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 CHEMICAL ENGINEERING TRANSACTIONS  
 

VOL. 54, 2016 

A publication of 

 
The Italian Association 

of Chemical Engineering 
Online at www.aidic.it/cet 

Guest Editors: Selena Sironi, Laura Capelli 
Copyright © 2016, AIDIC Servizi S.r.l., 
ISBN 978-88-95608-45-7; ISSN 2283-9216 

Biological Nitrous Oxide Abatement by Paracoccus 
denitrificans in Bubble Column and Airlift Reactors 

Osvaldo D. Frutosab, Irene Cortesa, Esther Arnaiza, Raquel lebreroa, Raúl Muñoza*  
a
 Department of Chemical Engineering and Environmental Technology, University of Valladolid, Dr. Mergelina, s/n, 47011, 

Valladolid, Spain. Tel. +34 983186424 
b
 Facultad de Ciencias Agrarias, Universidad Nacional de Asunción, Campus Ciudad de San Lorenzo, Paraguay. Tel. +595 

21585606 
mutora@iq.uva.es 
 
Nitrous oxide (N2O), with a global warming potential 300 times higher than that of CO2, represents 6.2 % of 
the total greenhouse gas emission inventory worldwide. Furthermore, N2O is considered the most critical O3-
depleting substance emitted in this XXI century. In spite of the environmental relevance of this pollutant, very 
little research on biotechnologies for the treatment of N2O emissions has been conducted. In this study, the 
potential of a bubble column (BCR) and an internal loop airlift (ALR) bioreactors of 2.3 L was evaluated for the 
abatement of N2O from industrial emissions from nitric acid plants along 62 days of operation. The systems 
were inoculated with a methylotrophic Paracoccus denitrificans strain (DSM 413) and continuously supplied 
with methanol as a carbon and electron donor source for the anoxic reduction of N2O. The simulated waste 
gas consisted of a N2 gas stream containing 1 ± 0.1 % of O2 and 3377 ± 312 ppmv of N2O at the inlet of the 
BCR and 1 ± 0.1 % of O2 with N2O concentration of 3617 ± 342 ppmv at the inlet of the ALR. This N2-laden 
stream was supplied at a constant flow rate of 110 ml min-1 in each reactor. The performance of the BCR was 
characterized by a steady state N2O removal efficiency (RE) of 87 ± 3 % with CO2 productions of 308 ± 36 g 
m-3 d-1 and total suspended solid (TSS) concentrations of 867 ± 109 mg L-1. On the other hand, the ALR 
showed a N2O RE of 88 ± 2 % with productions of CO2 of 346 ± 28 g m

-3 d-1 and TSS concentrations of 874 ± 
88 mg L-1. This work constitutes, to the best of our knowledge, the first systematic study of a biotechnology for 
the continuous abatement of N2O from nitric acid plants.  

1. Introduction 

The increasing concern about climate change and the steady rise of global temperature have attracted much 
attention in the scientific community. Scientists have confirmed that these environmental problems are 
produced by the rapid increase in atmospheric concentrations of greenhouse gases (GHGs), whose 
concentrations are 45 % higher than those prevailing in the preindustrial era (IPCC, 2014). Nitrous oxide 
(N2O), the third most important GHG with a global warming capacity 300 times larger than that of CO2 due to 
its larger atmospheric persistence (150 years), accounts for 6.2 % of the total GHG emitted globally. N2O is 
also one of the major source of stratospheric NOx and is considered as the most important ozone depleting 
substance emitted in this XXI century (Ravishankara et al., 2009). Agriculture is the most important source of 
anthropogenic N2O emissions, followed by industrial emissions and waste management process. The major 
N2O source in industrial processes is the production of nitric acid, whose global emissions can reach up to 400 
Kton of N2O per year (Pérez-Ramıŕez et al., 2003). The typical composition of a waste gas from nitric acid 
production can be represented by 100-3500 ppmv of NOx, 300-3500 ppmv of N2O, 1-4 % of O2 and 0.3-2 % of 
H2O.  
Several physical-chemical technologies are applied for the treatment of N2O emissions from nitric acid plants 
as end of pipe mitigation strategy. Nonselective catalytic reduction (NSCR) (Lee et al., 2011) is a typical 
technology applied in nitric acid plants nowadays. However, this system entails the consumption of a reducing 
agent such as hydrocarbons or ammonia and high temperature for N2O destruction. Furthermore, there are 
novel catalysts technologies that usually require the use of precious metals (Inger et al., 2013) and do not 
need a reducing agent, but the temperature required for its operation is higher than in NSCR. Thus, all those 

