and cheese production industrial plant are also presented in this
work. The reactor was a mesophilic continuously stirred tank
reactor with a working volume of 2500 m3. All the phys-
icalechemical and microbiological determinations were performed
in the same laboratory and with the same methods as for the
bench-scale UASB reactors.
3. Results and discussion
3.1. Shock experiments with laboratory-scale UASB reactors
Three baseline periods were established, corresponding to the
three periods before each of the applied shocks. The performance of
the intermittent systems operating under baseline conditions was
better than the observed for the continuous systems concerning
methanisation efficiency, in agreement with other reports
[47,52,53]. Under baseline conditions, the average values of this
parameter ranged 86%e90% and 53%e58%, for the intermittent and
the continuous systems, respectively. The average COD removal
efficiencies in baseline operation ranged 94%e96% for the inter-
mittent systems, and 85%e90% for the continuous systems. These
differences in operational results have been attributed to the
feedless phase of intermittent operation [54], in which the anaer-
obic biomass is forced to adapt to complex substrates difficult to
degrade (mainly fats and LCFA), by means of microbial population
shifts that raise the relative abundance of the Syntrophomonada-
ceae group [47] involved in the degradation of fatty substrates. In
fact, the relative abundance of the Syntrophomonadaceae group in
the microbial population developed in the intermittent systems
operating at baseline conditions ranged from 17% to 20%, while this
key microbial group was not present at detectable levels in the
continuous systems throughout all the experiments performed in
this work.
Results for the fat, hydraulic, and temperature shocks, applied to
continuous and intermittent UASB systems are discussed below.
Baseline and post-shock periods were immediately before and
immediately after the shocks, respectively. Values for the baseline
periods and for the post-shock periods are averages of 20 days or 5
intermittent cycles. Error bars represent standard deviations.
3.1.1. Fat shock
The applied fat shock consisted of a raise in average feed fat
content from 110 mg/L to 261 mg/L in the intermittent systems and
from 63 mg/L to 130 mg/L in the continuous systems. The shock
lasted for 20 days or 5 intermittent cycles. Average feed COD con-
centration and volumetric flow were maintained in order to keep a
constant OLR during the experiment.
Fats are substrates with higher methane production potential
than hydrocarbons and proteins. Theoretically, it would be ex-
pected that a raise in feed fat content would cause a raise in
methane production if inhibition thresholds are not reached. The
fat concentrations during the fat shock were above the inhibition
threshold value of 100 mg/L reported by Perle et al. [10]. Results in
Fig. 1a show that, opposite to the observed for the continuous
 C.S. Couras et al. / Energy 73 (2014) 866e874 869

Fig. 1. Results from the fat shock on a) methane production; b) COD removal; c) TSS in treated effluent. Baseline; Shock; Post-Shock. I ¼ Intermittent; C ¼ Continuous.

