Properties of neutral phosphate buffer extractable organic matter in soils revealed using size exclusion chromatography and fractionation with polyvinylpyrrolidone

Abstract

A neutral phosphate buffer (NPB) extraction method has been used to estimate the amount of available N in soil. However, the properties of soil NPB-extractable organic N have not been fully elucidated. The purpose of the present study was to characterize the properties of organic matter in the NPB extracts of soils. The NPB extracts were obtained from three soil samples, and the organic matter in the extracts was separated into three fractions according to its solubility in acid and adsorption onto polyvinylpyrrolidone (PVP). High-performance size exclusion chromatography (HPSEC) with ultraviolet (UV) and fluorescence detections was applied to the NPB extracts and their fractions. The HPSEC analysis of the NPB extract revealed the presence of a single broad peak, irrespective of the detection methods. The broad peak was identified as humic substances using the on-flow measurements of UV absorption spectra and fluorescence emission spectra. Among the fractions, the PVP-non-adsorbed fulvic acid (FA) fraction accounted for the largest proportion of organic C or N in the NPB extract, followed by the PVP-adsorbed FA and humic acid (HA) fractions. The peak of humic substances was observed for all fractions using HPSEC with the on-flow measurement of UV absorption and fluorescence emission spectra. The molecular weight of the humic substances varied with each fraction. When the Coomassie Blue-reactive substances (CBRS) were quantified using a Bradford protein assay, they were detected in the NPB extract and almost half were distributed in the PVP-non-adsorbed FA fraction. However, humic substances were considered to be the main constituents of CBRS in the soil NPB extract because of their reactivity with Coomassie Blue and the absence of proteinaceous materials. Furthermore, an incubation experiment revealed that the organic matter available to microorganisms was included in the HA and PVP-non-adsorbed FA fractions. Based on the HPSEC analysis of the NPB extracts and their fractions, it was observed that the humic substances in the NPB extract, particularly in the HA and PVP-non-adsorbed FA fractions, were available to microorganisms.     

Digestion of peptidic residues in humic substances by an alkali-stable and humic-acid-tolerant proteolytic activity in the gut of soil-feeding termites

Previous feeding experiments have shown that soil-feeding termites (Termitidae: Termitinae) preferentially mineralize the peptidic component of synthetic humic acids, but nothing was known about the mechanism involved in digestion. Here, we studied the hydrolysis of humus-stabilized peptides in gut extracts of Cubitermes orthognathus by measuring the release of radiolabel from ¹⁴C-peptide-labeled synthetic humic acids. Gut extracts exhibited proteolytic activity over a wide pH range (from 4 to 12) with a maximum at about pH 8. The highest activity was located in the gut section containing the midgut and the extremely alkaline (up to pH 12) mixed segment. Chemical hydrolysis at in situ pH (up to pH 12) was negligible. Proteolytic activity in the hindgut fluid was generally relatively low, but alkaline proteases dominated in the anterior hindgut. When compared to other alkaline proteases, the proteolytic activity of gut extracts had a higher alkali-stability and tolerance to humic acids than subtilisin and an alkaline protease of Streptomyces griseus. Gut extracts also hydrolyzed the peptidic component of synthetic humic acids more efficiently than the commercial enzymes. Together with previous results, this study strongly supports the hypothesis that soil-feeding termites mobilize and digest the peptidic component of organic nitrogen in soil humic substances by a combination of proteolytic activities and extreme alkalinity in their intestinal tract.     

Protease extraction from soil by sodium pyrophosphate and chemical characterization of the extracts

