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Effect of CO on Microbial Denitrification via Inhibiting Electron Transport and Consumption Rui Wan, Yinguang Chen, Xiong Zheng, Yinglong Su, and Mu Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05850 • Publication Date (Web): 26 Aug 2016 Downloaded from http://pubs.acs.org on August 28, 2016
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Environmental Science & Technology
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Effect of CO2 on Microbial Denitrification via Inhibiting Electron Transport
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and Consumption
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Rui Wan, Yinguang Chen*, Xiong Zheng, Yinglong Su, Mu Li
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(State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and
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Engineering, Tongji University, 1239 Siping Road, Shanghai 200092)
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*Corresponding author
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Tel: +86-21-65981263
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Fax: +86-21-65986313
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E-mail:
[email protected] 1
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Abstract Increasing anthropogenic CO2 emissions have been reported to influence global
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biogeochemical processes, however, in the literature the effects of CO2 on denitrification have mainly
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been attributed to the changes it causes in environmental factors, while the direct effects of CO2 on
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denitrification remain unknown. In this study, increasing CO2 from 0 to 30,000 ppm under constant
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environmental conditions decreased total nitrogen removal efficiency from 97% to 54%, but increased
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N2O generation by 240 fold.
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bacterial membrane and directly inhibited the transport and consumption of intracellular electrons by
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causing intracellular reactive nitrogen species (RNS) accumulation, suppressing the expression of key
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electron transfer proteins (flavoprotein, succinate dehydrogenase and cytochrome c) and the synthesis
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and activity of key denitrifying enzymes. Further study indicated that the inhibitory effects of CO2
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on the transport and consumption of electrons were caused by the decrease of intracellular iron due to
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key iron transporters (AfuA, FhuC and FhuD) being down-regulated. Overall, this study suggests
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that the direct effect of CO2 on denitrifying microbes via inhibition of intracellular electron transport
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and consumption is an important reason for its negative influence on denitrification.
CO2
Damaging cell integrity
e
CO2
Fe
Complex I
Cyt b Cyt c1
FMN
Cyt c
NAR
e Complex II
e
SDH
e
RNS
Supressing Iron transportation
CO2
Complex III
NIR
NOR
N2OR
RNS NO3-
NO2-
NO
N2O N2
Inhibiting electron consumption
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N2O (µg N/mgTN removal)
Inhibiting electron transport
TN removal efficiency (%)
A subsequent mechanistic study revealed that CO2 damaged the
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60
40 0.6 0.5 0.4 0.3 0.2 0.1 0.0
0
1000 3000 8000 30,000
CO2 concentration (ppm)
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TOC Art
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Introduction
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Atmospheric carbon dioxide (CO2) concentration has increased by more than 1.4-fold because of
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anthropogenic activity, such as the burning of fossil fuels and changes in land use associated with 2
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agricultural activities.1,
2
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concentrations because of microbial activity and plant growth.3 Direct injection of CO2 into the deep
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ocean has been suggested as a method to control the rise of anthropogenic CO2; however, this can
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result in ocean CO2 concentrations increasing to as high as 20,000 ppm, and the accidental emission of
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CO2 can even cause levels near “ocean disposal sites” to be much higher.4, 5
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of anthropogenic CO2 is absorbed by surface waters worldwide,6 which would affect water chemistry
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parameters such as pH, carbonate equilibrium, and mineral saturation state, ultimately influencing
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aquatic organisms.7-9
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decreased the growth of coccolithophore Emiliania huxleyi, while dissolved CO2 increased the toxic
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effects of copper on polychaete Arenicola marina because of acidification of aqueous environments.8, 9
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Denitrification is an important process of the nitrogen cycle in both terrestrial and marine
Soil CO2 levels are more than 10 times higher than atmospheric
Approximately one third
For example, variations in pH coupled with CO2 elevation are believed to have
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ecosystems.10, 11
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denitrification, but different observations have been reported.12, 13 For example, when the effects of
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CO2 on denitrification were studied in four European grasslands a significant decrease in
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denitrification was observed at a French site, but little change was observed at other sites.13
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conducted by Robinson and Conroy showed that the presence of CO2 favored denitrification because it
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decreased soil oxygen and increased soil water content.14
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levels could indirectly increase rhizosphere denitrifier activity by 3 to 24 times in plant roots as it
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increased root nonstructural carbohydrate accumulation and exudation.15
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environmental conditions caused by CO2 such as pH, soil oxygen content and labile carbon source are
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believed to be the main mechanism for the influence of CO2 on denitrification10, and variations of
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these experimental parameters might explain the different responses of denitrification to CO2 reported
It has been reported that increasing environmental CO2 levels affected microbial
A study
Smart et al. observed that increasing CO2
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in the literature.