systems, methane production in the intermittent systems raised

slightly when the feed fat content was raised. This difference in the
systems response to fat shock is probably due to the improved
substrate degradation occurring in the intermittent reactors as a
consequence of biomass adaptation to the complex substrates
present in the feed. The intermittent systems did not present sig-
nificant variations on the effluent COD removal efficiency (Fig. 1b)
or in the TSS (total suspended solids) washout (Fig. 1c) as a
consequent of the fat shock. However, the fat shock resulted in a
lowering of COD removal efficiency in the continuous systems. This
was probably due to the improved substrate degradation occurring
in the intermittent systems, resulting in an anaerobic sludge with
unaffected adsorption capacity, and leading to COD removal effi- Fig. 2. Methanisation with the fat shock experiment. Baseline; Shock; Post-
Shock. I ¼ Intermittent; C ¼ Continuous.
ciency similar to that observed for the baseline. This effect was not
observed in the continuous systems, where the uninterrupted
feeding did not allow the complete degradation of the adsorbed
complex substrates [55].
In the continuous systems, the effect of fat shock on suspended
solids (Fig. 1c) was more pronounced than the effect on effluent
soluble COD. The raise in feed fat content caused an increase in
effluent suspended solids of the continuous systems from an
average of 0.79 g/L to 1.30 g/L. A heavy accumulation of floating
biomass was observed at the top section of the continuous reactors.
This biomass flotation was also observed, although to a much lesser
extent, in the intermittent systems, since the feedless periods
diminished biomass flotation and washout.
The methanisation of the removed COD differed for the two
types of systems (Fig. 2). The continuous system suffered a loss in Fig. 3. VFA in effluent from the fat shock experiment. I ¼ Intermittent; C ¼ Continuous.
methanisation efficiency, from an average of 59%e34% during the B ¼ Baseline; S ¼ Shock; PS¼Post-Shock.
shock period, whereas the intermittent systems present a slight
increase in methanisation efficiency during the shock period, from
an average of 90%e92%. After the shock period, the intermittent In the intermittent systems, the fat shock caused a slight raise in the
reactors presented a methanisation efficiency similar to the relative abundance of the Syntrophomonadaceae group, during and
observed before the shock. The continuous reactors attained an after the shock. A slight raise in the relative abundance was also
average value of 48%, lower than the observed before the shock. detected for the Archaea microbial group. These results support the
Important differences were found in the VFA content of both sys- observed performances for biogas production of the continuous
tems subjected to the fat shock (Fig. 3). In baseline operation, acetic and intermittent reactors.
and propionic acids were the predominant acids in both systems,
although total VFA concentration was higher in the continuous
3.1.2. Hydraulic shock
systems. The fat shock caused a sharp rise in propionic acid con-
The hydraulic shock lasted for 12 h or half of the feeding period
centration from 28 to 105 mg/L (as acetic acid) in the continuous
of intermittent operation. This consisted in decreasing the average
systems. The surge of propionic acid is consistent with inhibitory
HRT from 11.3 h to 6.1 h in the intermittent systems, and from 12.0
effects of fats and fatty substrates upon anaerobic biomass. Other
to 6.1 h in the continuous systems. During the hydraulic shock the
acids present in the effluent from continuous systems were caproic
COD of the feed was halved in order to maintain a constant OLR
and n-butyric acids. In the intermittent systems, the fat shock
throughout the experiment: 10.9 g COD/L/day and 11.3 g COD/L/day
caused the appearance of n-butyric and i-valeric acids, and to a
for the intermittent and continuous systems respectively. The hy-
minor extent, of caproic acid.
draulic shock was severe for both continuous and intermittent
FISH results (Fig. 4a and b) show that the Syntrophomonadaceae
systems. Methane production (Fig. 5) in the intermittent systems
group, a key microbial group for the degradation of fatty substrates
diminished from an average of 0.89 L/h to an average of 0.26 L/h,
and maximizing biogas production, was absent in the continuous
equivalent to a 70% decrease. In the continuous systems the effect
reactors before, during, or after the shock. In these reactors, a
was even more severe, with methane production decreasing by
decrease on the relative abundance of Archaea (methanogenic)
76%, from an average of 0.50 L/h to an average of 0.12 L/h. The
microorganisms was also observed, during, and after the fat shock.
slightly higher resilience of methane production in the intermittent
870 C.S. Couras et al. / Energy 73 (2014) 866e874

Fig. 4. Microbial populations in the fat shock experiment; a) continuous system; b) intermittent system. Eubacteria excluding Syntrophomonadaceae; Syntrophomonadaceae;
Archaea. B ¼ Baseline; S ¼ Shock; PS ¼ Post-Shock.

Fig. 5. Results from the hydraulic shock a) methane production; b) COD removal; c) TSS in treated effluent. Baseline; Shock; Post-Shock. I ¼ Intermittent; C ¼ Continuous.

system may be attributed to the presence of microorganisms from
the Syntrophomonadaceae group (Fig. 8) and to a lower biomass
washout (Fig. 5c). The heavy TSS washout observed in the contin-
uous system was attributed to the combined effect of the high
upflow velocity and the presence of accumulated substrates on the
biomass surface, a typical result of continuous operation of UASB
reactors [55]. As in the intermittent reactors the biomass flocs were
not so heavily surrounded by adsorbed fatty substrates, the effects
of high upflow velocity were not so severe. The clean biomass
resulting from the intermittent operation also results in a higher
capacity for adsorption of the wastewater substrates, thus
explaining the higher COD removal efficiencies (Fig. 5b). It is worth
noting that the effluent COD for the continuous system decreased
slightly after the hydraulic shock as compared to baseline. This is
probably due to the effect of the shear forces of the ascending
liquid, resulting in the liberation of adsorbed substrates and
consequent higher availability of adsorption sites for substrate
removal. The effect of higher upflow velocity upon mass transfer
[35] is also to be considered.
In both systems, the methanisation efficiency was negatively
impacted by the hydraulic shock (Fig. 6), decreasing from 89% to
53% in the intermittent systems, and from 56% to 32% in the
continuous systems. During the hydraulic shock, the VFA concen-
tration in the effluents increased from 42 to 125 mg/L and from 69
to 182 mg/L (as AcH), in the intermittent and continuous systems,
respectively. In the intermittent systems, the major acids produced
due to the shock were n-butyric, i-butyric and i-valeric, whilst in
the continuous systems the major resultant acids were propionic,
n-butyric and n-valeric (Fig. 7). After the shock, the values of total
VFA concentration were 54 and 132 mg/L (as AcH), respectively for
the intermittent and continuous systems.
The results of biomass monitoring by FISH in the intermittent
systems (Fig. 8b) show that the microorganisms from the Syntro-
phomonadaceae group suffered washout, resulting in a decrease of
their relative abundance from 17% to 5%. According to Hwu et al.
[36] the bacteria that degrade LCFA are the most sensible to
washout caused by low HRT. After the shock, the relative abun-
dance of the Syntrophomonadaceae group raised to 10%. In the
continuous systems the microbial group that suffered washout due
to the hydraulic shock was the Archaea (methanogenic) group
(from 59% to 50%). After the shock, a slight recovery of relative

