Heterotrophic Nitrification in an Acid Forest Soil and by an Acid-Tolerant Fungus

Nitrate was formed from ammonium at pH 3.2 to 6.1 in suspensions of a naturally acid forest soil; the maximum rates of formation occurred at ca. pH 4 to 5. Nitrate was also formed from soil nitrogen in suspensions incubated at 50°C. Autotrophic nitrifying bacteria could not be isolated from this soil. Enrichment cultures produced nitrate in a medium with β-alanine if much soil was added to the medium, and nitrite but not nitrate was formed in the presence of small amounts of soil. Nitrification by these enrichments was abolished by eucaryotic but not procaryotic inhibitors. A strain of Absidia cylindrospora isolated from this soil was found to produce nitrate and nitrite in a medium with β-alanine at pH values ranging from 4.0 to 4.8. Nitrate production by A. cylindrospora required the presence of sterile soil. Free and bound hydroxylamine, hydroxamic acids, and primary aliphatic nitro compounds did not accumulate during the conversion of β-alanine to nitrite by the fungus. The organism also formed nitrite from ammonium in a medium containing acetate. We suggest that nitrification in this soil is a heterotrophic process catalyzed by acid-tolerant fungi and not by autotrophs or heterotrophs in nonacid microsites.     

Heterotrophic Nitrification by Arthrobacter sp

Arthrobacter sp. isolated from sewage oxidized ammonium to hydroxylamine, a bound hydroxylamine compound, a hydroxamic acid, a substance presumed to be a primary nitro compound, nitrite, and nitrate. The concentration of free hydroxylamine-nitrogen reached 15 mug/ml. The identification of hydroxylamine was verified by mass spectrometric analysis of its benzophenone oxime derivative. The bound hydroxylamine was tentatively identified as 1-nitrosoethanol on the basis of its mass spectrum, chemical reactions, and infrared and ultraviolet spectra. Hydroxylamine formation by growing cells was relatively independent of pH, but the accumulation of nitrite was strongly favored in alkaline solutions. The formation of hydroxylamine but not nitrite was regulated by the carbon to nitrogen ratio of the medium. The hydroxamic acid was the dominant product of nitrification in iron-deficient media, but hydroxylamine, nitrite, and 1-nitrosoethanol formation was favored in iron-rich solutions. Heterotrophic nitrification by Arthrobacter sp. was not inhibited by several compounds at concentrations which totally inhibited autotrophic nitrification.     

异养硝化作用酶学研究进展

对异养硝化作用作了简单的概述,着重从异养硝化作用和好氧反硝化作用的偶联机理、其中涉及的关键酶和相关基因几方面综述了异养硝化作用的研究进展,最后还提出了研究展望。     

异养硝化细菌脱氮特性及研究进展

异养硝化细菌能够在利用有机碳源生长的同时将含氮化合物硝化生成羟胺、亚硝酸盐、硝酸盐等产物, 多数还能同时进行好氧反硝化作用, 直接将硝化产物转化为含氮气体。因此, 这类细菌已成为废水处理中生物脱氮新工艺的重要研究对象。本文综述了目前所分离出的一些异养硝化菌的脱氮特性, 分析了各种环境条件如温度、pH、溶解氧、碳源类型、C/N以及抑制剂等对异养硝化菌的影响, 并介绍了异养硝化菌的应用现状及前景。     

Responses of Soil,Heterotrophic, and Autotrophic Respiration to Experimental Open-Field Soil Warming in a Cool-Temperate Deciduous Forest

How global warming will affect soil respiration (R _S) and its source components is poorly understood despite its importance for accurate prediction of global carbon (C) cycles. We examined the responses of R _S, heterotrophic respiration (R _H), autotrophic respiration (R _A), nitrogen (N) availability, and fine-root biomass to increased temperature in an open-field soil warming experiment. The experiment was conducted in a cool-temperate deciduous forest ecosystem in northern Japan. As this forest is subjected to strong temporal variation in temperature, on scales ranging from daily to seasonal, we also investigated the temporal variation in the effects of soil warming on R _S, R _H, and R _A. Soil temperature was continuously elevated by about 4.0°C from 2007 to 2014 using heating wires buried in the soil, and we measured soil respiratory processes in all four seasons from 2012 to 2014. Soil warming increased annual R _S by 32–45%, but the magnitude of the increase was different between the components: R _H and R _A were also stimulated, and increased by 39–41 and 17–18%, respectively. Soil N availability during the growing season and fine-root biomass were not remarkably affected by the warming treatment. We found that the warming effects varied seasonally. R _H increased significantly throughout the year, but the warming effect showed remarkable seasonal differences, with the maximum stimulation in the spring. This suggests that warmer spring temperature will produce a greater increase in CO₂ release than warmer summer temperatures. In addition, we found that soil warming reduced the temperature sensitivity (Q ₁₀) of R _S. Although the Q ₁₀ of both R _H and R _A tended to be reduced, the decrease in the Q ₁₀ of R _S was caused mainly by a decrease in the response of R _A to warming. These long-term results indicate that a balance between the rapid and large response of soil microbes and the acclimation of plant roots both play important roles in determining the response of R _S to soil warming, and must be carefully considered to predict the responses of soil C dynamics under future temperature conditions.     

