Waste and Biomass Valorization

https://doi.org/10.1007/s12649-018-0223-z

ORIGINAL PAPER

Denitrification of Nitric Oxide Using Hollow Fiber Membrane

Bioreactor; Effect of Nitrate and Nitric Oxide Loadings on the Reactor
Performance and Microbiology
Vahid Razaviarani1 · Melany Ruiz‑Urigüen2 · Peter R. Jaffé2

Received: 1 November 2017 / Accepted: 29 January 2018

© Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract
Nitric oxide (NO) removal from a gas stream containing ~500 ppm NO was studied in a hollow fiber membrane (HFM)
bioreactor. Compared to other biological NO removal methods the HFM bioreactor achieved NO removal rates that were as
good if not better, of up to 92% NO removal, under comparable loadings and reactor size. Results showed that a wastewater
stream containing organic carbon can be used as the electron donor to reduce the NO. Hence, combining biological NO
treatment with treatment of a wastewater containing organic carbon has may be an effective overall cost-reducing strategy.
The effect of different nitrate (NO−3 ) concentrations on the NO reduction rate was also evaluated, and results showed that
NO3− does enhance the NO removal rate. The reactor’s performance was studied under six different NO:NO−3 loading regimes
and the NO removal rate as well as the microbial denitrifier community in the reactor was tracked. Specifically, the relevant
genes responsible for each denitrification step were tracked during each different NO:NO−3 loading regime to the reactor.
Results showed that the denitrifying microbial community adjust rapidly to changes in the different N loadings, but overall
the performance of the reactor is robust and can withstand such variability in terms of NO, NO−3 and organic carbon removal.

Keywords  Nitric oxide (NO) · Hollow fiber membrane · Denitrification · Microbial community · Electron acceptor · Waste
treatment

Introduction
The equilibrium of the nitrogen cycle has been broken due
to human activities [1], causing several adverse environ-
mental and health impacts [2]. Before the Industrial Era,
the sources of all reactive nitrogen species (Nr), including
nitrogen oxides ­(NOx) came primarily from natural sources
(e.g. soil emissions and lightening), which were in balance
with nitrogen (N) losses through denitrification, a process
that returns nitrogen gas ­(N2) back to the atmosphere. By the
year 2010, the Nr formed by anthropogenic activity was at
Vahid Razaviarani and Melany Ruiz-Urigüen contributed equally
to this work.
* Peter R. Jaffé
jaffe@princeton.edu
1
Department of Chemical Engineering, American University
of the Middle East, 250 St, Egaila, Kuwait
2
Department of Civil and Environmental Engineering,
Princeton University, Princeton, NJ, USA
least two times larger than the rate of the natural terrestrial
formation, and the amount of N ­ 2 produced by denitrification
is only between 30 and 60% of the total Nr formed [3].
Nitric oxide (NO) is among the forms of Nr produced by
human activities including power generation, industry, tran-
sit, and biomass burning [1]. Typically, over 95% of N ­ Ox in
a combustion flue gas contains NO, while nitrogen dioxide
­(NO2) is mostly formed via photochemical reactions in the
atmosphere. These flue gases, NO and N ­ O2, are two major
air pollutants responsible for the formation of urban and
regional haze, acid rain, and ozone layer depletion, and can
contribute to climate change [4, 5]. Furthermore, exposure
to these compounds can cause severe health problems such
as respiratory illness [6]. ­NOx pollution is a problem in many
urban areas. Some megacities such as Beijing, Mexico and
New York City have had ­NOx concentration far beyond the
WHO standards [5], therefore, N ­ Ox pollution requires urgent
remediation and appropriate management.
Emissions of ­NOx have increased exponentially over the
past decades due to the rapid economic growth and energy
demand from fossil fuels. This has drawn much attention