Fig. 6. Methanisation with the hydraulic shock experiment. Baseline; Shock; Fig. 7. VFA in effluent from the hydraulic shock experiment. I ¼ Intermittent;
Post-Shock. I ¼ Intermittent; C ¼ Continuous. C ¼ Continuous. B ¼ Baseline; S ¼ Shock; PS ¼ Post-Shock.
 C.S. Couras et al. / Energy 73 (2014) 866e874 871

Fig. 8. Microbial populations in the hydraulic shock experiment; a) continuous system; b) intermittent system. Eubacteria excluding Syntrophomonadaceae; Syntrophomo-
nadaceae; Archaea. Shock; PS ¼ Post-Shock.

Fig. 9. Results from the temperature shock a) methane production; b) COD removal; c) TSS in treated effluent. Baseline; Shock; Post-Shock. I ¼ Intermittent; C ¼ Continuous.

abundance (to 55%) was observed for this microbial group. These
results correlate with the methane production observed in the
reactors.
3.1.3. Temperature shock
The temperature shock lasted for 12 days or 3 intermittent cy-
cles and consisted in a one step raise of the operating temperature
from (35 ± 1)  C to (55 ± 1)  C. The shock led to an increase in
biogas production in both systems (results not shown). A similar
effect was reported by Van Lier et al. [42]. However, the methane
percentage in the biogas was lowered in both systems. This
reduction was more severe in the continuous systems (67%e52%),
as compared to the intermittent systems (75%e67%). It would be
expected that a raise in the operational temperature might improve
the overall performance and biogas production of both systems,
since thermophile reactors have been described as more effective in
treating complex wastewaters compared to mesophile systems
[23,39]. However, the performance of both systems decreased,
considering all parameters monitored (Figs. 9e11). The most severe
effect was observed for the TSS washout, which may have been
caused by disaggregation of biomass flocs due to high temperature
[43] and due to the turbulence effects of the initial increase in
biogas production.
The temperature shock also resulted in a severe loss of micro-
organisms from the Syntrophomonadaceae group in the intermit-
tent reactors (Fig. 12b). The temperature shock generated
conditions that led to a reduction of the relative abundance of the
Syntrophomonadaceae group from an initial value of 20% to values
undetected by the FISH methodology used. Remarkably, after the
mesophile operation was resumed, the relative abundance of this
microbial group reached detectable levels (9%). This reduction of
the Syntrophomonadaceae group did not impact the biogas pro-
duction of the intermittent reactor. These results suggest that
methane production from substrates other than lipids/LCFA and/or
via metabolic pathways other than b-oxidation were affected by the
temperature shock at a more limited extent more limited than the
microorganisms from the Syntrophomonadaceae group. In the
continuous systems the Archaea microbial group was the most
susceptible to washout, with relative abundances declining from
60% to 47%, and recovering to 52% after the shock.

Fig. 10. Methanisation with the temperature shock experiment. Baseline; Shock; Fig. 11. VFA in effluent from the temperature shock experiment. I ¼ Intermittent;
Post-Shock. I ¼ Intermittent; C ¼ Continuous. C ¼ Continuous. B ¼ Baseline; S ¼ Shock; PS ¼ Post-Shock.
872 C.S. Couras et al. / Energy 73 (2014) 866e874

Fig. 12. Microbial populations in the temperature shock experiment; a) continuous system; b) intermittent system. Eubacteria excluding Syntrophomonadaceae; Syntro-
phomonadaceae; Archaea. B ¼ Baseline; S ¼ Shock; PS ¼ Post-Shock.

