ORIGINAL PAPER
Biological and technical study of a partial-SHARON reactor
at laboratory scale: effect of hydraulic retention time
A. Gonza
´
lez-Martı
´
nez
•
K. Caldero
´
n
•
A. Albuquerque
•
E. Hontoria
•
J. Gonza
´
lez-Lo
´
pez
•
I. M. Guisado
•
F. Osorio
Received: 26 April 2012 / Accepted: 9 June 2012
Ó Springer-Verlag 2012
Abstract This study was on the technical and biological
characteristics of a partial-SHARON submerged-filter
bioreactor of 3 L. The main focus was the influence of the
hydraulic retention time (HRT) on biofilms. For this pur-
pose, we used molecular tools based on the partial 16S
rRNA genes. The results showed that the HRT may affect
the nitrification processes of a bioreactor using synthetic
wastewater containing 600 mg/L of ammonia. It was found
that an HRT of 0.5 day transformed 100 % of the ammo-
nium into nitrite. However, when the HRT was decreased
to 0.4 day, there was a significant reduction (35 %) in the
quantity of ammonia transformed, which confirmed the
complexity of the system operation. Moreover, a PCR-
TGGE approach highlighted the differences observed. The
results obtained showed that an HRT of 0.5 day reduced
bacterial biodiversity in the biofilms, which were mainly
formed by Nitrosomonas and Diaphorobacter. In contrast,
an HRT of 0.4 day facilitated the formation of heteroge-
neous biofilms formed by nitrifying bacteria, such as
Nitrosomonas sp., Nitrosospira sp., and Nitrosovibrio sp.).
Keywords SHARON process Partial nitrification
Hydraulic retention time (HRT) Wastewater treatment
Submerged biofilter Nitrogen removal
Introduction
In the last 10 years, soaring population levels as well as a
corresponding growth in industrial activity have led to
increased amounts of wastewater in densely populated
areas. This surfeit of waste is having an extremely negative
impact on the environment. For example, high concentra-
tions of nitrogen, one of the main compounds in wastewater,
cause serious environmental problems such as oxygen
depletion and eutrophication [1]. The EU Water Framework
Directive 91/271/EEC clearly requires EU member states to
protect the environment from any adverse effects due to the
discharge of (untreated) urban and industrial waters. In this
context, new technologies, such as the partial-SHARON/
Anammox process, provide a cost-effective way to treat
highly contaminated effluent [1, 2]. This combined process
is an excellent alternative to conventional nitrification-
denitrification processes since it reduces the organic matter
(40 %) and oxygen (25 %) required for ammonia removal
in comparison to more conventional technologies [3].
In order to fully understand the biodiversity of biological
wastewater treatments, it is first necessary to identify the
microbiota present and analyze their numerical significance.
Culture-dependent methods have sometimes been regarded as
inadequate for the analysis of microbial communities in nat-
ural environments because of the high numbers of uncultur-
able bacteria. Furthermore, in recent years, molecular
methods, based on the sequencing of PCR-amplified the
partial 16S rRNA genes from DNA extracted from environ-
mental samples, have been widely used to reveal intrinsic
A. Gonza
´
lez-Martı
´
nez E. Hontoria F. Osorio (&)
Department of Civil Engineering, University of Granada,
Campus de Fuentenueva, s/n, 18071 Granada, Spain
e-mail: fosorio@ugr.es
A. Gonza
´
lez-Martı
´
nez K. Caldero
´
n E. Hontoria
J. Gonza
´
lez-Lo
´
pez I. M. Guisado F. Osorio
Institute of Water Research, University of Granada,
Granada, Spain
A. Albuquerque
Department of Civil Engineering and Architecture,
University of Beira Interior, Covilha, Portugal
123
Bioprocess Biosyst Eng
DOI 10.1007/s00449-012-0772-7
genetic biodiversity [4]. In particular, denaturing gradient gel
electrophoresis (DGGE) and temperature gradient gel elec-
trophoresis (TGGE) approaches yield large quantities of data
regarding the diversity of microorganisms in their natural
habitats. This has the advantage of permitting the taxonomic
classification of community members [5].
