Ecological Engineering 95 (2016) 384–389

Contents lists available at ScienceDirect

Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Denitrification aided by waste beer in anaerobic sequencing batch

biofilm reactor (AnSBBR)
Artur Mielcarek a , Joanna Rodziewicz a,∗ , Wojciech Janczukowicz a , Tomasz Dulski b ,
Slawomir Ciesielski b , Arthur Thornton c
a
University of Warmia and Mazury in Olsztyn, Department of Environment Engineering, Warszawska St. 117a, Olsztyn 10-719, Poland
b
University of Warmia and Mazury in Olsztyn, Department of Environmental Biotechnology, Słoneczna St. 45g, Olsztyn 10-709, Poland
c
Atkins, Woodcote Grove, Ashley Road, Epsom KT18, United Kingdom

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

Article history: The goal of research was to examine waste beer as a source of carbon in an anaerobic sequencing
Received 5 April 2016 batch biofilm reactor (AnSBBR). It was demonstrated that waste beer enhanced denitrification. Observed
Received in revised form 10 June 2016 biomass yield coefficient (Yobs ) was one size of magnitude lower than for suspended-biomass reactors
Accepted 20 June 2016
(0.03 ± 0.03 mgTSS mgCOD−1 ). The kinetic parameters (utilization of organic substrate and denitrifica-
tion rates) were lower during the start-up period (88.8 mgO2 L−1 h−1 and 17.37 mgN L−1 h−1 , respectively)
Keywords:
than during stable operation of the reactor (212.4 mgO2 L−1 h−1 and 34.66 mgN L−1 h−1 after 180 cycles).
Waste beer
The metagenomic results showed that the abundance of Alcaligenes decreased from 18.39% to 0.88% (in
External carbon source
Denitrification
180 cycle), whereas the read numbers of Trichococcus increased gradually from 2.93% to 52.26% (in 180
Biofilm reactor cycle). Trichococcus was the dominant microorganism (52.3%). The low value of Yobs most likely resulted
Biomass yield from the ability of Trichococcus to degrade complex organic compounds and extracellular polysaccharide
Metagenomic analysis substances (EPS), released from dead cells in deeper layers of the biofilm.
Trichococcus © 2016 Elsevier B.V. All rights reserved.

1. Introduction Considering the economic and environmental aspects, the use

Denitrification and biological dephosphatation depend on the
presence of organic compounds easily assimilable by microorgan-
isms. One solution used in wastewater treatment plants is the
introduction of an external organic carbon source to achieve a suit-
able C:N:P ratio. For wastewater treatment plant operators, the
presented solution should enable efficient denitrification at the
lowest possible biomass yield coefficient (Yobs ).
Recent studies on a sequencing batch biofilm reactor (SBBR)
indicated that this solution could be used for effective denitrifi-
cation and biological dephosphatation (Mielcarek et al., 2015a,b).
Sequencing batch biofilm reactors are superior to systems with
activated sludge because of their greater resistance to changes in
pollutant loads, the presence of toxic substances and more com-
pact design. In addition, biofilm reactors are also characterized by
a lower biomass growth and better sedimentation properties of
biofilm compared to activated sludge flocs (Helness and Qdegaard,
2001; Wilderer et al., 2001).
∗ Corresponding author.
E-mail address: joanna.rodziewicz@uwm.edu.pl (J. Rodziewicz).
of wastewater characterized by a high C:(N + P) ratio as an external
source of organic carbon appears to be the best solution. Wastew-
ater discharged from breweries is highly biodegradable and has a
high (over 25) ratio of BOD:(N + P). Among these products, beer after
the best-before date, as well as beer discarded during quality con-
trol, is particularly noteworthy. Breweries classify such products
as waste. Discarded beer can be used as an organic carbon source
during the biological removal of nitrogen and phosphorus (BNR). A
stream of wastewater with the best parameters for enhancing BNR
can be separated from a specific production department, while beer
unsuitable for consumption can be easily transported (Mielcarek
et al., 2013; Janczukowicz et al., 2013).
Considering the advantages of biological film, it seems rea-
sonable to use biofilm reactors for denitrification. However, the
implementation of this solution requires a prior understanding of
many aspects of the process. One of them is the kinetics of contami-
nant removal by microbial communities forming the biofilm (Hallin
et al., 2006; Osaka et al., 2006). This study was designed to obtain
novel information about microorganisms forming the biomass, and
changes in the whole microbial community during the process. The
identification of bacteria responsible for denitrification in a reactor

http://dx.doi.org/10.1016/j.ecoleng.2016.06.083
0925-8574/© 2016 Elsevier B.V. All rights reserved.
 A. Mielcarek et al. / Ecological Engineering 95 (2016) 384–389 385

