Published November 12, 2015
Journal of Environmental Quality
Special Section
Moving Denitrifying Bioreactors beyond Proof of Concept
Optimizing Hydraulic Retention Times in Denitrifying Woodchip
Bioreactors Treating Recirculating Aquaculture System Wastewater
Christine Lepine,* Laura Christianson, Kata Sharrer, and Steven Summerfelt
Abstract
The performance of wood-based denitrifying bioreactors to
treat high-nitrate wastewaters from aquaculture systems has
not previously been demonstrated. Four pilot-scale woodchip
bioreactors (approximately 1:10 scale) were constructed and
operated for 268 d to determine the optimal range of design
hydraulic retention times (HRTs) for nitrate removal. The
bioreactors were operated under HRTs ranging from 6.6 to 55 h
with influent nitrate concentrations generally between 20 and 80
mg NO3−–N L−1. These combinations resulted in N removal rates
>39 g N m−3 d−1, which is greater than previously reported. These
high removal rates were due in large part to the relatively high
chemical oxygen demand and warm temperature (~19°C) of the
wastewater. An optimized design HRT may not be the same based
on metrics of N removal rate versus N removal efficiency; longer
HRTs demonstrated higher removal efficiencies, and shorter HRTs
had higher removal rates. When nitrate influent concentrations
were approximately 75 mg NO3–N L−1 (n = 6 sample events), the
shortest HRT (12 h) had the lowest removal efficiency (45%) but
a significantly greater removal rate than the two longest HRTs
(42 and 55 h), which were N limited. Sulfate reduction was also
observed under highly reduced conditions and was exacerbated
under prolonged N-limited environments. Balancing the removal
rate and removal efficiency for this water chemistry with a
design HRT of approximately 24 h would result in a 65% removal
efficiency and removal rates of at least 18 g N m−3 d−1.
Core Ideas
• Woodchip bioreactor design parameters for aquaculture wastewater were developed.
• This application resulted in the highest N removal rates reported (39 g N m-3 d-1).
• Retention times differ for optimized removal efficiency versus
removal rate.
• Sulfate reduction intensified under prolonged N-limited environments.
Copyright © 2015 American Society of Agronomy, Crop Science Society of America,
and Soil Science Society of America. 5585 Guilford Rd., Madison, WI 53711 USA.
All rights reserved.
J. Environ. Qual.
doi:10.2134/jeq2015.05.0242
Freely available online through the author-supported open-access option.
Received 27 May 2015.
Accepted 4 Sept. 2015.
*Corresponding author ([email protected]; [email protected]).
E
nhanced-denitrification bioreactors are a
farm- or field-scale technology designed to address
increasing levels of reactive nitrogen (N) in the environment (Schipper et al., 2010a). Such N pollution from agricultural sources is a causal agent in eutrophication, hypoxia (dead
zones), and habitat degradation, resulting in a loss of biodiversity
in coastal waters worldwide (Galloway et al., 2003). Over the
past 20 yr, wood-based heterotrophic denitrifying bioreactors
have been used to mitigate nonpoint source N pollution associated with agricultural tile drainage and groundwater (Robertson
and Cherry, 1995; Woli et al., 2010; Christianson et al., 2012;
Schmidt and Clark, 2012) as well as N pollution from point
sources like greenhouses (Warneke et al., 2011). Other agricultural point sources, in particular land-based recirculating
aquaculture systems (RAS), stand to benefit greatly from such
relatively simple denitrification technologies (van Rijn et al.,
2006).
Fish protein consumption has increased 3.6% per year since
1961, which is double the worldwide population growth of 1.8%
per year (WHO, 2015). To meet this growth demand in the face
of increasingly stringent restrictions on wild ocean resources,
there is a market-based need for the aquaculture industry to
expand. As an efficient method for fish production, RAS are
highly productive due to their (i) controlled, bio-secure environment, allowing maximized fish production over relatively short
periods, and (ii) minimal water use, which makes them viable
solutions for areas where water is scarce or in areas near large urban
markets where strict water quality criteria exist (Summerfelt and
Vinci, 2007; Timmons and Ebeling, 2010; Wheaton and Singh,
1999). However, the minimal water use associated with RAS
technology concentrates wastes into (i) a solid waste/wastewater stream associated with solids settling/dewatering and (ii) an
effluent stream. The waste/wastewater stream typically contains
high concentrations of suspended solids and high biochemical
oxygen demand (BOD) and chemical oxygen demand (COD)
stemming from concentration and dewatering of fish manure
and uneaten fish feed using technologies such as gravity thickening settlers or inclined belt filters (Sharrer et al., 2010). The
effluent stream is generated from a small flushing or “exchange”
