CRISPR/Cas9-mediated mutation of Eil1 transcription factor genes affects exogenous ethylene tolerance and early flower senescence in Campanula portenschlagiana.

Improving tolerance to ethylene-induced early senescence of flowers and fruits is of major economic importance for the ornamental and food industry. Genetic modifications of genes in the ethylene-signalling pathway have frequently resulted in increased tolerance but often with unwanted side effects. Here, we used CRISPR/Cas9 to knockout the function of two CpEil1 genes expressed in flowers of the diploid ornamental plant Campanula portenschlagiana. The ethylene tolerance in flowers of the primary mutants with knockout of only one or all four alleles clearly showed increased tolerance to exogenous ethylene, although lower tolerance was obtained with one compared to four mutated alleles. The allele dosage effect was confirmed in progenies where flowers of plants with zero, one, two, three and four mutated alleles showed increasing ethylene tolerance. Mutation of the Cpeil1 alleles had no significant effect on flower longevity and endogenous flower ethylene level, indicating that CpEil1 is not involved in age-dependent senescence of flowers. The study suggests focus on EIN3/Eils expressed in the organs subjected to early senescence for obtaining tolerance towards exogenous ethylene. Furthermore, the observed allelic dosage effect constitutes a key handle for a gradual regulation of sensitivity towards exogenous ethylene, simultaneously monitoring possibly unwanted side effects.

Dose-dependent effects of sodium dodecyl sulfate and hydrogen peroxide treatments on methane emission from pig manure during storage.

Mitigation of methane (CH4) emissions from slurry pits within pig barns can be achieved through treatment of residual slurry left after frequent flushing of the slurry pits. In this study, dosages of additives such as sodium dodecyl sulfate (SDS) and hydrogen peroxide (H2O2) were optimized to achieve reduction in CH4 emissions from residual pig slurry during storage. In addition, the effects on emissions when both the treatments were combined and the effects of SDS treatment on slurry acidified with sulfuric acid (H2SO4) were studied in order to reduce CH4 and ammonia (NH3) emissions from residual pig slurry storage. A maximum of 98% and 70% reduction in CH4 emissions were achieved with SDS and H2O2 treatments, respectively. The combination of SDS and H2O2 did not increase efficiency in reducing CH4 emissions compared to SDS treatment alone. Whereas the application of SDS to slurry acidified with H2SO4 (pH 6.2) increased the CH4 mitigation efficiency by 15-30% compared to treating slurry with only SDS. The combined treatment (SDS + H2SO4) reduced NH3 emissions by 20% compared to treating slurry with H2SO4 (pH 6.2) alone. Hereby, combined treatment (SDS + H2SO4) can reduce both CH4 and NH3 emissions, with a reduced amount of chemicals required for the treatment. Hence, application of SDS at concentrations <2 g kg-1 to acidified slurry is recommended to treat residual pig manure in pig barns.

Simple Management Changes Drastically Reduce Pig House Methane Emission in Combined Experimental and Modeling Study.

Reducing methane from livestock slurry is one of the quickest ways to counteract global warming. A straightforward strategy is to reduce slurry retention time inside pig houses by frequent transfer to outside storages, where temperature and therefore microbial activity are lower. We demonstrate three frequent slurry removal strategies in pig houses in a year-round continuous measurement campaign. Slurry funnels, slurry trays, and weekly flushing reduced slurry methane emission by 89, 81, and 53%, respectively. Slurry funnels and slurry trays reduced ammonia emission by 25-30%. An extended version of the anaerobic biodegradation model (ABM) was fitted and validated using barn measurements. It was then applied for predicting storage emission and shows that there is a risk of negating barn methane reductions due to increased emission from outside storage. Therefore, we recommend combining the removal strategies with anaerobic digestion pre-storage or storage mitigation technologies such as slurry acidification. However, even without storage mitigation technologies, predicted net methane reduction from pig houses and following outside storage was at least 30% for all slurry removal strategies.

Effect of anaerobic digestion on odor and ammonia emission from land-applied cattle manure.