Fig. 1. A – changes in COD and total nitrogen during the tests; AMW, AMP1, AMP2 – collection of biofilm samples for microbiological analysis; B–D – tests on the kinetics of
utilization of waste beer and denitrification after 100, 140 and 180 cycles of operation.
386 A. Mielcarek et al. / Ecological Engineering 95 (2016) 384–389
supplied with waste beer will allow for easier control of the process
in the future.
2. Materials and methods
The reactor and synthetic wastewater used in the studies were
described by Mielcarek et al. (2015b) (pH = 7.59; COD = 20.0 mg L−1 ;
BOD = 8.0 mg L−1 ; total nitrogen = 80.00 mgN L−1 ; nitrate
nitrogen = 80.00 mgN L−1 ; total phosphorus = 14.02 mgP L−1 ;
orthophosphate = 14.00 mgP L−1 ). Waste beer was the only source
of organic carbon (COD = 113 000 mgO2 L−1 ). According to Henze
(1991) the concentration ratio of organic compounds to nitrogen
should be from 5 to 10 mgCOD mg−1 NNO3 . Activated sludge
from the mixing chamber of the “Łyna” Wastewater Treat-
ment Plant (WWTP) in Olsztyn constituted the inoculum. The
load of organic compounds on the surface of the filling was
3.98 × 104 mgO2 m−2 d−1 (1 × 106 mgO2 m−3 d−1 ). The oxygen
level in the reactor was below 0.1 mgO2 L−1 . The AnSBBR was
operated at a temperature of 20–22 ◦ C. Hydraulic retention time
in the reactor was 12 h. The lack of a sedimentation phase resulted
from the replacement of the whole treated wastewater together
with the exfoliated biofilm. A different procedure was applied in
the first stage of the operation (cycles 0–60), when only 50% of the
treated wastewater was removed from the reactor. The purpose of
this was to enable the colonization of the filling by microorganisms
present in the inoculum. The kinetics of the utilization of organic
compounds and denitrification were monitored after 100, 140
and 180 operation cycles. To minimize interference with the
process performance, the structure of the biofilm and its growth,
samples for metagenomic analysis were taken after 100 (AMP1,
the concentration of total nitrogen in the effluent from the reactor
below 2 mgN L−1 ) and 180 (AMP2, stable process parameters)
operation cycles and compared to a control sample of inoculum
(AMW, first day of operation).
2.1. Operation of the AnSBBR
In the study were measured pH value and temperature using
a CP-105 waterproof pH-meter (Elmetron, Poland);dissolved oxy-
gen using Oxi 330i/set (WTW, Germany),total nitrogen using Total
Organic Carbon Analyzer TOC-L CPH/CPN with TNM-L device (Shi-
madzu Corporation, Japan) for determination of total nitrogen with
the “oxidative combustion-chemiluminescence” method. Chemi-
cal oxygen demand (COD), ammonia nitrogen, nitrites, nitrates,
orthophosphate and dry mass according to APHA (1992) were also
determined for reactor effluent.
The biomass growth during one cycle was determined by fil-
tration of the whole volume of the treated wastewater through a
medium filter, followed by determination of the dry mass retained
on the filter. The measurements were carried out three times per
week following the cycle, after which no physicochemical analyses
were made.
2.2. DNA extraction
Genomic DNA was extracted from 0.2 g of semidry biofilm sam-
ples. DNA extracts were prepared in triplicates and pooled before
PCR amplification. DNA was purified using FastDNA Spin Kit for
soil (MP Biomedicals, USA) as per the instructions of the manufac-
turer. Qubit 2.0 Fluorometer (Invitrogen, USA) was used to obtain
accurate DNA quantification.
2.3. Library preparation and illumina sequencing
The microbial communities of the three analyzed samples were