The Conservation Fund Freshwater Institute, 1098 Turner Rd., Shepherdstown, WV
25443. Assigned to Associate Editor Louis Schipper.
Abbreviations: BOD, biochemical oxygen demand; COD, chemical oxygen
demand; DO, dissolved oxygen; DRNA, dissimilatory reduction of nitrate to
ammonium; HRT, hydraulic retention time; ORP, oxidation reduction potential; RAS,
recirculating aquaculture system; TAN, total ammonia nitrogen; TN, total nitrogen.
flow that is added to the fish production system to help dilute the
accumulation of nitrate (NO3-) in the fish tanks. Within RAS
unit processes, a biofilter is generally used to convert organic
N and total ammonia N (TAN) to nitrate to avoid accumulation of highly fish-toxic TAN and nitrite (NO2-) in the culture
tanks. Accumulation of nitrate in culture water has not historically been a concern for these systems, with levels as high as 400
mg NO3–N L-1 considered nontoxic (Timmons and Ebeling,
2010), although new recommendations encourage concentrations of <75 mg NO3-–N L-1 for optimal fish health (Davidson
et al., 2014). Both RAS outflow streams (wastewater and effluent) are suitable for application of a denitrification technology,
and, compared with many previous applications of wood-based
denitrifying bioreactors, treatment of recirculating aquaculture
outflows that stem from tightly controlled, often indoor, systems
provides an ideal opportunity to avoid variable flow rates and
seasonal temperature fluctuations that can affect woodchip bioreactor N-removal performance (Schipper et al., 2010a; Schmidt
and Clark, 2013). Although denitrification treatment of RAS
wastewater or effluent streams that leave the site as point source
discharge is an interesting opportunity for the treatment of point
source N pollution, recycling of woodchip bioreactor–treated
waters back to fish culture tanks in a tightly controlled RAS is
currently not recommended due to biosecurity and other fish
health uncertainties.
For woodchip bioreactor treatment of aquaculture wastewater to work successfully, several key design and operational
issues need to be addressed. Hydraulic retention time (HRT)
(bioreactor pore volume divided by flow rate) is a primary reactor design parameter, and engineering-based designs are essential
for preventing water from passing too quickly through a system
that minimizes nitrate removal (i.e., HRTs too short) or too
slowly (i.e., HRTs too long), which wastes the carbon investment as other reduction processes become active (e.g., sulfate
reduction and associated mercury methylation, methanogenesis)
(Robertson and Merkley, 2009; Shih et al., 2011). Reported
nitrate removal across HRTs varies widely due to differing environmental conditions and bioreactor system design. Influent
nitrate concentrations of 10 to 30 mg NO3-–N L-1 can be
reduced by approximately 50% with woodchip bioreactor HRTs
ranging from 6 to 30 h, a relatively wide range at groundwater
temperatures that generally range from approximately 10 to
15°C (Christianson et al., 2013; Moorman et al., 2015). Within
this span of influent concentrations, Woli et al. (2010) reported
that <3 h of HRT resulted in 33% reduction at a field site, and
Chun et al. (2009) found that 100% of nitrate was removed at
an HRT as low as 19 h (both woodchip bioreactors treating
groundwater). Higher influent concentrations tested in lab settings show that much greater HRTs may be required. Bock et al.
(2015) observed only a 13% concentration reduction from influent of 35 mg NO3-–N L-1 at 18 h with a woodchip-only control
column at 22°C, and Greenan et al. (2009) reported that 50%
reduction from an initial concentration of 50 mg NO3-–N L-1
required 2.8 d of HRT in a woodchip-packed column at 10°C in
an incubation chamber.
