Release Of Hydrogen Cyanide Research Articles

Mandelonitrile produced by commensal bacteria protects the Colorado potato beetle against predation

Larvae and adults of the Colorado potato beetle (Leptinotarsa decemlineata), a major pest of potato crops, display conspicuous coloration to advertise their toxicity to predators. However, the identity of the toxic compounds remains unclear. Here, we show that larvae and adults release toxic hydrogen cyanide (HCN) from the degradation of mandelonitrile and other cyano-compounds, which are produced by commensal bacteria. We isolate the bacterium Proteus vulgaris Ld01 from the insect’s gut, and show that it produces HCN and a mandelonitrile-producing cyanoglucoside, amygdalin. Knockout of a gene (hcnB) encoding putative hydrogen cyanide synthase impairs HCN production in P. vulgaris Ld01. Antibiotic treatment of larvae, to eliminate their commensal bacteria, leads to a substantial reduction of HCN emission in larvae and adults. HCN release by bacteria-deprived beetles can be restored by addition of mandelonitrile or by re-infection with P. vulgaris Ld01 (but not with its ∆hcnB1 or ∆hcnB2 mutants). Finally, we use dual-choice experiments to show that domestic chicks prefer to eat bacteria-deprived larvae over control larvae, larvae re-colonized with P. vulgaris Ld01, or mandelonitrile-injected larvae. Our work highlights the role of the beetle’s intestinal bacteria in the production of the cyanoglucoside amygdalin and its derived metabolites, including mandelonitrile and HCN, which protect the insect from predation.

Tissue-specific expression of HCN and its metabolic precursors in Trifolium repens

Cyanogenesis, the release of hydrogen cyanide (HCN), is governed by two loci, Ac and Li, in white clover ( Trifolium repens L.). Ac encodes a three gene region that regulates cyanogenic glucoside production, while Li encodes linamarase that hydrolyzes cyanogenic glucosides to produce HCN. We examined Ac and Li expression in leaf, floral, and root tissues of cyanogenic and acyanogenic plants, complemented by gene expression analysis, to understand tissue-specific expression of HCN metabolism. Ac metabolic expression was present in leaves, absent in roots, and rare in flowers. Li metabolic expression in leaves perfectly correlated with that in flowers but was present in roots regardless of linamarase presence in leaves, suggesting spatial enzyme expression variation due to a homologous gene copy. HCN expression in flowers was infrequently expressed at low concentrations in plants with functional Ac and Li. Gene expression analysis confirmed some tissue-specific expression patterns and also suggested more complex molecular regulation than previously described. This study provides the first detailed examination of cyanogenesis tissue specificity in white clover, a plant of agriculture significance. The varied expression of cyanogenic secondary metabolites across tissues indicates the need for research into how white clover regulates its defenses to conserve resources.

Evaluation of exposure to cyanogenic glycosides and potential hydrogen cyanide release in commercially available foods among the Korean population

The release of hydrogen cyanide (HCN) after food ingestion can pose a serious health risk to consumers. This study aimed to simultaneously quantify four cyanogenic glycosides (lotaustralin, prunasin, taxiphyllin, and dhurrin) using liquid chromatography–tandem mass spectrometry. The analysis scope extended beyond agricultural products to various consumer foods to estimate dietary exposure to cyanogenic glycosides and assess its risk levels. The major exposure sources are cassava chips (lotaustralin), apples (seeds) (prunasin and dhurrin), and Prunus mume axis (taxiphyllin). In addition to quantifying specific cyanogenic glycosides, this study proposed the development of a preliminary risk assessment framework based on the dietary exposure assessment and the calculation of theoretical levels of HCN derived from cyanogenic glycoside concentrations. In the absence of established guidelines for the permissible intake of foods containing cyanogenic glycosides, this study provides initial guidance for assessing the risks associated with a range of commonly consumed foods.

