土壤水解酶活性测定方法的研究进展

土壤水解酶直接参与有机物质的矿质化过程,对维持生态系统碳和养分的循环过程起重要作用。但不同研究者和实验室测定过程的差异,常给土壤酶测定方法的选定带来困难。针对这一问题,本文围绕土壤酶活性测定过程中样本贮存方式、酶底物选择以及培养条件等,对近二十多年来的研究成果进行了评述。土壤酶来源广泛,存在形式复杂,直接提取土壤酶还有很大的难度。目前土壤酶活性的测定主要是通过一定量底物在酶催化反应过程中的生成物或剩余物量来实现。基于对硝基酚衍生物为底物的分光比色法,因其成本低廉而被广泛使用。针对国际上未能建立统一的测定方法而导致研究数据之间难以进行比较的现状,作者认为现阶段土壤水解酶测定过程中应注意以下几点:1)尽量采用新鲜土样或短时间低温冷藏的土样;2)酶底物种类的选择应与国际接轨,采用饱和底物浓度使其反应速度接近最大值;3)缓冲液的pH值可与土壤pH值相近;4)4 h内培养的土壤酶,可忽略微生物抑制剂的使用。建议我国在近期内:1)应丰富土壤水解酶种类的研究;2)注重灵敏度高、需样量小以及培养时间短的荧光分析技术的应用;3)在强化典型土壤酶动力学特征研究基础上,尽快规范土壤酶活性的测定方法。     

Application of para-nitrophenol (pNP) enzyme assays in degraded tropical soils

Enzyme activities have been used as indicators of soil quality and changes in biogeochemical function due to management or perturbations. The objective of this study was to answer a number of methodological questions regarding sampling schemes, sample handling recommendations, and assay procedures to facilitate the use of enzyme assays in the tropical highlands of East Africa. We used para-nitrophenol (pNP) based substrates for five enzymes: β-glucosidase, cellobiohydrolase, chitinase, acid phosphatase, and alkaline phosphatase. In the first experiment, we examined sampling procedures and compared the results of determining enzyme activities on a plot using composite or discrete samples. Composite samples usually had higher activities than the means of individual cores (P<0.05), but relative ranking of sites was the same if analyses were based on composite or discrete samples. In the second experiment, we examined the effects of storage time and conditions on enzyme activity. Enzyme activity degraded rapidly in frozen samples, but was better maintained in samples stored at 4 °C. Phosphatase and cellobiohydrolase activity declined after 14 days of storage, while the activity of the other enzymes remained close to the values of fresh samples for 28 or more days. In the third experiment, we examined the effect of the addition of an antiseptic, toluene, to prevent bacterial growth during the assay. We found no consistent toluene effect (P>0.4), probably because the assays were of short duration and microbial growth was minimized. Finally, we looked at the incubation time necessary to produce reliable results. Phosphatases, with relatively high activities could reliably be determined in 2 h incubations, but the other enzymes had much lower activities and required longer incubation times for reliable determination. For the enzymes we looked at, 4 h was a good standard time for determining the activity of even the lowest activity enzymes. The results of this study provide practical guidelines for applying these enzyme assays in the degraded tropical soils.     

三峡库区消落带土壤酶活性特征

土壤酶驱动土壤生态系统养分的循环和控制生态系统的功能。选取三峡库区消落带三个海拔梯度的土壤,以从未淹没的样地作为对照,研究消落带土壤酶活性特征。结果表明,消落带区域土壤有机碳（OC）、微生物生物量碳（MBC）和所测定的四种酶[磷酸酶（PNP）、芳基硫酸酯酶（PNS）、N-乙酰-β-D-氨基葡萄糖苷酶（NAG）和β-糖苷酶（PAG）]的绝对活性（单位土壤干重的酶活性）和四种酶的几何平均数（GME）显著低于从未淹没的对照样地。在消落带区域,只有磷酸酶的绝对活性随着淹没持续时间的延长而降低,其他土壤三种酶的绝对活性在三个样地之间并无显著性的差异。土壤四种酶的几何平均数与有机碳、微生物生物量碳的变化相似,在海拔梯度较低的样地最低,其他两个较高海拔梯度的样地之间无显著性的差异。然而单位有机碳或微生物生物量碳的四种酶的具体活性的变化趋势与酶的绝对活性的变化并不一致。PNP的具体活性在所有测定的样地之间都无显著性的差异,而其他三个酶的具体活性是海拔最低的样地显著高于其他样地,其他样地之间并无显著性的差异。因而,这些结果意味着四种酶的几何平均数能够敏感指示三峡库区消落带水位消涨及其不同淹没持续时间对土壤质量的影响,是一个能够综合反映所有土壤酶活性对消落带水位消涨的响应的敏感指标。     