There has been no woodchip bioreactor HRT model calibrated specifically for the high nitrate concentrations and other
parameters (e.g., COD, dissolved oxygen, temperature) associated with recirculated aquaculture wastewater. This particular
water chemistry may help encourage denitrification along with
steady flows and constant temperature but may also lead to
excessive bacterial growth and clogging. Wood-based treatment
of aquaculture wastewater has been trialed at a small scale by
Saliling et al. (2007), although the woodchips were intended
to primarily serve as a colonization surface area and methanol
was added to fuel denitrification. Their simulated wastewater
initially ranged from 232 to 800 mg COD L-1 and from 50
to 200 mg NO3-–N L-1, and the methanol-enhanced systems
achieved >95% removal efficiency and removal rates as high as
1365 g N removed m-3 d-1. Other relevant previous applications of woodchip bioreactors include treatment of greenhouse
wastewater with nitrate concentrations between 200 and 300 mg
NO3-–N L-1 (Schipper et al., 2010b) and of septic effluent with
BOD concentrations as high as 150 mg BOD L-1 (Robertson
et al., 2005). Nitrex treatment of septic effluent in Canada has
used a design HRT of 1 to 10 d, with N removal typically >90%
(Robertson et al., 2005). The primary objective of this work was
to evaluate woodchip denitrifying bioreactor N-removal performance under varying HRTs given influent water chemistries
associated with RAS wastewater. This will help calibrate woodchip bioreactor design models specifically for recirculated aquaculture wastewater.
Materials and Methods
Bioreactor Experimental Design
Four pilot-scale woodchip bioreactors (3.8 × 0.76 × 0.76 m;
?1:10 scale based on surface footprint; i.e., the depth dimension
was not 1:10 scale) were constructed at The Conservation Fund’s
Freshwater Institute to evaluate N removal across a range of
HRTs (Fig. 1). Testing was completed in two phases to capture
a full range of potential design HRTs. During the Establishment
Period (Days 0–162), the four bioreactors were operated at
approximately 12, 24, 42, and 55 h HRT (1.68 ± 0.06, 0.85 ±
0.07, 0.47 ± 0.06, 0.36 ± 0.07 L min-1, respectively; mean ±
SD). The Loading Test Period (Days 169–268) allowed further
refining of the evaluation, with operation at 6.6, 12, 20, and 29 h
HRT (3.22 ± 0.35, 1.72 ± 0.24, 1.04 ± 0.07, and 0.71 ± 0.07 L
min−1, respectively).
The aboveground bioreactors were constructed of plywood
and lumber bracing and lined with plastic pond liner ( JPL-24,
Just Liners). Inlet and outlet manifolds (5.1 and 10.2 cm diameter PVC, respectively) with custom-drilled holes (4.0 and 5.5
cm diameter, respectively) and wrapped in plastic mesh (Pentair
N1020; 1.3-cm hole size) spanned the width of each bioreactor at the base of each system. The bioreactors were filled with
woodchips to a depth of 76 cm, and the 61-cm saturation level
was maintained by a static standpipe outside the bioreactors that
routed water through the base of each bioreactor’s downstream
wall (4-cm Uniseal fitting) to an effluent collection tank. The
locally sourced woodchips were classified by the vendor as “a 3
inch, hardwood blend” (Lowe Products) (no additional species
information available) and had an interpolated particle diameter
of approximately 1.2 cm at 50% of the cumulative distribution
(median diameter). Woodchip porosity was 70 ± 0.7% following
methods from Ima and Mann (2007), and dry-basis bulk density was 217 ± 11 kg m−3 (means ± SD). One pore volume based
on the bioreactor saturated volume was 1240 L (3.81 × 0.76 ×
Journal of Environmental Quality
Fig. 1. Schematic of four pilot-scale woodchip
bioreactors treating sodium nitrate–dosed
aquaculture wastewater. The bioreactor saturation level was maintained at 0.61 m depth.
0.61 m × 70% porosity). Three screened sampling wells (5.1-cmdiameter PVC) were located at 0.18, 1.74, and 3.57 m from the
bioreactor upstream wall in each bioreactor to provide access for
in situ measurements.
Aquaculture wastewater was generated on-site by the production of rainbow trout (Oncorhynchus mykiss) and Atlantic
salmon (Salmo salar). Waste was concentrated by a microscreen
drum filter and pumped into a series of gravity thickening settlers (i.e., settling cones). Overflow liquid from the settling cones
was directed into a supernatant holding tank, which was then
pumped to a mixing tank used to feed the four bioreactors (Fig.