Two arable soils and one pasture soil had previously been air-dried for 6 d and stored at room temperature. The enzyme activities remaining after this treatment were constant. The soils were then extracted with 140 mM sodium pyrophosphate at pH 7.1. Amino acid N and total organic C content of soils and soil extracts, together with humic and fulvic acids content of soil extracts were determined. Total organic C was determined in soil residues obtained after extraction. Chemical characterization of the organic matter of soils, soil extracts and soil residues was carried out by pyrolysis–gas chromatography (Py–GC). Protease activity was determined in soil extracts and soil residues by using three different substrates: N-benzoyl- -argininamide (BAA), specific for trypsin; N-benzyloxycarbonyl- -phenylalanyl -leucine (ZPL), specific for carboxypeptidases, and casein, essentially non-specific. Comparative studies between specific activities referred to organic C in soils, soil extracts and soil residues and their corresponding pyrogram composition, and also between total extracted or residual activity and the humine or unhumified organic matter content of the corresponding soil, allowed us to establish hypotheses about the type of organic matter the enzymes are associated with. From 12% to 21% of the soil organic C (33% to 39% of which were humic acids) and from 3% and 18% of amino acid N were extracted from soil using pyrophosphate. Py–GC analyses showed that pyrophosphate was effective in extracting condensed humic substances and glycoproteins and that the organic matter present in soil extracts was especially rich in intact or partially-decomposed fresh residues of carbohydrate origin and also in certain humus-associated proteins. Extracted BAA-hydrolysing activity accounted for 11% to 36% of the soil activity, depending on soil type. Extracted ZPL- and casein-hydrolysing activities were, with one exception, remarkably high, accounting for about 100% or even more of the soil activity, depending on soil type. According to the results BAA-hydrolysing proteases are probably mostly associated with highly condensed humus, ZPL-hydrolysing proteases with less condensed humic substances and casein-hydrolysing proteases with fresh organic matter.     

Comparison of microbiological methods for evaluating quality and fertility of soil

Routine soil testing procedures that are rapid and accurate are needed to evaluate C and N mineralization in agricultural soils in order to determine soil quality and fertility. Laboratory methods were compared for their usefulness in determining soil microbial biomass and potential activity in a Weswood silty clay loam (fine, mixed, thermic Fluventic Ustochrept) subjected to long-term tillage, crop sequence, and N-fertilizer management practices. The methods included basal soil respiration, net N mineralization during a 10-day incubation, soil microbial biomass C with the chloroform fumigation-incubation technique with and without subtracting a control value, soil microbial biomass N with the chloroform fumigation-incubation technique, substrate-induced respiration, and arginine ammonification. All methods were highly correlated with each other and, therefore, appear to adequately reflect soil microbial biomass and potential activity under laboratory conditions. The longer incubation times used with the basal soil respiration, N mineralization, and microbial biomass C and N assays resulted in higher correlations and lower variation among replications compared to the shorter incubation times used with substrate-induced respiration and arginine ammonification. The relatively rapid procedural time (3 h) required for the latter two assays could make these methods more attractive for routine soil testing, although multiple assays on the same sample may be necessary because these methods are less precise than the incubation methods that require 10 days.     

Bovine serum albumin and Triton X-100 greatly increase phosphomonoesterases and arylsulphatase extraction yield from soil

Extraction of arylsulphatase (aryS), acid (acP) and alkaline phosphomonoesterase (alkP) from six different soils using sodium pyrophosphate (0.14 M, pH 7.1) yielded brown extracts displaying enzymatic activity mostly below detection limit. Tris-HCl (50 mM, pH 7.5) gave an extraction yield, on average, lower than 0.5%, but addition of Triton X-100 or bovine serum albumine (BSA) to Tris buffer increased the extraction yield 2-8 times. When both Triton X-100 and BSA were added to the buffer, the extraction yield was more than additive and reached 2-13% for acP, 2-5% for alkP and 3-6% for aryS, depending on the soil. In addition, these extracts were colourless or at most light yellow, showing that besides the high yield enzymes were extracted along with negligible amounts of humic substances.     

Selective digestion of the peptide and polysaccharide components of synthetic humic acids by the humivorous larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

In order to identify the potential nutrient and energy sources of humivorous beetle larvae, we carried out feeding trials with soil supplemented with specifically ¹⁴C-labeled model humic acids synthesized by peroxidase-initiated radical polymerization, using the cetoniid beetle Pachnoda ephippiata (Coleoptera: Scarabaeidae) as a model organism. Ingestion of soil by the larvae significantly increased the mineralization of humic acids labeled in their peptide (HA-*peptide) or polysaccharide components (HA-*peptidoglycan and HA-*chitin), whereas the mineralization of humic acids labeled in the aromatic components (HA-*catechol) did not increase significantly. Mineralization was accompanied by a reduction of residual radiolabel in the acid-soluble fraction and an increase in the humic acid and humin fractions of the fecal pellets. During the gut passage, the residual label in peptide or polysaccharide components was transformed into acid-soluble products, especially in the alkaline midgut. High-performance gel-permeation chromatography demonstrated that the changes in solubility were accompanied by large changes in the molecular weight of the residual material. The amount of radiolabel derived from the peptide and polysaccharide components recovered from the larval body and hemolymph was significantly higher than that derived from the aromatic component, which supports the hypothesis that humivorous beetle larvae selectively digest the peptide and polysaccharide components of humic substances, whereas the aromatic components of humic substances are not an important source of nutrients and energy. This is also the first experimental evidence that also chitin and peptidoglycan, the major structural polymers in fungal and bacterial biomass, can be protected from microbial degradation in soil by a copolymerization with phenols and might contribute substantially to the refractory nitrogen pool in soil organic matter.     

