Increased CO2 Fixation Research Articles

Increased CO2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast

CO2 fixation plays a key role to make biobased production cost competitive. Here, we use 3-hydroxypropionic acid (3-HP) to showcase how CO2 fixation enables approaching theoretical-yield production. Using genome-scale metabolic models to calculate the production envelope, we demonstrate that the provision of bicarbonate, formed from CO2, restricts previous attempts for high yield production of 3-HP. We thus develop multiple strategies for bicarbonate uptake, including the identification of Sul1 as a potential bicarbonate transporter, domain swapping of malonyl-CoA reductase, identification of Esbp6 as a potential 3-HP exporter, and deletion of Uga1 to prevent 3-HP degradation. The combined rational engineering increases 3-HP production from 0.14 g/L to 11.25 g/L in shake flask using 20 g/L glucose, approaching the maximum theoretical yield with concurrent biomass formation. The engineered yeast forms the basis for commercialization of bio-acrylic acid, while our CO2 fixation strategies pave the way for CO2 being used as the sole carbon source.

24-Epibrassinolide alleviates drought effects in young Carapa guianensis plants, improving the hydraulic safety margin, gas exchange and antioxidant defence.

Climate change is increasing the frequency of extreme events such as droughts, limiting plant growth and productivity. Exogenous application of plant growth regulators, such as 24-epibrassinolide (EBR), might be a solution as this molecule is organic, eco-friendly, and biodegradable. This is the first research to examine possible roles of EBR on the hydraulic safety margin, physiological behaviour, and metabolism in Carapa guianensis Aubl. (Meliaceae) exposed to drought. C. guianensis is a widely distributed tree in tropical forests of the Amazon. The objective was to determine whether EBR can improve tolerance to water deficit in young C. guianensis by measuring hydraulic traits, nutritional, biochemical and physiological responses, and biomass. The experiment had four randomized treatments: two water conditions (control and water deficit) and two concentrations of EBR (0 and 100 nM EBR). EBR increased the water potential and hydraulic safety margin, increased CO2 fixation, and improved stomatal performance. EBR also stimulated antioxidant defences (SOD, CAT, APX, and POX). Overall, tretreatment with EBR improved drought tolerance of young C. guianensis plants.

An ATP-sensitive phosphoketolase regulates carbon fixation in cyanobacteria

Regulation of CO2 fixation in cyanobacteria is important both for the organism and global carbon balance. Here we show that phosphoketolase in Synechococcus elongatus PCC7942 (SeXPK) possesses a distinct ATP-sensing mechanism, where a drop in ATP level allows SeXPK to divert precursors of the RuBisCO substrate away from the Calvin–Benson–Bassham cycle. Deleting the SeXPK gene increased CO2 fixation particularly during light–dark transitions. In high-density cultures, the Δxpk strain showed a 60% increase in carbon fixation and unexpectedly resulted in sucrose secretion without any pathway engineering. Using cryo-EM analysis, we discovered that these functions were enabled by a unique allosteric regulatory site involving two subunits jointly binding two ATP, which constantly suppresses the activity of SeXPK until the ATP level drops. This magnesium-independent ATP allosteric site is present in many species across all three domains of life, where it may also play important regulatory functions.

The challenge of engineering Rubisco for improving photosynthesis.

Photosynthesis uses the energy of sunlight to convert water and atmospheric CO2 into sugars, providing food and oxygen for life. The fixation of atmospheric CO2 in this crucial biological process is mediated by the enzyme Rubisco. The inefficiencies of Rubisco have inspired researchers for decades to explore ways to improve its function with the goal of increasing crop yields [1-4], and more recently to combat global warming [5]. In this graphical review we highlight the challenges involved in engineering plant Rubisco, with a focus on the extensive chaperone requirement for its biogenesis. We discuss strategies for engineering the catalytic properties of Rubisco and for sequestering the enzyme in membraneless compartments to increase CO2 fixation.