1). The waste treatment system was previously described by
Tsukuda et al. (2015). In the mixing tank, the supernatant from
the settled fish waste was dosed with a concentrated sodium
nitrate solution to simulate nitrate concentrations experienced at
RAS facilities. The typical expected range is 60 to 80 mg NO3–N
L−1, although concentrations can be highly variable due to specific facility-based practices and flushing rates (van Rijn et al.,
2006). Some week-to-week nitrate concentration variation (e.g.,
±10 mg N L−1) in the supernatant wastewater was expected due
to changes in fish growth and water flushing requirements at this
production aquaculture facility; however, several equipment and
personnel oversights (e.g., dosing pump dial malfunction, dosing
calculations done in error) resulted in more variability than was
initially expected. Once dosed, the mixing tank solution was circulated into four individual treatment tanks of defined volume,
from where it was pumped into each bioreactor (Model# 6-CIA
115V, Little Giant Pump) over a period of less than 5 min on an
electronically controlled schedule (i.e., during the Establishment
Period, each pump kicked on one time per hour for generally about 3 min to pump each treatment’s predefined volume;
pumps kicked on two times per hour during the Loading Test).
Because only sodium nitrate was added to the mixing tank solution and because this solution was fairly quickly circulated to the
bioreactors (i.e., within 1 h after dosing), this procedure was not
expected to significantly alter other water quality parameters
besides nitrate in the aquaculture wastewater. Flow rates were
captured from each bioreactor on a weekly basis by filling containers of a known volume over a period of one pumping cycle.
The spring-sourced aquaculture system water was naturally alkaline (generally 200–400 mg CaCO3 L−1).
Initially, water quality sample collection timing was based on
flow through the reactors to normalize organic flushing parameters (e.g., COD) by cumulative pore volume eluted by each
Journal of Environmental Quality
HRT treatment. Thus, for the first 47 d, not all four bioreactors
had effluent samples collected at the same time. Beyond this day,
influent and effluent samples were collected concurrently weekly.
All collected samples were analyzed on-site for total nitrogen
(TN), TAN, NO2−–N, NO3−–N, sulfate, sulfide, COD, alkalinity, and pH following standard methods (APHA, 2005; Hach,
2003). Oxidation reduction potential (ORP), dissolved oxygen
(DO), and temperature were measured in the sampling wells
at least twice weekly (Hach HQ 40-d meter with ORP redox
MTC101 and LDO101-10; probes calibrated every 90 d).
Nitrogen removal rates (g N removed m−3 bioreactor d−1)
were calculated from the cumulative mass of NO3−–N removed
between two sample dates divided by the entire bioreactor
volume (length × width × depth of woodchips; 2.21 m3) and
the difference between the two sample dates (which is equivalent
to the difference in inflow and outflow NO3–N concentrations
times the bioreactor flow rate divided by the total bioreactor
volume). Alkalinity production and COD removal rates were
calculated similarly. Nitrogen removal efficiency was calculated
by dividing the difference of the inflow and outflow loads by the
inflow load. One-way ANOVA was used to assess significant differences between HRT treatments (a = 0.05).
Results and Discussion
Nitrate Removal
Nitrate removal occurred in all bioreactors over the course
of the experiment, with HRT means ranging from 6.6 to 55 h
(Fig. 2a). The initial influent nitrate concentration of 33.9 ± 7.37
mg NO3–N L−1 (mean ± SD) over the first 50 d was generally
reduced to <3.0 mg NO3–N L−1 in all four bioreactors due to
flushing of easily degradable carbon as evidenced by high effluent COD concentrations (Fig. 2e). The effluent contained >300
mg COD L−1 during first 28 d but was reduced to generally <80
mg COD L−1 and to values lower than the influent after Day 92
(influent mean ± SD, 83 ± 21 mg COD L−1, Day 92–268; n =
26). After this flushing period and when a higher N mass loading was applied (i.e., influent nitrate increased to consistently
between 71 and 78 mg NO3–N L−1 between days 113 and 146),
a relatively more steady-state measurement period was observed
where the bioreactors operating under the two shortest HRTs
did not continue to achieve complete nitrate removal (Fig. 2a).
Analyzed across the Establishment Period’s Measurement
Period (n = 6 sample events), the 12-h HRT treatment had the
Fig. 2. Influent and effluent
NO3−–N (a), total ammonia N
(b), NO2−–N (c), total N (d), and
chemical oxygen demand (e)
concentrations from pilotscale woodchip bioreactors.