Nutrient limitations to soil microbial biomass and activity in loblolly pine forests

We performed an assay of nutrient limitations to soil microbial biomass in forest floor material and intact cores of mineral soil collected from three North Carolina loblolly pine (Pinus taeda) forests. We added solutions containing C, N or P alone and in all possible combinations, and we measured the effects of these treatments on microbial biomass and on microbial respiration, which served as a proxy for microbial activity, during a 7-day laboratory incubation at 22 °C. The C solution used was intended to simulate the initial products of fine root decay. Additions of C dramatically increased respiration in both mineral soil and forest floor material, and C addition increased microbial biomass C in the mineral soil. Additions of N increased respiration in forest floor material and increased microbial biomass N in the mineral soil. Addition of P caused a small increase in forest floor respiration, but had no effect on microbial biomass.     

碱稳定污泥污水对土壤可提取有机碳和铜的影响

An incubation experiment was conducted to evaluate the potential for water contamination with sludge-derived organic substances and copper following land application of alkaline-stabilised sewage sludge. Two contrasting sludge-amended soils were studied. Both soils were previously treated with urban and ruralalkaline biosolids separately at sludge application rates of 0, 30 and 120 t ha^-1 fresh product. The air-driedsoil/sludge mixtures were wetted with distilled water, maintained at 40 % of water-holding capacity and equilibrated for three weeks at 4 ℃ before extraction. Subsamples were extracted with either distilled wateror 0.5 mol L^-1 K₂SO₄ solution. The concentrations of organic C in the aqueous and chemical extracts were determined directly with a total organic carbon (TOC) analyser. The concentrations of Cu in the twoextracts were also determined by atomic absorption spectrophotometry The relationship between the two extractable organic C fractions was examined, together with that between extractable organic C concentration and extractable Cu concentration. Application of alkaline biosolids increased the concentrations of soil mobile organic substances and Cu. The results are discussed in terms of a possible increase in the potential for leaching of sludge-derived organics and Cu in the sludge-amended soils.     

Characterization of water soluble organic matter in soils by size exclusion chromatography and fractionation with polyvinylpyrrolidone

Water extracts were obtained from four types of soils (Brown Lowland soil, Yellow soil with manure application for 6 years, non-allophanic Andosol, and allophanic Andosol), and the organic matter in the water extracts was fractionated according to the solubility in acid and adsorption onto polyvinylpyrrolidone (PVP). For the water extracts and their fractions, the amounts of organic C, total N, and anthrone-reactive C (ARC) were analyzed, and high performance size exclusion chromatography (HPSEC) was carried out. The PVP-non-adsorbed fulvic acid (FA) fraction accounted for the largest proportion of the total water-soluble organic C, ranging from 52% for the Yellow soil to 96% for the allophanic Amdosol, followed by the PVP-adsorbed FA and humic acid (HA) fractions. The water extract of the allophanic Andosol was characterized by the lack of HA fraction and a small proportion of PVP-adsorbed FA fraction. For all the water extract samples, more than 90% of the ARC was recovered in the PVP-non-adsorbed FA fraction. The proportion of ARC in the total organic C in the fraction was also highest in the PVP-non-adsorbed FA fraction. The molecular weight (MW) of the humic substances (HS) at peak maximum was estimated at 1,300 Da for the water extracts and their fractions from the Brown Lowland soil and non-allophanic Andosol samples by HPSEC using polyethylene glycols as MW standards. Manure application increased the MW of HS in the HA and PVP-adsorbed FA fractions. On the other hand, only a small amount of HS was found in the water extract of the allophanic Andosol by HPSEC.     