异养硝化⁃好氧反硝化复合菌剂在垃圾渗滤液处理中的应用

垃圾渗滤液中往往含有高浓度的有机物、氨氮等污染物。异养硝化-好氧反硝化型微生物能在脱氮的同时去除部分有机物,但目前对于相关混合菌剂直接应用于垃圾渗滤液处理的研究较少。从垃圾渗滤液中筛选出6株异养硝化-好氧反硝化菌株并组配成复合菌剂F6,探究菌剂在垃圾渗滤液中的脱氮效果。分别以单一菌株和复合菌剂F6为投放原料;以不同碳氮比、活性炭浓度、转速以及微量元素浓度为影响因素,研究复合菌剂对于垃圾渗滤液中氨氮(NH{}_{\mathrm{4}}^{+}-N)、总氮(total nitrogen, TN)、化学需氧量(chemical oxygen demand, COD)的去除性能。结果表明,提高碳氮比和微量元素浓度能够促进复合菌剂F6的降解效果;当接种量为10%、碳氮比为15%时,F6对NH{}_{\mathrm{4}}^{+}-N、TN、COD的去除率分别为74.69%、89.23%和83.50%,与不添加活性炭的处理相比,分别提高了约18.46%、20.97%和7.98%。复合异养硝化-好氧反硝化菌剂F6对高氨氮垃圾渗滤液去除NH{}_{\mathrm{4}}^{+}-N、TN、COD等方面具有良好的应用前景。     

Short- and Long-term Influence of Litter Quality and Quantity on Simulated Heterotrophic Soil Respiration in a Lowland Tropical Forest

Heterotrophic soil respiration (SR_H) alone can contribute up to 50% of total ecosystem respiration in tropical forests. Whereas the abiotic controls of SR_H have been extensively studied, the influence of plant traits is less well characterised. We used field experiments and a modelling approach to test the relative influence of plant traits on SR_H in lowland tropical forest in French Guiana. We measured leaf- and root litter traits for five common tree species and conducted a root decomposition experiment to evaluate the influence of root chemistry on decay rates. We measured SR_H in trenched plots and used our field measurements to parameterize and test the Century model of soil C dynamics. Overall, the Century model performed well in simulating SR_H, and species-specific root decomposition in Century corresponded well to decomposition rates measured in situ. Root litter characterized by low lignin-to-nitrogen ratios decomposed more rapidly than low-quality root litter during the first 6 months. Model runs over different time scales revealed that litter quality substantially influenced SR_H on an annual time-scale by determining the rates of root- and leaf litter decomposition. However, litter mass had an overriding influence on SR_H over the longer term in 20-year model runs. Synthesis Using simple plant trait data to parameterise the Century model, we were able to accurately simulate changes in SR_H in a lowland tropical forest. Our results suggest that this approach could be used to predict changes in tropical soil C dynamics under global change scenarios by including data on changes in plant productivity and C inputs to the soil (for example litterfall and root turnover).     

Nitrification and Autotrophic Nitrifying Bacteria in a Hydrocarbon-Polluted Soil

In vitro ammonia-oxidizing bacteria are capable of oxidizing hydrocarbons incompletely. This transformation is accompanied by competitive inhibition of ammonia monooxygenase, the first key enzyme in nitrification. The effect of hydrocarbon pollution on soil nitrification was examined in situ. In a microcosm study, adding diesel fuel hydrocarbon to an uncontaminated soil (agricultural unfertilized soil) treated with ammonium sulfate dramatically reduced the amount of KCl-extractable nitrate but stimulated ammonium consumption. In a soil with long history of pollution that was treated with ammonium sulfate, 90% of the ammonium was transformed into nitrate after 3 weeks of incubation. Nitrate production was twofold higher in the contaminated soil than in the agricultural soil to which hydrocarbon was not added. To assess if ammonia-oxidizing bacteria acquired resistance to inhibition by hydrocarbon, the contaminated soil was reexposed to diesel fuel. Ammonium consumption was not affected, but nitrate production was 30% lower than nitrate production in the absence of hydrocarbon. The apparent reduction in nitrification resulted from immobilization of ammonium by hydrocarbon-stimulated microbial activity. These results indicated that the hydrocarbon inhibited nitrification in the noncontaminated soil (agricultural soil) and that ammonia-oxidizing bacteria in the polluted soil acquired resistance to inhibition by the hydrocarbon, possibly by increasing the affinity of nitrifying bacteria for ammonium in the soil.     