Reconciling apparent variability in effects of biochar amendment on soil enzyme activities by assay optimization

We studied the effects of a biochar made from fast pyrolysis of switchgrass on four soil enzymes (β-glucosidase, β-N-acetylglucosaminidase, lipase, and leucine aminopeptidase) to determine if biochar would consistently modify soil biological activities. Thus, we conducted a series of enzyme assays on biochar-amended soils. Inconsistent results from enzyme assays of char-amended soils suggested that biochar had variable effects on soil enzyme activities, thus we conducted a second experiment to determine if biochar reacts predictably with either enzyme or substrate in in vitro reactions. Both colorimetric and fluorescent assays were used for β-glucosidase and β-N-acetylglucosaminidase. Seven days after biochar was added to microcosms of 3 different soils, fluorescence-based assays revealed some increased enzyme activities (up to 7-fold for one measure of β-glucosidase in a shrub-steppe soil) and some decreased activities (one-fifth of the unamended control for lipase measured in the same shrub-steppe soil), compared to non-amended soil. In an effort understand the varied effects, purified enzymes or substrates were briefly exposed to biochar and then assayed. In contrast to the soil assays, except for β-N-acetylglucosaminidase, the exposure of substrate to biochar reduced the apparent activity of the enzymes, suggesting that sorption reactions between substrate and biochar impeded enzyme function. Our findings indicate that fluorometric assays are more robust to, or account for, this sorption better than the colorimetric assays used herein. The activity of purified β-N-acetylglucosaminidase increased 50-75% following biochar exposure, suggesting a chemical enhancement of enzyme function. In some cases, biochar stimulates soil enzyme activities, to a much greater degree than soil assays would indicate, given that substrate reactivity can be impeded by biochar exposure. We conclude that the effects of biochar on enzyme activities in soils are highly variable; these effects are likely associated with reactions between biochar and the target substrate.     

Vertical gradients of potential enzyme activities in soil profiles of European beech,Norway spruce and Scots pine dominated forest sites

Management of forest sites has the potential to modulate soil organic matter decomposition by changing the catalytic properties of soil microorganisms within a soil profile. In this study we examined the impact of forest management intensity and soil physico-chemical properties on the variation of enzyme activities (β-glucosidase, β-xylosidase, α-glucosidase, phenol oxidase, N-acetyl-glucosaminidase, l-leucine aminopeptidase, phosphatase) in the topsoil and two subsoil horizons in three German regions (Schorfheide-Chorin, Hainich-Dün, Schwäbische Alb). The sandy soils in the Schorfheide-Chorin (SCH) showed lower ratios of the activity of carbon (C) acquiring enzymes (β-glucosidase) relative to nitrogen (N) acquiring enzymes (N-acetyl-glucosaminidase + l-leucine aminopeptidase), and activity of C acquiring enzymes relative to phosphorous (P) acquiring enzymes (phosphatase) than the finer textured soils in the Hainich-Dün (HAI) and Schwäbische Alb (ALB), indicating a shift in investment to N and P acquisition in the SCH. All enzyme activities, except phenol oxidase activity, decreased in deeper soil horizons as concentrations of organic C and total N did, while the decrease was much stronger from the topsoil to the first subsoil horizon than from the first subsoil to the second subsoil horizon. In contrast, phenol oxidase activity showed no significant decrease towards deeper soil horizons. Additionally, enzyme activities responsible for the degradation of more recalcitrant C relative to labile C compounds increased in the two subsoil horizons. Subsoil horizons in all regions also indicate a shift to higher N acquisition, while the strength of the shift depended on the soil type. Further, our results clearly showed that soil properties explained most of the total variance of enzyme activities in all soil horizons followed by study region, while forest management intensity had no significant impact on enzyme activities. Among all included soil properties, the clay content was the variable that explained the highest proportion of variance in enzyme activities with higher enzyme activities in clay rich soils. Our results highlight the need for large scale studies including different regions and their environmental conditions in order to derive general conclusions on which factors (anthropogenic or environmental) are most influential on enzyme activities in the whole soil profile in the long term at the regional scale.     