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Biological denitrification occurs intracellularly, via sequential bio-reductions of nitrate (NO3-) to
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nitrite (NO2-), nitric oxide (NO), nitrous oxide (N2O), and finally to nitrogen (N2). The core of these
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sequential bio-reductions depends on the electron transport and consumption system. In this system,
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electrons are delivered from nicotinamide adenine dinucleotide (NADH) via NADH dehydrogenase
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(complex I), the quinone pool, bc1 complex (complex III) and cytochrome c, then consumed during
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reductions of nitrate, nitrite, nitric oxide, and nitrous oxide, catalyzed by nitrate reductase (NAR),
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nitrite reductase (NIR), nitric oxide reductase (NOR), and nitrous oxide reductase (N2OR),
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respectively.10, 16, 17
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influence denitrification performance. However, the direct effects of CO2 on denitrification, especially
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electron transport and consumption, have not been reported to date.
Thus, any factors disturbing the transport and consumption of electrons would
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In this study, the direct effects of CO2 on denitrification and its mechanisms were investigated.
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First, the direct influences of CO2 on nitrate reduction and N2O emissions were studied under
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anaerobic conditions at pH 7.10 - 7.20 with sufficient carbon substrate. The causes for CO2 inhibition
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of denitrification and increasing N2O generation were then explored by analyses of the transport and
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consumption of electrons during denitrification. Finally, the reasons for the inhibitory effects of CO2
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on electron transport and consumption were identified by investigating the extracellular and
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intracellular iron and the iron transmembrane transport process. To our knowledge, this is the first
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study revealing the details of how CO2 directly affects denitrification.
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Materials and Methods
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Denitrifying Microorganism and Medium. Paracoccus denitrificans has been widely used in the
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literature as a model microbe of denitrification.18-20
In this study, P. denitrificans (ATCC 19367) was
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purchased from the American Type Culture Collection (Manassas, VA, USA), and routinely cultured
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under aerobic conditions in LB (Luria-Bertani) broth in an air-bath shaker (160 rpm) at 30°C.21 After
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cultivation for 24 h, the mixture was used as inoculum for subsequent experiments. The denitrifying
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medium was prepared in distilled water and modified from that described in the literature.18
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Specifically, it contained (g/L): NH4NO3 (0.86), KNO3 (1.08), sodium acetate (3.54), MgSO4·7H2O
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(0.1), trace elements (1 mL), KH2PO4 (2.44) and Na2HPO4 (4.65). The trace elements contained
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(g/L): Na2-EDTA (7.30), FeSO4·7H2O (2.50), MnCl2·4H2O (0.02), Na2MoO4·2H2O (0.242),
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CuCl2·2H2O (0.135), and ZnCl2 (0.34). The pH of the prepared denitrifying medium was adjusted to
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7.20 ± 0.05 with 6 M NaOH or HCl, then autoclaved at 121 °C for 15 min.
