Acetone problem in use of the acetylene blockage method for assessment of denitrifying activity in soil

Abstract

Studies to evaluate the acetylene (C₂H₂) blockage method for assessment of denitrifying activity in soil showed that the gas emerging from cylinders of purified C₂H₂ available commercially contains substantial amounts of acetone and that this impurity is readily utilized by denitrifying microorganisms for reduction of nitrate. They also showed that C₂H₂ enhanced denitrification of nitrate in soils even when it was purified to remove acetone, but that the effect of acetone‐free C₂H₂ on denitrifying activity was much smaller than that of acetone‐contaminated C₂H₂. These observations indicate that use of C₂H₂ not purified to remove acetone may lead to serious overestimation of denitrifying activity in soils by the C₂H₂ blockage method.     

Factors affecting the availability of organic carbon for denitrification of nitrate in subsoils

Summary Recent work in our laboratory indicated that the slow rate of denitrification in Iowa subsoils is not due to a lack of denitrifying microorganisms, but to a lack of organic C that can be utilized by these microorganisms for reduction of nitrate. To identify factors affecting the availability of leachable organic C in surface soils capable of promoting denitrification in subsoils, we studied the effects of freezing and drying and of plants and plant residues on the amounts of water-soluble organic C in surface soils and the ability of this organic C to promote denitrification in subsoils. We found that aqueous extracts of field-moist, frozen, and air-dried surface soils promoted denitrification in subsoils and that their stimulatory effects on denitrification were highly correlated (r=0.93) with their organic C contents and decreased in the order air-dried soils frozen soils >field-moist soils. But a detailed study of the effect of drying a surface soil to different water tensions indicated that drying of soils under natural conditions is not likely to lead to a substantial increase in their content of water-soluble organic C. Amendment of surface soils with corn or soybean residues led to a marked increase in the amount of organic C in aqueous extracts of the soils and in the ability of these extracts to promote denitrification in subsoils. These effects of plant residues could not be detected after incubation of residue-treated soils for a few days under aerobic conditions, but they increased markedly with an increase in the time of incubation from 1 to 10 days when residue-treated soils were incubated under anaerobic conditions. Analyses for organic acids indicated that this increase was largely due to fermentative production of acetic, propionic, and butyric acids by soil microorganisms. Growth chamber studies showed that growth of corn, soybean, wheat, and sorghum plants on surface soil did not significantly increase the organic C content of leachates of the soil or the ability of these leachates to promote denitrification in subsois. We conclude that plant residues are a major source of the leachable organic C in surface soils that is capable of promoting denitrification in subsoils.     

Schätzung der Denitrifikation in landwirtschaftlich genutzten Böden. I. Grundlagen

Estimating denitrification in agricultural soils: I. Basic assumptions Despite a common interest of soil scientists, groundwater hydrologists, agronomists, and climatologists in quantifying soil nitrogen losses through denitrification, these are often neglected when the nitrogen turnover of agricultural soils is modelled. This is the first of two papers describing a semi-quantitative denitrification model that has been derived from data collected from soils of a catchment area near Viersen in the Lower Rhine Valley in West Germany. A field study was conducted to study the soil environment of denitrifying microorganisms. The results of this study were used to evaluate the effect of soil water, soil air and soil temperature, of the amount and quality of the organic matter, and of the pH-value on denitrification. To this end an existing model to quantify denitrification was extended. Provided the seasonal nitrogen excess of a field is known, the model in its new form enables an estimation of the extent of field-specific denitrification rates. This paper describes the effects of the parameters under consideration on denitrification, the model components used to describe these parameters, and the development of the denitrification model.     

Availability of organic carbon for denitrification of nitrate in subsoils

Summary Previous work in our laboratory indicated that the slow rate of denitrification in Iowa subsoils is not due to a lack of denitrifying microorganisms, but to a lack of organic C that can be utilized by these microorganisms for reduction of NO ₃ ^– . This conclusion was supported by studies showing that drainage water from tile drains under agricultural research plots contained only trace amounts of organic C and had very little, if any, effect on denitrification in subsoils. Aqueous extracts of surface soils promoted denitrification when added to subsoils, and their ability to do so increased with increase in their organic C content. Amendment of surface soils with corn and soybean residues initially led to a marked increase in the amounts of organic C in aqueous extracts of these soils and in the ability of these extracts to promote denitrification in subsoils, but these effects were short-lived and could not be detected after incubation of residue-treated soils for a few days. We conclude from these observations that water-soluble organic C derived from plant residues is decomposed so rapidly in surface soils that very little of this C is leached into subsoils, and that this largely accounts for the slow rate of denitrification of nitrate in subsoils.     

