Ecological Engineering 82 (2015) 474–479

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

Denitrification on upflow-anaerobic filter filled with coconut shells

(Cocos nucifera)
Jenifer Clarisse Pereira da Silva, Adriano Luiz Tonetti* , Lays Paulino Leonel, Aline Costa
School of Civil Engineering, Architecture and Urbanism, FEC/UNICAMP, Avenida Albert Einstein, 951, Cidade Universitária “Zeferino Vaz”, Caixa Postal 6021,
CEP: 13083-852 Campinas, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Article history: This paper studied the denitrification in an upflow-anaerobic filter filled with coconut shells (Cocos
Received 22 January 2015 nucifera). We sought to remove nitrogen in decentralized systems, reducing the diffuse contamination of
Received in revised form 17 April 2015 groundwater and water bodies by nitrate. The upflow-anaerobic filter was operated under hydraulic
Accepted 23 May 2015
retention time (HRT) of 16 h and on the bottom, there was an inflow of a combination of raw sewage and
Available online 9 June 2015
nitrified effluents from an intermittent sand filter. The research was divided into 5 stages in which there
was an inflow of the following combination of raw sewage and nitrified effluent: Stage 1: 100.0%/0.0%;
Keywords:
Stage 2: 87.5%/12.5%; Stage 3: 75.0%/25.0%; Stage 4: 62.5%/37.5%, and Stage 5: 50.0%/50.0%. The upflow-
Sewage
Biofilm reactor
anaerobic filter promoted denitrification in all stages with a quick acclimatization of the denitrifying
Treatment biomass. In the most critical situation (Stage 5), in which there was an inflow of 46.9 mg L 1 of nitrate, a
Onsite transformation of 98% of such compounds was found. On the effluent, the nitrate concentration obtained
Nitrate did not exceed 1.0 mg L 1. So we found that a simple recirculation of nitrified effluent to anaerobic filters
Nitrogen can contribute to the reduction of diffuse pollution of nitrate in remote areas of large cities or in rural
areas.
ã2015 Elsevier B.V. All rights reserved.

1. Introduction denitrification in methanogenic systems is not well documented

Disposing nitrogen-rich effluents into water bodies is a public
health and environmental contamination issue. In the form of
nitrite combined with secondary amines, nitrogen may create
nitroamines, which are considered carcinogenic, teratogenic and
mutagenic products (USEPA, 1993). In the form of nitrate, it may
lead to a disease known as the blue baby syndrome. In the
environment, it causes nutrient enriching, leading to an excessive
growth of algae (Qin et al., 2012).
A form of preventing it from being disposed into water bodies
would be the wastewater treatment techniques that combine the
removal of nitrogen and carbonaceous matter in anaerobic
reactors. Such technology would be viable for developing countries
with warm climates. However, denitrification and methanogenesis
are mediated by different microbial populations requiring distinct
environmental conditions and consequently, an integration of
these processes might be problematic (Hendriksen and Ahring,
1996; Andalib et al., 2011). However, the occurrence of
* Corresponding author. Tel.: +55 19 35212369.
E-mail address: adriano@fec.unicamp.br (A.L. Tonetti).
(Andalib et al., 2011).
Baloch et al. (2006) and Tai et al. (2006) reported that the
production of alkalinity during the denitrification process can be
beneficially used to improve the stability of anaerobic reactors by
counteracting the decrease in pH, usually associated with acido-
genesis. However, in order for the denitrification to occur
satisfactorily, one must pay attention to the influent COD/NO3 N
ratio on the reactor. For Bernet et al. (1996), the complete
denitrification will only occur when the COD/NO3 N ratio is
greater than 3.4. Karim and Gupta (2003) found that denitrification
and methanogenesis may occur simultaneously with COD/
NO3 N ratios higher than 4.
The Brazilian Basic Sanitation Research Program (PROSAB) has
studied the sewage treatment of small and decentralized human
agglomerates using anaerobic filters. Anaerobic filters filled with
easily acquired packing materials were studied, such as bamboo
rings (Camargo and Nour, 2001), coconut shells (Cruz et al., 2013),
ceramic bricks and ground-up tires (Barros et al., 2011).
Cruz et al. (2013) verified that coconut shells showed a high
resistance to biological degradation and empty bed volume
(81.3  2.7%) and a specific surface area (100.3  14.8 m2 m 3)
compatible with synthetic materials. In addition, the effluent