Bioreactor hydraulic retention times in hours by testing
period are indicated in the
legend.
lowest removal efficiency (45%) (Table 1). However, because this
bioreactor received the highest hydraulic and N loading rate, it
also resulted in a removal rate that was significantly greater than
the two longest HRT treatments (39 g N m−3 d−1) (Table 1). The
24-h HRT treatment also demonstrated a significantly greater
removal rate than the longest HRT treatment (32 g N m−3 d−1)
(Table 1). This Measurement Period evaluation (i.e., Table 1)
was only performed during the end of the Establishment phase
because this period provided the longest record of consistent
influent nitrate concentrations. Removal rates were generally
greater than the highest reported rate of 22 g N m−3 d−1 (David
et al., 2015; Schipper et al., 2010a) (Table 1) due to the high
influent nitrate loads combined with HRTs sufficient to facilitate anoxic conditions (i.e., not too short), high COD loading
in the wastewater, and warm water temperatures (mean ± SD,
18.9 ± 1.4°C; n = 6). Warneke et al. (2011) reported a maximum removal rate of 11.2 g N m−3 d−1 for treatment of greenhouse wastewater with influent nitrate concentrations of 100 to
250 mg NO3–N L−1 at comparable water temperatures (15.5–
23.7°C). The 99% nitrate removal efficiency in the 42- and 55-h
HRT treatments indicated that denitrification was N limited
Journal of Environmental Quality
measurement period in the 12- and 24-h HRT treatments as well
as in the three longest HRT treatments toward the end of the
experiment (Fig. 2a, c). Regardless of internal TAN and NO2−
cycling, overall reductions in TN were consistently achieved
across all bioreactors.
The water temperature of these greenhouse-run experiments significantly declined from 14.6 to 23.5°C during the
Establishment Period to 12.6 to 16.2°C during the Loading
test (t test; p < 0.001) (Fig. 3a). Bioreactor effluent generally
contained <1 mg DO L−1, which was greatly reduced from the
influent (5.7–9.1 mg DO L−1) (Fig. 3b). Oxidation reduction
potentials of less than −300 mV measured within 30 cm of the
bioreactor outlets indicated that conditions were extremely
reduced in all the bioreactors during the early stages of the experiment (Fig. 3c). Influent ORP was recorded 25 times during the
Establishment Period and averaged −24 mV (range, −133 to 45
mV; data not shown). As influent nitrate loading was gradually
increased and the HRTs were reduced for the loading test, ORPs
more targeted for denitrification (i.e., +50 to −50; Gerardi,
2007) were observed near the outlet of each bioreactor. The two
longest HRT treatments (20 and 29 h) of the loading test period
showed the least reduced and most oxygenated conditions (i.e.,
the most positive ORPs and DOs); this was an unexplained
observation and was contrary to the observed greatest nitrate
concentration reductions in these bioreactors during this time.
Heterotrophic denitrification produces alkalinity, and here
alkalinity production increased with increasing HRT, thereby
also increasing nitrate removal, across the entire study (Fig.
3d). Influent alkalinity was inherently high due to the limestone karst-sourced spring water used in the aquaculture growout systems. Alkalinity production rates across the bioreactors
were significantly different between treatments during the measurement period (Table 1). Theoretical alkalinity production
rates based on the stoichiometric ratio of 3.57 mg alkalinity
(as CaCO3) produced for each milligram of NO3–N reduced
(Tchobanoglous et al., 2003; van Rijn et al., 2006) were within
10% of the observed production rates (Table 1). However, sulfate reduction also produces alkalinity (reduction of 1 mol
SO42− yields 100 mg CaCO3) (van Rijn et al., 2006), and this
may account for some of the difference in effluent alkalinity concentrations between treatments. The dip in effluent alkalinity
on Day 78 coincided with a decrease in influent nitrate loading,
leading to complete nitrate and sulfate removal and thus limited
alkalinity production.
and resulted in an excess of reducing conditions and sulfate
reduction (i.e., a hydrogen sulfide “rotten egg” smell).
The wastewater influent COD:NO3–N ratio averaged 1.26:1
during the measurement period (range, 0.86:1–1.66:1; n = 6).
The optimal range for denitrification is 3:1 to 6:1 (van Rijn et al.,
2006); thus, the COD in the wastewater was not sufficient alone
to completely fuel denitrification. This COD load accounted for
roughly a maximum of 42% of the COD required for denitrification (or 1.26 divided by 3.0, assuming this to be the optimal
range). Extrapolating this, if the wastewater COD load was fueling at most 42% of observed N removal rates, the woodchips
themselves would have accounted for removal rates of at least
22.6, 18.6, 14.4, and 10.4 g N m−3 d−1 for bioreactors 1, 2, 3,
and 4, respectively (i.e., removal rates in Table 1 multiplied by
1 minus 0.42). These “corrected” rates are more consistent with
previously reported N removal rates for woodchip bioreactors.