Heterotrophic Fixation of CO2 in Soil

The occurrence of heterotrophic CO₂ fixation by soil microorganisms was tested in several mineral soils differing in pH and two artificial soils (a mixture of silica sand, alfalfa powder, and nutrient medium inoculated with a soil suspension). Soils were incubated at ambient (∼0.05 vol%) and elevated (∼5 vol%) CO₂ concentrations under aerobic conditions for up to 21 days. CO₂ fixation was detected using either a technique for determining the natural abundance of ¹³C or by measuring the distribution of labeled ¹⁴C-CO₂ in soil and bacteria. The effects of elevated CO₂ on microbial biomass (direct counts, chloroform fumigation extraction method), composition of microbial community (phospholipid fatty acids), microbial activity (respiration, dehydrogenase activity), and turnover rate were also measured. Heterotrophic CO₂ fixation was proven in all soils under study, being higher in neutral soils. The main portion of the fixed CO₂ (98–99%) was found in extracellular metabolites while only ∼1% CO₂ was incorporated into microbial cells. High CO₂ concentration always induced an increase in microbial activity, changes in the composition of the microbial community, and a decrease in microbial turnover. The results suggest that heterotrophic CO₂ fixation could be a widespread process in soils.     

Effects of Organic Acids on Heterotrophic Nitrification by Alcaligenes faecalis OKK17

Factors affecting heterotrophic nitrification by Alcaligenes faecalis OKK17, which was isolated from sewage sludge, were examined. Specific nitrifying activity increased as the pH increased up to 8.5. Most of the nitrogenous compounds (88%) in the culture supernatant were converted to hydroxylamine or nitrite at pH 9 but 87% of them remained as ammonium at pH 7. These results imply that the substrate for heterotrophic nitrification is ammonium and that the organism oxidizes ammonium to lower its toxic effect. Although the addition of acetate to a defined medium increased growth of the bacterium up to C/N = ca. 6, the accumulation of nitrification products almost paralleled the growth and the specific nitrifying activity decreased. Pyruvate and oxaloacetate increased the specific nitrifying activity six- to eightfold compared with the other organic acids examined, but the key enzyme activities in the glyoxylate cycle were not increased. Acetate, glyoxylate, and malonate did not increase the specific nitrifying activity, but they increased the enzyme activities. These results imply that the involvement of acetate metabolism in the heterotrophic nitrification is unlikely.     

Variation in Heterotrophic and Autotrophic Nitrifier Populations in Relation to Nitrification in Organic Soils

The occurrence of heterotrophic and autotrophic nitrifiers in Pahokee muck and the role of these organisms in the ecosystem were assessed by surveying their population densities under different field conditions and by observing the relationship of these populations with aerobic bacteria and soil moisture. Heterotrophic nitrifier populations varied from 2.0 × 10⁵ to 3.8 × 10⁶ bacteria per cm³ of muck in surface fallow (bare) Pahokee muck during the annual cycle. This population decreased 40-fold between the surface and the 60- to 70-cm depths of soil. Similar variations were noted with autotrophic nitrifier populations. Significant correlations were found between heterotrophic nitrifiers and both soil moisture and aerobic bacteria. These relationships did not exist for the autotrophic nitrifiers. In soil that had been heated to kill the autotrophic nitrifiers, while preserving a population of the heterotrophs, and then amended with sodium acetate or ammonium sulfate or both, no nitrate or nitrite accumulated, although significant increases in heterotrophic nitrifiers were detected. In unheated control soil, nitrate plus nitrite-N increased from 14.3 to 181 μg/g of wet soil, and 48 μg of nitrite-N per g was produced. These data suggest that the autotrophic nitrifiers were the sole population responsible for nitrification in Pahokee muck.     