土壤供氮能力测试方法与指标

研究了土壤供氮能力的主要测试方法和指标,包括培养矿化法、化学提取法、起始矿质氮指标和电超滤法测定指标等。对各种方法和指标的发展应用作了叙述,并对有关方法和指标的效果进行了比较。     

Isoelectric focusing of β-glucosidase humic-bound activity in semi-arid Mediterranean soils under management practices

Changes in β-glucosidase enzyme–humic complexes and conventional parameters (pH, total organic C, total N, water-soluble C, and bulk density) were studied in an almond-cropped soil prone to erosion under a rehabilitation practice. The experimental plan included three soil slopes (0%, 2%, and 6%) and two type of fertilization (organic and mineral), with sampling of rhizosphere and inter-row soils. The enzyme humic complexes were extracted by pyrophosphate, purified by ultrafiltration of the organic extracts on molecular mass exclusion membranes (mol wt > 10⁴) and fractionated by isoelectric focusing technique (IEF). The IEF on polyacrylamide rod gels with a restricted pH gradient ranging between 6.0 and 4.0 gave five humic bands on the basis of the little differences of their electric charges (pI). Under both organic and mineral fertilization, β-glucosidase activity bound to the fractionated humic substances, especially in the pH range 4.5–4.2 of the rhizosphere soil, was higher than that of the inter-row soil. This also occurred in 6% slope where the enzyme activity was lower than in soil with lower slopes. The higher number of the β-glucosidase active humic bands in rhizosphere than inter-row soil, particularly for the 0% slope, may be due to the presence of humic molecules capable of preserving the enzyme molecules in the active form, other than to the higher microbial activity synthesizing and releasing the tested enzymes.     

Acceleration of N mineralization by release of enzymes and substrates from soil mineral particles with phosphates

A study was conducted to develop an improved method for measuring organic N (net) mineralization in which chemical extraction takes place in combination with suspension incubation in ammonia-absorbing membrane bottles. To obtain direct evidence of the extent to which extracted organic N is mineralizable, the extraction suspension was further incubated immediately after the extraction procedure with mild and selective extractants. In this ‘extraction incubation’ method, extraction continues during the incubation but only relatively easily mineralizable organic matter is released. Standard incubation is usually carried out in sealed N₂-flushed bottles. However, when phosphate or pyrophosphate soil suspensions are incubated, mineralization is much higher than in soil water suspensions. Further, accumulation of ammonia+(ammonium) and other gases, i.e. CO_2, can affect the reaction rate and final reaction equilibrium in the sealed incubation flask. It was to avoid these effects that the membrane method was developed. With this procedure, the flask is closed with an ammonia-absorbing membrane permeable to other gases. Water, phosphate and pyrophosphate suspensions were incubated at 37 °C in sealed bottles (SB), in sealed N₂ gas-flushed bottles (SBN₂), and in bottles with ammonia-trapping filters (MB). The maximum amount of released during 10 days' incubation was 133.0 mg kg⁻¹ in the water, 208.0 mg kg⁻¹ in the phosphate and 454.1 mg kg⁻¹ in the pyrophosphate suspension (soil total C content 6.2% and N 0.25%). During incubation in phosphate and pyrophosphate suspensions, the mobilization was nearly linear in membrane bottles. The variation between replicates was also smallest in these bottles. It was concluded that membrane bottles were best suited to incubation when mobilization reactions were accelerated with phosphate or pyrophosphate extractants. The method was easy to perform and gave results with good replicability.     

Degradation and humification of maize straw in soil microcosms inoculated with simple and complex microbial communities

Microbial communities are responsible for soil organic matter cycling and thus for maintaining soil fertility. A typical Orthic Luvisol was freed from organic carbon by thermal destruction at 600°C. Then the degradation and humification of ¹⁴C‐labelled maize straw by defined microbial communities was analysed. To study the role of microbial diversity on the humification of plant material, microcosms containing sterilized soil were inoculated with a natural microbial community or with microbial consortia consisting of bacterial and fungal soil isolates. Within 6 weeks, 41 ± 4% of applied ¹⁴C‐labelled maize straw was mineralized in the soil microcosms containing complex communities derived from a soil suspension, whilst the most efficient communities composed of soil isolates mineralized less than 35%. The humification products were analysed by solution state ¹³C‐NMR‐spectroscopy and gel permeation chromatography (GPC). The analyses of humic acids extracts by solution state ¹³C‐NMR‐spectroscopy revealed no difference in the development of typical chemical functional groups for humic substances during incubation. However, the increase in specific molecular size fractions of the extracted humic acids occurred only after inoculation with complex communities, but not with defined isolates. While it seems to be true that redundancy in soil microbial communities contributes to the resilience of soils, specific soil functions may no longer be performed if a microbial community is harshly affected in its diversity or growth conditions.     