An Approach to Understanding the Hydration of Cement-Based Composites Reinforced with Untreated Natural Fibers

The use of untreated natural fibers to reinforce cementitious composites improves their environmental friendliness, resulting in a more sustainable material. Moreover, the influence of the untreated natural fibers on the hydration process of Portland cement composites presents some uncertainties. According to the literature, the most usual tests to analyze the degree of hydration of cement composites are the differential thermal and thermogravimetric analyses (TGA/dTGA). Several authors propose to analyze data methods to establish the degree of hydration of cement composites. This paper presents the TGA/dTGA test carried out on mortar samples with and without fibers at age 2, 3, 7, 14, and 28 days. The degree of hydration was calculated according to Bhatty’s method. To characterize the raw materials, the quantitative chemical was determined using scanning electron microscopy and energy dispersive X-ray spectroscopy (SEM–EDX). The main findings of this study were that the presence of untreated natural hemp fibers in the OPC composites increased the hydration degree by 9%. The presence of fibers affected the formation of several components. Thus, their presence increased the formation of monosulphate, reduced portlandite, did not affect ettringite, and increased the formation of calcite, thereby improving the sustainable footprint due to the increased CO2 fixation.

Bacillus H47 triggers Olea europaea metabolism activating DOXP and shikimate pathways simultaneously and modifying leaf extracts' antihypertensive activity.

Improvement of plant adaptation by beneficial bacteria (PGPB) may be achieved by triggering multiple pathways to overcome the environmental stress on plant’s growth cycle, activating plant’s metabolism. The present work reports the differential ability of three Bacillus strains to trigger olive tree metabolism, among which, only H47 was outstanding increasing iridoid and flavonol concentration. One-year old olive seedlings grown open air, under harsh conditions of water shortage in saline soils, were root-inoculated with three Bacillus PGPB strains throughout a 12-month period after which, photosynthesis was determined; photosynthetic pigments and bioactive secondary metabolites (iridoids and flavonols) were analyzed, and a study of gene expression of both pathways involved was undertaken to unravel molecular targets involved in the activation. All three strains increased plant fitness based on photosynthetic values, increasing energy dissipation capacity to lower oxidative stress; only H47 increased CO2 fixation and transpiration. Bacillus H47 was found to trigger synthases in the DOXP pathway (up to 5-fold in DOXP-synthase, 3.5-fold in Iridoid synthase, and 2-fold in secologanin synthase) associated to a concomitant increase in iridoids (up to 5-fold in oleuropein and 2-fold in its precursor secologanin). However, despite the 2-fold increases detected in the two predominant flavonols, gene expression was not enhanced, suggesting involvement of a pulse activation model proposed for innate immunity. Furthermore, the activity of leaf extracts to inhibit Angiotensin Converting Enzyme was evaluated, to explore further uses of plant debris with higher added value. Despite the increases in iridoids, leaf extracts from H47 did not increase ACE inhibition, and still, increased antihypertensive potential in oil obtained with this strain is to be explored, as leaves are the source for these metabolites which further translocate to fruits. In summary, Bacillus H47 is an effective strain to increase plant adaptation to dry and saline environments, activates photosynthesis and secondary metabolism in olive tree.

Renewal of nanofibers in Chlorella fusca microalgae cultivation to increase CO2 fixation

This study explored strategies to increase the CO2 fixation ability of microalgae by renewing polymeric nanofibers in cultures of Chlorella fusca LEB 111. Nanofibers composed of 10% (w v−1) polyacrylonitrile (PAN)/dimethylformamide (DMF) containing 4% (w v−1) iron oxide nanoparticles (NPsFe2O3) were added to photobioreactors. The nanomaterial was renewed in the test cultures as follows: renewal only on day 7; renewal only on day 15; or renewal on both days 7 and 15 (i.e., double renewal). The highest biomass concentration (2.53 g L−1) and CO2 biofixation rate (141.5 mg L−1 d−1) were obtained by cultivating with double renewal, resulting in values 21.6% and 23% higher, respectively, than those obtained by cultivation without renewal. The application of nanofiber renewal in the cultivation of C. fusca LEB 111 shows the potential to increase CO2 biofixation, which may contribute to reducing the atmospheric concentrations of this main greenhouse gas intensifier.