For each shock experiment performed in this work (fats, hy- Syntrophomonadaceae was present with a relative abundance of
draulic and temperature shocks), a ManneWhitney (non-para- 2% (SD ¼ 1.7%, n ¼ 18), whilst Eubacteria excluding Syntropho-
metric) statistical test was performed to check the statistical monadaceae and Archaea had relative abundances of 57%
significance of the differences in relative abundance of the different (SD ¼ 13%, n ¼ 18) and 41% (SD ¼ 17%, n ¼ 18), respectively. The
microbial groups between operating conditions for each pair of intermittent operation resulted in improved performance of the
reactors. For each pair of reactors all the differences in the relative reactor (Table 2) and in the presence of the Syntrophomonadaceae
abundance of the various microbial groups from baseline, to shock microbial group, although in a lower relative abundance than
or post-shock conditions were found to be significant for a signif- detected in the intermittent lab-scale UASB reactors. According to
icance level of 5%. published results [46,56] a longer feedless period in intermittent
Table 1 resumes the effects of the applied shocks upon the operation improves biomass adaptation to fatty substrates and
relative abundance of the Syntrophomonadaceae microbial group consequently results in higher COD removal and methanisation
in the intermittent systems. In terms of this key microbial group, efficiencies. The low frequency and the short duration of the
the severity of the applied shocks may be classified as: feedless periods in the industrial reactor (phase 2) explains the
fats < hydraulic < temperature. lower relative abundance of the Syntrophomonadaceae in com-
parison to what was detected in the lab-scale UASB reactors.
These results show that the intermittent operation favors the
3.2. Full-scale anaerobic reactor development of the key microbial group Syntrophomonadaceae in
a full-scale anaerobic reactor treating dairy wastewater subjected
In other research works done by our group we used several to transient operational conditions.
biomass samples collected throughout a period of more than 3
years from an anaerobic industrial reactor in a dairy wastewater
treatment plant located in the North of Portugal. Although the 4. Conclusions
wastewater passed through an equalization tank some variations
occurred in the operational conditions of the reactor. The func- In this work we have compared the performance of continuous
tioning of the reactor can be divided in two phases (see Table 2). In and intermittent UASB reactors when subjected to fat, hydraulic, or
phase 1 the anaerobic reactor worked in a continuous mode and temperature shocks, with a special focus on its effects on the key
presented several problems caused by the fatty materials in the microbial group Syntrophomonadaceae, detected only in the
wastewater, like encapsulation of biomass flocs and consequent intermittent systems. This microbial group plays a key role in the
biomass washout. In the second phase, due to an alteration in the degradation of methane-rich substrates, like lipids and LCFA. Syn-
production schedule, the reactor was operated in an intermittent trophomonadaceae are also considered a key microbial group for
mode in which the feed was interrupted during one day of the week
(Sundays). The data presented in Table 2 show that in phase 2 the
reactor had an improved performance as compared to phase 1 in Table 2
what concerns COD removal and methanisation efficiency. The Operational data for an industrial scale anaerobic reactor treating dairy wastewater
from a milk processing and cheese production industry.
results from FISH determinations on the biomass developed in the
industrial reactor during phase 1 showed that the Syntrophomo- Parameter Average SD Max Min n
nadaceae microbial group was not present in detectable levels in Phase 1 (continuous)
the biomass and the relative abundance of Eubacteria and Archaea Volumetric flow (m3/d) 476.3 58.3 701 223 93
were 63% (SD ¼ 12%, n ¼ 27) and 37% (SD ¼ 10%, n ¼ 27), respec- O&G (g/m3) 130 26 204 92 113
OLR (g COD/m3/d) 398 65.2 674 165 85
tively. The results from determinations on the biomass developed
COD removal (%) 71 14 89 52 87
in the industrial reactor during phase 2 show that the Methanisation of removed COD (%) 55 28 71 18 50
VSS (g/L)a 11.9 1.3 14.1 8.8 33
Phase 2 (intermittent)
Table 1 Volumetric flow (m3/d) 552.5 77.4 886 337 196
Effects of operational shocks upon detectable levels of Syntrophomonadaceae mi- O&G (g/m3) 145 49 211 72 97
crobial group in intermittent UASB reactors treating dairy wastewater (percentages OLR (g COD/m3/d) 468 98.3 850 190 123
refer to variations from baseline relative abundance). COD removal (%) 92 11 96 84 110
Methanisation of removed COD (%) 80 16 97 63 98
Shock Shock effect (%) Recovery level (%)
VSS (g/L)a 16.5 3.2 23.3 10.3 55
Fats þ11.8 112
SD ¼ standard deviation; Max ¼ maximum value; Min ¼ minimum value;
Hydraulic 85.7 57
O&G ¼ oils and greases; n ¼ number of observations.
Temperature 100 45 a
VSS measured at the bottom sampling port of the reactor.
 C.S. Couras et al. / Energy 73 (2014) 866e874 873

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