13
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globally to the enactment of stringent regulations to control
and decrease the ­NOx emitted to the environment. Of these
oxides, ­NO2 is quite soluble in water and can be removed
easily from the off-gas stream by simple scrubbing and strip-
ping methods, while NO is fairly insoluble in water and can-
not be separated easily [7], unless it is removed very rapidly
from the water phase via reaction.
Conventional chemical and physical N ­ Ox controls such as
selective catalytic reduction, selective non-catalytic reduc-
tion, adsorption and scrubbing are currently used to control
­NOx emissions. However, these technologies are cost inten-
sive and produce large amounts of hazardous by-products
such as ammonia and particulate matter [8].
Biological ­NO x removal technologies have recently
drawn much attention due to the lower operating costs,
lower energy demands and absence of secondary pollutants
requiring further treatment [9]. Enhanced NO removal using
biological processes has been reported in several studies.
Direct transfer of NO gas to the liquid phase followed by
biological reduction of NO has also been studied by several
researchers. NO removal via biotrickling filter (BTF) was
demonstrated by Wei et al. [10] who studied thermophilic
(~50 °C) aerobic denitrification process in the removal of
NO from gas streams. In this work, a novel aerobic denitri-
fier Chelatococcus daeguensis was isolated from the biofilm
of an on-site BTF and inoculated in a 2500 mL BTF. Results
showed that with the inlet NO concentration between 100
and 500 ppm the BTF system could remove NO above 80%
at the presence of 8% oxygen. Hiroyasu et al. [11] studied
NO removal in a bubble-column type of reactor seeded by
the microalgae, Dunaliella tertiolecta. The NO removal
percentage was about 60% in a continuous culture under
light condition. Jun et al. [12] used an anaerobic rotating
drum biofilter to examine the NO removal percentage from
an off-gas stream. They reported a removal efficiency of 60
and 85% when the NO concentration was varied between 67
and 323 ppm, respectively.
Transfer of NO from the gas to the liquid phase can be
enhanced significantly in the presence of Fe(II)EDTA2−. In
the ­BioDeNOx process, ­NOx is absorbed by the chelated
Fe(II)EDTA2− in a physicochemical reactor. This step is
fast and results in an increased mass transfer rate of ­NOx.
Subsequently, in the second step, biological reduction of
­NOx to ­N2 via denitrification occurs when an adequate
amount of external electron donor, e.g. ethanol, is available
[13]. Though, a stable operation of the ­BioDeNOx process
is much more complex where the two microbial reactions,
Fe(III)EDTA− reduction and NO reduction, compete for the
electron donor supplied to the bioreactor. It was reported
that when the redox potential of the bioreactor remained
well below − 140 mV, the complete regeneration of Fe(II)
EDTA2− as the key reaction resulted in a stable NO removal
efficiency from the process. This successful continuous
13
Waste and Biomass Valorization
NO removal is achieved when the NO and Fe(III) loading
rates are equal to or lower than the NO and Fe(II) reduc-
tion rates, respectively [14]. The deficiency of the external
electron donor (e.g. ethanol) causes a decline in the Fe(II)
EDTA2− regeneration rate resulting in an increase in redox
potential. This could hamper the capacity of the NO mass
transfer and therefore reduces the absorption efficiencies
for gaseous NO in the physicochemical process. Van der
Maas et al. [14] demonstrated that when the concentration
of ethanol declined below 1 mM the NO removal efficiency
dropped dramatically from the gas phase. Van der Maas et al.
[15], also observed that exceeding ethanol as the sole exter-
nal electron donor in the bioreactor feed lowers the redox
potential below − 300 mV and the process is outcompeted by
other biological competitors, e.g. sulfate reducing bacteria
and methanogens. Therefore, to achieve a stable NO removal
efficiency through the ­BioDeNOx process, a proper control
of external electron donor supply is critical. Furthermore, it
should be noted that the cycling of the Fe(II)EDTA2− in the
­BioDeNOx process is a closed system, hence it is exceed-
ingly difficult to use waste-water containing organic carbon
as the electron donor.
To overcome the limitations of the low solubility of NO
in water, membrane bioreactors have been employed, and
among the different types of membrane reactors, hollow
fiber membrane (HFM) bioreactors possesses a number of
advantages over the other forms of membranes such as high
packing density and large surface contact area [16]. Zhang
et  al. [16] treated NO from a simulated flue gas stream
through the denitrification process using a HFM bioreac-
tor. These researchers reported the maximum NO removal
efficiency of 86% for a gas stream with 1000 ppm NO and a
gas flow rate of 20 mL/min. Min et al. [17] used a nitrifying
biofilm in a 500 mL-HFM bioreactor to remove NO from
combustion gases. A maximum NO removal efficiency of
74% was reported at the NO concentration of 100 ppm and
the flow rate of 100 mL/min. They demonstrated that the
NO removal efficiency remained almost constant regard-
less of the gas composition or temperature. Enhanced NO
removal through the HFM bioreactor has been reported to be
due to the improved support for the microbial population as
well as the increased surface area for the NO mass transfer.
Although, nitrification of NO using the HFM bioreactor has
largely been examined in previous studies [17, 18], to the
authors knowledge, only one study has been conducted on
the NO denitrification process in these reactors [16].
Denitrification of NO through various technologies has
been reported to range from 68 to 92% removal percentage
in previous studies [19–21], as shown in Table 3.
The performance and stability of the NO removal through
the denitrification process depends on a series of sequential
biological reactions ­(NO3−⟶NO2−⟶NO⟶N2O⟶N2)
catalyzed by four types of enzymes encoded by
Waste and Biomass Valorization

functional genes of the same name: nitrate reductase (Nar),
nitrite reductase (Nir), nitric oxide reductase (Nor), and
nitrous oxide reductase (Nos). Furthermore, microbial stud-
ies on the denitrification of NO linked to the performance
of HFM bioreactor when altering the N composition and
concentrations have yet to be investigated. To sustain the
microbial community for denitrification, the pH in bioreac-
tor must be monitored and controlled to remain near the
optimum value of 8.0 as suggested by Niu et al. [22]. Zhang
et al. [16] observed a significant increase in NO removal
efficiency when pH increased from 6 to 8, and a decline
when the pH further increased from 8 to 9. The denitrifica-
tion activity conducted outside of the ideal pH range could
result in the accumulation of intermediate products includ-
ing nitrite and nitrous oxide [23, 24].
A possible advantage of HFM-based biological NO
removal, versus a closed system like the B ­ ioDeNOx pro-
cess, is that NO can be used as an electron acceptor for the
degradation of wastewater contaminated with biodegradable
organic compounds, hence resulting in energy savings dur-
ing the wastewater treatment because of decreased needs for
aeration. Various wastewaters containing organic carbon can
be used as the electron donor for the denitrification process.
These wastewaters could contain varying degrees of N in
the form of nitrate ­(NO3−-N). Higher ­NO3−-N loads might
result in additional production of NO during denitrification,
and hence make the reactor less efficient in its gas-phase
NO removal rate, or vice versa, it could stimulate denitrifier
activity and enhance the overall NO removal rate. Therefore,
one of the key objectives of this study was to investigate the
effects of ­NO3−-N loading on the biological NO removal
through the denitrification process in a HFM bioreactor, to
optimize the performance of such a reactor, and to determine
if ­NO3−-N levels are of concern in the selection of waste-
water used for the reduction of NO. This goal was achieved
by altering the N­ O3−-N concentration in the feed solution
and the NO flow rates supplied to the membrane assembly
of the reactor, and examining the reactor’s performance and
microbial ecology once steady state was reached.
Materials and Methods
Bioreactor Setup and Operation
The denitrifying HFM bioreactor system is shown in Fig. 1.
The experiment was conducted in parallel in two separate
bioreactors, HFM setup and a semi-continuous bioreactor