First comprehensive higher level phylogeny of Zygaenidae (Lepidoptera) including estimated ages of the major lineages and a review of known zygaenid fossils

AbstractZygaenidae, also known as burnet, forester, smoky, or leaf skeletonizer moths, are a family of mainly diurnal moths well known for their aposematic colouration and the ability to release hydrogen cyanide as a defence mechanism. So far, few attempts have been made to understand the evolutionary history of the global zygaenid fauna. Here, we inferred the most comprehensive molecular phylogeny for Zygaenidae to date and estimated the lineage timing‐of‐divergence with a Bayesian approach. Building on earlier work, we significantly increased the taxon and gene sampling for the family, which here included data from 30 gene fragments, recovered from public databases or newly sequenced, for almost 30% of the species representing 92 genera (49%) and all five subfamilies. We recovered strong support for the monophyly of Zygaenidae, Chalcosiinae, and Zygaeninae. Procridinae were recovered as monophyletic with low support, whereas the monophyly of Callizygaeninae remains untested as we sampled only one of the two genera. In the core dataset, we recovered Procridinae as sister to Callizygaeninae + Chalcosiinae. This large clade is the sister lineage to Zygaeninae. The position of Inouelinae could not be resolved. The lineage leading to the extant Zygaenidae appears to have diverged in Late Cretaceous (ca. 86 Ma), while the divergence among the subfamilies occurred several million years before the Cretaceous–Paleogene mass extinction event (ca. 66 Ma). Additionally, we provide a review of known fossil Zygaenidae as Appendix S1. Our results form a strong basis for future studies of zygaenid biosystematics, including their ecology, evolution, and behaviour.

Amelioration of growth of maize (Zea mays L.) seedling using plant growth promoting bacteria

This research was aimed to screen plant growth promoting bacteria (PGPB) from soil and study its effect on maize plant growth. PGPB were isolated from Saurashtra coastal region soil and cultured in Nitrogen fixing Ashby’s medium to find potent of PGPBs, we conducted a thorough screening process, assessed their abilities in phosphate and zinc solubilization, siderophore production, hydrogen cyanide (HCN) release and the antifungal activity was performed against Fusarium oxysporum, a pathogenic fungus. These tests helped us identify bacteria with plant growth-promoting characteristics for plants. Bacterial isolates which provided better results were sequenced and sequences were submitted to NCBI. Bacterial isolates selected for application on maize in primary screening showed most treated seeds increased the seedling vigor of maize. In the latter stages of screening where bacterial consortia were developed from primary and secondary screening. In 30 days, the experiment in maize plant height, number of leaves, chlorophyll content and anatomy was analysed. All the bacterium consortiums displayed an increase in height (24.75%), number of leaves (47.77%) and total chlorophyll content (23.59%) as compared to the control maize plant. Additionally, microscopic examination of the treated plants showed improved growth, especially in the increased starch grain content in the leaves, stems and roots. Out of the eleven PGPB consortia, 3 specific PGPB consortia in this study have significantly substantiated the growth of maize plants as evidenced by the comprehensive analysis of anatomical features.

Biodegradation of Mandelonitrile Using Bacillus sp.: A Potential Approach for Environmental Remediation

Mandelonitrile, a cyanogenic compound, poses environmental risks due to its toxicity and potential to release hydrogen cyanide. The potential technique for removing mandelonitrile from polluted environments is biodegradation. In this study, we investigated the isolation and identification of bacterial strains from soil samples for the biodegradation of mandelonitrile. The selective enrichment culture technique was used for isolation, using mandelonitrile as the sole carbon and nitrogen source. The isolated strain was identified and named Bacillus sp based on morphological studies and biochemical analysis. MN1. Further degradation studies demonstrated the efficiency of the isolated strain in degrading mandelonitrile under optimum conditions. Bacillus sp. MN1 can degrade 0.5% v/v mandelonitrile completely into mandelic acid and ammonia under optimum conditions of 30 °C and pH 7.5 within five days of incubation. The enzyme assay and HPLC analysis show that mandelonitrile degradation by Bacillus sp. MN1 follows the Nitralase pathway, mandelic acid, and ammonia. This research study reveals the potential application of Bacillus sp. in the bioremediation of mandelonitrile-contaminated sites, highlighting the importance of microbial biodegradation in environmental management strategies.