Capability of carbon fixation in bicarbonate-based and carbon dioxide-based systems by Scenedesmus acuminatus TH04

Carbon sequestration using microalgae can be performed from bicarbonate (a product of carbon dioxide capture) and/or carbon dioxide. To implement either process successfully, the capability of microalgal strains is the most important criteria. In this work, a thermophilic microalgal Scenedesmus acuminatus TH04 was investigated to optimize its growth and carbon fixation in bicarbonate-based and carbon dioxide-based reactor systems. Culture variables including temperature, light intensity, pH, and aeration rate, concentration of NaHCO3, Na2CO3, CO2 and triethylenetetramine (TETA) were examined. The S. acuminatus TH04 strain achieved the highest biomass production and bicarbonate fixation efficiency of 1.7 g/L and 100 %, respectively, with 4.2 g NaHCO3/L. In a single photobioreactor (PBR), the maximum biomass production and CO2 fixation efficiency of 4.7 g/L and 9.8 % were achieved under the optimal conditions of CO2 of 5% (v/v), aeration rate of 0.1 vvm, TETA of 5 mM and Na2CO3 of 50 mM. Remarkably, a sequence of ten PBRs increased CO2 fixation efficiency by S. acuminatus TH04 to 93.9 %. The S. acuminatus TH04 strain demonstrated a highly potential capability in simultaneous production of biomass and carbon fixation from HCO3− and CO2 as carbon sources.

Improving light distribution and light/dark cycle of 900 L tangential spiral−flow column photobioreactors to promote CO2 fixation with Arthrospira sp. cells

The light distribution and light/dark cycle were improved in 900 L tangential spiral-flow column photobioreactors (TSCP) to promote CO2 fixation with Arthrospira sp. cells. Solar irradiation model was employed in CFD simulation to investigate light distribution and light/dark cycle in flow field composed of culture medium, CO2 bubbles and Arthrospira sp. cells under actual sunlight irradiation considering geolocation and time. An accurate way to divide light/dark zone based on saturate light intensity and light intensity field was adopted for the first time. When Arthrospira sp. cell concentration increased from 0.1 to 0.9 g/L, light/dark cycle frequency of cells firstly increased from 0.650 Hz to 0.868 Hz and then decreased to 0.117 Hz. Intracellular chlorophyll a content and carotenoids content of Arthrospira sp. cells in TSCP were 6% and 41% higher than those in conventional bubble column photobioreactor. This promoted cellular photosynthesis and stress resistance, which contributed to increase CO2 fixation rate of Arthrospira sp. cells by 59%. When CO2 aeration rate, CO2 volume concentration, and circulating pump power were 0.210 L/min, 15%, and 30 W, chlorophyll a content, helix pitch, and CO2 fixation rate of Arthrospira sp. cells all reached peak values of 8.769 mg/L, 78.26 μm and 0.358 g/L/d, respectively.

Fed-batch cultivation with CO2 and monoethanolamine: Influence on Chlorella fusca LEB 111 cultivation, carbon biofixation and biomolecules production

The aim of this study was to evaluate the interaction between the periodic addition of monoethanolamine (MEA) and CO2 during the cultivation of Chlorella fusca LEB 111. For this purpose, MEA has been added in abiotic assays, followed by fed-batch cultures with that green alga and the absorbent. BG-11 medium shown a higher potential of CO2 absorption with MEA addition, and the bicarbonate was the chemical species of carbon prevailing in the chemical equilibrium. The periodic addition of MEA did not reduce the kinetics of growth, promoted a higher accumulation of DIC (81.4 mg L−1) in the medium and protein (44.0% w w−1) and lipid (30.8% w w−1) concentrations in the biomass of C. fusca LEB 111. Therefore, it was demonstrated that fed-batch culture with MEA increased CO2 fixation and the biomolecule synthesis as proteins and lipids.

Gas exchange and photosynthetic light response curves in nematode-infected tomato plants treated with Thuya occidentalis

The use of homeopathic formulations on plants may cause changes in plant metabolism and result in disease control by inducing resistance. This work aimed to study the physiological responses of tomato plants infected with nematode (Meloidogyne incognita) and treated with highly diluted preparations of Thuya occidentalis, a homeopathic remedy. The different treatments included T. occidentalis 6CH, 24CH, and 50CH (Centesimal Hahnemanian), a water control (with nematode and without treatment), and an absolute control (no treatment and no nematode). The assay was conducted in a greenhouse using a randomized block design with four replications. Gas exchange parameters were measured at specific times before and after nematode inoculation. Photosynthetic rate, stomatal conductance, transpiration, leaf internal CO2 concentration, and leaf temperature were measured at various photosynthetic photon flux densities. No significant difference was found in the different gas exchange specific measure parameters between the treatments. Untreated infected plants showed an increase in net photosynthesis and in the carboxylation capacity, as shown by the light response curve. Treatment with T. occidentalis 24CH inhibited the increase in CO2 fixation in tomato plants inoculated with M. incognita, leading to a similar behavior as healthy plants, independently of the photon density.