High ammonia (NH3) and odor emission can occur after land application of liquid animal manure. This study was aimed at evaluating NH3 loss and odor nuisance after field application of cattle manure and how it is affected by two anaerobic digestion strategies: i) digestion of cattle manure alone and ii) digestion with catch crops and dilution by water. A system of dynamic chambers with online measurements of NH3 and odorous compounds (summarized as odor activity value, OAV) was used. Two experiments were conducted under different temperature conditions. The results demonstrated that anaerobic digestion did not affect NH3 loss but did decrease OAV. Addition of catch crops and water to the digestion process reduced both NH3 loss and OAV. Cool temperature in one of the experiments had a large effect on both NH3 and odor emissions, and at high temperature the differences between treatments increased.

Effect of storage and field acidification on emissions of NH3, NMVOC, and odour from field applied slurry in winter conditions.

Land spreading of liquid animal manure (slurry) is a major source of atmospheric emissions. Ammonia (NH3) emission is of concern, as it is one of the main contributors to ambient air pollution and nitrogen deposition. Storage and field acidification of the slurry prior to application is used to mitigate NH3 emission, but the effect of acidification on emissions of odorous non-methane volatile organic compounds (NMVOC) has not been investigated, and there is a scarcity of data investigating the effect of field acidification. Four field experiments, two with cattle slurry and two with pig slurry, were performed. Ammonia and NMVOC emissions were measured simultaneously in a system of dynamic chambers and online measurements by cavity ring-down spectroscopy (CRDS) and proton-transfer-reaction mass spectrometry (PTR-MS). The system allowed for a high time resolution and low variation. All four experiments were performed under cold conditions (<10°C average temperature). Storage and field acidification significantly lowered the NH3 emission by 79 ± 18% and 30 ± 6% on average, respectively. The NMVOC cumulative emission increased by 202 ± 133% and 17 ± 16% on average after storage and field acidification, respectively, even if the increase was only significant for storage acidification. Storage acidification significantly increased the emissions of odour at most measuring times. The increases of cumulative NMVOC emissions and odour was primarily caused by higher emissions of volatile fatty acids.

Low-Cost Fluorescence Sensor for Ammonia Measurement in Livestock Houses.

Measurements of ammonia with inexpensive and reliable sensors are necessary to obtain information about e.g., ammonia emissions. The concentration information is needed for mitigation technologies and documentation of existing technologies in agriculture. A flow-based fluorescence sensor to measure ammonia gas was developed. The automated sensor is robust, flexible and made from inexpensive components. Ammonia is transferred to water in a miniaturized scrubber with high transfer efficiency (>99%) and reacts with o-phthalaldehyde and sulfite (pH 11) to form a fluorescent adduct, which is detected with a photodiode. Laboratory calibrations with standard gas show good linearity over a dynamic range from 0.03 to 14 ppm, and the detection limit of the analyzer based on three-times the standard deviation of blank noise was approximately 10 ppb. The sampling frequency is 0.1 to 10 s, which can easily be changed through serial commands along with UV LED current and filter length. Parallel measurements with a cavity ring-down spectroscopy analyzer in a pig house show good agreement (R2 = 0.99). The fluorescence sensor has the potential to provide ammonia gas measurements in an agricultural environment with high time resolution and linearity over a broad range of concentrations.

Synergistic Tannic Acid-Fluoride Inhibition of Ammonia Emissions and Simultaneous Reduction of Methane and Odor Emissions from Livestock Waste.

Gaseous emissions from livestock production are complex mixtures including ammonia, methane, volatile organic compounds (VOC), and H2S. These contribute to eutrophication, reduced air quality, global warming, and odor nuisance. It is imperative that these gases are mitigated in an environmentally sustainable manner. We present the discovery of a microbial inhibitor combo consisting of tannic acid and sodium fluoride (TA-NaF), which exhibits clear synergistic inhibition of ammonia production in pure bacteria culture and in pig manure while simultaneously inhibiting methane and odorant (H2S and VOC) emissions. In laboratory headspace experiments on pig manure, we used proton-transfer-reaction mass spectrometry and cavity ring-down spectroscopy to measure the effect of TA-NaF on gaseous emissions. Ammonia emission was reduced by more than 95%, methane by up to ∼99%, and odor activity value by more than 50%. Microbial community analysis and gas emission data suggest that TA-NaF acts as an efficient generic microbial inhibitor, and we hypothesize that the synergistic inhibitory effect on ammonia production is related to tannic acid causing cell membrane leakage allowing fluoride ions easy access to urease.