Preference of dhurrin‐free sorghum by ewes

AbstractSorghum [Sorghum bicolor (L.) Moench] is a resilient forage crop due to its drought tolerance and adaptation to low‐N environments. Sorghum produces a cyanogenic glucoside called dhurrin. The breakdown of dhurrin leads to the release of hydrogen cyanide (HCN), which can cause cyanide toxicity in livestock. Dhurrin‐free sorghum lines have been developed through chemical mutagenesis by mutagenizing the gene for the first enzyme, CYP79A1, in the biosynthetic pathway. The CYP79A1 mutation was bred into sorghum lines to create a dhurrin‐free experimental hybrid. Grazing preference of ewes was assessed when allocated to the dhurrin‐free hybrid and three commercial hybrids. Near isogenic lines (NIL), contrasting in dhurrin production, were also compared for grazing preference. Forage mass was measured before and after grazing to determine the amount of forage mass grazed by the ewes. An unmanned aerial vehicle (UAV) was flown to quantify changes in normalized difference vegetation index (NDVI) over time for each hybrid. The nutritive values of the hybrids were also evaluated. The dhurrin‐free hybrid was grazed 19% and 13% more (p‐value ≤ 0.05) in comparison to the commercial hybrids for the second and third grazing cycles in 2019 and 2020. The NIL Tx623 bmr6 CYP79A1, was grazed 20% more than Tx623 bmr6 in two grazing cycles in 2020. Remote sensing data showed a similar pattern with the dhurrin‐free hybrid having the largest decline in NDVI for three grazing cycles in 2019. Nutritive value of the dhurrin‐free hybrid was similar to the two hybrids with the brown midrib (bmr) trait.

Harnessing the anti-cancer potential of linamarin: A computational study on design and hydrolysis mechanisms of its derivatives

Cassava extracts containing cyanogenic compounds demonstrate anticancer properties. The cyanogenic glucoside linamarin found abundantly in cassava can release hydrogen cyanide (HCN) upon hydrolysis, a potent cytotoxin. However, linamarin's hydrolysis mechanism by human enzymes is poorly delineated and constitutes a bottleneck for therapeutic development. This study aimed to investigate linamarin's hydrolysis mechanism by human β-glucosidase and identify structural derivatives with enhanced hydrolytic potential using density functional theory calculations. Results revealed α-anomeric derivatives as promising, with leaving group ability and steric bulk strongly governing hydrolysability. We identified several linamarin analogs with predicted rapid hydrolysis kinetics that may enable swift cytotoxic HCN release against cancer cells. This investigation enriches understanding of cyanogenic glycoside reactivity to facilitate their development as targeted antineoplastic agents. The identified derivatives set the groundwork for experimental evaluation of enhanced linamarin-inspired compounds as innovative cancer therapeutics.

Experimental investigation and numerical modelling of CO and HCN release during the combustion of flexible polyurethane foam

The combustion of flexible polyurethane foam (FPUF) in fires releases relatively large amounts of carbon monoxide (CO) and hydrogen cyanide (HCN), threatening the lives of those exposed thereto. It is therefore necessary to use computational fluid dynamics method to explore the distribution of CO and HCN in a fire involving the combustion of FPUF. However, the release of CO and HCN varies significantly with the combustion conditions, which makes the numerical modelling challenging. In the present research, the yields of CO and HCN during the combustion of a non-flame-retardant FPUF were determined using a steady-state tube furnace (SSTF) facility, considering the effect of the ventilation condition. Meanwhile, a pyrolysis and combustion model of FPUF was constructed based on the fire dynamics simulator (FDS), and a four-step chemistry model was proposed to deal with the reactions in the gas phase. The experimental results showed that the yields of CO and HCN both increased with the limitation of ventilation, which was more pronounced in under-ventilated conditions. Meanwhile, the numerical simulation applying the four-step chemistry model predicted the variations in the yields of CO and HCN under ventilated conditions. In addition, when the appropriate kinetic parameters were applied to the reactions governing the oxidation of CO and HCN, the comparative results indicated that the global average deviations between the predicted and experimental yields could be reduced to 13.61 % (CO) and 8.19 % (HCN), respectively.

Antineoplastic effects of cassava-cyanide extract on human glioblastoma (LN229) cells

Several natural compounds reduce tumour cell growth and metastasis by inducing programmed cell death. Cassava (Manihot esculenta Crantz) contains cyanogenic glycosides such as, linamarin and lotaustralin, can be enzymatically cleaved by linamarase to release hydrogen cyanide (HCN), which can have therapeutic benefits against hypertension, asthma, and cancer. We have developed a technology for isolating bio-active principles from cassava leaves.The present study is designed to analyze the cytotoxic effect of cassava cyanide extract (CCE) against human glioblastoma cells (LN229). The treatment of CCE demonstrated a dose dependent toxicity on glioblastoma cells. At higher concentration tested, the CCE (400 μg/mL) was found to be cytotoxic, reducing the cell viability to 14.07 ± 2.15% by negatively influencing the mitochondrial activity, and lysosomal and cytoskeletal integrity. Coomassie's brilliant blue staining confirmed cells' morphological aberration after 24 h of treatment with CCE. Moreover, DCFH-DA assay and Griess reagent showed an increase in ROS but a decrease in RNS production at a concentration of CCE. Flow cytometry analysis revealed that CCE interfered with G0/G1, S, and G2/M stages of the cell cycle of glioblastoma, and Annexin/PI staining indicated a dose-dependent increase in cell death, confirming the toxic nature of CCE on LN229 cells. These findings suggest that cassava cyanide extract has potential as an antineoplastic agent against glioblastoma cells, which is an aggressive and difficult-to-treat type of brain cancer. However, it is important to note that the study was conducted in vitro, and further research is necessary to assess the safety and efficacy of CCE in vivo. Additionally, it is essential to establish the optimal dose and potential side effects before considering its use as a therapeutic agent.