Modeling and analysis of flux distribution and bioproduct formation in Synechocystis sp. PCC 6803 using a new genome-scale metabolic reconstruction

Cyanobacteria are prokaryotes capable of performing oxygenic photosynthesis. This makes them an attractive candidate for genetic engineering to produce commercially important chemicals. However, optimally harnessing this potential requires an understanding of metabolic regulation in cyanobacteria under photoautotrophic conditions. Here we present an updated genome-scale metabolic network reconstruction (iSynCJ816) of Synechocystis sp. PCC 6803. This updated model, containing 816 genes and 1045 reactions, builds upon previously published models. New features include an unconstrained photo-respiratory reaction mechanism and a mechanism to account for changes in energy absorption from light at different wavelengths. We used Flux Balance Analysis (FBA) to calculate the flux distribution within iSynCJ816 and compared in silico predictions with values obtained by previous in vivo metabolic flux analyses in Synechocystis sp. PCC 6803. A qualitative growth comparison of 167 gene-deletion mutants with experimental studies resulted in an accuracy rate between 70 and 79%. We used the model to estimate both the maximum theoretical yield of each metabolite and the feasibility of engineering Synechocystis to increase CO2 fixation. We found that it is theoretically possible to increase CO2 fixation by up to 35% from wild-type levels. We also carried out a dynamic flux balance analysis of fluxes throughout a light-dark cycle and obtained results that qualitatively matched experimental observations.

Phytoplankton Plastid Proteomics: Cracking Open Diatoms to Understand Plastid Biochemistry Under Iron Limitation

Diatoms, such as Thalassiosira pseudonana, are important oceanic primary producers, as they sequester carbon dioxide (CO₂) out of the atmosphere, die, and precipitate to the ocean floor. In many areas of the world’s oceans, phytoplankton, such as diatoms, are limited in growth by the availability of iron (Fe). Fe is an essential nutrient for phytoplankton, as it is central in the electron transport chain component of photosynthesis. Through this study, we examined if Fe-limitation makes a significant difference in the proteins expressed within the chloroplast, the power source for diatoms. Here, we utilized a new plastid isolation technique specific to diatoms and completed 14 mass spectrometry experiments to determine how many proteins transit the plastid membrane, if there are any differences in the expressed proteomes within the plastid grown under Fe-replete and Fe-limited conditions, and what those differences are. Over 900 unique proteins were identified from the isolated plastids, and cluster analyses of the data verified that statistical differences are present between the Fe-replete and Fe-limited growth conditions. Furthermore, our plastid proteome is in agreement with many of the recognized proteins previously discovered in land plant plastids, suggesting the isolation method followed by proteomic mass spectrometry is valid and sensitive. Through the isolation and analysis of plastid proteins, as shown here, scientists can now better identify which nutrients and/or trace metals directly affect diatom photosynthetic capacity so they can design better experiments to increase CO₂ fixation rates.

Overexpression of a Calvin cycle enzyme SBPase improves tolerance to chilling-induced oxidative stress in tomato plants

Sedoheptulose-1,7-bisphosphatase (SBPase) is a critical enzyme involved in photosynthetic carbon fixation in the Calvin cycle. Here, we report the effects of SBPase overexpression on the tolerance to chilling-induced oxidative stress in transgenic tomato (Solanum lycopersicum) plants. In transgenic plants overexpressing SBPase, CO2 fixation and carbohydrate accumulation were increased in comparison with equivalent wild-type plants. SBPase was found to be susceptible to oxidative stress and the activity was substantially inhibited by reactive oxygen species both in vivo and in vitro. In response to chilling stress, production of H2O2 was increased in parallel with the reduction in SBPase activity in tomato plants, however, transgenic plants maintained significantly higher SBPase activity than wild-type plants did. Under chilling stress, compared with wild-type plants, transgenic plants were found to have increased CO2 fixation and reduced electrolyte leakage. The overall tolerance could be ascribed to the enhancement of photosynthetic carbon fixation, the reductions in the level of H2O2 and the increased accumulation of carbohydrate in transgenic plants. Collectively, our data suggest that high level of SBPase activity gives an advantage to photosynthetic carbon fixation and tolerance to chilling-induced oxidative stress in tomato plants. This work presents a case study that an individual enzyme in the Calvin cycle may be a useful target for genetically engineering stress tolerance in horticultural crops.