The single reactor system for high-activity ammonia
removal over nitrite (SHARON) process was described in
detail by Hellinga et al. [6], who proposed the partial-
SHARON technology. The partial-SHARON process is a
modification of the traditional SHARON process, in which
100 % of the ammonium is converted into nitrite. In contrast,
the partial-SHARON process, as its name implies, consists of
a partial nitritation. More specifically, only 50 % of the
ammonium is converted to nitrite. Thisprocess was developed
for the elimination of ammonium by the ‘‘nitrite route’’ [7].
When the partial-SHARON process is used in combination
with the Anammox process, nitrogen removal takes place in
two steps. According to Van Dongen et al. [8], the Anammox
process achieves an optimal performance with an ammo-
nium–nitrite mixture of 50 % ammonium and 50 % nitrite.
For this reason, the Anammox process has to be preceded by a
partial-SHARON process involving a partial nitrification.
Molecular techniques have been used to provide a
broader vision of the different biotechnological systems in
wastewater treatment as shown in recent studies (e.g., [4]).
These techniques have been used to obtain a wide range of
data regarding microbiota in their habitats. In fact, they
facilitate the study of non-cultivable bacteria by specifying
the microbial populations that carry out these processes
[9, 10]. For this reason, this research analyzed the fol-
lowing: (1) the hydraulic retention time (HRT) in a partial-
SHARON reactor in which submerged filters were used to
remove nitrogen; (2) the effect of the HRT on the structure
of the bacterial community. In our study, molecular fin-
gerprinting tools (PCR-TGGE) and scanning electron
microscope (SEM) were used to evaluate the structure of
the bacterial community.
Materials and methods
The SHARON bioreactor: bench-scale plant
The bench-scale plant used in our experiments consisted of
a plastic SHARON bioreactor with a volume of 3 L. It was
constructed as a submerged biofilter with PVC carriers
(BioFlow 9). A schematic diagram of the experimental
plant is shown in Fig. 1. The bioreactor received synthetic
wastewater [2] from a peristaltic pump, and was operated
in continuous flow.
The operating conditions in the bioreactor (i.e., HRT,
pH, dissolved oxygen concentration, and temperature) were
monitored every 24 h in order to verify that they remained
stable. Four 15-cm air diffusers at the bottom of the vessel
supplied oxygen from an air pump to ensure that the
oxygen concentration in the bioreactor was maintained at
2 mg/L. All of the experimental work was performed at a
pH of 7.5 and a temperature of 35 °C[11, 12], thanks to an
adjustable thermostat.
Inoculation of the pilot plant
The partial-SHARON bioreactor was inoculated with
mixed liquor from an aerobic reactor located in the Los
Vados urban wastewater treatment plant (Granada, Spain).
The mixed liquor was recirculated for 3 days until a bio-
film formed on the surface of the plastic carriers used in the
construction of the submerged biofilter. After inoculation,
synthetic wastewater was fed into the bioreactor.
Synthetic wastewater
The synthetic wastewater [2] used in our study simulated
the leachate from an anaerobic digester, since it contained a
high concentration of ammonium and was low in organic
matter (see Table 1).
To prepare the synthetic wastewater, 24 L of distilled water
was poured inside the 60-L tank along with the exact quantity
of the chemical compounds that made up the synthetic sewage
medium. All components were then mixed and dissolved.
The influent was continuously fed into the bioreactor by a
peristaltic pump (Watson Marlow s-520) that pumped the
synthetic wastewater at different flow rates.
Physico-chemical parameters
The physico-chemical parameters analyzed in our study
were the following: pH, dissolved oxygen concentration,
temperature, and nitrogen concentration in its various
Fig. 1 Diagram of the pilot-scale partial-SHARON bioreactor used
in the experiments. 1 Synthetic wastewater tank; 2 peristaltic pump; 3
oxygen diffusers (porous plates); 4 air pump; 5 thermostat; 6 tank of
NaOH 0.1 M for pH control; 7 tank of H
2
SO
4
0.1 M for pH control; 8
pH meter; 9 partial-SHARON bioreactor stuffed with carriers
Bioprocess Biosyst Eng
123
inorganic forms (ammonium, nitrite, and nitrate). Samples
were taken every 24 h because of the slow growth of
ammonia-oxidizing bacteria [8].