                               
 
 

 

 
   

                                                  
DOI: 10.3303/CET1654049

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Please cite this article as: Frutos O.D., Cortes I., Arnaiz E., Lebrero R., Munoz R., 2016, Biological nitrous oxide abatement by paracoccus 
denitrificans in bubble column and airlift reactors, Chemical Engineering Transactions, 54, 289-294  DOI: 10.3303/CET1654049 

289



physical-chemical technologies require the preheating of the tail gas to be treated, resulting in a considerable 
energy consumption since nitric acid waste gas is typically emitted at ambient temperature (Wu et al., 2015). 
Furthermore, some reports have pointed out that NSCR technology can even emit CH4 as a result of an 
incomplete fuel burning for the treatment of N2O emissions in nitric acid plants (Environmental Protection 
Agency, 2010). 
Biological technologies have demonstrated promising features such as robustness, cost efficiency and 
environmentally friendliness for the treatment of industrial waste gases (Estrada et al., 2011). In spite of the 
above advantages, no biotechnological system has been ever evaluated for the abatement of N2O emissions 
from nitric acid plants. N2O denitrification seems to be the most favorable biological pathway for degradation 
of N2O, where this GHG is an obligate intermediate in the reduction steps of NO3

- or NO2
- to N2 during organic 

matter oxidation in wastewater treatment plants. Thus, since nitric acid emissions are mainly composed of 
N2O, N2 and trace levels of O2, denitrification can be an attractive alternative for the abatement of N2O 
provided a cheap source of organic carbon and electron donor for heterotrophic bacteria (Frutos et al., 2016, 
2014). 
Bubble column (BCR) and internal loop airlift (ALR) bioreactors have been consistently proven as low cost 
alternative technologies for N2O abatement. These bioreactor configurations are pneumatically agitated by a 
gas phase bubbled from the bottom, resulting in a low energy consumption. Moreover, simplicity in 
construction with no moving parts and high gas-liquid mass transfer rates constitute also key advantages over 
conventional stirred tank reactors (Chisti and Moo-Young, 1989; Fu et al., 2007; Merchuk et al., 1994). ALRs 
differ from BCRs in the presence of an inner tube that separates the gas bubbling in the inner part (riser) from 
an external part (downcomer) promoting liquid recirculation. The ALR configuration has been previously 
studied for the removal of nitrogen in a sequential nitrification-denitrification process (Guo et al., 2005) where 
aerobic-anoxic environments are combined in a single reactor. 
In this context, a BCR and an ALR inoculated with a denitrifying strain of Paracoccus denitrificans (DSM 413) 
were studied and compared systematically for a continuous N2O abatement in a simulated emission of a nitric 
acid plant for a period of 62 days. 