扩散法测定土壤无机氮~(15)N丰度方法优化研究

扩散法与质谱测定技术相结合,被广泛应用于环境、生态和农业领域中土壤、水等样品中无机氮15N同位素丰度的测定。为建立一套可快速、准确测定土壤无机氮15N同位素丰度的扩散培养体系,针对土壤样品无机氮量的变化特点,从培养温度、培养时间、试剂选择和用量等方面对扩散条件进行优化。结果发现,对于大部分无机氮浓度大于2 mg L~(-1)的土壤样品,20 ml土壤提取液,在不小于250 ml的蓝盖瓶中,悬挂两张各滴加了10μl 1 mol L~(-1)草酸的滤纸,加入0.1 g的Mg O,25℃下以140 r min-1的转速振荡培养24 h即可完成对样品中NH4+-N的扩散与回收;换入2张加酸滤纸继续摇培48 h可基本去除残余的NH4+-N;再换入2张加酸滤纸并加入0.1 g的戴氏合金振荡培养24 h即可。对于无机氮浓度低于2 mg L~(-1)的土壤提取液,需用50 ml提取液按以上条件进行扩散培养即可保证测定结果的准确性。本方法大大缩短了扩散法所需的实验周期,实现在一份样品内同时完成NH4+-N和NO3--N的扩散与回收,减少了样品的需要量,并通过优化Mg O、戴氏合金(Devarda’s alloy)的用量减少杂质氮可能带来的污染。     

玉米植株残体培肥土壤的研究

采用土培试验对玉米植株残体施人后土壤理化性质及有机质组成的变化进行了分析。结果表明，土壤加入玉米植株残体后，土壤微生物生物量碳和氮含量提高，土壤的阳离子交换量增强，松结合态、联结合态和紧结合态腐殖质的含量增加，稳结合态腐殖质的相对含量下降，土壤中五碳糖和六碳糖的含量升高，土壤胶体活性变强。玉米秸秆和根茬对土壤的培肥作用存在差异。     

Humic materials from an organic soil: A comparison of extractants and of properties of extracts

Five extraction procedures and thirteen extracting reagents, which included dipolar aprotic solvents, organic chelating agents, pyridine, ethylenediamine, sodium hydroxide, ion-exchange resins and two salts (sodium pyrophosphate and ethylenediamine hydrochloride), were used to extract humic materials from an organic soil. Extractabilities increased in the general order: salts < organic chelating agents < dipolar aprotic solvents < pyridine < ethylenediamine = sodium hydroxide, and the amounts of the soil organic matter extracted by the reagents in the series ranged from 13 to 63%. Gel chromatography techniques indicated that extracts in dipolar aprotic solvents were predominantly of intermediate and low molecular-weight values, and it is suggested that the more highly oxidised soil humic materials were extracted in these. The more efficient solvents extracted materials with a range (high—low) of molecular-weight values. Data from elemental analysis and from E.S.R. measurements indicated that ethylenediamine altered the chemical nature and the composition of extracts. Dipolar aprotics, by the same criteria, were found not to alter the humic extracts, and can be regarded as mild reagents for the extraction of a less representative (of the total) fraction of soil organic matter. Sodium hydroxide in solution, despite its oxidation effects, was the best of the reagents tested for isolating extracts which were representative of a wide range of soil humic substances.     

Effect of humic amendments on inorganic N, dehydrogenase and alkaline phosphatase activities of a Mediterranean soil

Dehydrogenase activity, alkaline phosphatase activity and NH₄ ⁺, NO₂ ⁻ and NO₃ ⁻ concentrations were monitored in an aridisol treated with three commercially available humic amendments. The materials were of plant residue, lignite and peat origins. The humus plant residues, fulvic acids, with a high content of Kjeldahl-N, sustained high enzyme activities and highest levels of NH₄ ⁺, NO₂ ⁻ and NO₃ ⁻. Humus lignite (mainly humic acids) produced the highest dehydrogenase activity, whereas the alkaline phosphatase activity was not as high as that amendment with humus plant residues. The lower activity of alkaline phosphatase could not be attributed to the higher P content of humus lignite. Nitrification was also low, probably due to the low N content of this fertilizer. The amendment of humus peat origin (only humic acids) did not increase enzyme activity or inorganic N concentrations of soil. Our results show that although these materials are widely utilized and recommended as microbial and plant activators, they all behave very differently, and the effects on soil microbiological activity cannot be predicted solely on the basis of their humic and/or fulvic acid contents.     