Additional N Species and Water Chemistry Parameters
Total N generally mirrored nitrate in the influent and effluent because nitrate was the primary N species present due to the
supplemental dosing system (fraction of TN occurring as nitrate
in the influent [mean ± SD], 91 ± 26%; n = 66) (Fig. 2a, d).
During the initial phase, when nitrate was frequently removed
to below 3 mg NO3–N L−1, effluent TN was mainly composed
of TAN and some organic N. An initial flush of ammonium has
similarly been reported by Healy et al. (2015). Total ammonium
N concentrations in the effluent were elevated above the influent throughout the experiment for all treatments (Fig. 2b). High
removal of total suspended solids across all bioreactors (generally >90%; data not shown) may have led to waste-associated N
cycling within the bioreactors and may account for a portion of
the elevated effluent TAN levels. An increase in TAN production
was observed coincident with a spike in influent nitrate loading
and alkalinity production (Days 50–75). After oxygen depletion
occurs within a system, nitrate is reduced to either molecular
nitrogen (i.e., denitrification) or ammonium (i.e., dissimilatory
reduction of nitrate to ammonium [DRNA]), depending on the
environmental conditions and bacteria present. This may have
been an indication of DRNA because (i) nitrate was nearly completely removed during this time and (ii) the available carbon
relative to nitrate was still likely relatively high during this initial time period, a condition thought to favor this process over
denitrification (Tiedje, 1994). Nitrite production was observed
coincident with the incomplete nitrate reduction during the
Table 1. Nitrate-N and chemical oxygen demand removal and alkalinity as CaCO3 production from four hydraulic retention times during the
Measurement Period.
HRT†
h
12 ± 0.4
24 ± 1.9
42 ± 4.1
55 ± 3.6
NO3–N removal
efficiency
NO3–N removal
rate
Observed alkalinity
production rate
Theoretical alkalinity
production rate‡
Chemical oxygen demand
removal rate
%
45 (9)b§
71 (8)ab
99 (0.5)a
99 (0.9)a
g N m-3 d-1
39 (11)a
32 (5)ab
–¶
–¶
g CaCO3 m-3 d-1
152 (28)a
110 (24)b
85 (18)bc
61 (14)c
g CaCO3 m-3 d-1
139
115
89
64
g COD m-3 d-1
66 (33)a
31 (16)ab
12 (8.0)b
8.0 (5.1)b
† Hydraulic retention time. Values are average (± SD) for each treatment across the entire Establishment Period.
‡ Based on 3.57 mg alkalinity as CaCO3 produced for each mg NO3–N reduced.
§ Results are mean (SD). Means with the same letter are not significantly different (a = 0.05; n = 6 sample events).
¶ Nitrate removal rates were N limited and were thus excluded from the table. For reference, these mean rates were 25 (3)bc and 18(2)c g N m−3 d−1 for
the 42- and 55-h HRT bioreactors, respectively.
Journal of Environmental Quality
Fig. 3. Air and water temperature (a)
and influent and effluent dissolved
oxygen (DO) (b), oxidation reduction
potential (c), alkalinity (d), and pH (e)
from pilot-scale woodchip bioreactors.
Oxidation reduction potential and
DO were measured in the monitoring
well approximately 25 cm from the
bioreactor end. Bioreactor hydraulic
retention times are indicated in the
legend.
The wastewater influent was generally within the optimum
denitrification pH range of approximately 7 to 9 (influent range,
7.22–7.96; mean ± SD, 7.76 ± 0.15) (Lu et al., 2014). Bioreactor
effluent pH was initially lower likely due to flushed organics (Fig.
3e). Slower flow rate treatments exhibited the lowest effluent pH
for longer periods, potentially because this start-up flushing took
more time and therefore there may have been some additional
processing of organic acids within these high HRT bioreactors.
Loading and Temperature Impacts on N Removal
Data from across all periods showed increased N removal rates
at increased N influent loading (Fig. 4a, b; data from only Day
113 onward to avoid carbon flushing effects). That is, at lower
HRTs (i.e., higher flow rates and greater hydraulic loading; Fig.