Identification and Characterization of Lipolytic Enzymes from a Peat-Swamp Forest Soil Metagenome

In this work, a metagenomic library was generated from peat-swamp forest soil obtained from Narathiwat Province, Thailand. From a fosmid library of approximately 15,000 clones, six independent clones were found to possess lipolytic activity at acidic pH. Analysis of pyrosequencing data revealed six ORFs, which exhibited 34–71% protein similarity to known lipases/esterases. A fosmid clone, designated LP8, which demonstrated the highest level of lipolytic activity under acidic conditions and demonstrated extracellular activity, was subsequently subcloned and sequenced. The full-length lipase/esterase gene, estPS2, was identified. Its deduced amino acid was closely related to a lipolytic enzyme of an uncultured bacterium, and contained the highly conserved motif of a hormone-sensitive family IV lipase. The EstPS2 enzyme exhibited highest activity toward p-nitrophenyl butyrate (C₄) at 37 °C at pH 5, indicating that it was an esterase with activity and secretion characteristics suitable for commercial development.     

Effects of Forest Age on Soil Autotrophic and Heterotrophic Respiration Differ between Evergreen and Deciduous Forests

We examined the effects of forest stand age on soil respiration (SR) including the heterotrophic respiration (HR) and autotrophic respiration (AR) of two forest types. We measured soil respiration and partitioned the HR and AR components across three age classes ∼15, ∼25, and ∼35-year-old Pinus sylvestris var. mongolica (Mongolia pine) and Larix principis-rupprechtii (larch) in a forest-steppe ecotone, northern China (June 2006 to October 2009). We analyzed the relationship between seasonal dynamics of SR, HR, AR and soil temperature (ST), soil water content (SWC) and normalized difference vegetation index (NDVI, a plant greenness and net primary productivity indicator). Our results showed that ST and SWC were driving factors for the seasonal dynamics of SR rather than plant greenness, irrespective of stand age and forest type. For ∼15-year-old stands, the seasonal dynamics of both AR and HR were dependent on ST. Higher Q₁₀ of HR compared with AR occurred in larch. However, in Mongolia pine a similar Q₁₀ occurred between HR and AR. With stand age, Q₁₀ of both HR and AR increased in larch. For Mongolia pine, Q₁₀ of HR increased with stand age, but AR showed no significant relationship with ST. As stand age increased, HR was correlated with SWC in Mongolia pine, but for larch AR correlated with SWC. The dependence of AR on NDVI occurred in ∼35-year-old Mongolia pine. Our study demonstrated the importance of separating autotrophic and heterotrophic respiration components of SR when stimulating the response of soil carbon efflux to environmental changes. When estimating the response of autotrophic and heterotrophic respiration to environmental changes, the effect of forest type on age-related trends is required.     

一株异养硝化细菌的分离及系统发育分析

从大棚土壤中分离到一株异养型硝化细菌,命名为菌株HN,分离菌株为革兰氏染色阳性,球状或杆状。菌落颜色为橙红色。该菌株能以乙酰胺为唯一碳源和氮源进行氨化作用和硝化作用并产生亚硝酸。以硝酸钠为氮源时能进行反硝化作用。部分长度的16S rDNA 序列分析表明,分离菌株HN与Rhodococcus ruber 具有99％相似性。并用PHYLIPS程序将该菌株与报道的相关硝化细菌进行系统发育进化分析。本文首次报道Rhodococcus sp.HN为异养型亚硝化细菌。     

Radiocarbon-Based Assessment of Heterotrophic Soil Respiration in Two Mediterranean Forests

The amount of soil organic carbon (SOC) released into the atmosphere as carbon dioxide (CO₂), which is referred to as heterotrophic respiration (Rh), is technically difficult to measure despite its necessity to the understanding of how to protect and increase soil carbon stocks. Within this context, the aim of this study is to determine Rh in two Mediterranean forests dominated by pine and oak using radiocarbon measurements of the bulk SOC from different soil layers. The annual Rh was 3.22 Mg C ha^?1 y^?1 under pine and 3.13 Mg C ha^?1 y^?1 under oak, corresponding to 38 and 31% of the annual soil respiration, respectively. The accuracy of the Rh values was evaluated by determining the net primary production (NPP), as the sum of the Rh and the net ecosystem production measured by eddy covariance, then comparing it with the NPP obtained through independent biometric measurements. No significant differences were observed, which suggested the suitability of our methodology to infer Rh. Assuming the C inputs to soil to consist exclusively of the aboveground and belowground litter and the C output exclusively of the Rh, both soils were C sinks, which is consistent with a previous modeling study that was performed in the same stands. In conclusion, radiocarbon analysis of bulk SOC provided a reliable estimate of the average annual amount of soil carbon released to the atmosphere; hence, its application is convenient for calculating Rh because it utilizes only a single soil sampling and no time-consuming monitoring activities.     