2. Materials and Methods 

2.1 Chemicals and mineral salt medium 
All chemicals for mineral salt medium (MSM) preparation were purchased from PANREAC (Barcelona, Spain) 
with a purity of at least 99%. The MSM used in the experimentation was composed of (g L-1): Na2HPO4·12H2O 
6.16, KH2PO4 1.52, MgSO4·7H2O 0.2, CaCl2 0.02, NH4Cl 1.5, and 10 mL L

−1 of a trace element solution 
(containing per liter: EDTA 0.5 g, FeSO4·7H2O 0.2 g, ZnSO4·7H2O, 0.01 g, MnCl2·4H2O 0.003g, H3BO3 0.03 
g, CoCl2·6H2O 0.02 g, CuCl2·2H2O 0.001 g, NiCl2·6H2O 0.002 g, NaMoO4·2H2O, 0.003 g). The final pH of the 
MSM was 7. A cylinder of 40 L of 50,000 ppmv of N2O in N2 was purchased from Abelló Linde S.A. 
(Barcelona, Spain) as well as the 40 L cylinder of pure N2 needed to create the simulated emission. 

2.2 Microorganism cultivation 
A lyophilised methylotrophic strain of Paracoccus denitrificans (DSM 413) was purchased from DSMZ 
(Braunschweig, Germany). The bacterium was cultivated in 2 sterilized flasks with 0.5 L of MSM with methanol 
(1 %v/v) as the sole carbon and energy source under aerobic conditions for 3 weeks. 

2.3 Experimental set up and operational conditions 
A BCR of 42 cm of height (H) and 9 cm of inner diameter (ID), and an ALR of the same dimensions with a 
concentric draft tube (5.5 cm ID, 29.5 cm H) located at 4 cm from the bottom of the reactor were inoculated 
with 0.5 L of methylotrophic inoculum and filled with MSM to a working volume of 2.3 L, resulting in an initial 
total suspended solid (TSS) concentration of 56 mg L-1 in both bioreactors. The simulated nitric acid gas 
emission was prepared by mixing 50,000 ppmv of N2O in N2, air from a compressor and pure N2. The gas 
mixture resulted in a BCR inlet gas N2O concentration of 3377 ± 342 ppmv with 1 ± 0.1 % of oxygen, while the 
inlet gas of the ALR was composed of 3617 ± 342 ppmv of N2O and 1 ± 0.1 % of oxygen. Both the BCR and 
ALR were supplied with a gas inlet flow rate of 110 mL min-1, which correspond to a gas empty bed residence 
time (EBRT) of 17 ± 0.6 and 16 ± 1.2 min, respectively. Pure methanol (CH3OH) was injected in the gas line 
by means of a syringe pump in a sample port filled with fiberglass wool to facilitate its evaporation at a flow 
rate of 1.9 mL d-1, which resulted in a daily CH3OH loading rate of 661 g m

-3 d-1. A detailed diagram of the 
experimental setup is presented in Figure 1. Prior to inoculation, an abiotic test was conducted under abiotic 
conditions with MSM in order to assess the potential abiotic elimination of N2O by photolysis or adsorption. 
The concentrations of N2O, CO2, and O2 were periodically monitored by GC-ECD and GC-TCD at both inlet 
and outlet gas sampling ports of the bioreactors. The total organic carbon (TOC), total nitrogen (TN), dissolved 

290



CH3OH and TSS were measured three times per week before the replacement of 300 mL of fresh MSM to 
replenish nutrient concentrations. The systems were operated in a controlled temperature room at 25 ºC. 

Mixed gas inlet

Treated gas outlet

F

F

N2

N2O

Air compresor

1 2

3

3

4

5

6

7
7

7
7

8 9

F

3

Figure 1: Schematic diagram of the experimental setup: (1 and 2) N2O and N2 gas cylinder, respectively, (3) 
mass flow controller, (4) mixing chamber, (5) methanol syringe pump, (6) gas flowmeter, (7) gas sampling 
port, (8) bubble column and (9) internal loop airlift reactor.   