identified by amplifying and sequence analysis of the V3-V4 region
of 16S rRNA gene from the metagenome. 16S rRNA gene frag-
ment was amplified using Illumina recommended PCR primers.
These primers were created by adding Illumina adapter overhang
nucleotide sequences to the PCR primers given by Klindworth et al.
(2012). PCR amplification was performed accordingly to Illumina
protocol using Platinum® Taq DNA Polymerase Hiigh Fidelity (Ther-
moFisher Scientific). Amplicons were indexed using Nextera® XT
Index Kit accordingly to producer’s instruction. DNA was sequenced
on an Illumina MiSeq instrument using 2 × 250 paired-end mode.
For the sequencing Miseq reagent kit v3 (Illumina, San Diego, USA)
was used.
The sequencing results were recorded as FASTQ files and
uploaded to the MetaGenome Rapid Annotation Subsystems Tech-
nology (MG-RAST) server for analysis (Meyer et al., 2008).
The Illumina metagenomic datasets are available at MG-RAST
under accession numbers 4613174.3 (AMW), 4613172.3 (AMP1)
and 4613173.3 (AMP2).
3. Results
The study demonstrated that waste beer can be used to enhance
the denitrification process in AnSBBR. Tests revealed changes in the
kinetics of wastewater treatment and structure of the microbial
community.
3.1. Physicochemical studies
The first stage of the research included start-up of the reactor
and the formation of biofilm on the filling. The concentration of total
nitrogen in the effluent from the reactor after 86 cycles (43 days)
was below 2 mgN L−1 (Fig. 1A). The thickness of the biofilm was
2 mm at the edges of discs to 20 mm near the axis of rotation.
The rate of denitrification and utilization of organic com-
pounds during the operation cycle was determined based on a
zero-order equation. The denitrification rate in the 100 cycle was
17.37 mgN L−1 h−1 , and a slight accumulation of nitrites was found
in the 4th hour of the cycle, reaching the level of 2.81 mgN L−1 .
The rate of organic compound utilization was 88.8 mgO2 L−1 h−1
(Fig. 1B).
Between cycles 86 and 180, the concentration of total nitro-
gen in treated wastewater was stable and below 2 mgN L−1 . A
gradual decrease in COD was found in the effluent from the
reactor up to about 140 cycle (Fig. 1A). The mean COD concentra-
tion was 101.5 ± 15.6 mgO2 L−1 between cycles 100 and 140, and
54.7 ± 8.8 mgO2 L−1 between cycles 141 and 180.
The denitrification rate was 35.11 mgN L−1 h−1 and
34.66 mgN L−1 h−1 during tests carried out after 140 and 180
cycles, respectively. The rate of organic compound utilization was
205.6 mgO2 L−1 h−1 and 212.4 mgO2 L−1 h−1 during tests carried
out after 140 and 180 and cycles, respectively (Fig. 1C, D). The
profile of changes in the concentrations of total nitrogen and
organic compounds, as well as the parameters of the treated
wastewater indicated that during that period the reactor reached
a stable performance and maximum efficiency of wastewater
treatment. The concentration of total phosphorus between cycles
141 and 180 was 11.14 ± 0.57 mgP L−1 (removal efficiency 20.4%).
Excessive biomass is one of the products generated during
biological treatment of wastewater. Despite the stable opera-
tion of the reactor between cycles 141 and 180, the biomass
yield coefficient (Yobs ) was characterized by a high variability
and was 0.03 ± 0.03 mgTSS mgCOD−1 , which corresponded with
0.19 ± 0.16 mgTSS mgN−1 . This results from the specific perfor-
mance of biofilm reactors (Grady et al., 1999). It was stated that
the removal of one mg of nitrogen required 5.7 mg of organic com-
pounds, expressed as COD.
 A. Mielcarek et al. / Ecological Engineering 95 (2016) 384–389 387