Extractability and Subsequent Biodegradation of PAHs from Contaminated Soil

The biodegradation of polyaromatic hydrocarbons (PAHs) has been well documented; however, the biodegradation of PAHs in contaminated soil has proved to be problematic. Sorption of PAHs to soil over time can significantly decrease their availability for extraction much less than for biodegradation. In this study the ability of various organic solvents to extract PAHs from coal tar-contaminated soil obtained from former manufactured gas plant (MGP) sites was investigated. Solvents investigated included acetone/hexane, dichloromethane, ethanol, methanol, toluene, and water. The extraction of MGP soils with solvents was investigated using soxhlet extraction, multiple soxhlet extractions, sonication, and brief agitation at ambient temperature with a range of solvent concentrations. Of particular interest was the documentation of the recalcitrance of PAHs in weathered MGP soils to extraction and to bioremediation, as well as to demonstrate the ease with which PAHs extracted from these soils can be biodegraded. The efficiency of extraction of PAHs from MGP soils was found to be more dependent upon the choice of solvent. The environmentally-benign solvent ethanol, was shown to be equal to if not better than acetone/hexane (the EPA recommended solvent) for the extraction of PAHs from MGP soils, brief contact/agitation times (minutes) using small quantities of ethanol (2 volumes or less) can achieve nearly quantitative extraction of PAHs from MGP soils. Moreover aqueous slurries of an MGP soils experienced less than 10% biodegradation of PAHs in 14 days while in the same period about 95% biodegradation was acieved using PAHs extracted from this soil by ethanol and subsequently added to aqueous bacterial suspensions.     

The influence of soil pH on denitrification: progress towards the understanding of this interaction over the last 50 years

Results from the pioneering research on the interactions between pH and denitrification in soil from the 1950s to the present are reviewed, the changing perceptions of this complex relationship are discussed, and the current status of the subject is assessed. Facets of this relationship that are analysed in detail include the direct or indirect influence of pH on overall denitrification rates in soils, changes in the composition of gaseous products that depend on pH, methods for measuring the process, the concept of an optimum pH for denitrification, and the adaptation of microbial denitrifying communities to acidic environments. The main conclusions to be drawn are as follows. Total gaseous emissions to the atmosphere (N₂O, NO and N₂) have repeatedly been shown to be less in acidic than in neutral or slightly alkaline soils. This may be attributable to smaller amounts of organic carbon and mineral nitrogen available to the denitrifying population under acid conditions rather than a direct effect of low pH on denitrification enzymes. Numerous laboratory and field studies have demonstrated that the ratio N₂O:N₂ is increased when the pH of soils is reduced. The relation between soil pH and potential denitrification as determined by various incubation methods remains unclear, results being influenced both by original conditions in soil samples and by unknown changes during incubation. The concept of an optimum pH for denitrification has been frequently proposed, but such a term has little or no meaning without reference to specific attributes of the process.     

Assessing the Potential of Organic Solvents on Total Petroleum Hydrocarbon Extraction from Diesel-Contaminated Soils

The total petroleum hydrocarbon (TPH) extraction potential of organic solvents including dichloromethane (DCM), pentane, hexane, methanol, ethanol, propanol, and acetone was investigated along with the effect of water content in solvents for their efficiency of extraction. The extent of TPH extraction was analyzed using various extraction schemes (i.e., solvent/solid ratio, treatment time, extraction method, solvent/water ratio) to better understand the physical and chemical factors controlling TPH release from contaminated soils. More TPH was extracted with increasing solvent/solid ratio and increasing time. The extent of TPH extracted also varied depending on the extraction method, solvent type, and solvent/water ratio, but was highest when using the total extraction method and 100% DCM. However, the efficiency of TPH extraction decreased dramatically with the increase in the water content in organic solvents. The results also showed that TPH extraction using DCM was the best option for achieving cost-effective, eco-friendly outcomes along with remediation goals. DCM used in solvent extraction to remediate diesel-contaminated soils showed low toxicity, low cost, high recycling potential, and high efficiency compared to the other solvents tested in this study.     