 

2.4 Analytical procedures 
The concentration of N2O was measured in a Bruker Scion 436 Gas chromatograph with an Electron Capture 
Detector (GC-ECD) (Palo Alto, USA) equipped with a HS-Q packed column (1 m x 2 mm ID x 3.18 mm OD) 
(Bruker, USA). Injector, detector and oven temperatures were set at 100, 300, and 40 °C, respectively. 
Nitrogen was used as the carrier gas at 20 mL min−1. External standards prepared in volumetric bulbs (Sigma-
Aldrich, USA) were used for N2O quantification. The concentrations of CO2 and O2 were determined in a 
Bruker 430 gas chromatography (Palo Alto, USA) coupled with a thermal conductivity detector and equipped 
with a CP-Molsieve 5A (15 m x 0.53 µm x 15 µm) and a CP-PoraBOND Q (25 m x 0.53 µm x 10 x µm) 
columns. The oven, injector and detector temperatures were maintained at 40, 150 and 200 ºC, respectively. 
Helium was used as the carrier gas at 13.7 mL min-1, while external standards prepared from calibration 
mixtures were used for CO2 quantification.  
TOC and TN concentrations were measured using a TOC-VCSH analyser (Shimadzu, Tokyo, Japan) coupled 
with a total nitrogen chemiluminescence detection module (TNM-1, Shimadzu, Japan). Dissolved CH3OH 
concentration was determined in a Gas chromatograph coupled to a Flame Ionization Detector (Bruker 3900, 
Palo Alto, USA) equipped with a SupelcoWax (15 m × 0.25 mm × 0.25 µm) capillary column. Injector and 
detector temperatures were maintained at 200 and 250 ºC, respectively. Nitrogen was used as the carrier gas 
at 1 mL min−1 while H2 and air were fixed at 30 and 300 mL min

−1, respectively. N2 was used as the make-up 
gas at 25 mL min−1. The determination of TSS concentration was performed according to standard methods 
(APHA, 2005) and pH was periodically monitored with using a pH/mV/°C meter (pH 510 Eutech Instruments, 
Nijkerk, the Netherlands). 
The results were statistically analysed to compare the performance of the bioreactors with an analysis of 
variance (ANOVA) with 95 % of confidence level and Tukey`s honest significance test. 

3.  Results and discussions 

The abiotic test conducted prior inoculation showed a negligible (<3 %) adsorption or photolysis of N2O. The 
pH of the BCR remained at 6.65 ± 0.13 with a dissolved oxygen (DO) concentration of 0.11 ± 0.14 mg L-1 for 
the entire experimentation period, while the ALR presented a pH of 6.64 ± 0.13 with a DO concentration of 

291



0.06 ± 0.08 mg L-1. The first ten days of operation were characterized by a gradual increase in the removal 
efficiency (RE) of N2O in both systems (Figure 2A and 2B). This was likely due to the need for an adaptation 
period where the P. denitrificans synthetized the enzymes necessary for the anoxic degradation of CH3OH. At 
this point, it is important to stress that the cultivation of the strain during inoculum preparation was carried out 
aerobically. The inlet and outlet N2O concentrations of the ALR were 3617 ± 342 and 420 ± 69 ppmv, 
respectively (Figure 2A), which represented a steady state RE of 88 ± 2 %. On the other hand, the BCR 
showed a steady state N2O RE of 87 ± 3 % with inlet and outlet N2O concentrations of 3377 ± 342 and 441 ± 
74 ppmv, respectively (Figure 2B). Similar results were observed in a previous work (Frutos et al., 2016) where 
the abatement of diluted N2O (≈100 ppmv) in air and simultaneous wastewater treatment were evaluated 
under a gas EBRT of 40 min in a bioscrubber. However, the results here reported represent the highest N2O 
RE observed during continuous operation (50 days of steady state removal) in biological systems. The 
statistically evaluated data showed unexpected significant differences between both bioreactors, where 
slightly higher N2O REs were observed in the ALR likely due to the lower DO (0.06 mg L

-1) recorded, which 
promoted the reduction of N2O by the denitrifying bacteria.  

 

Figure 2: Time course of the Inlet (circle), outlet (square) and removal efficiencies (triangle) of N2O in the 
ALR (A) and BCR (B). Verticals bars represent the standard deviation from duplicate measurements. 