Copper Adsorption on Lignin for the Removal of Hydrogen Sulfide.

Lignin is currently an underutilized part of biomass; thus, further research into lignin could benefit both scientific and commercial endeavors. The present study investigated the potential of kraft lignin as a support material for the removal of hydrogen sulfide (H2S) from gaseous streams, such as biogas. The removal of H2S was enabled by copper ions that were previously adsorbed on kraft lignin. Copper adsorption was based on two different strategies: either directly on lignin particles or by precipitating lignin from a solution in the presence of copper. The H2S concentration after the adsorption column was studied using proton-transfer-reaction mass spectrometry, while the mechanisms involved in the H2S adsorption were studied with X-ray photoelectron spectroscopy. It was determined that elemental sulfur was obtained during the H2S adsorption in the presence of kraft lignin and the differences relative to the adsorption on porous silica as a control are discussed. For kraft lignin, only a relatively low removal capacity of 2 mg of H2S per gram was identified, but certain possibilities to increase the removal capacity are discussed.

Application of Proton-Transfer-Reaction Mass Spectrometry (PTR-MS) and 33S Isotope Labeling for Monitoring Sulfur Processes in Livestock Waste.

Reduced sulfur compounds emitted from livestock production cause odor nuisance for local residents. The microbial processes responsible for this are not well described in swine manure and a method for monitoring the biological processes is necessary to develop strategic abatement technologies. In this study, Proton-Transfer-Reaction Mass Spectrometry and isotope-labeled sulfate were combined and applied to elucidate the sulfur processes in swine manure with high time resolution. We successfully monitored reduction of isotope 33S labeled sulfate into corresponding 33S hydrogen sulfide and found that some of the 33S hydrogen sulfide was further methylated into 33S methanethiol. The isotope patterns in reduced sulfur compounds together with usage of inhibitors enabled us to calculate a sulfate reduction rate of 1.03 ± 0.18 mM/day equivalent to 76.9 ± 3.0% of total hydrogen sulfide emissions. Cysteine degradation constituted 20.2 ± 2.7% of the total hydrogen sulfide produced and the remaining hydrogen sulfide came from demethylation of methanethiol and dimethyl sulfide. Another source to methanethiol, besides hydrogen sulfide methylation, was methionine degradation, which contributed with 78.3 ± 2.5% of the methanethiol production, whereas the remaining 21.7 ± 2.5% came from hydrogen sulfide methylation. This study suggests, therefore, that emissions of odorous sulfur compounds from swine manure can be reduced by inhibiting methionine degradation and sulfate reduction.

Mechanisms of Loss of Agricultural Odorous Compounds in Sample Bags of Nalophan, Tedlar, and PTFE.

Alteration of the chemical composition of odor samples during storage in polymer sample bags can significantly impair the accuracy of subsequent odor evaluations. To overcome or minimize this effect, the mechanisms determining compound loss must be more thoroughly understood. The present study examines the storage stability of a selection of key odorants from livestock production in polymer sample bags of Nalophan, Tedlar, and polytetrafluoroethylene (PTFE). The compounds included are acetic acid, butanoic acid, propanoic acid, 3-methylbutanoic acid, hydrogen sulfide, methanethiol, dimethyl sulfide, trimethylamine, and 4-methylphenol. The fate of the unrecovered compound fractions is clarified by means of thermal desorption and concentric double bags, allowing estimation of the magnitude of losses due to adsorption and diffusion, respectively. The degree of recovery was found to be PTFE > Tedlar > Nalophan, and smaller ratios of bag surface area to sample volume improved the recovery significantly. Furthermore, PTFE bags were found far superior for maintaining the original sample humidity and for storing 4-methylphenol. Analysis of sample humidity, partitioning coefficients, and thermal desorption suggested that the loss in PTFE bags was mainly controlled by adsorption, whereas for Nalophan and Tedlar, compound loss is a combined effect of adsorption and diffusion. It is suggested to heat the bags when evacuating the sample for analysis, as this was found to improve the recovery significantly. For a 5-L PTFE bag, all odorants could be found at concentration levels between 71.6 and 98.8% even after 48 h of storage when heated to 57°C prior to analysis.