Phases of denitrification following oxygen depletion in soil

The short-term response of soil denitrification to reduced aeration was studied using the acetylene inhibition method for the assay of denitrification. Two distinct phases of denitrification rate were observed. An initial constant rate, termed phase I, was not decreased by chloramphenicol, was increased slightly or not at all by organic carbon amendment, and lasted for 1–3 h. Phase I was attributed to the activity of pre-existing denitrifying enzymes in the soil microflora. Following phase I the denitrification rate increased; chloramphenicol inhibited this increase. In soils without organic-C amendment a second linear phase, termed phase II, was attained after 4–8 h of anaerobic incubation. The linearity of this phase was attributed to the full derepression of denitrifying enzyme synthesis by the indigenous population and to the lack of significant growth of denitrifiers. Phase I rate was dependent on the initial or in situ aeration state of the soil sample; phase II was not. Therefore, phase I may be more directly related to field denitrification rates.Denitrification rate changes following water saturation of soils in aerobic atmospheres were also examined. Rates were greatly increased by wetting but only after a lag of several hours. Our interpretation is that following wetting of natural soils, anaerobic or partially anaerobic conditions are established by respiration and reduced O₂ diffusion rate; this first eliminates O₂ inhibition then derepresses the synthesis of denitrifying enzymes. Although denitrifying enzymes are apparently present even in relatively dry soils, their activity is low until O₂ inhibition is eliminated. From this evidence we reason that most N is lost from soils during brief periods beginning a few hours after irrigation or a rainfall.     

Denitrification activity in the vadose zone beneath a sludge‐amended semi‐arid soil

Abstract

Soil cores were collected to a depth of 14 m from a Southwest semi‐arid soil amended with either anaerobically digested sludge or inorganic fertilizer. Twenty sections partitioned from each core were characterized for their physical and chemical properties. Denitrification potential was estimated in each core section in the laboratory using the acetylene reduction method. The sludge‐amended soil had significantly higher denitrification rates within and below the root zone than the fertilizer‐amended soil. Additionally, significant correlation values were obtained in both cores between denitrification rates and particle size distribution, moisture, and total organic carbon (C). Sludge applications in semi‐desert soils may add much needed organic C in the soil profile. This additional soluble organic C may help control nitrate (NO₃) ground water pollution by providing substrate C for denitrifying bacteria below the root zone.     

长期施肥对土nirS型反硝化细菌的影响及其与N2O排放的关系

土壤反硝化作用是土壤N₂O产生的重要过程,亚硝酸盐还原酶(NIR)催化的亚硝态氮(NO^-₂)还原为一氧化氮(NO)是反硝化作用的关键环节,研究长期施肥对反硝化微生物的影响及其与N₂O排放的关系对于全面理解土壤反硝化过程具有重要意义。基于28年的旱作雨养长期施肥试验,通过常规监测、定量PCR和高通量测序等探讨了长期不同施肥(不施肥CK、偏施肥的单施氮肥N和氮钾配施NK、以及氮磷钾平衡施肥NPK)下土N₂O排放和nirS反硝化细菌群落特征及两者之间的关系。结果表明:长期化肥施用(N,NK和NPK)均显著提高了N₂O累积排放量,其中平衡施肥(NPK)最高。长期化肥施用对nirS基因丰度和nirS型反硝化细菌的α-多样性无显著影响,但长期平衡施用化肥提高了uncultured_bacterium_2303和Rhodanobacter_sp._D206a的相对丰度,降低了unclassified_k_norank_d_Bacteria和unclassified_p_Proteobacteria的相对丰度,从而改变了nirS型反硝化细菌的群落结构组成。雨养旱作条件下,土壤有机碳(SOC)、全氮(TN)、有效磷(AP)和pH等土壤性质是土nirS型反硝化细菌群落结构组成变化的主要影响因素。土nirS型反硝化细菌群落结构组成对土壤N₂O排放具有显著影响,而nirS基因丰度和nirS型反硝化细菌多样性并没有显著影响。     

Mineralization of organic contaminants under aerobic and anaerobic conditions in sludge-soil mixtures