http://dx.doi.org/10.1016/j.ecoleng.2015.05.007
0925-8574/ ã 2015 Elsevier B.V. All rights reserved.
 J.C.P. Silva et al. / Ecological Engineering 82 (2015) 474–479
produced in anaerobic filters filled with such shells reached a
77  50 mg L 1 COD, with an efficiency of 81  38%.
There has been a considerable effort to couple denitrification
and methanogenesis in a single reactor in both suspended and
attached growth systems (Andalib et al., 2011). However, the great
majority of the papers on denitrification in anaerobic reactors are
related to UASB reactors. As for example, Tai et al. (2006) found
that the synchronous production of methane and nitrogen gases
demonstrated the potential of combined methanogenesis and
denitrification in a UASB reactor. The operation of the integrated
system demonstrated that a combined carbon and nitrogen
removal was technically feasible with more than 87 and 96% of
total COD and BOD removal, 46–79% of TN removal, and 49–86% of
denitrification efficiency, depending on the HRTs and recycle
ratios. Tai et al. (2006) concluded that combined carbon and
nitrogen removal can occur in the UASB reactor without addition of
an external carbon source.
Cruz et al. (2013) stated that nevertheless, the anaerobic filters
are more easily constructed than a UASB reactor, requiring only a
tank and packing material. The packing material makes it difficult
to washout the sludge and particulate organic matter, ensuring
better effluent quality even with the existence of large fluctuations
in the flow, characteristic of small treatment systems. The packing
material also assists in the proper sewage flow distribution,
hampering the formation of short circuits. In comparison, the UASB
reactor requires the gas–liquid separator, which requires greater
care in its design and construction. Additionally, in such a system, if
the sewage distribution system is not built and maintained
correctly, there will be the risk of forming preferential flow
channels, impairing treatment.
Therefore, the objective of this paper was to evaluate the
denitrification on an upflow-anaerobic filter using coconut shells
as the packing material. For such, several nitrified effluent and raw
sewage ratios were used as the influent for the reactor.
2. Material and methods
The research was developed at the School of Civil Engineering,
Architecture and Urbanism of Unicamp. The raw sewage (RS) came
Fig. 1. Schematic of the studied system.
475
from the university and was directed toward the up-flow-
anaerobic filters (Fig. 1). The effluent was directed to sand filters,
whose role was to create the nitrified effluent and which were built
according to the study developed by Tonetti et al. (2012).
To build the sand filters, cylindrical boxes with an inner size of
1.00 m were used. The bed was constituted in three layers. The first
was 0.20 m deep and consisting of gravel with an effective size (ES)
of 16.12 mm and uniformity coefficient (UC) of 1.89. Right on top,
there was another layer consisting of 0.05 m of gravel (ES of
7.51 mm and UC of 1.66). The last layer consisted of 0.75 m of sand
(ES of 0.17 mm and UC of 3.14). The sand filters received a
200 L m 2 day 1 hydraulic loading rate.
The anaerobic filter and sand filter were only used to produce
the anaerobic effluent and nitrified effluent. These reactors have
been investigated in other studies (Cruz et al., 2013; Tonetti et al.,
2012).
In this research, the strategy of adding synthetic nitrate to raw
sewage was not adopted. This is because in the future, a
recirculating sand filter effluent to the anaerobic reactor will be
studied.
3. Denitrifying upflow-anaerobic filter (DAF)
As shown in Fig. 1, the raw sewage (RS) and the effluent
produced by intermittent sand filters (ISF) were sent to a
denitrifying anaerobic filter (DAF). In this reactor, the packing
material used was coconut shells from the Cocos nucifera species.
Before they were placed inside the cylinder, such shells were
divided into four sections (Fig. 2).
The denitrifying anaerobic filter was built with stainless steel
shaped as a cylinder and with a total volume of 500 L (Fig. 3). It was
1.68 m high, and its diameter was 0.76 m. The conic-shaped bottom
was separated from the section filled with coconut shells by a
bamboo grid.
Denitrification was evaluated in this anaerobic filter. In the
study, different combinations of raw sewage and nitrified effluents
from intermittent sand filter were applied (Table 1). The hydraulic
retention time used in the denitrifying anaerobic filter (DAF) was
16 h.
476 J.C.P. Silva et al. / Ecological Engineering 82 (2015) 474–479
Fig. 2. Coconut shells of the Cocos nucifera species (Cruz et al., 2013).