Key Odorants from Pig Production Based on Improved Measurements of Odor Threshold Values Combining Olfactometry and Proton-Transfer-Reaction Mass Spectrometry (PTR-MS).

Analytical measurements of odorants in combination with odor threshold values is an alternative to sensory measurements that can be used to evaluate abatement technologies for pig production facilities. The purpose of the present study was to estimate odor threshold values for key odorants found in pig house air. A new method was applied where an olfactometer was used to dilute the sample air and the concentrations of odorants presented to the panelists at the dilutions steps were measured by proton-transfer-reaction mass spectrometry (PTR-MS). The results demonstrate that the odor threshold values of acetic acid, butanoic acid, and 4-methylphenol are considerably lower than reported previously, whereas the values of hydrogen sulfide, methanethiol and dimethylsulfide were comparable. Consequently, acetic acid, butanoic acid, and 4-methyl-phenol will have a larger influence on odor from pig production facilities than previously assumed. The results highlight the necessity for directly measuring exposure concentrations when determining odor threshold values.

Relevance of n-butanol as a reference gas for odorants and complex odors.

Odor analysis by olfactometry relies on the use of n-butanol as a reference compound for standardizing the selection of human panelists. This requires that human sensitivity towards n-butanol is correlated to sensitivity towards other odorants as well as complex odor mixtures. However, there is limited evidence in the literature of such correlations. In this work, datasets from three odor laboratories were investigated in order to clarify this. All panels routinely analyzed n-butanol and H2S samples. Two of the laboratories analyzed samples from pig production or industry, whereas one laboratory determined odor threshold values for typical pig production odorants. Non-significant correlations were observed in most cases and odor threshold values for structurally related compounds were not well correlated. The work presented strongly indicates that the sensitivity of odor panelists towards n-butanol is not well transferred to other odorants or odor samples. Furthermore, minimization of variance by using n-butanol is not transferable to other odorants or environmental samples. Thus, the harmonization of human panelists for odor analysis based on n-butanol does not appear to result in harmonization with respect to other odorants or odor samples.

Effects of Dilution Systems in Olfactometry on the Recovery of Typical Livestock Odorants Determined by PTR-MS.

The present study provides an elaborate assessment of the performance of olfactometers in terms of odorant recovery for a selection of odorants emitted from livestock houses. The study includes three different olfactometer dilution systems, which have been in use at accredited odor laboratories. They consist of: (i) a custom-built olfactometer made of glass tubes, (ii) a TO8 olfactometer, and (iii) an Olfacton dilution system based on a mass flow controller. The odorants include hydrogen sulfide, methanethiol, dimethyl sulfide, acetic acid, butanoic acid, propanoic acid, 3-methylbutanoic acid, 4-methylphenol, and trimethylamine. Furthermore, n-butanol, as the reference gas in the European standard for olfactometry, EN13725, was included. All measurements were performed in real time with proton-transfer-reaction mass spectrometry (PTR-MS). The results show that only dimethyl sulfide was almost completely recovered in all cases, while for the remaining compounds, the performance was found to vary significantly (from 0 to 100%) depending on the chemical properties of the compounds, the concentration levels, the pulse duration, and the olfactometer material. To elucidate the latter, the recovery in different locations of the TO8 olfactometer and in tubes of different materials, that is, poly-tetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), stainless steel and SilcoTek-coated steel, were tested. Significant saturation effects were observed when odorants were in contact with stainless steel.