Background and Main Features  The mineralization of eight organic chemicals (surfactants, substituted aromatic compounds, di(2-ethylhexyl)phthalate and phenanthrene) was examined in sludge-soil mixtures under aerobic, denitrifying and methanogenic conditions. Results and Discussion  Most of the chemicals were extensively or partially mineralized under aerobic conditions with mineralization half-lives between 1.5 and 12.5 days. Linear tridecyl tetra ethoxylate, di(2-ethylhexyl)phthalate and 2,4-dinitrophenol were also mineralized partially under denitrifying conditions. No mineralization of the chemicals was observed under methanogenic conditions, with the exception of a minor mineralization of linear tridecyl tetraethoxylate. Conclusion  This study indicates that the examined organic chemicals may be rapidly degraded in sludge-amended fields under aerobic conditions, and that some of the chemicals may also be degraded during denitrification. Recommendations and Outlook  When investigating the degradation of sludge-bound chemicals in soil, it is relevant to consider both aerobic and anaerobic soil regimes due to spatial and temporal variations in the redox conditions within sludge and soil. The approach presented in this article may be used for evaluation of the long-term fate of sludge-bound chemicals in soil.     

Characterization of humic substances obtained from calcareous soils from Greece with various extractants. I. Chemical characterization

Humic substances of the Ah horizons of five differently calcareous soils from Greece under different vegetative covers were successively extracted with diethylether, acetone, ethanol and dioxane. Following these extractions, remaining humic substances were extracted with 0,1N NaOH. Humic substances were also directly extracted with 0,1N NaOH. The extracts of the organic solvents were separated by thin layer chromatography (T.L.C.) on silicagel plates, while the NaOH extracts were characterized by elemental and functional group analyses. The T.L.C. revealed that ?-acceptors and phenols were present in all extracts, while ?-donors and lignines were present only in the dioxane extracts. Lipides were present in the diethylether, dioxane and acetone extracts. Sugars were found only in the acetone extracts and reducing substances in the acetone and dioxane extracts. Some analytical characteristics of the humic substances were associated with the types of vegetative cover and/or soil properties. Humic acids (HA's) extracted with 0,1N NaOH from soil samples previously treated with organic solvents, generally varied in their carbon (C), carboxyl (COOH) and ash contents as compared with those extracted directly.     

Combined effects of soil waterlogging and compaction on rice (Oryza sativa L.) growth, soil aeration, soil N transformations and 15N discrimination

 The combined effects of soil compaction and soil waterlogging on the growth of two rice cultivars (Oryza sativa L., cultivars Kanto 168 and Koshihikari) and soil N transformations were studied in pots. Although waterlogging eliminated initial differences in mechanical resistance between compacted and loose soils, Kanto 168 and Koshihikari roots had, respectively, less biomass and a lower porosity if soil was compacted prior to waterlogging. The cause for this was probably established before waterlogging. Redox values showed that upland soils were well aerated. Loose waterlogged soils contained oxic sites, but compacted waterlogged soils did not. Potential denitrification was stimulated by waterlogging and, to a larger extent, by plant presence. Waterlogging lowered potential nitrifying capacities, by competition between plants and micro-organisms for NH₄ ⁺ rather than by oxygen shortage. Compaction prior to waterlogging benefited the potential nitrifying capacity of soils with either cultivar and the potential denitrifying capacity for soils with Koshihikari. Compaction had no effect on nitrification or denitrification in upland soils. N recoveries were low, especially in pots without plants, as a result from sampling strategy and N loss. On day 42/43 after potting, total δ¹⁵N values of waterlogged pots were positive, whereas after 22 days all pots had negative total δ¹⁵N values. Final δ¹⁵N values of plant parts from waterlogged and upland soils were positive and negative, respectively. Although the δ¹⁵N values generally accorded well with the other results, they did not support higher N losses from compacted waterlogged soils than from loose waterlogged soils with plants, as suggested by potential denitrifying activities. Received: 4 February 2000     

Denitrification enzyme activity is limited by soil aeration in a wastewater-irrigated forest soil