Uncomposted Sheep and Cow Manures as a Source of Commercial Humic Amendments: An Exploratory Study

Liquid humic amendments are widely used in many countries as an enhancer of crop production (particularly root growth). While leonardite is the main substrate for obtaining these products, there is growing concern about obtaining them from plant-derived residual materials, rich in lignin and polyphenolics, thus making possible a residue valorization. Composting is a necessary step for converting these residues into a source of humic substances. In arid countries, obtaining humic products from sources other than leonardite faces two main challenges: (i) often such countries are devoid of forested areas or tree crops capable of supplying lignin-rich residues in large amounts and (ii) water is scarce (or very expensive if obtained by desalination) and therefore composting on an industrial scale is very difficult. Thus it is essential to find alternative sources capable of yielding humic or humic-like compounds without any previous composting. Here we present the main results obtained using sheep and cow manure, two materials widely available in the Kingdom of Saudi Arabia (KSA). Both were extracted using potassium hydroxide (KOH) at a variety of concentrations (from 0.25 to 2 M), times (from 20 to 120 min), temperatures (from 100 to 150 °C), and pressures. Sheep manure, much easier to extract than cow manure, appears the more promising as a source of commercial humic amendments. No lengthy extraction times are needed to obtain alkaline extracts; 20 min yields only slightly less humic-like matter than 2 h. The relative abundance of humic-like substances in the extract tends to decrease with time, but some optical indicators of humification (E₄/E₆ ratio, absorption at 280 nm, ratios of absorption at 253/203 and 253/220 nm) tend to increase. Polysaccharides, the dominant form of organic matter in the original manure, strongly resist alkaline extraction: their prior hydrolysis may be necessary to increase the yields of humic-like matter from the studied manures.     

Changes in microbial community structure in soil as a result of different amounts of nitrogen fertilization

The changes in size, activity and structure of soil microbial community caused by N fertilization were studied in a laboratory incubation experiment. The rates of N fertiliser applied (KNO₃) were 0 (control), 100 and 2,000 μg N g⁻¹ soil. Despite no extra C sources added, a high percentage of N was immobilized. Whereas no significant increase of microbial C was revealed during incubation period, microbial growth kinetics as determined by the substrate-induced growth-response method demonstrated a significant decrease in the specific growth rate of microbial community in soil treated with 2,000 μg N g⁻¹ soil. Additionally, a shift in microbial community structure resulting in an increase in fungal biomarkers, mainly in the treatment with 2,000 μg N g⁻¹ soil was visible.     

缓释复合肥料对土壤和黑麦草氮素营养的影响

采用恒温培养和盆栽生物试验研究了非包膜缓释复合肥料对土壤氮素养分(铵态氮、硝态氮、碱解氮、微生物量氮和固定态铵)和黑麦草氮素营养的效应。结果表明,在恒温培养条件下,各施肥处理土壤铵态氮含量随培养时间均表现为先升高后降低的趋势,且中后期缓释复合肥料处理(SRCF)高于普通复合肥料处理(CCF);缓释复合肥料SRCF1处理土壤硝态氮含量始终低于普通复合肥料CCF1处理,缓释复合肥料SRCF2处理土壤硝态氮含量在初期较高、中后期较低;SRCF各处理土壤碱解氮和微生物量氮含量变化均表现为增加-降低-增加-降低趋势,分别在培养第21d和105d时出现峰值;土壤固定态铵含量变化较小,但总体上以SRCF处理高于CCF处理。在等养分比例盆栽试验中,SRCF1-3处理黑麦草株高、生物量、干重和氮素养分吸收量均高于CCF1处理,SRCF有利于提高黑麦草产量、氮素吸收和利用率;黑麦草氮素吸收量与培养土壤铵态氮、微生物量氮含量呈负相关,而与硝态氮和固定态铵含量呈正相关。