In constant pH, oxygen, and temperature conditions, two
experiments were performed at different HRTs (0.4 and
0.5 day) with a view to analyzing the evolution of inor-
ganic nitrogen concentration in the bioreactor and also the
microbial diversity in the biofilm. Table 2 shows the con-
ditions of both experiments.
pH
The pH was measured directly in the bioreactor at 8-h
intervals, using a pH meter (Crison GLP 91) [15]. The
equipment was adjusted daily with buffer solutions of pH
4.0 and 7.0.
Dissolved oxygen concentration
The dissolved oxygen concentration in the bioreactor was
determined by means of a pulse oximeter (CRUCIBLE
OXI320), which was calibrated according to the manu-
facturer’s instructions.
Determination of ammonium, nitrite, and nitrate
Concentrations of the various inorganic forms of nitrogen
(nitrite, nitrates and ammonium) were measured daily at
the entry and exit points of the partial-SHARON bioreactor
with an ionic chromatograph Metrohm. Nitrite and nitrate
levels were measured with an anion column Metrosep A
supp-4-250, and ammonium levels, with a cation column
Metrosep C 2-150. A carbonate/bicarbonate solution was
used as an eluent. Calibration curves of known concen-
trations of ammonium, nitrite, and nitrate (10, 500 and
1,000 mg/L) were also analyzed daily.
DNA extraction and PCR amplification of partial
bacterial 16S rRNA genes
DNA was extracted from the biofilm that formed in the
submerged biofilter. This was done by vortexing approxi-
mately 200 mL of plastic carriers from the biofilters with a
saline solution, and then centrifuging them to obtain the
biofilm fraction. Samples (approx. 200 mg) from the bio-
film were collected with the FastDNA Kit and the Fast-
Prep24 apparatus (MP-BIO, Germany).
Polymerase chain reaction (PCR) amplification was
performed in two steps, following other research on TGGE
and DGGE fingerprinting [4, 9]. One microliter (2–5 ng) of
the DNA extracted was used as a template for all the PCRs.
At the first PCR, the template was diluted 1:10. High-
performance liquid chromatography (HPLC)-purified oli-
gonucleotides were purchased from Sigma. AmpliTaq
Gold polymerase (Applied Biosystems, Life Technologies,
Carlsbad, CA, USA) was used for all PCRs, which were
performed in an Eppendorf Master Cycler (Eppendorf,
Hamburg, Germany). Primers and conditions for each of
the PCR reactions were those described in Molina-Mun
˜
oz
et al. [9]. The final PCR products were cleaned and/or
concentrated (when required) using Amicon Ultra-0.5 mL
Centrifugal Filters (Eppendorf, Hamburg, Germany). Ten
microliters (60–100 ng DNA) were loaded into each well
for TGGE.
TGGE analysis
TGGE was performed using a TGGE Maxi system
(Whatman-Biometra, Goettingen, Germany). The denatur-
ing gels (6 % polyacrylamide [37.5:1 acrylamide:bis-
acrylamide], 20 % deionized formamide, 2 % glycerol, and
8 M urea) were prepared and run with 29 Tris–acetate-
EDTA buffer. All chemicals were purchased from Sigma
Aldrich (St. Louis, MO, USA). The temperature gradient
was optimized at 43–63 °C[9]. The bands were visualized
by silver staining with the Gel Code Silver Staining kit
(Pierce, Thermo Fisher Scientific, Rockford, IL, USA).
Various PCR reactions were tested, and different TGGE
gels were run to check the reproducibility of the results.
Analysis of TGGE fingerprints
The band patterns generated by TGGE were normalized,
compared, and clustered by using the Gel Compar II v.
5.101 software (Applied Maths, Belgium). For cluster
Table 1 Composition of the
synthetic wastewater in g/L
used in the experiments
Chemical g/L
(NH
4
)
2
SO
4
2.35
NaHCO
3
3.25
CaCl
2
0.30
KH
2
PO
4
0.07
MgSO
4
0.02
FeSO
4
7H
2
O 0.009
H
2
SO
4
0.005
Table 2 Conditions of the partial-SHARON bioreactor in experi-
ments 1 and 2
Parameter Experiment 1 Experiment 2 References
Oxygen demand (mg/L) 1.5 1.5 [13]
pH 7.5 7.5 [14]
Temperature (°C) 35 35 [3]
HRT (days) 0.5 0.4
Bioprocess Biosyst Eng
123
analysis, the TGGE profile was compared by means of a
band assignment independent method (Pearson product-
moment correlation coefficient) as well as a method based
on band presence/absence (Dice coefficient). In reference
to band assignment, a 1 % band position tolerance (relative
to the total length of the gel) was applied [4]. Dendrograms
relating band pattern similarities were automatically cal-
culated with unweighted pair group method with arithmetic
mean (UPGMA) algorithms. The significance of UPGMA
clustering was estimated by calculating the cophenetic
correlation coefficients.