 In land-based wastewater treatment systems (LTS), denitrification is an important nitrogen removal process. We investigated the factors limiting the denitrifying population in a forested LTS, by studying the individual and combined effects of soil aeration, water content, nitrate and carbon on denitrification enzyme activity (DEA). The size of the soil denitrifying population in the LTS appeared to be limited by soil aeration, and limiting oxygen availability increased the denitrifying population above that observed in the field. Furthermore, we found that wastewater irrigation altered the short-term response of denitrifiers to anaerobic soil conditions. Under low oxygen conditions, denitrifiers in the wastewater-irrigated soils produced enzymes sooner and at a greater rate than soils without a history of wastewater irrigation. We propose that the size of the denitrifying population cannot be expected to be large in free-draining, coarsely textured soils even when provided with additional nitrogen and water inputs. Received: 11 October 1999     

Characterization of humic substances obtained from calcareous soils from Greece with various extractants. II. Physical characterization

Humic substances from the Ah horizons of five differently calcareous soils from Greece were successively extracted with organic solvents and/or 0.1 N NaOH and they were characterized by UV/VIS/IR spectroscopy and differential thermal analysis (DTA). Diethylether, acetone and dioxane extracts contained non-humic substances such as aliphatic and aromatic ethers, esters, alcoholes and long aliphatic chains, while low molecular weight humic substances were found in the ethanol extracts. All the extracts were found to contain phenolic and ?-acceptor compounds. Some differences in the physical characteristics of the humic substances were associated with the kinds of vegetation. The IR-spectra showed no essential differences between HA's and FA's extracted directly with NaOH or after previous soil extraction with organic solvents. HA's extracted directly by NaOH from soil samples under grass had a higher degree of condensation and thermal stability and generally higher carbon contents than those from soils under pine, cultivation and sedges.     

The effect of oxygen,pH and organic carbon on soil-layer specific denitrifying capacity in acid coniferous forest

Emissions of N₂O from acid coniferous forest soils are found to be low and considered to be due to nitrification rather than denitrification. Recently we have demonstrated soil-layer specific denitrification in a Scots pine forest in the Netherlands. N₂O production, in the presence of high concentrations of acetylene, was detected in the intact needle fraction but was absent in the fragmentation layer of this forest soil. To identify the factors regulating denitrification activity, in the present study the effects of oxygen, pH and organic carbon were investigated in the needle and fragmentation fraction of acid coniferous forest soils. Under natural circumstances denitrification in the Scots pine needles was higher than in Douglas fir needles and absent in fragmentation material. Under anaerobic conditions comparable N₂O production in the two soil types was found in needle suspensions of both forest types, indicating that differences in anaerobic microsites were responsible for different N₂O production under aerobic circumstances. Denitrifying capacity was absent in the fragmentation layer; under anaerobic circumstances little N₂O was produced. Neither an addition of available carbon (glucose and succinate) nor an increase in pH revealed a denitrifying capacity comparable to that observed in needles. The increase in pH, under anaerobic circumstances, was most effective on N₂O production in the fragmentation material. The denitrifying capacity in the fragmentation layer remained low during short-term incubation under optimal conditions. This indicates the presence of a low denitrifying population, most likely due to aerobic conditions, low pH and low available organic carbon. Although the significance of N₂O production under natural conditions remains speculative, this study seeks to clarify soil-layer specific denitrifying activity in acid coniferous forest soils.     

Biological and chemical properties of arable soils affected by long-term organic and inorganic fertilizer applications

 Using soils from field plots in four different arable crop experiments that have received combinations of manure, lime and inorganic N, P and K for up to 20 years, the effects of these fertilizers on soil chemical properties and estimates of soil microbial community size and activity were studied. The soil pH was increased or unaffected by the addition of organic manure plus inorganic fertilizers applied in conjunction with lime, but decreased in the absence of liming. The soil C and N contents were greater for all fertilized treatments compared to the control, yet in all cases the soil samples from fertilized plots had smaller C:N ratios than soil from the unfertilized plots. The soil concentrations of all the other inorganic nutrients measured were greater following fertilizer applications compared with the unfertilized plots, and this effect was most marked for P and K in soils from plots that had received the largest amounts of these nutrients as fertilizers. Both biomass C determined by chloroform fumigation and glucose-induced respiration tended to increase as a result of manure and inorganic fertilizer applications, although soils which received the largest additions of inorganic fertilizers in the absence of lime contained less biomass C than those to which lime had been added. Dehydrogenase activity was lower in soils that had received the largest amounts of fertilizers, and was further decreased in the absence of lime. This suggests that dehydrogenase activity was highly sensitive to the inhibitory effects associated with large fertilizer additions. Potential denitrification and anaerobic respiration determined in one soil were increased by fertilizer application but, as with both the microbial biomass and dehydrogenase activity, there were significant reductions in both N₂O and CO₂ production in soils which received the largest additions of inorganic fertilizers in the absence of lime. In contrast, the size of the denitrifying component of the soil microbial community, as indicated by denitrifying enzyme activity, was unaffected by the absence of lime at the largest rate of inorganic fertilizer applications. The results indicated differences in the composition or function of microbial communities in the soils in response to long-term organic and inorganic fertilization, especially when the soils were not limited. Received: 10 March 1998     