Changing Feeding Regimes To Demonstrate Flexible Biogas Production: Effects on Process Performance, Microbial Community Structure, and Methanogenesis Pathways.

Flexible biogas production that adapts biogas output to energy demand can be regulated by changing feeding regimes. In this study, the effect of changes in feeding intervals on process performance, microbial community structure, and the methanogenesis pathway was investigated. Three different feeding regimes (once daily, every second day, and every 2 h) at the same organic loading rate were studied in continuously stirred tank reactors treating distiller's dried grains with solubles. A larger amount of biogas was produced after feeding in the reactors fed less frequently (once per day and every second day), whereas the amount remained constant in the reactor fed more frequently (every 2 h), indicating the suitability of the former for the flexible production of biogas. Compared to the conventional more frequent feeding regimes, a methane yield that was up to 14% higher and an improved stability of the process against organic overloading were achieved by employing less frequent feeding regimes. The community structures of bacteria and methanogenic archaea were monitored by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA and mcrA genes, respectively. The results showed that the composition of the bacterial community varied under the different feeding regimes, and the observed T-RFLP patterns were best explained by the differences in the total ammonia nitrogen concentrations, H2 levels, and pH values. However, the methanogenic community remained stable under all feeding regimes, with the dominance of the Methanosarcina genus followed by that of the Methanobacterium genus. Stable isotope analysis showed that the average amount of methane produced during each feeding event by acetoclastic and hydrogenotrophic methanogenesis was not influenced by the three different feeding regimes.

Application of PTR-MS for measuring odorant emissions from soil application of manure slurry.

Odorous volatile organic compounds (VOC) and hydrogen sulfide (H2S) are emitted together with ammonia (NH3) from manure slurry applied as a fertilizer, but little is known about the composition and temporal variation of the emissions. In this work, a laboratory method based on dynamic flux chambers packed with soil has been used to measure emissions from untreated pig slurry and slurry treated by solid-liquid separation and ozonation. Proton-transfer-reaction mass spectrometry (PTR-MS) was used to provide time resolved data for a range of VOC, NH3 and H2S. VOC included organic sulfur compounds, carboxylic acids, phenols, indoles, alcohols, ketones and aldehydes. H2S emission was remarkably observed to take place only in the initial minutes after slurry application, which is explained by its high partitioning into the air phase. Long-term odor effects are therefore assessed to be mainly due to other volatile compounds with low odor threshold values, such as 4-methylphenol. PTR-MS signal assignment was verified by comparison to a photo-acoustic analyzer (NH3) and to thermal desorption GC/MS (VOC). Due to initial rapid changes in odorant emissions and low concentrations of odorants, PTR-MS is assessed to be a very useful method for assessing odor following field application of slurry. The effects of treatments on odorant emissions are discussed.

Quantifying contribution of synthrophic acetate oxidation to methane production in thermophilic anaerobic reactors by membrane inlet mass spectrometry.

A unique method was developed and applied for monitoring methanogenesis pathways based on isotope labeled substrates combined with online membrane inlet quadrupole mass spectrometry (MIMS). In our study, a fermentation sample from a full-scale biogas plant fed with pig and cattle manure, maize silage, and deep litter was incubated with 100 mM of [2-(13)C] sodium acetate under thermophilic anaerobic conditions. MIMS was used to measure the isotopic distribution of dissolved CO2 and CH4 during the degradation of acetate, while excluding interference from water by applying a cold trap. After 6 days of incubation, the proportion of methane derived from reduction of CO2 had increased significantly and reached up to 87% of total methane, suggesting that synthrophic acetate oxidation coupled to hydrogenotrophic methanogenesis (SAO-HM) played an important role in the degradation of acetate. This study provided a new approach for online quantification of the relative contribution of methanogenesis pathways to methane production with a time resolution shorter than one minute. The observed contribution of SAO-HM to methane production under the tested conditions challenges the current widely accepted anaerobic digestion model (ADM1), which strongly emphasizes the importance of the acetoclastic methanogenesis.