Cyanogenesis, a Plant Defence Strategy against Herbivores.

Plants and phytophagous arthropods have coevolved in a long battle for survival. Plants respond to phytophagous feeders by producing a battery of antiherbivore chemical defences, while herbivores try to adapt to their hosts by attenuating the toxic effect of the defence compounds. Cyanogenic glucosides are a widespread group of defence chemicals that come from cyanogenic plants. Among the non-cyanogenic ones, the Brassicaceae family has evolved an alternative cyanogenic pathway to produce cyanohydrin as a way to expand defences. When a plant tissue is disrupted by an herbivore attack, cyanogenic substrates are brought into contact with degrading enzymes that cause the release of toxic hydrogen cyanide and derived carbonyl compounds. In this review, we focus our attention on the plant metabolic pathways linked to cyanogenesis to generate cyanide. It also highlights the role of cyanogenesis as a key defence mechanism of plants to fight against herbivore arthropods, and we discuss the potential of cyanogenesis-derived molecules as alternative strategies for pest control.

HCN-induced embryo arrest: passion fruit as an ecological trap for fruit flies.

Fruit flies are important economic pests of fruits, vegetables, and nuts all over the world. In this study, a permanent ecological trap, which was created by the ovicidal effect of phytogenic hydrogen cyanide (HCN) liberated from passion fruits due to oviposition by fruit flies and can be used in the pest management, were determined. Observation of fruit fly eggs in Passiflora within the passion fruit cultivation region in southern China, from Aug 2019 to Oct 2020 showed that the exotic Passiflora attracted the native fruit flies to oviposit, but the eggs could not hatch. Using classical staging to categorize embryonic development and fumigation assays, we show that oviposition by fruit fly on passion fruits, release HCN from the cyanogenic mesocarp. Exposure of the eggs to HCN causes arrest of embryonic development and finally the death of eggs. Our results reveal that the life cycle of fruit fly in Passiflora is interrupted at the egg stage. Consequently, we predict that this ecological trap may be permanent. Extensive cultivation of the Passiflora vine as a dead-end trap crop may be an effective avenue to reduce populations of fruit fly pests. © 2023 Society of Chemical Industry.

Dhurrin stability and hydrogen cyanide release in dried sorghum samples

Dhurrin stability and hydrogen cyanide release in dried sorghum samples

Pengaruh Penambahan Zat Kapur dan Lama Perendaman Terhadap Kadar Sianida pada Singkong (Manihot Esculanta Crantz)

Cassava (Manihot esculenta Crantz) or also known as cassava has a high carbohydrate content and has become the third staple food source after maize and rice. Cassava also contains linamarin and lotaustralin poisons which belong to the cyanogenic glycoside group. Cyanogenetic glycosides are compounds found in plant foodstuffs and are potentially very toxic because they decompose and release hydrogen cyanide. This study aims to determine the cyanide acid content in cassava before being soaked in lime solution and to determine the cyanide acid content in cassava after soaking in lime solution with concentrations of 0.25%, 0.50%, 0.75%, 1% and 2% for 2 hours, 4 hours, and 6 hours. Using argentometry method. Soaking with lime concentrations of 0.25%, 0.50%, 0.75%, 1% and 2% does not affect the cyanide acid levels, which affects the cyanide levels, namely the long soaking time which is 2 hours can reduce the cyanide acid levels. up to 50%, soaking time of 4 hours reduced 62.5% and soaking time of 6 hours reduced up to 75% the level of cyanide acid contained in cassava. Based on the research, it can be concluded that the soaking time greatly affects the decrease in cyanide levels found in cassava. Suggestions should try measurements using instrumentation methods.