土壤酶学硏究进展

土壤酶学是研究土壤酶活性及其相关特性的科学,是一门介于土壤生物学和生物化学之间的边缘交叉学科。土壤酶在土壤生态系统的物质循环和能量流动方面具有主要的作用。本文综述了土壤酶学发展简史、土壤酶的来源、分类、功能及分布特征等,总结了土壤理化性质、施肥、放牧、土壤微生物、施用稀土元素、草地生长年限、不同土地利用和耕作方式及其他因素对土壤酶活性的影响,对于加深理解生态系统中的物质循环、土壤酶的生态重要性以及土壤生态系统退化机理有重要作用。最后,结合本项目组的研究对土壤酶学的研究前景进行了展望。     

Soil enzyme activity: a brief history and biochemistry as a basis for appropriate interpretations and meta-analysis

Enzyme activity as a method for soil biochemistry and microbiology research has a long history of more than 100 years that is not widely acknowledged in terms of adherence to strict assay protocols and the interpretation of results. However, in the recent past, there is a growing lack of recognition of the historic advancements among researchers that use soil enzymology. Today, many papers are being published that use methods that either do not follow exact protocols as originally vetted in the research literature or individual labs use their own method that has not been optimized for pH, co-factors, substrate concentrations, or other conditions. This is of particular concern for fluorogenic substrates and microplate methods. Furthermore, there is a lack of understanding of the origin and location of a given enzyme being studied. Notably, regardless of the enzyme, it is too often assumed that enzyme activity equals microbial activity—which is not the case for most hydrolytic enzyme assays. Because as established by Douglas McLaren in the 1950s, a considerable amount of activity can come from catalytic enzymes stabilized in the soil matrix but that are no longer associated with viable cells (known as abiontic enzymes). In summary, today, many papers are using imperfect methods and/or misinterpret enzyme activity data that at a minimum confounds cross paper studies and meta-analysis. However, most importantly, lack of historical perspectives and ignoring strict protocols cause redundancy and fundamentally undermine the discipline and understanding of soil microbiology/biochemistry when enzymology methods are used.     

Hydrolase enzyme activities in a successional gradient of biological soil crusts in arid and semi-arid zones

In arid and semi-arid regions, pioneer organisms form complex communities that penetrate the upper millimetres of the bare substrate, creating biological soil crusts (BSC). These thin crusts play a vital role in whole ecosystem functioning because they enrich bare surfaces with organic matter, initiate biogeochemical cycling of elements, modify hydrological cycles, etc., thus enabling the ground to be colonized by vascular plants. Various hydrolase enzymes involved in the carbon (cellulase, β-glucosidase and invertase activities), nitrogen (casein-protease and BAA-protease activities) and phosphorus (alkaline phosphomonoesterase activity) cycles were studied at three levels (crust, middle and deep layers) of three types of BSCs from the Tabernas Desert (SE Spain), representing an ecological gradient ranging from crusts predominated by cyanobacteria to crusts predominated by lichens (Diploschistes diacapsis, Lepraria crassissima). All enzyme activities were higher in all layers of all BSCs than in the bare substrate. The enzymes that hydrolyze low molecular weight substrates were more active than those that hydrolyze high molecular weight substrates (cellulase, casein-protease), highlighting the pioneering characteristics of the BSCs. The hydrolytic capacity developed in parallel to that of ecological succession, and the BSCs in which enzyme activity was highest were those under L. crassissima. The enzyme activity per unit of total organic C was extremely high; the highest values occurred in the BSCs formed by cyanobacteria and the lowest in those formed by lichens, which indicates the fundamental role that the primary colonizers (cyanobacteria) play in enriching the geological substrate with enzymes that enable degradation of organic remains and the establishment of more developed BSCs. The results of the study combine information on different enzyme activities and provide a clear vision of how biogeochemical cycles are established in BSCs, thus confirming the usefulness of enzyme assays as key tools for examining the relationship between biodiversity and ecosystem function in biological soil crusts.     