4a) and at higher influent N concentrations at a consistent HRT
(Fig. 4b), N removal rates increased. The exponential decay relationship between HRT and N removal rate indicated there may
be a “change-point” HRT for a given water chemistry and temperature where each additional unit decrease of HRT merited
a relatively greater gain in removal rate (Fig. 4a). For example,
these change points occurred at HRTs of 13.4 and 14.5 h for the
Establishment Period and the Loading Test Period, respectively
(Fig. 4a). These were the HRTs at which the rate of change, or
derivative, of the regression (Table 2) was approximately −1.0,
indicating that a one-unit decrease in HRT gained a one-unit
increase in removal rate. This type of relationship also showed
there was no additional notable removal rate benefit of increased
HRTs beyond these given change points. An exponential relationship between HRT and N removal rate has previously been
demonstrated by Healy et al. (2015). For design purposes, this
indicated the optimal HRT for this type of wastewater was less
than approximately 15 h to maximize the mass of N removed
per volume of bioreactor. However, this HRT only resulted in
<60% N removal efficiency (Fig. 4c). Using a longer design HRT
Journal of Environmental Quality
Fig. 4. Pilot-scale bioreactor nitrate removal rate (a, b, d) and removal efficiency (c) across
hydraulic retention times (HRTs) (a, c), influent nitrate concentrations at HRTs of 10.2 to
12.9 h (b), or water temperature (d).
would be slightly less efficient in terms of bioreactor volume (i.e.,
g N removed per m3), but if a bioreactor must achieve a 90%
removal efficiency to meet effluent water quality permit criteria,
for example, a longer design HRT would be necessary. In this
case, 75 and 90% removal efficiencies required design HRTs of
at least 33 and 48 h, respectively (Table 2). Selection of a 24-h
design HRT would result in a 65% modeled removal efficiency
and modeled removal rates of at least 18 g N m−3 d−1; this example of design parameter selection allows a compromise between
these two N-removal performance metrics by not falling too far
to the right of the change point in Fig. 4a while still providing
notable (e.g., >50%) N treatment efficiency in Fig. 4b.
When N loading was increased via changes in influent nitrate
concentration rather than flow rate manipulation (i.e., Fig. 4b
rather than Fig. 4a), significant relationships showed removal
rates increased at increasing influent N concentrations (Table
2). For these data spanning a relatively consistent range of 10.2
to 12.9 h HRT, this indicates that first-order kinetics existed.
It is thought denitrification within woodchip
bioreactors follows Michaelis–Menten kinetics (Schipper et al., 2010a), but both zero-order
(Robertson, 2010; Schmidt and Clark, 2013)
and first-order kinetics have been reported
(Christianson et al., 2012; Chun et al., 2009).
Modeling both the Establishment Period and
the Loading Test Period together resulted in
a reasonably strong correlation (R2 = 0.46; p <
0.0001) (dashed line in Fig. 4b).
Removal rates tended to be lower for the
Loading Test versus the Establishment Period
(e.g., Fig. 4a, b), which may have been caused
by differences in water temperature (mean
± SD, 19.5 ± 1.8 and 13.8 ± 1.0°C for the
Establishment Period and the Loading Period,
respectively). However, when data were sorted
to analyze across a relatively consistent HRT
(10.2–12.6 h) and influent nitrate concentration
(64–79 mg NO3–N L−1), removal rates were not
significantly different between the two periods (p
= 0.272) (Fig. 4d). Nevertheless, there was a significant relationship across the selected dataset
(Fig. 4d) (R2 = 0.25; p = 0.0431; removal rate =
18.47 × e (0.047 × Temperature)), which allowed calculation of a Q10 = 1.6, or the removal rate increased
by a factor of 1.6 for every 10°C increase. This
is similar to previous Q10 values of 1.6 and 2.0
reported by Cameron and Schipper (2010) and
Warneke et al. (2011), respectively, but lower
than those reported by Van Driel et al. (2006)
and Schmidt and Clark (2013) (i.e., >2.7).
Sulfate Reduction
Significant sulfate reduction and sulfide production occurred
for the two bioreactors operating with the longest HRTs during
nearly the entire experiment (Fig. 5a, b). The highest effluent sulfide concentrations of 13,230 to 27,760 mg S−2 L−1 occurred early
in the experiment, when effluent COD concentrations still indicated the occurrence of flushing particularly from the long HRT
treatments and when water temperatures were relatively high (i.e.,
Days 57–71, effluent 51–205 mg COD L−1, 21.0 ± 1.4°C [mean
± SD]). Samples before these dates were not analyzed for sulfide.
A spike of 208 mg NO3–N L−1 used to induce non-nitrate limited
conditions in all four bioreactors on Day 155 resulted in a notable
decrease in both sulfate reduction and sulfide production. Sulfate
reduction was not notably observed toward the end of the Loading
Test Period when no systems were nitrate limited.