(5) (8) (1) Hollow fiber membrane

(2) NO cylinder
(3) N2 cylinder
(1)
(4) Gas flow meter
(5) NO analyzer
(6) Mixing tank (MT)
(7) (7) Pump
(8) pH controller
(9) pH probe
(6) (10) Stirrer plate
(11) Stirrer bar
(9) (12) Substrate tank

(11)

(12)
(10)

(4)

(4)

(2) (3)

Fig. 1  Schematic of HFM bioreactor system

13

(BR), the latter used as a control reactor, both operated
at room temperature (25 ± 1 °C), following the different
N feeding protocols shown in Table 1. During the entire
experiment one bioreactor (BR) received ­NO3−-N as the
only source of N while the other was operated as the main
bioreactor (HFM setup, Fig. 1) and received varying loads
of ­NO3−-N and NO as N sources in various stages, as shown
in Table 1. A PermSelect hydrophobic polydimethylsiloxane
(PDMSXA-2.1) hollow-fiber membrane module (MedArray
Inc., Ann Arbor, MI, USA) was used to transfer NO from the
gas to the aqueous phase, serving also as a support for the
microbial population conducting the reduction of NO. The
module contained 30,000 fibers with an active surface area
of 2.1 m2. The inner and outer diameters of the fibers were
0.19 and 0.3 mm, respectively. The effective shell volume
was 190 mL which was about 38% of the total bioreactor’s
working volume. Initially, both bioreactors were filled with
500 mL of acclimated biomass and the reactor headspace
was purged with argon gas for 2 min. They were fed with
the same feed compositions used in the acclimation phase,
mixed thoroughly using magnetic stirrers, and were oper-
ated at a hydraulic retention time (HRT) of 6 h and a solids
retention time of 40 days. During the experiment, the HFM
bioreactor received NO gas supplied from gas cylinder as
N source in addition to ­KNO3− present in the feed solution.
­ O3−-N and NO
During the operation, the effects of varying N
loadings were determined in six stages as shown in Table 1.
Preliminary Performance of the HFM Reactor
The performance of the HFM in the transfer of NO from
the gas to the liquid phase was first examined using a series
of measurements in the absence of biomass. The removal
percentage of NO was measured using tap water in the shell
side and NO gas (500 ± 30 ppm) in the lumen side of the
HFM. At room temperature (~25 °C), the total gas flow rate
(NO and ­N2 as makeup gas) was maintained at 1 L/min, and
the liquid flow rate varied within the acceptable ranges sug-
gested for the HFM operation. The liquid circulation flow
rate was increased progressively from 0.24 to 2.15 L/min,
Table 1  Bioreactors substrate Parameters BR
loading characteristics
NO3−-N (g/L) 4.6
NOa (L/day) – 144
TOC (g/L) 17.8
Duration (day) 137
BR Batch (bio)Reactor, HFM hollow fiber membrane bioreactor
a
 The concentration of NO was maintained at 500 ± 30 ppm using ­N2 as the makeup gas
13
Waste and Biomass Valorization
and the maximum NO transfer at the optimum water flow
rate was determined in the absence of biomass.
Inoculum Acclimation
A fresh batch of mixed culture (about 1000 mL) containing
denitrifying bacteria was collected from the effluent of a
landfill leachate treatment facility in Princeton, New Jer-
sey. The acclimation was conducted for 40 days to obtain
initial mixed liquor volatile suspended solids (MLVSS) of
4500–5000 mg/L. The nutrient composition for biomass
acclimation was prepared based on the stoichiometry reac-
tion of heterotrophic denitrification. The HRT was kept at
6 h to maintain the working volume and biomass concen-
tration persistent. The acclimation process was deemed
complete when the changes in the effluent of MLVSS and
­NO3− values measurements over a 5-day period were < 5%.
At this point, the acclimated biomass was separated in half,
one-half was destined as the seed for the main bioreactor,
the HFM setup, and the second one destined as the seed
for the BR. The composition of the nutrient solution fed
to the bioreactors was as follows: 150 mg/L ­CH3COONa,
100 mg/L ­KNO3, 17 mg/L ­KH2PO4, and 8 mg/L ­K2HPO4.
Simulated flue gas was a mixture of NO (500 ppm in ­N2,
v/v) and ­N2 (99.95%).
Analytical Methods
Effluent samples were collected at steady-state conditions in
each stage and were stored for subsequent analyses. Steady
state operation was attained when coefficients of variation of
effluent ­NO3−-N, NO, total organic carbon (TOC), and pH
were < 5%. Nitrite was measured according to the Nitrite-
Diazotization (10019) method using a HACH DR3900
spectrophotometer. An Eco Physics Inc. CLD 60 series NO
analyzer was used to measure the inlet and outlet NO con-