Fig. 3. Upflow-anaerobic filter schematic (Tonetti et al., 2012; Cruz et al., 2013).
The following samples were collected twice a week: raw
sewage, intermittent sand filter effluent and denitrifying anaerobic
filter effluents. The raw sewage and intermittent sand filter effluent
sampling was conducted immediately before the input on the
denitrifying anaerobic filter. The analyses of pH, chemical oxygen
demand (COD), total Kjeldahl nitrogen (TKN), ammoniacal nitro-
gen, nitrate, nitrite and alkalinity were performed according to
Standard Methods for the Examination of Water and Wastewater
(APHA et al., 2012).
The coconut shells were inside the reactor for over two years.
This may be due to the consumption of easily degradable
compounds (Cruz et al., 2013). The portion of the husk consisting
of fibers has its degradation hampered by the existence of cellulose
and woody material, which confers high stability to the biological
action (Ohmiya et al., 2005). Therefore, this material was stabilized
and did not interfere in the biological process of removing organic
Table 1
Nitrified effluent and raw sewage application stages in the denitrifying anaerobic filter (DAF).
Influent source Stage 1
Raw sewage (%) 100
Nitrified effluent (%) 0 12.5
Raw sewage to nitrified effluent ratio – 7.0/1.0
matter or denitrification. The shells only served as support for the
biofilm.
4. Results
In Stage 1 of the study, 100% of raw sewage was applied to the
denitrifying upflow-anaerobic filter (DAF). The objective was to
evaluate the performance of the anaerobic reactor without the
nitrified effluent. The reactor showed a similar behavior (Table 2)
to the upflow-anaerobic filters studied by Cruz et al. (2013) and
Camargo and Nour (2001). In this case, a removal of 63% of COD
was found, which is close to the typical values for anaerobic
reactors (Foresti, 2002).
In Stage 2, there was an inflow of nitrified effluent together with
raw sewage. The study considered as DAF influent the result of the
raw sewage and nitrified effluent combination (RS + ISF combina-
tion). It was observed that even by increasing the nitrate
concentration throughout the stages, the DAF was highly efficient
in transforming such a compound. In Stage 5, where there was an
inflow of a combination of 50% raw sewage and 50% nitrified
effluent, the influent had a nitrate concentration of 47.7  11.4 mg
L 1, while the effluent had a concentration of only 0.89  1.0 mg
L 1. This refers to a 98.1% nitrate transformation.
Even with such an increase of nitrate concentration on the
influent, in every stage the DAF reached a stable state after a short
period of time. In Stage 2, 95% reduction of the concentration of
NO3 N was reached after 60 h of the first nitrified effluent
application. This shows the quick acclimatization of the denitrify-
ing biomass. Akunna et al. (1994) found a greater period for such
acclimatization, reaching 120 h.
A possible explanation for this rapid acclimatization of biomass
may be the fact that a reactor that was in operation for over two
years was used, treating the same wastewater. Moreover, according
Achak et al. (2009), even with the sand filter having a large air
capacity, denitrification can occur in anaerobic microcosms. When
the reactor is supplied with wastewater the process of denitrifica-
tion takes place in the anoxic zones. Thus, the nitrified effluent
could already have a small amount of denitrifying bacteria adapted
to the wastewater. When these bacteria reached the anaerobic
filter, they encountered conditions for their rapid development.
As a consequence of the nitrate transformation, the total
nitrogen concentration was also reduced (Fig. 4). In Stage 2, the
influent Total-N concentration was 90.8  14.6 mg L 1 and the
effluent concentration was 68.5  4.7 mg L 1, with an average
reduction of 23.3  9.3%. In Stage 5, the average reduction reached
59.4  11.4%. Huang et al. (2007) found that the combined UASB–AS
reactor system achieved an efficient removal of TKN (100%) and TN
(54–77%) from piggery wastewater. Ahn et al. (2007) combined an
anaerobic upflow bed filter with a membrane-aerated bioreactor
and obtained a 46% removal of nitrogen.
During each stage, there was no significant difference between
the influent and effluent concentration of N–NH3 (Fig. 5). This
indicates that an assimilatory reduction, in which the nitrate is
transformed into ammoniacal nitrogen, did not occur. Such results
are in agreement with the research developed by Akunna et al.
(1994). The authors found that 24 h after introducing the nitrate
into an anaerobic reactor, the ammonia production was
Stage 2 Stage 3 Stage 4 Stage 5
87.5 75.0 62.5 50.0
25.0 37.5 50.0
3.0/1.0 1.7/1.0 1.0/1.0
 J.C.P. Silva et al. / Ecological Engineering 82 (2015) 474–479 477