Range-weighted richness indices (R
r
), which estimate
the level of microbial diversity in environmental samples,
were calculated, based on the total number of bands in each
TGGE pattern (N) and the temperature gradient (°C)
between the first and last band of each pattern (Tg), fol-
lowing Marzorati et al. [16]. The resulting values were
divided by 100 [5] to keep an order of magnitude analo-
gous to that of the R
r
index, as originally described for
DGGE in Marzorati et al. [16].
Pareto-Lorenz distribution curves rendered a graphical
representation of the evenness of the bacterial communities
in the different samples, based on the TGGE fingerprints
[16]. The bands in each TGGE lane were ranked from
highest to lowest based on intensity levels. The cumulative
normalized band intensities for each TGGE lane were
plotted against their respective cumulative normalized
number of bands. The curves were numerically interpreted
by the functional organization index (F
o
), given by the
horizontal y-axis projection on the intercept with the ver-
tical 20 % x-axis line [16]. The calculation of the F
o
indexes permitted the evaluation of the functional redun-
dancy of the microbial communities analyzed by finger-
printing methods [16].
DNA reamplification and sequencing
Portions of individual bands on silver-stained TGGE gels
were picked up with sterile pipette tips, placed in 10 lLof
filtered autoclaved water, and 3 lL of the resulting DNA
suspensions were used for reamplification with the appro-
priate primers. The PCR products were electrophoresed in
agarose gels and purified with the Qiaex-II kit (Qiagen,
Hamburg, Germany). The recovered DNA was directly
used for automated sequencing in an ABI PRISM 3100
Avant Genetic Analyzer (Life Technologies, CA, USA).
Bacterial community analysis
The DNA sequences were analyzed and compared with the
biocomputing tools provided online by the National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.
gov). Sequence similarity analysis was performed with the
BLASTn program [17]. ClustalX v. 2.0.3 software was
used for the alignment of the DNA sequences. The
graphical distribution of the main bacterial groups found is
shown in this article.
Scanning electronic microscopy
The biofilm formed in the submerged biofilter was ana-
lyzed by scanning electron microscopy (SEM). Individual
pieces of plastic carriers from the biofilter were fixed with
glutaraldehyde (5 % v/v) in a 0.2 M sodium cacodylate
buffer (pH 7.1), washed, and post-fixed in OsO
4
, before
being dehydrated with graded ethanol solutions (10, 30, 50,
70, 90, and 100 % ethanol). All chemicals were purchased
from Sigma–Aldrich (St. Louis, MO, USA). The samples
were transferred to fresh 100 % ethanol and critical point-
dried from liquid carbon dioxide at 36.1 °C and 7.37 Pa, using
a Samdri 780B apparatus (Tousimis, Rockville, USA). Sam-
ples were coated with gold before being examined by variable
pressure scanning electron microscopy (VP-SEM), model
LEO 1430VP-SEM.
Results and discussion
Physico-chemical parameters at different HRT
Experiment 1: HRT of 0.5 day
The partial-SHARON bioreactor was fed with synthetic
wastewater at a constant flow rate of 4.16 mL/min and an
HRT of 0.5 day. The concentration of ammonium, nitrate,
and nitrite was measured at the entry and exit points of the
system. These results are shown in Fig. 2.
As can be observed in Fig. 2, after 5 days of operation,
100 % of the ammonium was converted to nitrite. After
this period, the partial-SHARON bioreactor stabilized and
maintained its high capacity for biotransformation. How-
ever, the higher nitrite concentration caused a sharp drop in
the pH of the bioreactor. To correct this, it was necessary to
add small amounts of NaOH 1 % (p/v), which kept the pH
value at 7.5.