centrations. The inlet NO flow rate balanced with ­N2 was
controlled using two calibrated Cole-Parmer Aluminum/
Glass flow meters. The pH in the reactors was monitored
and controlled continuously using an automatic BlueLab pH
HFM
Stage
1 2 3 4 5 6
4.6 4.6 4.6 4.6 2.3 0
288 0 288 288 288
17.8 17.8 17.8 17.8 17.8 17.8
20 25 20 27 30 15
Waste and Biomass Valorization
controller. The ­NO3−-N was measured with the Nitrate-HR
(10020) method using a HACH DR/3900 spectrophotom-
eter. For TOC and total dissolved nitrogen (TN) analyses,
samples were filtered through 0.2 µm nylon filter syringes
and then measured using a Shimadzu TOC/TN analyzer. All
analyses were performed in triplicate.
Denitrifiers Composition Analysis
Sludge Sampling and DNA Extraction
A 5 mL sludge sample was collected from the acclimated
biomass prior to starting the reactors operations. From the
HFM bioreactor, 13 mL were sampled from the mixing
tank during stages 1–6. At the end of the operation period,
6.5 mL from the biofilm in the HFM compartment were
obtained via destructive sampling of the HFM. From the
BR, 13 mL of sludge sample were obtained at the end of
the last stage. Total genomic DNA was extracted using Fast
DNA® Spin kit for soil (Biomedical, USA) according to
the manufacturer’s instructions. The sludge samples were
initially centrifuged at 10,000 rpm for 10 min and the super-
natant was decanted carefully to obtain the pellet of biomass
for DNA extraction. All the DNA samples were preserved at
− 20 °C until further analysis.
Denitrifier Quantification Analysis
Gene abundances of relevant functional genes have been
shown to serve as a tool to predict the capacity of an eco-
system to carry out a given process such a denitrification
[25]. In this study we chose to detect denitrifying bacteria by
targeting relevant genes responsible for each denitrification
step: narG for nitrate reduction, nirS for nitrite reduction as
it has been shown to be the gene present in denitrifiers better
adapted to waterlogged systems [25], CnorB for NO oxida-
tion, and nosZ for ­N2O oxidation. Quantification of total
Table 2  Primer sets used to Target gene Primers
target different bacteria groups
narG narG328F
narG497R
nirS nirS1F
nirS3R
CnorB cnorB2F
cnorB7R
nosZ nos1527F
nos1773R
Bacterial 16s rDNA 1055F
1392R
F forward and R reverse primers
and denitrifying bacteria present in the samples were deter-
mined by Quantitative PCR (qPCR) using specific primer
sets reported in the literature (Table 2), which amplify the
genes encoding narG, nirS, CnorB and nosZ enzymes. Fur-
thermore, to compare biomass production and sustainability
over time, a total bacteria count was conducted by amplify-
ing a region of the 16S rDNA gene using the primer set
1055F/1392R. The qPCR assays were carried out using the
Applied Biosystems StepOnePlus™ Real Time PCR system.
Each qPCR mixture (20 µL) was composed of 10 µL of
SYBR Premix Ex Taq® II 2X (Takara, Japan), 0.8 µL of
each forward and reverse 10 µM primer, and DNA tem-
plate. Thermal cycling conditions were initiated for 30 s
at 95 °C, followed by 40 cycles of 5 s at 94 °C, 30 s of
annealing according to primers’ specific temperature indi-
cated in Table 2, and 30 s at 70 °C. Finally, a melting curve
analysis for SYBR Green assay was run to distinguish the
targeted PCR product from the non-targeted PCR product.
Each qPCR reaction was run in triplicate and included nega-
tive controls and a standard curve; the last one consisting of
serial dilutions of known numbers of copies of DNA of the
gene per volume. Standards were generated via PCR using
TaKaRa Ex Taq® (Takara, Japan) following the manufac-
tures protocol, by amplification of each targeted gene using
the primers indicated in Table 2 and total genomic DNA
from Thiobacillus denitrificans (ATCC 25259C-5) as the
template. The standards’ single amplicon purity and size
was confirmed by 0.8% agarose gel electrophoresis and then
purified using QIAquick® PCR Purification Kit (Qiagen).
Qubit 2.0® (Invitrogen, USA) was used to measure DNA
concentration which was then used to calculate the standards
concentration according to each amplicon size.
Primer sequence (5′–3′) Annealing tem- Reference
perature (°C)
GAC​AAA​CTT​CGC​AGCGG​ 61 [26]
TCA​CCC​AGG​ACG​CTG​TTC​ 61
CCT​AYT​GGC​CGC​CRC​ART​ 57 [27]
GCC​GCC​GTC​RTG​VAGGAA​ 57
GAC​AAG​NNNTAC​TGG​TGGT​ 57 [28]
TGNCCRTGNGCNGCNGT 57
CGC​TGT​TCHTCG​ACA​GYCA​ 56 [29]
ATR​TCG​ATC​ARC​TGBTCGTT​ 56
ATG​GCT​GTC​GTC​AGCT​ 55 [30]
ACG​GGG​CGG​TGT​GTAC​ 55
13