Biochar Decelerates Soil Organic Nitrogen Cycling but Stimulates Soil Nitrification in a Temperate Arable Field Trial

Biochar production and subsequent soil incorporation could provide carbon farming solutions to global climate change and escalating food demand. There is evidence that biochar amendment causes fundamental changes in soil nutrient cycles, often resulting in marked increases in crop production, particularly in acidic and in infertile soils with low soil organic matter contents, although comparable outcomes in temperate soils are variable. We offer insight into the mechanisms underlying these findings by focusing attention on the soil nitrogen (N) cycle, specifically on hitherto unmeasured processes of organic N cycling in arable soils. We here investigated the impacts of biochar addition on soil organic and inorganic N pools and on gross transformation rates of both pools in a biochar field trial on arable land (Chernozem) in Traismauer, Lower Austria. We found that biochar increased total soil organic carbon but decreased the extractable organic C pool and soil nitrate. While gross rates of organic N transformation processes were reduced by 50–80%, gross N mineralization of organic N was not affected. In contrast, biochar promoted soil ammonia-oxidizer populations (bacterial and archaeal nitrifiers) and accelerated gross nitrification rates more than two-fold. Our findings indicate a de-coupling of the soil organic and inorganic N cycles, with a build-up of organic N, and deceleration of inorganic N release from this pool. The results therefore suggest that addition of inorganic fertilizer-N in combination with biochar could compensate for the reduction in organic N mineralization, with plants and microbes drawing on fertilizer-N for growth, in turn fuelling the belowground build-up of organic N. We conclude that combined addition of biochar with fertilizer-N may increase soil organic N in turn enhancing soil carbon sequestration and thereby could play a fundamental role in future soil management strategies.     

Nitrogen Redox Metabolism of a Heterotrophic, Nitrifying-Denitrifying Alcaligenes sp. from Soil

Metabolic characteristics of a heterotrophic, nitrifier-denitrifier Alcaligenes sp. isolated from soil were further characterized. Pyruvic oxime and hydroxylamine were oxidized to nitrite aerobically by nitrification-adapted cells with specific activities (V_max) of 0.066 and 0.003 μmol of N × min⁻¹ × mg of protein⁻¹, respectively, at 22°C. K_m values were 15 and 42 μM for pyruvic oxime and hydroxylamine, respectively. The greater pyruvic oxime oxidation activity relative to hydroxylamine oxidation activity indicates that pyruvic oxime was a specific substrate and was not oxidized appreciably via its hydrolysis product, hydroxylamine. When grown as a denitrifier on nitrate, the bacterium could not aerobically oxidize pyruvic oxime or hydroxylamine to nitrite. However, hydroxylamine was converted to nearly equimolar amounts of ammonium ion and nitrous oxide, and the nature of this reaction is discussed. Cells grown as heterotrophic nitrifiers on pyruvic oxime contained two enzymes of denitrification, nitrate reductase and nitric oxide reductase. The nitrate reductase was the dissimilatory type, as evidenced by its extreme sensitivity to inhibition by azide and by its ability to be reversibly inhibited by oxygen. Cells grown aerobically on organic carbon sources other than pyruvic oxime contained none of the denitrifying enzymes surveyed but were able to oxidize pyruvic oxime to nitrite and reduce hydroxylamine to ammonium ion.     

A Strain of Pseudomonas sp. Isolated from Piggery Wastewater Treatment Systems with Heterotrophic Nitrification Capability in Taiwan

A high concentration of NH₄⁺ in piggery wastewater is major problem in Taiwan. Therefore, in our study, we isolated native heterotrophic nitrifiers for piggery wastewater treatment. Heterotrophic nitrifier AS-1 was isolated and characterized from the activated sludge of a piggery wastewater system. Sets of triplicate crimp-sealed serum bottles were used to demonstrate the heterotrophic nitrifying capability of strain AS-1 in an incubator at 30°C. All serum bottles contained 80 mL medium, and the remainder of the bottle headspace was filled with pure oxygen. The experimental results showed that 2.5 ± 0.2 mmol L⁻¹ NH₄⁺ was removed by 58 hours, and, eventually, 1.5 ± 0.5 mmol L⁻¹ N₂ and 0.2 ± 0.0 mmol L⁻¹ N₂O were produced. The removal rate of NH₄⁺ by the strain AS-1 was 1.75 mmol NH₄⁺ g cell⁻¹ h⁻¹. This strain was then identified as Pseudomonas alcaligenes (97% identity) by sequencing its 16S rDNA and comparing it with other microorganisms. Thus, strain AS-1 displays high promise for future application for in situ NH₄⁺ removal from piggery wastewater.