Isomerization of trans-astaxanthin to cis-isomers in organic solvents.

The isomerization of trans-astaxanthin to cis-isomers in organic solvents was investigated. trans-Astaxanthin was dissolved in dimethyl sulfoxide, dichloromethane, chloroform, acetone, methanol, acetonitrile, and a mixture of dichloromethane and methanol (25:75) respectively, and heated at 35 degrees C followed by analyzing cis- and trans-astaxanthins in the solutions using HPLC. The isomerization rates of trans-astaxanthin were dependent on the solvent, and the following order was found: dichloromethane > chloroform > the mixture of dichloromethane and methanol (25:75) > methanol > acetonitrile > acetone > dimethyl sulfoxide. In different solvents, the relative contents of 9-cis- and 13-cis-astaxanthins formed during isomerization were different. In all solvents, 13-cis-isomer was the main cis-isomer from trans-astaxanthin. trans-Astaxanthin dissolved in dichloromethane or chloroform was very readily isomerized to cis-isomers, especially for dichloromethane, in which a maximum isomerization percentage was found and an equilibrium practically was reached after an appropriate time interval. Results also indicated that a higher temperature could promote markedly the isomerization of trans-astaxanthin.     

Solvent-based washing removes lipophilic contaminant interference with phospholipid fatty acid analysis of soil communities

Phospholipid fatty acid (PLFA) analysis is an informative method for characterising and quantifying changes in the phenotypic profile of the soil microbial community when soils are exposed to chemical toxicants and other xenobiotics. However, where such materials are lipophilic, a range of non-polar compounds can be co-extracted with PLFAs and can consequently mask PLFA chromatograms. We found this to be the case with the lipophilic anti-microbial compound triclosan, which can enter the soil via the addition of sewage sludge. A simple method of washing soil in solvent prior to extraction was developed in order to remove triclosan without altering the relative abundance of PLFAs. Three contrasting soils were spiked with 500 mg kg⁻¹ of triclosan before being washed with methanol (MeOH), dichloromethane (DCM), hexane or aqueous solutions of these solvents. PLFAs were then extracted and analysed. All treatments were found to remove triclosan effectively, allowing all peaks to be identifiable. Whilst the polar solvents MeOH and DCM significantly altered the relative abundance of extracted fatty acids in most of the soils tested, soil washing with a small quantity of hexane was able to remove triclosan whilst best preserving the fidelity of the PLFA profiles.     

Cr3+污染对稻田土壤的反硝化细菌种群数量及其活性影响的研究

在实验室条件下,对黄松稻田土壤、紫色稻田土壤和红壤稻田土的反硝化细菌(DenitrifyingBacteria,DNB)种群数量及其反硝化活性,以及Cr2(SO4)36H2O处理4周后Cr3+污染对DNB种群数量及其反硝化活性影响进行了研究。结果表明,紫色稻田土壤、黄松稻田土壤和红壤稻田土的DNB种群数量每克干土分别为(59.04～157.59)104cfu、(42.89～108.97)104cfu和(32.14～75.30)104cfu。在每千克干土中加入200mgCr3+时,对稻田土的DNB种群数量及其活性有明显的抑制作用,但稻田土壤还能通过自身的能力,在培养3周后完全恢复稻田土壤的DNB种群数量及其活性。随着Cr3+加入量的增加,Cr3+对DNB的毒性越来越大,稻田土壤通过自身来恢复DNB种群数量及其活性越来越困难,但可通过外界力量(如调节土壤pH)逐渐恢复稻田土壤的生物活性。