Multiple-substrate enzyme assays: a useful approach for profiling enzyme activity in soils?

This study focuses on the applicability of multiple-substrate enzyme assays to simultaneously determine various soil enzyme activities within one assay. Mineral soils from agricultural field sites differing in soil properties and management were used to optimise substrate composition and concentration of 4-methylumbelliferone and 7-amino-4-methylcoumarin derivatives as model substrates. In contrast to conventional assays, enzyme activity was measured at soil pH since optimum pH is not more applicable using a multiple-substrate approach. Furthermore, enzyme activity was not calculated from the product formed but from substrate decrease. After incubation the added substrates were re-extracted, separated by high-performance liquid chromatography and quantified by UV-absorption at 320 nm. This approach allows simultaneous measurement of the activity of β-d-glucosidase, N-acetyl-β-d-glucosaminidase, β-d-glucuronidase, β-d-xylosidase, phosphomonoesterase, sulfoesterase and leucine-aminopeptidase within one assay and with sufficient accuracy. However, incomplete re-extraction due to adsorption of substrates to the soil matrix was observed. In addition, certain competitive inhibition effects due to chemically similar substrates were found. Compared to conventional methods, no distinct differences in enzyme activity profile were detected, with both assays—conventional and multiple-substrate approach—leading to similar differentiation among the investigated soils. In conclusion, the multiple-substrate approach may serve as time-saving alternative to standard enzyme assays in mineral soils. Certainly, since the multiple-enzyme assay is conducted at soil pH, the procedure leads to reduced comparability of soils with contrasting pH values.     

南方红壤丘陵区土壤侵蚀-沉积作用对土壤酶活性的影响

土壤酶与土壤矿质营养元素循环、能量转移等密切相关。明确土壤酶对土壤侵蚀—沉积作用的响应机制,有助于进一步把握土壤侵蚀在全球碳循环中的作用。通过分析湘中红壤丘陵区松林坡面侵蚀区及沉积区土壤酶活性的变化特征,揭示了酶活性与土壤主要养分因子之间的关系,并在此基础上深入探讨了土壤侵蚀—沉积作用对土壤酶活性的影响。结果表明:沉积区绝大多数土层土壤有机碳(soil organic carbon,SOC)、全氮(total nitrogen,TN)、可溶性有机碳(dissolved organic carbon,DOC)、脲酶、酸性磷酸酶及过氧化氢酶活性均要显著高于侵蚀区。土壤沉积作用明显提高了土壤养分含量及酶活性。其次,侵蚀区与沉积区土壤养分含量及酶活性差异在侵蚀干扰较为严重的表层(0~30 cm)土壤表现较为明显,随着土壤深度的增加差异逐渐减小。侵蚀区与沉积区SOC、TN、DOC及酶活性均随土壤深度的增加呈现总体下降的趋势。相关性分析表明,土壤脲酶、酸性磷酸酶、过氧化氢酶之间及其与SOC、TN、DOC之间均存在极显著正相关关系(p0.01)。此外,偏冗余分析结果进一步表明SOC是解释土壤酶活性动态变化的主要因子,其解释量达7.5%,侵蚀诱导SOC在坡面的再分布是影响土壤酶活性的重要途径之一。     