RNA Extraction, RT‑qPCR and Gene Expression
Quantification
To determine shifts on narG and nirS gene transcripts due
to ­NO3−-N loading changes (4.6, 2.3, 0 and 9.2 g/L) under
constant NO loading (288 L/day), RNA was extracted from
the HFM Reactor’s Mixed Tank samples obtained at the end
of stages 4–6. Additionally, RNA analysis was carried out
on a sample of the biofilm formed on the membrane fibers,
for this purpose the reactor had to be destructively sampled.
PureLink® RNA Mini Kit (Invitrogen, USA) was used to
extract RNA from 15 mL of sludge from the mixing tank.
The sludge samples were centrifuged at 10,000 rpm for
15 min, the supernatant was discarded and the formed pellet
was used as the substrate for the RNA extraction, accord-
ing to the manufacturer’s instructions. Immediately after the
RNA extraction, cDNA of nirS and nosZ genes were pro-
duced via reverse transcription using qScript™ One-Step
SYBR® Green qRT-PCR kit, ROX™ (Quanta Biosciences,
USA), and corresponding primer sets indicated in Table 2.
Statistical Analysis
To determine if the samples show a significant difference
between gene quantity and gene expression at each stage,
ANOVA and MANOVA-Pillai’s trace statistical analysis
were run using R software (R Development Core Team,
2014).
Results and Discussion
HFM Reactor Performance without Biomass
The performance of the HFM was investigated when no bio-
mass was present in the reactor. The experiment was con-
ducted at constant NO flow rate, 1 L/min, and various water
7.0
6.0
5.0
NO removal (%)
4.0
3.0
2.0
1.0
0.0
0 0.5 1 1.5
Liquid flow rate (L/min)
Fig. 2  NO removal percentage at various liquid flow rates in the
absence of biomass
13
Waste and Biomass Valorization
flow rates, as shown in Fig. 2. As indicated in literature [17,
18], the mass transfer of poorly soluble gases trough mem-
branes is more dependent on the liquid phase turbulence
than on the gas phase turbulence, hence, increase in liquid
velocity, or flow rate, improved the gas transfer across the
membrane between the gas and liquid phases, as also seen
in our results. NO removal without biomass reached a maxi-
mum of 6.22%.
HFM Bioreactor Performance
The NO removal rate in the gas stream and the N ­ O3−-N
removal rate in the liquid stream were monitored carefully
throughout the experiment. The experiment was conducted
in six stages (Table 1). During stage 1, the concentration
of ­NO3−-N in the feed was 4.6 g/L (corresponding to 100%
loading shown in Fig. 3), the gas flow rate was set to 144 L/
day with a NO concentration of 518 ppm, using ­N2 as the
makeup gas. Once steady state conditions were reached, the
NO concentration in the gas outlet decreased to 50 ppm.
This represents a NO removal of over 90% at the gas-phase
with residence time of 2 min. At this stage, the NO and
­NO3−-N removal rates were 0.46 and 18.37 g/L day in the
HFM bioreactor, respectively. During stage 2, the gas flow
rate was increased to 288 L/day while the inlet NO concen-
tration (~525 ppm) as well as the N ­ O3−-N loading remained
−
unchanged (100% N ­ O3 -N loading). Monitoring continued
until steady state conditions were reached. A NO removal of
81% was observed at the gas-phase residence time of 1 min,
which corresponds to the reduction of NO concentration
from 525 ppm in the inlet to 100 ppm measured in the outlet.
Therefore, during stage 2, at the constant N­ O3−-N loading
(100%), doubling the NO flow rate to 288 L/day resulted in
a 76% increase in the NO removal rate relative to that during
stage 1. This represents a NO removal rate of 0.81 g/L day
in the HFM bioreactor. During stage 3, the N ­ O3−-N loading
remained constant (100%) while no NO was introduced to
the system. As shown in Fig. 2, ­NO3−-N removal remained
almost constant indicating that the addition of NO to the
bioreactor does not influence the removal rate of ­NO3−-N.
After a 20-day period of operating without NO loading,
NO was introduced again to the HFM during stage 4 with
a flow rate of 288 L/day corresponding to a gas-phase resi-
dence time of 1 min. During this stage, NO removal rate
reached 0. 66 g/L day when the ­NO3−-N loading remained
unchanged. A NO removal efficiency of 68% was achieved
during stage 4, which is about 13% lower than during stage
2. These results indicate that although there was a slight
2 2.5
reduction in the NO removal during stage 4 compared to
stage 2, the system had recovered significantly after no NO
was provided. This is also explained by the bacterial gene
population dynamics which showed continuous presence
of all denitrifying genes throughout those stages (Fig. 4).
Waste and Biomass Valorization

Fig. 3  Nitrate and NO removal 40

rates at various stages. The 1.4
NO3-N (HFM) NO3-N (BR) NO (HFM)
accompanying table shows the 35
NO and ­NO3−-N loadings for 1.2

NO3-N removal rate (g/Ld)

the HFM bioreactor at each 30
stage. The BR reactor had no

NO removal rate (g/Ld)

1.0
NO input but constant ­NO3−-N 25
loading of 4.6 g/L
0.8
20

0.6
15

10 0.4

5 0.2

0 0.0
1 2 3 4 5 6
Stage

Stage 1 2 3 4 5 6
NO (L/d) 144 288 0 288 288 288
(mole/d) 2.4 4.8 0.0 4.8 4.8 4.8
NO3--N loading (%) 100 100 100 100 50 0
(mole/d) 0.66 0.66 0.66 0.66 0.33 0.0