Table 2
Mean values for the evaluated parameters.
1 1 1 1
Stage Effluent COD (mg O2 L ) TKN-N (mg L ) NO3 N (mg L ) NO2 N (mgL )
1 RS 837  295 98.5  19.5 3.1  1.0 0.1  0.1
ISF 49  32 15.2  9.2 67.1  19.8 0.7  1.3
DAF 291  127 84.8  12.6 1.5  0.8 0.1  0.1
2 RS 643  171 91.6  18.8 2.3  1.0 0.1  0.1
ISF 41  18 1.8  2.2 57.8  12.1 1.8  2.4
Combination (RS + ISF) 568  149 80.4  16.5 9.3  2.1 0.2  0.3
DAF 173  79 68.0  6.3 1.1  1.9 0.1  0.2
3 RS 926  384 93.7  18.5 2.2  1.1 0.1  0.1
ISF 22  11 0.7  0.4 64.0  9.3 0.3  0.4
Combination (RS + ISF) 700  288 82.1  16.2 17.7  2.4 0.1  0.1
DAF 139  58 51.8  4.7 1.2  1.1 0.1  0.1
4 RS 1224  437 90.4  28.5 2.8  1.0 0.1  0.1
ISF 39  13 0.4  0.1 76.3  9.7 1.3  1.3
Combination (RS + ISF) 779  273 79.1  24.9 30.4  3.9 0.5  0.5
DAF 172  66 43.0  5.0 1.4  0.4 0.1  0.1
5 RS 1428  843 91.4  26.9 2.8  3.0 0.1  0.1
ISF 55  41 1.2  1.4 91.1  21.5 0.7  0.4
Combination (RS + ISF) 742  420 80.2  23.5 47.7  11.4 0.3  0.2
DAF 121  30 33.8  3.7 0.89  1.0 0.1  0.2