Phenol oxidase, peroxidase and organic matter dynamics of soil

Extracellular enzymes mediate the degradation, transformation and mineralization of soil organic matter. The activity of cellulases, phosphatases and other hydrolases has received extensive study and in many cases stoichiometric relationships and responses to disturbances are well established. In contrast, phenol oxidase and peroxidase activities, which are often uncorrelated with hydrolase activities, have been measured in only a small subset of soil enzyme studies. These enzymes are expressed for a variety of purposes including ontogeny, defense and the acquisition of carbon and nitrogen. Through excretion or lysis, these enzymes enter the environment where their aggegrate activity mediates key ecosystem functions of lignin degradation, humification, carbon mineralization and dissolved organic carbon export. Phenol oxidases and peroxidases are less stable in the environment than extracellular hydrolases, especially when associated with organic particles. Activities are also affected, positively and negatively, by interaction with mineral surfaces. High spatiotemporal variation obscures their relationships with environmental variables and ecological process. Across ecosystems, phenol oxidase and peroxidase activities generally increase with soil pH, a finding not predicted from the pH optima of purified enzymes. Activities associated with plant litter and particulate organic matter often correlate with decomposition rates and potential activities generally increase with the lignin and secondary compound content of the material. At the ecosystem scale, nitrogen amendment alters the expression of phenol oxidase and peroxidase enzymes more broadly than culture studies imply and these responses correlate with positive and negative changes in litter decomposition rates and soil organic matter content. At the global scale, N amendment of basidiomycete-dominated soils of temperate and boreal forest ecoystems often leads to losses of oxidative enzyme activity, while activities in grassland soils dominated by glomeromycota and ascomycetes show little net response. Land use that leads to loss of soil organic matter tends to increase oxidative activities. Across ecosystems, soil organic matter content is not correlated with mean potential phenol oxidase and peroxidase activities. A multiple regression model that includes soil pH, mean annual temperature, mean annual precipitation and potential phenol oxidase activity accounts for 37% of the variation in soil organic matter (SOM) content across ecosystems (n = 63); a similar model for peroxidase activity describes 32% of SOM variance (n = 43). Analysis of residual variation suggest that suites of interacting factors create both positive and negative feedbacks on soil organic matter storage. Soils with high oxygen availability, pH and mineral activity tend to be substrate limited: high in situ oxidative activities limit soil organic matter accumulation. Soils with opposing characteristics are activity limited: low in situ oxidative activities promote soil organic matter storage.     

Enzyme activity in soil: Location and a possible role in microbial ecology

The activity of any particular enzyme in soil is a composite of activities associated with various biotic and abiotic components, e.g. proliferating cells, latent cells, cell debris, clay minerals, humic colloids and the soil aqueous phase. The location of the enzyme is at least partially determined by such factors as the size and solubility of its substrate, the species of microorganism, and the physical and chemical nature of the soil colloids. However, enzymes may change location with time, for example, many hydrolases are intracellular sensu stricto but are also found associated with cell debris and clay and organic colloids. There are difficulties in quantifying the various activities, but this may be possible by employing different types of assays, the prudent use of controls and the study of crude enzyme extracts from soil.Enzymes bound to clay and humic colloids (the immobilized or accumulated enzyme fraction) have a residual activity not found in enzymes free in the soil aqueous phase. However, the mere adsorption of enzymes to soil surfaces does not guarantee subsequent activity, and it appears that some mechanism of association with the humic polymer offers the best form of protection, yet permits the retention of enzyme activity.The catalytic activity of extracellular enzymes is discussed and a possible relationship between soil microorganisms, exogenous substrates and immobilized enzymes is suggested.     

施用有机肥对土壤生物性状影响的研究进展

施肥是农业生态系统中的重要一环,因土壤生物特性如土壤酶活力、微生物量、呼吸以及生物多样性等对外来扰动的灵敏性优于理化特性而在近几年受到了广泛关注。长期配施有机肥能显著调节土壤营养环境,提高微生物碳氮含量,降低代谢呼吸商值并提高多种土壤酶的活力和土壤生物多样性,为作物稳产高产创造良好的土壤生态环境,而化肥施用的效果恰相反。土壤生物特性的变动关系到土壤质量、农业生产的产量以及生态系统的稳定,本文综述了近几年国内外关于施用有机肥对土壤生物性质影响的研究结果。     

Soil moisture pre-treatment effects on enzyme activities as indicators of heavy metal-contaminated and reclaimed soils