Fig. 4  a Denitrification path- a Denitrificaon pathway and enzymes analyzed.

way, substrates and enzymes.
b Dynamics of gene families
involved in denitrification dur- narG nirS CnorB nosZ
ing the different stages of NO
and ­NO3−-N loading. Values NO3- NO2- NO N 2O N2
show the mean and SD (n = 3)

b Denitrifying genes dynamics

1.E+10

narG nirS CnorB nosZ

1.E+08
DNA copies/mL of sample

1.E+06

1.E+04

1.E+02

1.E+00
1 2 3 4 5 6
Stages

From stages 4–6 the NO flow rate remained constant at
288 L/day while the ­NO3−-N loading varied. During stage
5, ­NO3−-N loading was decreased by 50% compared to that
of the previous loadings. At this loading, as shown in Fig. 3,
the NO removal rate was 0.40 g/L day, a reduction of 39%
in the NO removal efficiency compared to stage 4. During
stage 6, the ­NO3−-N loading was discontinued (0% loading)
and NO loading remained unchanged. Interestingly, when
no ­NO3−-N was introduced to the HFM bioreactor, the NO
removal rate was further reduced by about 23% relative to
that of stage 5, and reached 0.31 g/L day. This loading pro-
tocol showed the sensitivity of the denitrifier activity to the
­NO3−-N loading as indicated by the RNA quantification,
further discussed in “Denitrifier Quantification Analysis”

13
 Waste and Biomass Valorization

a Denitryfing genes quanficaon section. The nirS and nosZ gene transcripts showed a sig-
nificant decrease (p < 0.05) as the ­NO3−-N loading decreased
1.E+12
NarG+NirS CnorB+NosZ from 100 to 50 and 0 mg/L day uring stages 4, 5 and 6
1.E+11 respectively (Fig. 5).
Results show that NO was removed at different concen-
1.E+10 ­ O3−-N, even in the absence of nitrate,
trations of influent N
DNA copies /mL

and that increasing N ­ O3−-N loading resulted in an increase

1.E+09 in NO removal.
During the entire experiment, the concentration of
1.E+08 ­NO3−-N was well below the detection level. Several condi-
tions resulting in N­ 2O generation throughout the denitri-
1.E+07
fication process have been stated in the literature. Among
them, low pH and organic carbon deficiency are of major
1.E+06
Aclimmated MT HFM BR reasons resulting in ­N2O emissions. It has been hypothesized
seed that organic carbon scarcity results in the N ­ 2O accumula-
Inial stage Final stage tion [31], and it was revealed that the ­N2O reductase kinetic
is strongly dependent on the pH level [32]. Therefore, to
b Total 16s rRNA quanficaon minimize possible ­N2O emission in this study, the pH level
1.E+12
was kept at an optimum level of 8.0 ± 0.1, and ­CH3COONa
1.E+11
as a carbon source was continuously introduced to the
DNA copies /mL

1.E+10 bioreactors.
1.E+09 A comparison of the results from other studies to those
1.E+08 obtained from the present study is shown in Table 3. This
1.E+07 comparison shows that the membrane reactor configura-
1.E+06 tion, as applied here, combined with the use of wastewater
Aclimmated MT HFM BR as the reductant achieves among the highest NO removal
seed
efficiencies.
Inial stage Final stage

Denitrifier Quantification Analysis

Fig. 5  Number of amplified genes per mL of sludge sampled from the
acclimated biomass used as a seed for the reactor at the initial stage,
Denitrification is a sequential biological reaction catalyzed
and at the end of the operation from the mixing tank and HFM com-
partment from the HFM bioreactor and from the Batch Reactor (BR), by four types of enzymes: nitrate reductase (Nar), nitrite
the latter was run in parallel without NO loadings. a Denitrifying reductase (Nir), nitric oxide reductase (Nor) and nitrous
genes quantification, and b 16s rDNA quantification. Values show the oxide reductase (Nos) as summarized in Fig. 4a. Hence, an
mean and SD (n = 3)

Table 3  Description of previous studies of biological NO removal systems

Bioreactor T (°C) Volume (mL) NOin (ppm) Rta (min) QNOin (mL/min) REb (%) Reference

HFMb 22 ± 2 500 500 ± 30 2–1 100–200 92–81 Present study

HFMb 30 800 1000 0.5 20 86 [16]
HFMc 20 500 100 5 100 74 [17]
Jet-BioDeNOxb 21 20,000 500 – 500 92 [20]
BioDeNOxb 50 5000 100–500 – 1000 90–68 [19]
Biotrickling ­filterb 50 2500 100–500 1.92 1000 80 [10]
CABRb 50 4000 0–500 – 2000 90 [21]
Bubble ­columnd 25 4000 300 26.7 150 50–60 [11]
Rotating ­biofiltere 10–30 3000 67–323 1.44 1600 60–85 [12]
a
 Gas residence time
b
 Denitrification process
c
 Nitrification process
d
 Microalgea, Dunaliella tertiolecta, was seeded
e
 Anaerobic rotating biofilter