RS: Raw Sewage; ISF: Intermittent sand filter; DAF: Denitrifying upflow-anaerobic filter; TKN: Total Kjendal nitrogen.

Fig. 4. Total nitrogen influent and effluent concentration in the denitrifying anaerobic filter (DAF).

Fig. 5. Influent and effluent concentration of ammoniacal nitrogen in the denitrifying anaerobic filter (DAF).

insignificant, and the reduction of N-nitrate into nitrogen gas
prevailed. They stated that dissimilatory nitrate reduction to
ammonia took place in anaerobic digestion only during fermenta-
tion and that nitrate conversion to ammonia was greatly
minimized with non-fermentable organic carbon sources.
In Stage 1 (raw sewage application only), the COD removal was
equal to 63% (Table 2). After introducing the nitrified effluent, there
was an increase in efficiency, reaching 75  12% in Stages 2–5. Such
results may indicate that the organic matter consumption by
denitrifying bacteria is not inhibiting methanogenesis. In
478 J.C.P. Silva et al. / Ecological Engineering 82 (2015) 474–479

comparison, by using a sludge blanket reactor, Lee et al. (2004)

found a reduction in the COD removal after adding the nitrified
effluent. For Andalib et al. (2011), in biofilm processes, where
diffusion gradients can evolve, resulting in distinct environmental
conditions, methanogenesis and denitrification has reportedly
been observed to coexist. Lin and Chen (1995) state that in a co-
immobilized mixed culture system of denitrifying bacteria and
methanogenic microbes contained in polyvinyl alcohol gel beads, it
was demonstrated that methanogenesis was active inside the
beads where nitrate is absent, but denitrifiers grew on the surface
of the beads.
Based on the calculation suggested by the USEPA (1993), in
which a 1.00 g nitrate reduction is equivalent to a 2.86 g reduction
of O2, it is believed that between 5 and 23% of the influent COD
removal in DAF occurred during the reduction of NO3 N to N2.
Such COD removal percentage grew at every stage, following the
nitrified effluent proportion increase applied to DAF (Table 3).
Therefore, in Stage 5, possibly 77% of organic matter removal in
terms of COD was promoted during methanogenesis, while 23%
was removed during the nitrate reduction. Such results are in
agreement with the research developed by Huang et al. (2007).
These authors concluded that the presence of methane in a UASB
reactor showed that methanogenesis occurred at the same time as
denitrification.
For Huang et al. (2007), low values for the COD/NOx N ratio
will tend to result in a prevalence of the microbial group
responsible for denitrification, leading to a lack of substrate for
methanogenic microorganisms. For Bernet et al. (1996), the total
denitrification will only occur when the COD/NO3 N ratio is
greater than 3.4. Karim and Gupta (2003) found that denitrification
and methanogenesis may occur simultaneously with COD/
NO3 N ratios greater than 4.
Akunna et al. (1992) and Rustrian et al. (1997) stated that
denitrification increases as the COD/NO3 N relation is reduced.
For these authors, the denitrifying activity is hampered when the
COD/NO3 N relation is below 10 or at a nitrate concentration
above 2000 mg L 1. This occurs due to the accumulation of
intermediate denitrification products, such as NO2 and N2O. Such
intermediate products may inhibit the methanogenic activity,
compromising the system’s global efficiency (Andalib et al., 2011).
In this research (Fig. 6), the COD/NO3 N relation varied
between 15.64  7.30 and 62.05  16.16 averages. Therefore, such
values were adequate for denitrification, leading to the creation of
an effluent with low nitrate concentrations in every stage studied
(Table 2).
Akunna et al. (1992) and Andalib et al. (2011) reported methane
production without denitrification at COD/NO3 N greater than
53, with glucose as substrate in a CSTR of combined acidogenesis
and methanogenesis. In Stage 2, a mean COD/NO3 N ratio equal
to 62.05  16.16 was found. Even so, no reduction in the
denitrifying efficiency was found (Table 2). Therefore, we may
say that the great variety of compounds in the raw sewage may
favor denitrification, regardless of the COD/NO3 N ratio.
Another parameter that may be used to evaluate denitrification
is alkalinity. According to Metcalf and Eddy Inc. (2003), during the
NO3 N reduction to N2, a theoretical gain of 3.57 mg CaCO3 per
Fig. 6. COD/nitrate ratio.
Table 4
Total alkalinity on influent and effluent DAF.
1 1
Stages Influent (mg CaCO3 L ) Effluent (mg CaCO3 L )
1 292  54 355  42
2 164  36 399  47
3 114  25 355  24
4 146  29 361  31
5 149  24 382  37
reduced mg NO3 N occurs. In this study, an increase in the
alkalinity occurred in all stages (Table 4).
Since denitrification produces gaseous nitrogen, there was a
concern that the formation of such gas could cause the sludge on
the anaerobic filter to be dragged off. Therefore, the concentration
of suspended solids (TSS) was constantly monitored at the DAF
effluent. In Stages 1, 2, 3, 4, and 5, the mean concentrations were
35  24; 22  11; 18  08; 23  10 and 15  06 mg L 1, respectively.
No significant difference was found between these mean values.
This indicates the non-interference of denitrification for the
biomass to be dragged off in the upflow-anaerobic filter. Possibly,
in UASB reactors, where the biomass remains scattered, such
dragging may compromise the quality of the effluent.
The nitrite concentration during the entire analytical period
and at all collection points never exceeded 2.2 mg L 1. Therefore,
there was no accumulation of nitrogen in the reactor that could
compromise denitrification. Under normal conditions for nitrifi-
cation, ammonia and nitrite oxidation rates are reported to be
highly coupled, both in rates as in space, preventing nitrite
accumulation (Philips et al., 2002).
However, wastewater treatment plants frequently fail to
establish stable nitrification, which is often attributed to the slow
growth of nitrifying bacteria (Philips et al., 2002). For Hongwei
et al. (2009) nitrite accumulation can be caused by microbial
communities with different characteristics for nitrate and nitrite
reduction.
Schuch et al. (2000) observed that a higher alkaline pH (>7.8)
resulted in a lower denitrification rate and in an increasing nitrite
concentration in the effluent. On the other hand, Cao et al. (2013)
concluded that nitrite accumulation was more serious at low pH