Development of a stable genetic system for Chlorella vulgaris—A promising green alga for CO2 biomitigation

Chlorella vulgaris has been proposed as a promising green alga for CO2 biomitigation due to its attractive traits of high growth rate, excellent CO2 fixation ability and broad industrial applications. Nevertheless, the genetic engineering of this alga is still in its infancy. In the present study, a stable and efficient transformation system was established for C. vulgaris CBS 15-2075, a robust strain with great potential for CO2 biomitigation. The antibiotic sensitivity spectrum of this organism was evaluated, and the nptII gene was selected as a dominant selectable marker for genetic transformation. The selectable marker, together with an enhanced green fluorescent protein (EGFP) gene, was delivered into C. vulgaris protoplasts by using a PEG-mediated method, giving a transformation efficiency of 356±30cfu per μg vector DNA. Molecular characterization and live-cell fluorescence microscopy demonstrated that the EGFP gene was stably integrated into C. vulgaris genome and expressed in the cytoplasm of transformed cells. Taken together, we for the first time established a stable and useful genetic toolkit for the industrially important microalga C. vulgaris, which will facilitate to a great extent the future rational genetic manipulation for strain improvement to increase CO2 fixation capacity and may as well provide valuable insights into other Chlorella species. Besides, the success in EGFP expression and live-cell fluorescence detection in C. vulgaris will be useful in molecular and cell biology for protein subcellular localization.

The effect of nutrients on carbon and nitrogen fixation by the UCYN-A-haptophyte symbiosis.

Symbiotic relationships between phytoplankton and N2-fixing microorganisms play a crucial role in marine ecosystems. The abundant and widespread unicellular cyanobacteria group A (UCYN-A) has recently been found to live symbiotically with a haptophyte. Here, we investigated the effect of nitrogen (N), phosphorus (P), iron (Fe) and Saharan dust additions on nitrogen (N2) fixation and primary production by the UCYN-A-haptophyte association in the subtropical eastern North Atlantic Ocean using nifH expression analysis and stable isotope incubations combined with single-cell measurements. N2 fixation by UCYN-A was stimulated by the addition of Fe and Saharan dust, although this was not reflected in the nifH expression. CO2 fixation by the haptophyte was stimulated by the addition of ammonium nitrate as well as Fe and Saharan dust. Intriguingly, the single-cell analysis using nanometer scale secondary ion mass spectrometry indicates that the increased CO2 fixation by the haptophyte in treatments without added fixed N is likely an indirect result of the positive effect of Fe and/or P on UCYN-A N2 fixation and the transfer of N2-derived N to the haptophyte. Our results reveal a direct linkage between the marine carbon and nitrogen cycles that is fuelled by the atmospheric deposition of dust. The comparison of single-cell rates suggests a tight coupling of nitrogen and carbon transfer that stays balanced even under changing nutrient regimes. However, it appears that the transfer of carbon from the haptophyte to UCYN-A requires a transfer of nitrogen from UCYN-A. This tight coupling indicates an obligate symbiosis of this globally important diazotrophic association.

Nitrogen fixation, hydrogen production and N2O emissions

Flynn, B., Scott, N. and Dong, Z. 2014. Nitrogen fixation, hydrogen production and N2O emissions. Can. J. Plant Sci. 94: 1037–1041. H2 is a by-product of the nitrogenase reaction. Exposure to H2 is linked to increased N2O production, increased CO2 fixation and plant growth promotion in soil. The effects of H2 exposure on soil were observed using controlled H2 gas treatments and field trials with legumes. In field trials, increased N2O production was observed in soil adjacent to legume nodules and inoculation of H2-oxidizing isolates led to increased N2O emissions in corn fields. Many H2-oxidizing isolates tested positive for key denitrification genes, indicating a connection between H2 uptake and N2O emissions. H2 treatment significantly increased copy number of the nitrite reductase (nirK) gene suggesting increased denitrification as the source of N2O. There was also a significant increase in copy number and expression of the RubisCO (cbbL) gene in soil. H2-oxidizing bacterial isolates (JM63 and JM162a) were found to promote plant growth, increasing tiller number and yield in spring wheat and barley. Combined results of T-RFLP and 16S rDNA clone libraries analysis revealed bacterial community structure changes in response to H2 treatment, primarily with increases to the Gammaproteobacteria and Betaproteobacteria groups. The results of these studies help provide a better understanding of the soil bacterial community's responses to H2 exposure and may lead to the development of a commercially viable plant growth promoting inoculant.