Effect of aryl phosphates on toxicity of combustion gases of flame‐retardant polycarbonate/acrylonitrile butadiene styrene blends according to EN 45545 railway standard

SummaryIn this study, the toxicity of combustion gases of polycarbonate/acrylonitrile butadiene styrene (ABS) blends that include aryl phosphates as flame‐retardants (FRs) was analyzed according to the European railway standard EN 45545‐2 (NBS chamber + FTIR). FRs have a significant influence on the evolution of the toxicity of gases generated during the combustion process. In the experiment, the asphyxiant hydrogen cyanide (HCN) was detected at the beginning of combustion (4 min of testing) as a product of ABS degradation. CO was generated throughout the test (8 min) because of the incomplete combustion of both the ABS and PC fractions. The presence of aryl phosphates promoted the inhibition of the flame. The reaction of PO·radicals in the gas phase resulted in OH·scavenging and a higher release of HCN and CO. The results suggest that aryl phosphates act in the first 4 min and do not have an effect later. FRs with lower thermal stability exhibited lower heat release and flame propagation but generated more toxic gases. This effect is attributed to the higher activity of the flame‐retardant in the gas phase. Further, additional fire performance parameters, including thermal stability (thermogravimetric analysis), flammability (UL94), and heat and smoke generation (cone calorimeter), were studied. It was found that aryl phosphates reduced the fire hazard, prevented the spread of the flame, reduced heat generation, increased the time to ignition, and, at the same time, promoted the emission of toxic gases that differ in function of the selected flame‐retardant.

Dhurrin increases but does not mitigate oxidative stress in droughted Sorghum bicolor

Main conclusionDroughted sorghum had higher concentrations of ROS in both wildtype and dhurrin-lacking mutants. Dhurrin increased in wildtype genotypes with drought. Dhurrin does not appear to mitigate oxidative stress in sorghum.Sorghum bicolor is tolerant of high temperatures and prolonged droughts. During droughts, concentrations of dhurrin, a cyanogenic glucoside, increase posing a risk to livestock of hydrogen cyanide poisoning. Dhurrin can also be recycled without the release of hydrogen cyanide presenting the possibility that it may have functions other than defence. It has been hypothesised that dhurrin may be able to mitigate oxidative stress by scavenging reactive oxygen species (ROS) during biosynthesis and recycling. To test this, we compared the growth and chemical composition of S. bicolor in total cyanide deficient sorghum mutants (tcd1) with wild-type plants that were either well-watered or left unwatered for 2 weeks. Plants from the adult cyanide deficient class of mutant (acdc1) were also included. Foliar dhurrin increased in response to drought in all lines except tcd1 and acdc1, but not in the roots or leaf sheaths. Foliar ROS concentration increased in drought-stressed plants in all genotypes. Phenolic concentrations were also measured but no differences were detected. The total amounts of dhurrin, ROS and phenolics on a whole plant basis were lower in droughted plants due to their smaller biomass, but there were no significant genotypic differences. Up until treatments began at the 3-leaf stage, tcd1 mutants grew more slowly than the other genotypes but after that they had higher relative growth rates, even when droughted. The findings presented here do not support the hypothesis that the increase in dhurrin commonly seen in drought-stressed sorghum plays a role in reducing oxidative stress by scavenging ROS.

Structural characterization of Linum usitatissimum hydroxynitrile lyase: A new cyanohydrin decomposition mechanism involving a cyano-zinc complex

Hydroxynitrile lyase from Linum usitatissimum (LuHNL) is an enzyme involved in the catabolism of cyanogenic glycosides to release hydrogen cyanide upon tissue damage. This enzyme strictly conserves the substrate- and NAD(H)-binding domains of Zn2+-containing alcohol dehydrogenase (ADH); however, there is no evidence suggesting that LuHNL possesses ADH activity. Herein, we determined the ligand-free 3D structure of LuHNL and its complex with acetone cyanohydrin and (R)-2-butanone cyanohydrin using X-ray crystallography. These structures reveal that an A-form NAD+ is tightly but not covalently bound to each subunit of LuHNL. The restricted movement of the NAD+ molecule is due to the “sandwich structure” on the adenine moiety of NAD+. Moreover, the structures and mutagenesis analysis reveal a novel reaction mechanism for cyanohydrin decomposition involving the cyano-zinc complex and hydrogen-bonded interaction of the hydroxyl group of cyanohydrin with Glu323/Thr65 and H2O/Lys162 of LuHNL. The deprotonated Lys162 and protonated Glu323 residues are presumably stabilized by a partially desolvated microenvironment. In summary, the substrate binding geometry of LuHNL provides insights into the differences in activities of LuHNL and ADH, and identifying this novel reaction mechanism is an important contribution to the study of hydroxynitrile lyases.