Table 3
COD consumption during denitrification.

Stage Influent COD Theoretical COD required for denitrification Removed COD % of theoretical removal of COD during denitrification
(mg L 1) (mg L 1) (mg O2 L 1)
2 568  149 26 395  151 5%
3 700  288 33 561  291 6%
4 779  273 89 607  282 13%
5 742  420 136 620  410 23%
 J.C.P. Silva et al. / Ecological Engineering 82 (2015) 474–479
than at high pH, regardless whether the pH of the mixed liquor was
stabilized during denitrification.
The major importance of the pH at such, it usually influences
other parameters playing even a greater role in nitrite accumula-
tion; among others, the balance of ammonium and ammonia in
water is dependent on the pH, and the effect of free ammonia
seems to be more pronounced.
In this study the pH was always near neutrality (7.12  0.48).
This shows that the system was able to maintain optimum
conditions for denitrification throughout the analytical period.
5. Conclusions
The upflow-anaerobic filter with coconut shell as packing
material (C. nucifera) was efficient in reducing the organic matter
and denitrification. Even with an influent consisting of 50% sewage
and 50% nitrified effluent, an effluent with low nitrate concen-
trations, never higher than 1.5 mg L 1 was produced.
The biomass in the denitrifying upflow-anaerobic filter showed
quick acclimatization, even when there was an increase in the
nitrate load applied. During the denitrification process, no solids
were dragged off from the reactor by the gases created. This shows
the robustness of upflow-anaerobic filters and their feasibility to
be used as a denitrifying reactor to treat wastewater.
Acknowledgments
The authors would like to thank CNPq (the Brazilian National
Council for Scientific and Technological Development) for the
scholarships granted, in addition to FAPESP (São Paulo Research
Foundation) and FINEP (Studies and Projects Financing Agency) for
financing this study. The authors would also like to acknowledge
the service of the Writing Space/General Coordination of UNICAMP
for helping translate the original manuscript.
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