Table 2. Regression relationships from Fig. 4 for hydraulic retention time (Fig. 4a, c) and influent nitrate N concentration (Fig. 4b) modeled against N
removal rate and removal efficiency for pilot-scale bioreactors treating aquaculture wastewater. Nitrogen-limited points are not included in regression analysis.
HRT†
Influent NO3–N
concentration
Establishment period (14.6–23.5°C)
Regression
R2
(-1.25 × HRT)
) 0.12
removal rate = 34.8 + (15,270,000 × e
removal efficiency = 33.5 + 1.23 × HRT
0.75
removal rate = −109 + 2.01 × influent nitrate
0.51
concentration
† Hydraulic retention time.
Journal of Environmental Quality
p value
<0.0001
<0.0001
0.072
Loading test period (12.6–16.2°C)
Regression
R2
(-0.22 × HRT)
0.53
removal rate = 17.3 + (111.2 × e
)
removal efficiency = 39.9 + 1.05 × HRT
0.16
removal rate = 5.9 + 0.41 × influent nitrate 0.36
concentration
p value
<0.0001
<0.0001
0.0007
Fig. 5. Influent and effluent sulfate
(a) and sulfide (b) (log scale) concentrations from pilot-scale woodchip
bioreactors. Bioreactor hydraulic
retention times by testing period are
indicated in the legend.
Sulfate reduction was clearly related to both N limitation and
highly reduced conditions in excess of −300 mV ORPs (Fig. 6a,
b). The longer HRT treatments (Fig. 6a, green square and blue
upright triangle symbols) exhibited the most sulfate reduction
as these bioreactors more consistently achieved 100% nitrate
removal. Shih et al. (2011) recommended bioreactors be operated to maintain no less than 0.5 mg NO3–N L−1 in the outflow
to minimize the potential for sulfate reduction and associated
mercury methylation. Interestingly, the bioreactor operating
under the shortest HRT achieved 100% nitrate removal during
several sample events (Fig. 6a, red circles on right side of graph),
but less notable sulfate reduction occurred. This may indicate
that sulfate reduction becomes exacerbated under prolonged
N-limiting conditions. Sulfide production was more evident
when sulfate reduction occurred to a greater extent (Fig. 6c).
Sulfate production was also observed, primarily in the shortest
HRT treatment (i.e., negative sulfate reduction in Fig. 6a and b,
primarily red circles). Although sulfate reduction in woodchip
bioreactors operating under highly reduced conditions is a wellestablished phenomenon, this signifies additional potential for
internal sulfur cycling when N removal is not complete under
short HRTs. Nevertheless, in this case, these concentration
increases were typically small (Fig. 5).
Fig. 6. Comparison of sulfate reduction versus
nitrate reduction (a) or oxidation reduction
potential (ORP) values (b) (optimal ORP range for
denitrification of −50 to 50 mV noted in the gradient) and sulfate reduction versus sulfide production
(c) (note log scales).
Journal of Environmental Quality
Conclusions
Pilot-scale woodchip bioreactor treatment of RAS wastewater resulted in some of the highest N removal rates reported to
date (≥39 g N removed m−3 d−1) due to high nitrate and COD
loading inherent to this water, relatively warm water temperatures, and consistent flow rates. This application of woodchip
bioreactors provides the opportunity for a more tightly controlled design compared with treatment of nonpoint source
nitrate flows, such as agricultural drainage.
Manipulating HRTs to span 6.6 to 55 h demonstrated that the
optimal HRT to maximize N removal rate may not be the same
the optimal HRT to maximize N removal efficiency. Balancing
these two removal metrics at a design HRT of approximately
24 h would result in a 65% removal efficiency and removal rates
of at least 18 g N m−3 d−1. Longer HRT designs may maximize
removal efficiency but may also result in sulfate reduction/sulfide production under highly reduced, N-limited conditions.
Sulfate reduction was observed in the bioreactors under such
highly reduced conditions and was exacerbated under prolonged
N-limited environments. Heterotrophic denitrification, as well
as sulfate reduction, resulted in notable alkalinity production.
Overall, woodchip bioreactor denitrification treatment of relatively high COD RAS wastewater is a useful application of this
simple water treatment technology.
Acknowledgments
This work was supported by Tides Canada, Vancouver, British Columbia
and the USDA Agriculture Research Service (Agreement No. 59-19300-046).
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