13
Waste and Biomass Valorization
investigation on how these genes respond when the NO and
­NO3−-N loading to the reactors changed during each stage
can provide additional insights on the reactor’s performance.
The dynamics of these genes during the different phases of
the reactor’s performance are shown in Fig. 4b.
The effects of ­NO3−-N and NO loadings on the micro-
bial community structure was studied on the samples col-
lected from the mixing tank (MT) at the end of each stage
(1–6), from the HFM compartment at the end of the opera-
tion period via destructive sampling (Fig. 1), and from the
BR, the latter only received ­NO3−-N as N source.
Results show that all the denitrifying genes vary sig-
nificantly (MANOVA p < 0.01) between stages. However,
the statistical analysis cannot determine the precise effect
of each loading regime. Therefore, we also analyze them
separately.
During stages 1–3, the results of the specific gene DNA
quantification showed that changes in NO loading while
keeping the N ­ O 3−-N inflow unchanged did not signifi-
cantly alter the dynamics of the denitrifying genes as the
experiment progressed. Additionally, as shown in Fig. 3,
during stages 1–3, there was no change in the ­NO 3−-N
removal capacity, suggesting that NO was not a competing
substrate over ­NO3−-N. Moreover, Fig. 4 shows that the
levels of CnorB, the gene responsible for NO reduction,
remained fairly constant during the experiment, even in
the absence of NO. This suggests that NO reduction was
also occurring in response to the ­NO3−-N denitrification.
Furthermore, one can see from the results (Fig. 6) that the
higher the NO load the more TOC was removed, demon-
strating our initial hypothesis that biological removal of
NO could assist the removal of organic compounds in the
treatment of wastewater.
Throughout stages 4 to 6, when NO flow was kept
constant and ­NO3−-N input concentrations changed from
4.6 to 2.3 and 0 g/L at stages 4, 5 and 6 respectively, the
genes codifying for narG and nirS decreased at each stage
Fig. 6  TOC and TN removal for
140
the different operational stages,
analyzed from samples taken
120
from the HFM Reactor’s Mixed
Removal rate (g/Ld)
Tank 100
80
60
40
20
0
1 2
(MANOVA p < 0.001). Quantified narG showed a decrease
of over 90% from stage 4 to 5 and 55% from stage 5 to 6.
NirS DNA quantification showed a decrease of 20% going
from stage 4 to 5 and of 33% from 5 to 6. The effect of var-
iating ­NO3−-N concentrations also showed changes on the
amount of CnorB and nosZ quantified DNA (MANOVA
p < 0.05).
Quantifying functional genes using DNA amplification is
a useful method to determine the overall microbial composi-
tion at different stages and in the different reactors. However,
RNA quantification is a more optimal approach to capture
the immediate microbial response to changes in the reac-
tor’s operation. Furthermore, nirS and nosZ genes have been
shown to be a good predictor of denitrification potential rates
of the systems [25]. Therefore, we quantified the amount of
transcripts of nirS and nosZ genes during stages 4–6 from
HFM Reactor’s Mixed Tank samples.
Decrease of N ­ O3−-N resulted in the decrease of nirS and
nosZ gene transcripts (Fig. 7). The major change is cap-
tured when no N ­ O3−-N is provided in the feed, nirS and
nosZ mRNA decrease by 31 and 17% respectively. Statisti-
cal analysis denote that ­NO3−-N loading regimes affected
both nirS and nosZ expression (MANOVA p < 0.1); and the
ANOVA analysis showed equal significant response from
both nirS and nosZ expression (p < 0.05). This indicates that
the changes in ­NO3−-N loading have similar effect on the
activity of both genes. The results suggest that the micro-
bial community can rapidly adjust gene transcription as a
response to changes in their environment, and that denitri-
fication of the N gas forms are driven by the NO inflow
and by the byproducts of N ­ O3− denitrification. The ratio of
nirS:nosZ during stages 4, 5, and 6 was 2.839, 2.829, and
2.333 respectively.
NO was directly delivered via the gas phase to the HFM
compartment through the hollow part of each membrane
thread (lumen part), where it was transferred to the aque-
ous phase (shell side) across the membrane. A biofilm can
TOC removal rate TN removal rate
3 4 5 6
Stage
13
 Waste and Biomass Valorization

Fig. 7  Number of copies of RNA response to NO3- loading changes under constant NO load
nirS and nosZ cDNA/mL of
sample, used for quantifying 2.0E+08 6.0E+07

Copies of nosZ cDNA/mL of sample

Copies of nirS cDNA/mL of sample
­ O3−-N loading
the effect of N nirS nosZ
changes, stage 4: 4.6 g/L, stage 1.6E+08 5.0E+07
5: 2.3 g/L, and stage 6: 0 g/L,
while at constant NO loading 4.0E+07
of 288 L/day. Values show the 1.2E+08
mean and SD (n = 3) 3.0E+07
8.0E+07
2.0E+07
4.0E+07 1.0E+07