When the biotransformation capacity of ammonium into
nitrite in submerged biofilters was compared with that of
other systems such as conventional partial-SHARON bio-
reactors [8, 12], the results showed that submerged biofilters
have higher levels (three times higher) of biotransformation.
Thehightransformationcapacityofsubmerged-biofilter
systems should be regarded as an important operational factor
for the development and future design of partial-SHARON/
Anammox systems, which can be applied to the treatment
of effluents with high nitrogen content such as landfill
leachate [18].
Bioprocess Biosyst Eng
123
Experiment 2: HRT of 0.4 day
Experiment 2 was performed at an HRT of 0.4 day and a
constant flow rate of 5.20 mL/min of synthetic wastewater.
In the same way as in experiment 1, the concentration of
ammonium, nitrate, and nitrite was measured at the entry
and exit point of the partial-SHARON bioreactor. The
results are shown in Fig. 3.
As can be observed in Fig. 3, the transformation of
ammonium into nitrite reached 60 % after 5 days of
operation. After this period, the partial-SHARON biore-
actor stabilized, and its capacity for the biotransformation
of ammonia to nitrite remained constant. The increased
nitrite concentration caused a sharp drop in the pH level of
the bioreactor. To correct this, it was necessary to add
small amounts of NaOH 1 % (p/v) to maintain the pH
value at 7.5.
The results obtained in the submerged-biofilter partial-
SHARON system showed that working at experimental
conditions of temperature (35 °C), oxygen concentration
(1.5 mg/L), pH (7.5), and HRT from 0.5 to 0.4 days, an
evident reduction in the biotransformation of ammonium to
nitrite was observed when the HRT was decreased. When
the bioreactor was operating at an HRT of 0.5 day, 100 %
of the ammonium was converted to nitrites, whereas when
the bioreactor was operating at an HRT of 0.4 day, only
60 % of the ammonium was converted to nitrites. How-
ever, undetectable amounts of nitrates were produced at the
exit point of the partial-SHARON bioreactor. This low
capacity of transformation of ammonium to nitrate in the
bioreactor can be due to the operational conditions of the
system that increase the biological activity of the ammo-
nium-oxidizing bacteria and decrease the biological activ-
ity of the nitrite-oxidizing bacteria. In this sense, according
to the bacterial community analysis obtained in our study
(described below), the use of an HRT of 0.5 days, deter-
mined the production of highly specialized biofilms mainly
integrated by Nitrosomonas sp., which are very effective in
the oxidation of ammonium into nitrite.
According to Van Dongen et al. [8], the optimal
ammonium and nitrite ratio in the effluents in partial-
SHARON systems for their combination with Anammox
bioreactors is 50 % ammonium and 50 % nitrite. In this
context, our data suggest that in submerged-biofilter par-
tial-SHARON systems, the ammonium–nitrite ratio can be
modified by the HRT. Moreover, the results obtained in our
experiments show that the submerged-filter technology
applied to partial-SHARON processes increased the
transformation of ammonium into nitrite and decreased the
time required for the start-up of the bioreactors. This is
evident when the data obtained in submerged-biofilter
systems are compared with other technologies [12, 18, 19].
Study of the bacterial diversity in the partial-SHARON
bioreactor
The structure of bacterial communities was analyzed by
TGGE fingerprinting. The prevalent TGGE bands indicated
the phylogenetic groups. The sequencing of the TGGE
bands revealed that the prevalent bacteria populations were
developmentally close to Proteobacteria and specifically to
Alphaproteobacteria, Betaroteobacteria, Gammaproteo-
bacteria, and Deltaproteobacteria. The bacteria populations
in the partial-SHARON bioreactor varied, depending on
operational conditions. Accordingly, the PCR-TGGE
method showed significant differences in the structure of
the bacteria community at HRTs of 0.5 and 0.4 day (see
Fig. 4). The Pearson coefficient-based analysis permitted
the identification of four clusters corresponding to the
different treatments analyzed. On the other hand, the Dice
Fig. 2 Values of ammonium and nitrite expressed as total nitrogen
detected in the effluent of a partial-SHARON bioreactor over time
with an HRT of 0.5 day
Fig. 3 Values of ammonium and nitrite expressed as total nitrogen
detected in the effluent of a partial-SHARON bioreactor over time
with an HRT of 0.4 day
Bioprocess Biosyst Eng
123