Fig. 2. Class level affiliations assigned to contigs with 16 S rRNA genes in analyzed samples. Only classes with abundance higher than 1.0% are given.

Table 1
Metagenomic sequences statistic derived from MG-RAST platform (QF – quality filtering).

Sample Total reads Reads (post QF) Mean sequence length (post QF) Mean GC percent (post QF) ␣ diversity

AMW 284,097 260,504 313 ± 158 bp 55 ± 4% 80.88

AMP1 274,441 251,022 399 ± 97 bp 53 ± 4% 8.63
AMP2 319,776 296,915 330 ± 163 bp 53 ± 4% 8.37

3.2. Microbial community composition Table 2

Phylogenetic affiliations of the microbial reads in percent identified at the genus
level (at >0.3% abundance of assigned sequences) by Illumina sequencing of the 16S
The analysis found that the bacterial community structure of rRNA genes in the analyzed samples.
the activated sludge used as inoculum was different from that of
Genus AMW AMP1 AMP2
biofilm from AnSBBR. Significant differences were also registered
for samples of biofilm taken after 100 (AMP1) and 180 (AMP2) days Trichococcus 2.93 13.04 52.26
from the beginning of the process. Desulfosporosinus 0.007 0.17 0.94
Legionella 0.001 0.13 0.43
In order to analyze the changes in the microbial community
Simplicispira 0.003 0.22 0.31
structure, we compared the phylogenetic profiling of two samples Alcaligenes 0.05 18.39 0.88
taken during process performance with the control sample repre- Flavobacterium 0.24 4.13 0.45
senting activated sludge from WWTP. The statistics of the analyzed Parabacteroides 0.05 1.73 0.20
Rhodanobacter 0.0002 1.22 0.27
sequences are summarized in Table 1.
Comamonas 0.03 1.09 0.17
Taxonomic affiliation was assigned to contigs with rRNA genes Pseudomonas 0.03 1,04 0.23
based on comparison with the M5RNA database. The alpha diver- Chryseobacterium 0.009 1,01 0.67
sities for samples AMW, AMP1 and AMCP were 80.88, 8.63 and Xanthomonas 0.01 0.99 0.12
8.37, respectively. Rarefraction curves for each of the samples were Castellaniella 0.004 0.59 0.17
Rhodococcus 0.95 0.01 0.27
asymptotic, suggesting that the majority of bacterial taxons were
Arthrobacter 3.87 0.01 0.27
revealed. Both alpha diversity and rarefraction curves were exam- Riemerella 0.84 0.55 0.08
ined using MG-RAST. Desulfovibrio 0.01 0.001 0.64
Bacteria constitute the dominant domain (95.0–99.7 of the total Eubacterium 0.31 0.01 0.43
Prolixibacter 0.10 0.001 0.43
community) in all samples. At class level, 51 classes were found in
Sphingobacterium 0.04 0.01 0.31
AMW, 29 classes in AMP1 and 35 classes in AMP2 sample.
The proportions of the 6 major classes are presented in Fig. 2.
Contigs affiliated with Actinobacteria, Bacilli and Betaproteobacteria
constituted the majority of the communities. Actinobacteria pre-
dominated in the AMW sample (23.9% of the total community), ria (1.47–5.83%), Clostridia (0.32–2.09%), and Gammaproteobacteria
whereas in samples AMP and AMP2, this class was detected at a (1.26–4.0%).
much lower level (0.22 and 0.37, respectively). The contigs number Bacterial diversity and abundance were analyzed more specif-
of the Bacilli class was gradually increasing, starting from 6.27% ically at the genus level (Table 2). The sample of activated sludge
in AMW, through 13.2% in AMP1 and up to 52.8% in a sample (AMW), characterized by the highest indices of biodiversity, was
taken at 180 day of the process. Betaproteobacteria was dominated predominated by two genera: Arthrobacter (3.9%) and Trichococcus
in AMP1 sample, where it was counted at the level of 21.5%, a much (2.9%). The abundance of other genera was much lower. The AMP1
lower number of these bacteria were detected in AMW (4.05%) and sample was dominated by Alcaligenes (18.39%) and Trichococcus
AMP2 (2.31%) samples. A minor fraction of the total communities (13.02%), other genera with abundance above 1% were: Flavobac-
were represented by the contigs affiliated with classes: Flavobacte- terium, Parabacteroides, Rhodanobacter, Comamonas, Pseudomonas
and Chryseobacterium. The sample of biofilm taken at 180 day
(AMP2) was predominated by Trichococcus (Bacilli), which consti-
388 A. Mielcarek et al. / Ecological Engineering 95 (2016) 384–389
tuted almost 53% of the total community. The abundance of other
genera was lower than 1%.