The steady state production of CO2 was reached after 10 days of operation, when the systems reached stable 
N2O REs (Figure 3A). The BCR showed a production of CO2 of 308 ± 36 g m

-3 d-1, while the ARL CO2 
production was 346 ± 28 g m-3 d-1 (Figure 3A). The statistical analysis of the CO2 production data showed 
significant differences between both bioreactors. The biomass concentration achieved under steady state after 
30 days of operation remained very similar in both reactors at TSS concentrations of 867 ± 109 mg L-1 in the 
BCR and 874 ± 88 mg L-1 in the ALR (Figure 3B). No significant differences were observed between both TSS 
concentrations. The dissolved CH3OH and TOC concentrations gradually increased up to day 30, when 
steady values were observed concomitant with the TSS concentration stabilization. Thereafter, the 

292



concentrations of CH3OH and TOC in the BCR remained stable at 1115 ± 99 and 390 ± 38 mg L
-1, 

respectively. Similarly, the concentrations of CH3OH and TOC in the ALR remained at 967 ± 96 and 348 ± 28 
mg L-1, respectively (Figure 3C and 3D). 

 

Figure 3: Time course of CO2 production (A), total suspended solid concentration (B), dissolved CH3OH 
concentration (C) and TOC concentration (D) in the ALR (circle) and BCR (square). 

The higher N2O RE and CO2 production in the ALR under same biomass concentrations observed in both 
bioreactors confirmed the slightly better performance of the ALR over the BCR. Thus, the greater N2O RE of 
the ALR can be attributed to the particular configuration of this bioreactor, which promoted the maintenance of 
anoxic conditions due to the liquid recirculation in the downcomer (non aerated). Others authors have 
previously proposed ALRs as a platform for the removal of contaminants in processes that require both 
aerobic and anoxic conditions. Thus, Dhamole et al. (2009) used a 42 L ALR for the simultaneous elimination 
of COD in the riser (aerated part) and the denitrification of nitrate in the downcomer (anoxic part). Similarly, 
Zhang and Wei (2013) used a 47 L airlift reactor for the successful development of simultaneous nitrification 
and denitrification treating synthetic wastewater. 
The results here reported showed that biotechnologies may be an interesting alternative to physical-chemical 
technologies for the abatement of nitrous oxide, exhibiting similar N2O REs and without the production of 
undesirable secondary pollutants. Furthermore, the biotechnologies here studied were characterized by the 
low energy consumption, simple configuration and operation. On other hand, the use of specific 
microorganisms capable of producing high added-value by-products such as biopolymers during the 
simultaneous abatement of pollutant may be an interesting approach to improve the cost-effectiveness of 
these technologies. The greatest limitation of the systems here proposed was the high gas EBRT (>15 min) 
required to obtain high elimination due the poor solubility of N2O, which results in large volume reactors with 
the consequent increase in capital cost. Therefore, more studies devoted to optimize bioreactor design and 
operational strategies are necessary in order to overcome the N2O mass transfer limitations typically 
encountered during the off-gas abatement of this GHG. 

4. Conclusions 

Bubble column and internal loop airlift bioreactors are considered low cost technologies due to their low power 
consumption and maintenance and therefore represent a promising platform for the biological abatement of 
N2O. To the best of our knowledge, this is the first systematic study where two biotechnologies are analysed 
for the treatment of N2O emissions from nitric acid plants. The study showed the high N2O RE of both BCR 
and ALR along 62 days of operation. The latter presented a better performance for the elimination of N2O 
likely due to its particular configuration, which allowed the maintenance of the anoxic conditions required for 
the complete reduction of N2O to N2 by P. denitrificans.  

293



Acknowledgments 

This research was supported by the Spanish Ministry of Economy and Competitiveness (CTQ2012-34949 and 
Red NOVEDAR CTQ2014-51693-REDC projects) and the European Union through the Erasmus Mundus 
Program BABEL and FEDER Funding Program.  

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