Evaluation of clay aggregate biotrickling filters for treatment of gaseous emissions from intensive pig production.

Treatment of ventilation air from livestock production by biological airfiltration has emerged as a cost-effective technology for reduction of emissions of odorants and ammonia. Volatile sulfur compounds from livestock production include H2S and methanethiol, which have been identified as potentially important odorants that are not removed sufficiently by biological air filters. Light-expanded clay aggregates (Leca(®)) is a biotrickling filter material that contains iron oxides, which can oxidize H2S and methanethiol, and thus potentially may help to remove these two compounds in biological air filters. This study used on-line PTR-MS measurements to investigate the performances of two Leca(®) biotrickling filters (abraded Leca(®) filter and untreated Leca(®) filter) for removal of odorants and ammonia emitted from an experimental pig house. The results indicated that the abraded Leca(®) filter had a similar or slightly better capability for removing odorants than the untreated Leca(®) filter. This may be due to the enlargement of the surface area by the friction process. The volatile sulfur compounds, however, were not removed efficiently by either of the two Leca(®) filters. Kinetic analysis of a ventilation controlled experiment during the first period indicated that Grau second-order kinetics could be applied to analyze the removal of sulfur compounds and other odorants, whereas the Stover-Kincannon model could only be applied to analyze the removal of odorants other than sulfur compounds, partly due to the limitation of mass transfer of these compounds in the biotrickling filters. In the last measurement period, a production of dimethyl disulfide and dimethyltrisulfide coinciding with strongly enhanced removal of methanethiol was observed for the untreated filter. This was assumed to be enhanced by an incidence of low local air velocity in the filter and indicated involvement of iron-catalyzed reactions in the removal of sulfur compounds.

Recovery of odorants from an olfactometer measured by proton-transfer-reaction mass spectrometry.

The aim of the present study was to examine the recovery of odorants during the dilution in an olfactometer designed according to the European standard for dynamic olfactometry. Nine odorants in the ppmv-range were examined including hydrogen sulfide, methanethiol, dimethyl sulfide, acetic acid, propanoic acid, butanoic acid, trimethylamine, 3-methylphenol and n-butanol. Each odorant was diluted in six dilution steps in descending order from 4,096 to 128 times dilutions. The final recovery of dimethyl sulfide and n-butanol after a 60-second pulse was only slightly affected by the dilution, whereas the recoveries of the other odorants were significantly affected by the dilution. The final recoveries of carboxylic acids, trimethylamine and 3-methylphenol were affected by the pulse duration and the signals did not reach stable levels within the 60-second pulse, while sulfur compounds and n-butanol reach a stable signal within a few seconds. In conclusion, the dilution of odorants in an olfactometer has a high impact on the recovery of odorants and when olfactometry is used to estimate the odor concentration, the recoveries have to be taken into consideration for correct measurements.

Regenerative one-stage catalytic absorption process with cupric ions for removal of reduced sulfur compounds in polluted air.

Reduced volatile sulfur compounds emitted from e.g. livestock production and biogas production facilities contribute to general air pollution and local odour nuisance. Improved technologies are required to mitigate the emissions of both hydrogen sulfide and organic sulfur compounds. The present study examines the oxidative absorption of reduced sulfur compounds, i.e. hydrogen sulfide, methanethiol and dimethyl sulfide in a wet oxidation process with cupric chloride. It was found that this process efficiently removes both hydrogen sulfide and methanethiol with removal efficiencies >94% under all process conditions tested, while the removal of dimethyl sulfide was in the range 20-40%. The main products determined were dimethyl disulfide, dimethyl trisulfide and elemental sulfur. It was shown that the process was more efficient than the similar process with ferric ions and higher removal could be obtained with lower residence times. Furthermore, though employing cupric ion as metal catalysts results in the production of gaseous sulfur compounds, it is estimated that this process is efficient for deodorization due to the higher odour threshold values of the product compounds and the pH range is optimal for gas streams containing CO2.