Heavy metal contamination can inhibit soil functions but it is often difficult to determine the degree of pollution or when soil reclamation is complete. Enzyme assays offer potential as indicators of biological functioning of soils. However, antecedent water content of soil samples may affect the outcome of biological measurements. In Mediterranean regions, for much of the year ‘field moist’ surface soil can have water content similar to that of air-dry samples. The objectives of this study were to: (1) determine the sensitivity of a range of enzyme assays to detect the degree of pollution from a heavy metal mine spill; (2) evaluate rewetting field-dry soil as a pre-treatment for enzyme assays; and (3) test multivariate analysis for improving discrimination between polluted, reclaimed and non-polluted soils. The Aznalcóllar mining effluent spill provided a unique opportunity to address these objectives. This accident released toxic, heavy metal-contaminated (As, Bi, Cd, Cu, Pb, Tl, Zn…) and acid tailings into the Guadiamar watershed (SW Spain) in 1998, severely affecting the riparian zone along more than 4000 ha. Contaminated soils were collected from the highly polluted upper watershed and less polluted lower watershed along with reclaimed soil at both sites. Enzyme activities (phosphatases, arylsulfatase, β-glucosidase, urease and dehydrogenase) were assessed on both field-moist samples and soils rewetted to 80% of water-holding capacity and then incubated at 21 °C for 7 d prior to the assay. The reclaimed soils had higher activities than polluted soils but, typically, 1.5-3 times lower levels of activity than the non-polluted soil. Regardless of the moisture pre-treatment, all enzymes showed significant effects due to pollution, with urease and β-glucosidase showing the greatest discrimination between degrees of contamination. In general, rewetting field-dried soils increased activities on non-polluted and reclaimed soils which improved discrimination with polluted soils. Another method to increase the potential of soil enzyme activities to detect soil contamination could be to combine them in multivariate analysis, which provides a more holistic representation of the biochemical and microbial functionality of a soil.     

新型肥料对全球三大粮食作物产量和土壤生物学活性影响的Meta分析

  【目的】  随着近年来农业发展方式从资源消耗型向绿色生态型的转变,发展绿色新型肥料成为一大热门。已有研究大多关注新型肥料对三大粮食作物 (小麦、玉米和水稻) 产量、氮吸收和氮利用效率的影响,但关于新型肥料对土壤生物学活性影响的系统研究相对较少。本研究旨在整合已有的研究结果,定量分析新型肥料对三大作物产量和土壤生物学活性的影响,进而为加快新型肥料的研发与推广提供科学依据。  【方法】  本研究数据来源于“中国知网 (CNKI) ”及“Web of Science”数据库,以“小麦”、“玉米”、“水稻”、“产量”、“微生物量”、“酶活性”、“新型肥料”为主要关键词检索相关的田间试验文献,共筛选出文献29篇,包含32个独立试验,共545组数据。以施用传统化学肥料为对照组,施用新型肥料为试验组,采用Meta分析的方法,整合分析施用新型肥料对作物产量、土壤微生物量及酶活性的影响。  【结果】  与施用传统化学肥料相比,施用新型肥料显著提高三大作物产量、地上部吸氮量和氮肥利用效率,分别提高8.4%、9.9%和36.8%。施用新型肥料显著增加土壤微生物量氮 (14.6%),但对土壤微生物量碳无显著影响。施用新型肥料显著提高了土壤磷代谢酶活性 (8.6%) 和氧化相关酶活性 (5.7%),但对土壤碳代谢酶活性和氮代谢酶活性无显著影响。  【结论】  施用新型肥料提高了三大作物产量、地上部吸氮量和氮肥利用效率,同时增加了土壤微生物量氮、土壤磷代谢酶活性和氧化相关酶活性,提高了农田生态系统土壤生物学活性。     

Soil enzyme activity in an old-growth northern hardwood forest: Interactions between soil environment,ectomycorrhizal fungi and plant distribution