Simultaneous Quantitative Detection of HCN and C2H2 in Combustion Environment Using TDLAS

Emission of nitrogen oxides (NOx) and soot particles during the combustion of biomass fuels and municipal solid waste is a major environmental issue. Hydrogen cyanide (HCN) and acetylene (C2H2) are important precursors of NOx and soot particles, respectively. In the current work, infrared tunable diode laser absorption spectroscopy (IR-TDLAS), as a non-intrusive in situ technique, was applied to quantitatively measure HCN and C2H2 in a combustion environment. The P(11e) line of the first overtone vibrational band v1 of HCN at 6484.78 cm−1 and the P(27e) line of the v1 + v3 combination band of C2H2 at 6484.03 cm−1 were selected. However, the infrared absorption of the ubiquitous water vapor in the combustion environment brings great uncertainty to the measurement. To obtain accurate temperature-dependent water spectra between 6483.8 and 6485.8 cm−1, a homogenous hot gas environment with controllable temperatures varying from 1100 to 1950 K provided by a laminar flame was employed to perform systematic IR-TDLAS measurements. By fitting the obtained water spectra, water interference to the HCN and C2H2 measurement was sufficiently mitigated and the concentrations of HCN and C2H2 were obtained. The technique was applied to simultaneously measure the temporally resolved release of HCN and C2H2 over burning nylon 66 strips in a hot oxidizing environment of 1790 K.

Regulation of dhurrin pathway gene expression during Sorghum\xa0bicolor development

Main conclusionDevelopmental and organ-specific expression of genes in dhurrin biosynthesis, bio-activation, and recycling offers dynamic metabolic responses optimizing growth and defence responses in Sorghum.Plant defence models evaluate the costs and benefits of resource investments at different stages in the life cycle. Poor understanding of the molecular regulation of defence deployment and remobilization hampers accuracy of the predictions. Cyanogenic glucosides, such as dhurrin are phytoanticipins that release hydrogen cyanide upon bio-activation. In this study, RNA-seq was used to investigate the expression of genes involved in the biosynthesis, bio-activation and recycling of dhurrin in Sorghum bicolor. Genes involved in dhurrin biosynthesis were highly expressed in all young developing vegetative tissues (leaves, leaf sheath, roots, stems), tiller buds and imbibing seeds and showed gene specific peaks of expression in leaves during diel cycles. Genes involved in dhurrin bio-activation were expressed early in organ development with organ-specific expression patterns. Genes involved in recycling were expressed at similar levels in the different organ during development, although post-floral initiation when nutrients are remobilized for grain filling, expression of GSTL1 decreased > tenfold in leaves and NITB2 increased > tenfold in stems. Results are consistent with the establishment of a pre-emptive defence in young tissues and regulated recycling related to organ senescence and increased demand for nitrogen during grain filling. This detailed characterization of the transcriptional regulation of dhurrin biosynthesis, bioactivation and remobilization genes during organ and plant development will aid elucidation of gene regulatory networks and signalling pathways that modulate gene expression and dhurrin levels. In-depth knowledge of dhurrin metabolism could improve the yield, nitrogen use efficiency and stress resilience of Sorghum.

Operando diffuse reflectance infrared detection of cyanide intermediate species during the reaction of formaldehyde with ammonia over V2O5/WO3-TiO2

Hydrogen cyanide (HCN) is a highly poisonous gas that can form through the reaction between formaldehyde and ammonia (NH3) in the exhaust. While this detrimental side reaction has been the subject of recent catalytic studies under selective catalytic reduction conditions, the molecular mechanism leading to the release of HCN remains far from elucidated. Here, we report the detection of the cyanide intermediate species by operando diffuse reflectance infrared spectroscopy, thereby providing unambiguous evidence that the HCN-forming reaction occurs on the catalyst surface. Formaldehyde likely reacts from the gas phase with pre-adsorbed NH3 to form HCN. Although formate species were formed abundantly upon formaldehyde introduction, they were not responsible for the high level of HCN emissions observed. TiO2 alone is sufficient to catalyze the side reaction, but VOx and/or WOx sites increase HCN production by preventing the formation of inactive polyamide species. These mechanistic insights should serve as a basis for understanding the chemistry responsible for the side reaction so that mitigation measures can be put forward.