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Effects of CO2 on Denitrification. According to the IPCC A2-SRES emission trajectory, the partial
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pressure of CO2 in atmosphere will reach 1020 ppm, and higher level of CO2 may arise near the
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“ocean disposal sites”.4
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240, and 30,000 ± 315) of CO2 on denitrification were investigated. Specifically, 0 ppm was set as
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the control, 1000 ppm represented the predictable CO2 concentration in the atmosphere by the year of
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2100, and 3000, 8000 and 30,000 ppm were selected as the concentrations of CO2 near deep ocean
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disposal sites or sources of accidental emission.5
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serum bottles, that were divided equally into five groups to investigate the effects of five concentration
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levels of CO2 on denitrification. Five standard gas mixtures (CO2 and N2) containing 0, 1000, 3000,
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8000 and 30,000 ppm CO2, were purchased from a gas cylinder supplier (ChunYu Special Gases Co.,
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Ltd. Shanghai, China). To obtain accurate CO2 levels, 50 mL of the prepared denitrification medium
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were added into individual serum bottles in each group, after which they were pre-equilibrated for 30
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min with headspace CO2 at corresponding concentration levels. It should be noted that the pH of the
In this study, the effects of five levels (0, 1000 ± 130, 3000 ± 210, 8000 ±
Batch experiments were conducted in 30 identical
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denitrifying medium varied between 7.10 and 7.20 in all bottles because of the use of phosphate buffer
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(KH2PO4 and Na2HPO4).
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denitrifying microorganisms were inoculated immediately, then all bottles were sealed with rubber
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stoppers and placed in an air-bath shaker (120 rpm) with medium temperature of 28 ± 1°C, and one
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bottle from each test group was then selected randomly every 4 h for analysis.
Under corresponding concentration level of CO2 atmosphere, the
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Electron Transport System Activity (ETSA) Assay. The electron transport system activity of the
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bacteria was determined by reducing 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium
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chloride (INT, a kind of exogenous electron acceptor) to formazan (INF), which was modified from
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the literature.22
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the bacteria were harvested by centrifugation at 3500 g for 15 min, rinsed twice with phosphate
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buffered saline (PBS, 50 mM, pH 7.4) buffer, and then resuspended in 50 mM PBS with a final OD600
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of 1.0. Next, 200 µL of INT (0.5%) and 0.2 mg NADH were added into 1 mL of bacterial culture
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harvested from one of the tests. The mixture was subsequently incubated at 30°C for 30 min in the
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dark, after which 100 µL of formaldehyde were added to terminate the reaction. The samples were
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subsequently centrifuged at 10,000 g for 3 min to collect the cells.
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were used to extract the INF from the bacteria twice, after which the mixed INF extract was measured
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spectrophotometrically at 490 nm against a solvent blank. The ETSA was calculated according to the
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following formula:
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After culturing for 24 h under different CO2 atmospheres (0, 3000 and 30,000 ppm),
ETSA ( µg O 2 ⋅ g -1 protein ⋅ min -1 ) =
Next, 500 µL of 96% methanol
ABS 490 V 32 1 × 1 × × 15.9 V0 × t 2 m
(1)
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where, ABS490 is the sample absorbance, 15.9 is the specific absorptivity of INT–formazan, V0 and V1
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are the initial volume of bacteria and the total volume of methanol (mL), t is the incubation time (min),
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32/2 is the constant for transformation of µmol INT-formazan to µg O2, and m is the protein
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concentration per milliliter of bacteria (mg protein/mL bacteria).
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Effect of CO2 on Intracellular Iron Concentration. Intracellular iron levels were determined by
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fluorescence assay with calcein acetoxymethyl ester (Calcein-AM, Dojindo Molecular Technologies,
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Inc. Shanghai, China) as the fluorescent probe.23-25
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levels, the cells of P. denitrificans were harvested by centrifugation at 3500 g for 15 min, then rinsed
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twice with HEPES buffer solution (20 mM HEPES, 153 mM NaCl, 5 mM KCl, and 5 mM glucose)
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according to the manufacturer’s instructions. The bacteria were subsequently resuspended in the
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HEPES buffer solution with a final OD600 of 1.0, after which 50 µL calcein-AM (1 mM, dissolved by
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dimethyl sulphoxide) was added into 1 mL of the bacteria suspension.