Plants and soil microbes produce extracellular enzymes (EE) that catalyze the hydrolysis of nitrogen (N) and phosphorus (P) containing compounds in soil and other enzymes involved in degradation of lignin and cellulose. We explored whether soil enzyme activity involved in carbon (C), N and P cycling were correlated with plant distribution, soil chemical conditions and the identity of fungi colonizing tree roots in an old growth forest remnant. Terminal restriction fragment length polymorphism (TRFLP) was used to determine the presence of root fungi and standard fluorometric analysis was used to determine soil enzyme activities. Soil enzymes were consistently positively correlated with soil C and N, but not CN ratio. Soil P was also correlated with enzyme activity during both June and September sampling. We saw no significant relationships between herbaceous plant cover and enzyme activity in June, but there were significant positive correlations between α-glucosidase and herbaceous plant coverage in September. We also found that some enzymes were significantly correlated with the identity of fungi colonizing tree roots separated from the soil cores. Chitinase and β-glucosidase were positively correlated with the genera Russula and Piloderma while chitinase was negatively correlated with Amanita and Entoloma. In addition, phosphatase was positively correlated with Russula, Meliniomyces and Solenopezia. Our results suggest that enzyme activity in old growth forest soils are affected by a variety of environmental factors, and that herbaceous plants and some root fungi may be associated with sites of elevated or decreased decomposition potential and nutrient cycling.     

Enzyme additions as a tool to assess the potential bioavailability of organically bound nutrients

Potential enzyme activities in soil and water samples are measured by addition of an excess amount of suitable substrate and subsequent determination of product release. If the approach is reversed and an excess of enzyme is added, substrate availability becomes rate-limiting and the maximum release of product indicates the availability of a given substrate in a sample. This approach has been used in a range of studies using phosphatase enzyme additions to soil, manure and sediment extracts, soil suspensions, and lake and sea water (n = 41). Significantly fewer studies have used enzymes from the carbon, nitrogen and sulfur cycles (n = 14). In this review, the methodological aspects of enzyme additions are discussed using examples from studies in which enzymes from the phosphorus cycle were used. A meta-analysis performed for various soil extracts and water samples revealed that the majority of studies (75th percentile) indicate availability of organic phosphorus to enzymatic hydrolysis of up to 60%, with crude phytase preparations showing the lowest substrate specificity and greatest release of phosphorus. Compared to addition of enzymes from the phosphorus cycle, lower substrate degradation was generally achieved by addition of enzymes from the carbon, nitrogen and sulfur cycles to soil suspensions and soil organic matter extracts. Enzyme additions can be a valuable tool in process research, provided all the necessary controls are included and assay conditions optimized to ensure that the reaction reaches completion. Recommendations for the development of a standard protocol are made.     

葡萄糖添加对不同演替阶段森林土壤微生物及酶化学计量特征的影响

糖类作为一种重要的根系分泌物,如何影响土壤微生物及酶化学计量特征目前尚不清楚,制约着人们对上述过程的认识。为探究糖类对森林生态系统中土壤微生态环境和酶活性的影响,以黄土高原子午岭地区森林演替先锋(山杨林)、中期(油松林)和气候顶级群落(辽东栎林)为研究对象,通过野外土壤采样、添加3种不同浓度的葡萄糖(0.1,0.5,1 g/kg干土),和室内培养的方法,研究了葡萄糖添加对土壤微生物量和酶活性及化学计量的影响。结果表明:(1)随着森林演替,土壤中速效磷(SAP)、硝态氮(NO^-₃-N)等速效养分显著降低。在葡萄糖影响下,3种林地土壤全氮(TN)降低,山杨林土壤C/N值显著大于油松林和辽东栎林。(2)随着森林演替,山杨林和辽东栎林微生物碳(MBC)和微生物氮(MBN)的值显著大于油松林,均为先减小后增加。随葡萄糖浓度增加,3种林地MBC和MBN均增加,微生物(MBP)呈波动趋势。(3)山杨林和辽东栎林土壤酶活性均显著大于油松林。随葡萄糖浓度增加,油松林和辽东栎林4种酶活性均为先增加后减小。BG/(LAP+NAG)、BG/AP和(LAP+NAG)/AP辽东栎林的值最大,山杨林和辽东栎林的值显著大于油松林。(4)RDA分析表明,土壤酶、微生物量及其酶活性分别与有机质(SOC),MBC有着显著性正相关关系,土壤酶活性(除NAG)与pH值均呈显著负相关。本研究说明碳输入增加了3种林分土壤的激发效应,土壤微生物量增加,降低了土壤TN含量,改变了土壤养分化学计量,进而加剧了油松林地微生物N限制和辽东栎林地微生物P限制,影响根际土壤有机质分解和养分代谢等过程,从而影响了森林演替过程。