4. Discussion
Wastewater from breweries contains significant amounts of dis-
solved organic contaminants and suspended solids (Shao et al.,
2008; Tabatabaei et al., 2010), which explains its high chemi-
cal oxygen demand (COD) (Feng et al., 2009). COD values for
brewery wastewater are usually 1000–4000 mgO2 L−1 , with BOD5
up to 1500 mgO2 L−1 (Janhom et al., 2009). An important factor
determining the quality of wastewater is the loss of beer dur-
ing its production (8.01–14.96%), as this significantly increases the
concentration of contaminants in raw wastewater (Shao et al.,
2008). The qualitative composition of mixed wastewater dis-
charged from breweries is similar to the composition of the final
product (beer) (Janczukowicz et al., 2013). Mielcarek et al. (2013)
and Janczukowicz et al. (2013) suggested that this type of wastew-
ater would be useful as an external source of organic carbon for
BNR.
In addition, the biomass yield coefficient in the present study
was one size of magnitude lower than for suspended-biomass reac-
tors (Randall et al., 1992; Nyberg et al., 1996; Lee et al., 1997;
Metcalf and Eddy, 2003; Janczukowicz and Rodziewicz, 2013).
This most likely resulted from anaerobic wastewater treatment
and the involvement of biofilm, and thus the low increase in
biomass yield and longer retention of biomass in the reactor.
Because of the significant thickness of the biofilm, the organic
substrate and nitrates were only available in its superficial lay-
ers. Bacterial cells and ESP probably decomposed in deeper, older
layers of the biofilm containing no substrates, thus the released
organic compounds could be supplied to superficial layers con-
taining nitrates (electron acceptors). Because of this, denitrification
occurred in the AnSBBR simultaneously with the decomposition
of excessive biomass and, thus, the level of biomass was lower in
the effluent from the reactor. Rapid consumption of waste beer
within the first 2 h of the cycle made the EPS the main source
of organic compounds for another 10 h. Mayhew and Stephenson
(1997) indicated that the biomass yield coefficient can be reduced
through the promoted growth of protozoa, nematodes, rotifers or
oligochaeta which, due to predation, reduce the biomass increase.
In our study, the biomass reduction could be most likely attributed
to the previously-mentioned ability of Tichococcus to degrade com-
plex organic compounds and EPS.
The metagenomic results showed that some genera showed
progress and others declined during the process. The abundance
of Alcaligenes decreased from 18.39% in AMP1 to 0.88% in AMP2
sample, whereas the numbers of Trichococcus increased gradually
from 2.93% in inoculum, through 13.04% in AMP1 sample and up to
52.26% in AMP2 sample. It seems that the conditions in the stud-
ied reactor were more suitable for Trichococcus because its cells
preempted Alcaligenes during the process. The domination of Tri-
chococcus must be the result of the important role of these bacteria
in the reactor. Accordingly to Hao et al. (2015) Trichococcus is able to
degrade a variety of complex organic compounds, such as polysac-
charides, glucose and yeast extract into simple molecules such as
acetate, which could be more suitable for other bacteria. Moreover,
Trichococcus is a producer of esterases, lipases and phosphohydro-
lases and it therefore often occurs in extracellular polysaccharide
substances (EPS) (Liu et al., 2002).
5. Conclusions
Studies on denitrification in an AnSBBR after the addi-
tion of waste beer as the only carbon source demonstrated
that under stable operation the biomass yield coefficient
(Yobs ) was 0.03 ± 0.03 mgTSS mgCOD−1 (corresponding to
0.19 ± 0.16 mgTSS mgN−1 ).
The waste beer effectively aids denitrification process in AnS-
BBR and promotes the development of Trichococcus genus bacteria
(52,3%).
Discarded beer, because of its properties should not be classi-
fied as waste because it could be used in municipal wastewater
treatment plants as an external source of organic carbon.
Acknowledgment
This study was financed under Project No. 18.610.008-300 of the
University of Warmia and Mazury in Olsztyn, Poland.
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