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incubated at 37 °C for 30 min and washed with HEPES buffer solution three times before being
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determined by flow cytometer (BD AccuriC6; BD Biosciences, Franklin Lakes, NJ, USA). Samples
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were illuminated with an air-cooled argon ion laser (488 nm), and the fluorescence emission was
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detected at 515 ± 20 nm for Calcein-AM.23, 24
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µL bacteria suspension was collected and analyzed using the BD Accuri C6 Software (version
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1.0.264.21, BD Biosciences, Franklin Lakes, NJ, USA).
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Other Analytical Methods. Reactive oxygen species (ROS) and reactive nitrogen species (RNS)
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were detected by Cellular ROS/RNS Detection Assay Kit (Abcam Co., UK) according to the
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manufacturer’s instructions.
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chromatography (7820A, Agilent Technologies) with an electron capture detector as previously
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described,21 and their sum was recorded as the total N2O generation. The protein content was
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detected by the Coomassie Brilliant Blue G-250 method using bovine serum albumin (BSA) as a
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standard after the cells were ultrasonificated for 5 min in an ice-water bath and then centrifuged (10,000 g,
After culturing for 24 h under different CO2
The aliquot mixture was
The fluorescence of approximately 10,000 cells or 200
N2O in gas and liquid was determined by head space gas
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5 min, 4°C) to remove cell fragments.26
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screen for variations in protein expressions, NO2--N, NO3--N, lactate dehydrogenase (LDH),
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extracellular metal ion and morphology of cells) were detailed in Supporting Information (SI).
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Statistical Analysis. All tests were carried out in triplicate and the results were expressed as the
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mean ± standard deviation.
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analysis of variance with the Least-Significant Difference post-hoc test (SPSS 20.0, IBM SPSS,
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Armonk, NY).
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Results
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Effect of CO2 on Denitrification Performance.
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N2O and total nitrogen (TN, the sum of nitrate and nitrite) removal efficiency at different CO2
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concentrations.
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N/L in the control test, but it reached 29.6, 51.5, 82.9 and 123.6 mg N/L following treatment with 1000,
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3000, 8000 and 30,000 ppm CO2, respectively.
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significant accumulation of nitrite in all CO2 tests. Specifically, the final nitrite level increased from
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0.75 to 13.6 mg N/L as the CO2 concentration increased from 0 to 30,000 ppm. Similarly, the total
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amount of N2O generated was very low in the control test (0.002 µg N/mg TN removed), but was
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0.026, 0.063, 0.199 and 0.479 (µg N/mg TN removed) following treatment with 1000, 3000 8000 and
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30,000 ppm CO2, which was approximately 13, 32, 100 and 240 fold of the control, respectively
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(Figure 1c). As shown in Figure 1d, the TN removal efficiency decreased from 97% (control) to 88%
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(1000 ppm CO2), 80% (3000 ppm), 68% (8000 ppm), and 54% (30,000 ppm). These findings
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indicate that CO2 had an obvious negative effect on the denitrification process that occurred in a
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concentration-dependent manner and led to the accumulation of nitrite and emission of N2O.
Other analyses (such as iTRAQ LC-MS/MS employed to
Significant differences among groups were identified by one-way
Figure 1 presents the variations of nitrate, nitrite,
After the experiment was over 24 h, the final nitrate concentration was only 8.1 mg
Moreover, as shown in Figure 1b, there were
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Figure 1. Effects of CO2 on the variations of nitrate (a), final nitrite accumulation (b), final N2O
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generation (c), and total nitrogen removal efficiency (d) during denitrification. Error bars represent
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standard deviations of triplicate tests.
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Effect of CO2 on Integrity of the Bacteria Membrane. In this study, the analysis of LDH release,
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an indicator of cell membrane damage,20 was investigated (Figure 2a). Compared with the control,
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the exposure to 1000 ppm CO2 had no remarkable influence on the release of LDH. However, the
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release of LDH was significantly increased (p