Exclusion of solar UV components improves growth and performance of Amaranthus tricolor varieties

A field experiment was conducted to study the influence of exclusion of solar UV components on the growth responses, gas exchange rates, chlorophyll fluorescence, pigmentation, carboxylation enzymes and foliage yield of four Amaranthus tricolor varieties; Arka arunima, Arka suguna, Pusa lalchaulai and Pusa kiran. The plants were grown in specially designed UV exclusion chambers, wrapped with filters that excluded UV-B (<315nm), UV-A/B (<400nm) or transmitted ambient UV or lacked filters. Plants grown under ambient UV radiation (280–400nm) were compared with those grown without UV-B or UV-A/B. The results indicated increased plant height, leaf weight ratio, specific leaf weight and foliage yield in all the four varieties of A. tricolor grown without UV-B and UV-A/B compared with those grown under ambient UV. Exclusion of UV significantly increased the total chlorophyll while Chl a/b ratio decreased. The efficiency of PS II (Fv/Fm), rate of photosynthesis and stomatal conductance significantly enhanced along with a remarkable increase in Carbonic anhydrase, PEP carboxylase and total soluble proteins. Thus UV excluded plants have higher reducing power and increased CO2 fixation and decreased UV-B absorbing compounds, channelling the additional fixation of carbon towards the improvement of yield in A. tricolor varieties. The response for UV exclusion was higher in variety Arka arunima and Arka suguna compared with Pusa kiran and Pusa lalchaulai. UV sensitivity of the A. tricolor varieties was in the following sequence – Pusa lalchaulai>Pusa kiran>Arka suguna>Arka arunima. Thus Arka arunima and Arka suguna were most sensitive and Pusa lalchaulai and Pusa kiran the least sensitive varieties to ambient level of UV. The least sensitive varieties may be used in the development of UV-B tolerance mechanisms and also developing new varieties tolerant to higher levels of UV-B in future attempts in plant breeding programmes.

Metabolic engineering of Propionibacterium freudenreichii: effect of expressing phosphoenolpyruvate carboxylase on propionic acid production.

Propionic acid is currently produced mainly via petrochemicals, but there is increasing interest in its fermentative production from renewable biomass. However, the current propionic acid fermentation process suffers from low product yield and productivity. In this work, the gene encoding phosphoenolpyruvate carboxylase (PPC) was cloned from Escherichia coli and expressed in Propionibacterium freudenreichii. PPC catalyzes the conversion of phosphoenolpyruvate to oxaloacetate with the fixation of one CO2. Its expression in P. freudenreichii showed profound effects on propionic acid fermentation. Compared to the wild type, the mutant expressing the ppc gene grew significantly faster, consumed more glycerol, and produced propionate to a higher final titer at a faster rate. The mutant also produced significantly more propionate from glucose under elevated CO2 partial pressure. These effects could be attributed to increased CO2 fixation and resulting changes in the flux distributions in the dicarboxylic acid pathway.

Nitrogen fixation, hydrogen production and N2O emissions

Flynn, B., Scott, N. and Dong, Z. 2014. Nitrogen fixation, hydrogen production and N2O emissions. Can. J. Plant Sci. 94: 1037-1041. H2 is a by-product of the nitrogenase reaction. Exposure to H2 is linked to increased N2O production, increased CO2 fixation and plant growth promotion in soil. The effects of H2 exposure on soil were observed using controlled H2 gas treatments and field trials with legumes. In field trials, increased N2O production was observed in soil adjacent to legume nodules and inoculation of H2-oxidizing isolates led to increased N2O emissions in corn fields. Many H2-oxidizing isolates tested positive for key denitrification genes, indicating a connection between H2 uptake and N2O emissions. H2 treatment significantly increased copy number of the nitrite reductase (nirK) gene suggesting increased denitrification as the source of N2O. There was also a significant increase in copy number and expression of the RubisCO (cbbL) gene in soil. H2-oxidizing bacterial isolates (JM63 and JM162a) ...