0.0E+00 0.0E+00
4 5 6
Stage

The difference in loading and type of the N source intro-

duced to the HFM compartment, the HFM Reactor’s Mixed
Tank and the BR is translated to the difference in the number
of denitrifying genes detected. The acclimated biomass at
the initial stage, used as the seed for the HFM and BR, had
a slight higher amount of genes responsible for ­NO3− and
­NO2− reduction (narG + nirS = 5.53 × 1010 copies of DNA/
mL) than the number of genes responsible of NO and N ­ 2O
10
reduction (CnorB + nosZ = 1.98 × 10 copies of DNA/mL)
(Fig. 5a). At the end of the operation, the HFM had over six
times more total biomass than the initial seed and three times
more total quantified biomass than in the BR (Fig. 5b). Fur-
thermore, the HFM bioreactor showed a higher density of all
denitrifying genes compared to the initial biomass and the
BR. The HFM bioreactor had seven times more DNA copies
of CnorB and nosZ than the initial biomass, and 1.9 × 103
more than the BR. These results show a clear difference in
the microbial composition between the two types of reactors,
HFM bioreactor and BR, and between the two compartment
that make up the HFM bioreactor, in which the HFM com-
partment is where most of the biomass accumulates. It is
also at HFM where the genes responsible for the reduction to
the N gas forms are enriched. Data shows a clear connection
between the type of N.
Fig. 8  a Schematic of NO flow through HFM thread and biofilm
­ O3− concentration change over
formed over it. b Scheme of NO and N
distance
Conclusions

then develop on the wet surface of each thread, and as the
NO diffuses away from the surface, it allows for the biofilm
to grow, reducing the NO that diffuses from the membrane
and utilizing the organic carbon that diffuses into the bio-
film from the bulk liquid. N­ O3−-N from the bulk liquid may
also diffuse into the biofilm (Fig. 8). This explains why the
microbial composition in the biofilm is very different from
that in the HFM Reactor’s Mixed Tank (Fig. 5a).
Results presented here show that a HFM bioreactor can
achieve the same NO removal efficiency as the ­BioDeNOx
process. HFM reactors in the absence of biomass are not
capable of removing substantial amounts of NO from the gas
phase. The microbial population colonizing the membrane
on the wet side of the membrane, and reducing the NO, is
required to maintain a steep diffusion gradient needed for
high NO transfer across the membrane. Results without bio-
mass showed a maximum of 6.22% NO transfer from the gas

13
Waste and Biomass Valorization
to the liquid phase, however, when biomass was introduced
to the HFM bioreactor, the NO removal reached 92%.
Although higher gas loading rates to the HFM bioreactor
might result in a higher NO removal rate, the NO removal
efficiency decreases as the gas flow rate increases if the
removal rate does not increase linearly. For example, over
90% NO removal was achieved in stage 1 (at a gas resi-
dence time of 2 min), corresponding to a NO removal rate of
0.46 g/L day. By doubling the NO flow rate while maintain-
ing the N­ O3−-N loading constant during stage 2, 81% NO
removal was achieved with a NO removal rate of 0.81 g/L
day.
Based on the results of this work, additional research
should be conducted to determine the optimal membrane
characteristics that maximizes the overall NO removal in
HFM bioreactors, since a 2-min HRT for the gas phase,
that achieved 90% NO removal might be too large for many
applications dealing with high gas flow rates.
This study has shown that NO can be an effective elec-
tron acceptor for the biodegradation of organic carbon under
denitrifying conditions and that as the NO load increases
the organic carbon degradation also increases. Furthermore,
results showed that the overall NO removal benefits from
higher ­NO3−-N loads, which rather than acting as a com-
peting substrate with NO, stimulates denitrifier activity and
enhances the overall NO removal rate.
By focusing on the microbial community, this study has
shown that denitrifiers can sustain their population under
different ­NO3−-N and NO loadings, including at the absence
of NO or N ­ O3−-N, and that the HFM bioreactor’s microbial
community adjust rapidly to changes in the different N load-
ings, hence having a reactor with varying loadings of NO/
NO3− might influence NO removal as discussed above, but
overall the perforce of the reactor is robust and can with-
stand such variability.
DNA quantification is a good method to determine the
overall microbial composition as it will detect any DNA pre-
sent in a cell regardless of the cell’s state. Whereas, RNA
quantification is useful as it can capture the bacterial activ-
ity, meaning, that it detects only live functioning bacteria.
RNA captured the quick response in the decrease of both
nirS and nosZ where nosZ decreased faster (as shown by
the decreasing nirS:nosZ) due to the decreasing of N ­ O3−-N
−
in the feed. However, since during stage 6 the ­NO3 -N was
discontinued one might have expected not to detect nirS. At
this point it is not clear if this is because the reactor did not
run long enough at these stages for nirS to reach steady state,
even though the chemical fluxes were at steady state, or tran-
scription is not shut down completely while denitrifiers are
active even in the absence of N ­ O3−. Overall, we show that
the Mixing tank of the HFM bioreactor can support a stable
and active microbial population.
Although we have shown here that NO can easily be
removed in biological reactors such as described here, a
major challenge, which was not addressed is that ­NOx gases
from combustion processes are emitted at high temperatures
(i.e. ~200 °C). For the biological treatments the temperature
requires to be reduced to a level that makes the biological
reduction of NO feasible. Following that treatment, the gases
would have to be heated again (i.e. ~150 °C) to have suffi-
cient buoyancy. This was not addressed in this project and it
might limit its potential application until a cost effective way
of rapidly changing the temperature can be implemented.
Alternatively, biological reduction of NO can be applied to
gas streams with an ambient temperature, or gas streams
that do not have to be reheated before being released to the
environment.
Acknowledgements  Funding for this work was provided by the Hebei
Huafeng Coking & Power Co. The seed was provided by Dr. Eugenio
Giraldo from NuOrganics LLC.
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