两种测定土壤微生物量氮方法的比较初探

用氯仿熏蒸-0.5mol/L的K2SO4直接浸提,280nm紫外比色法和熏蒸-淹水培养法测定了20种有机质、全氮和速效氮差异较大的土样的土壤微生物量N。研究结果表明,两种方法测得20种土样的土壤微生物量N数值呈极显著正相关;280nm紫外比色法操作步骤简单、产生误差的环节少、测定所需时间短、且测定数据比熏蒸-淹水培养法有更好的重现性。初步认为,280nm紫外比色法来反映土壤微生物量的大小。结果还表明,两种方法的测定结果都与土壤的全氮含量呈极显著正相关关系,与有机碳含量有一定的正相关关系,与速效氮无明显的相关关系;但在不同的土壤类型上,与全氮、有机碳和速效氮的相关性有所不同。用280nm紫外比色法测定两种土壤的新鲜和风干样的微生物生物量的结果说明,可用风干土经预培养后测定土壤微生物生物量。风干土样的预培养时间初步确定为10天。     

熏蒸浸提法测定碱性土微生物生物量碳初探

为探究熏蒸浸提法测定碱性土壤微生物生物量碳(microbial biomass carbon,MBC)的最优熏蒸时间和浸提液浓度,本研究采用9种熏蒸时间、2种K_2SO_4浸提液浓度对不同有机质含量的碱性土壤MBC进行了测定。结果表明:(1)对于碱性土壤,熏蒸时间的选择与土壤有机质含量有关,土壤有机质含量越高,MBC提取值达到稳定的熏蒸时间越长。建议测定高有机质土壤(≥60 g/kg)MBC时熏蒸时间不少于24 h;测定中、低有机质土壤(≤30g/kg)MBC时熏蒸时间不少于18 h。(2)土壤有机质含量影响K2SO4浸提液的提取效率,测定高、中有机质土壤MBC时需要0.5 mol/L K_2SO_4浸提液进行提取;在低有机质土壤MBC测定中,0.25 mol/L K_2SO_4浸提液即可。(3)对于未知有机质含量的碱性土壤建议氯仿熏蒸时间至少为24 h,K_2SO_4浸提液浓度为0.5 mol/L。     

紫外分光光度法测定农田土壤微生物生物量氮可行性初探

采用氯仿熏蒸浸提-紫外分光光度法和消化法比较测定了田间定位试验不同施肥处理土壤、添加植物残体土壤、添加葡萄糖土壤的微生物生物量碳、氮(SMBC,SMBN)。结果表明,当土壤微生物生物量氮含量较高时(>20 mg kg-1),采用分光光度法与消化法测定的SMBN具有显著正相关关系(P<0.05),但当SMBN量较低时(<20 mg kg-1)时,分光光度法测定与消化法测定的SMBN没有显著相关性。当土壤中添加麦秸和玉米秸时,土壤浸提液颜色较深(黄色),不适合采用分光光度法测定SMBN。因此,熏蒸提取–分光光度法测定SMBN,仅适于土壤浸提液无色透明、且SMBN含量较高的土壤。     

土壤微生物体氮测定方法的研究

用熏蒸-0.5mol/LK2SO4 直接浸取NH4+-N法 (简称薰蒸 铵态氮法 ) ,熏蒸 淹水培养法和熏蒸 通气培养法测定了有机质、全氮和C/N比差异较大的 15种土壤的铵态氮增量 (FN)。结果表明 ,它们之间有极显著的正相关 ,在反映土壤微生物体氮上有相同趋势。两种培养方法测定的FN 近乎一致 ,由此而计算的微生物体氮也几乎相同。对红油土铵态氮法测定值仅为两种培养法的 1/ 10。把铵态氮法中的FN 校正后 ,其结果与 2种培养法测定的微生物体氮同样近乎一致。用 3种方法测定的微生物体氮均与土壤有机碳存在显著正相关性。淹水培养和铵态氮法水分条件易控制 ,又无NH3的挥发损失 ,比通气培养法更加优越。对培养试验和长期肥料定位试验的土样测定结果表明 ,土壤中易矿化新鲜有机物料也会使熏蒸 淹水培养法测定的FN 显著下降 ,由此而计算的微生物体氮也显著减少 ,但熏蒸 铵态氮法测定的FN 不受新鲜有机物质的影响。与土壤微生物数目进行比较后发现 ,土壤中含易分解有机物质少或微生物体氮含量低时 ,选用熏蒸 淹水培养法测定误差小 ;当土壤中富含新鲜有机物质时 ,熏蒸 铵态氮法测定的结果更加可靠。用这两种方法在同类土壤上测定的FN 的比值相对稳定 ,微生物体氮 (BN)的平均比值为 0.98～1.01,不受施肥的影响     

水稻土微生物生物量的测定方法

土壤微生物生物量常被作为植物所需营养元素的转化因子和资源库而日益受到人们的重视^[1]。而测定土壤微生物生物量的方法当首推氯仿熏蒸一培养法^[2]。该法是根据被杀死的微生物细胞因矿化作用而释放的CO₂量的激增来估计微生物生物量的。     

放牧与围栏内蒙古针茅草原土壤微生物生物量碳、氮变化及微生物群落结构PLFA分析

以内蒙古贝加尔针茅草原、大针茅草原和克氏针茅草原为研究对象,采用氯仿熏蒸法和磷脂脂肪酸（PLFA）分析方法研究了放牧与围栏条件下内蒙古针茅草原土壤微生物生物量和群落结构特征的变化情况。研究表明放牧与围栏草地土壤微生物生物量和群落结构差异显著。氯仿熏蒸法分析结果表明内蒙古针茅草原土壤微生物生物量碳的含量介于166.6-703.5mg·kg^-1之间,微生物生物量氮含量介于30.34-92.15mg·kg^-1之间,其中贝加尔针茅草原土壤微生物生物量碳、氮最高,大针茅草原次之,克氏针茅草原则最低。放牧条件下,贝加尔针茅草原、大针茅草原土壤微生物生物量碳、氮显著低于围栏草地,克氏针茅草原则无显著变化。PLFA分析结果显示,内蒙古针茅草原土壤微生物群落PLFAs种类、含量丰富,共检测出28种PLFA生物标记磷脂脂肪酸,并且以直链饱和脂肪酸和支链饱和脂肪酸为主,相对含量占总量的2/3左右,其中贝加尔针茅草原土壤微生物含量最丰富,其围栏样地土壤的PLFA含量达到27.3nmol·g-1,大针茅草原和克氏针茅草原依次降低。围栏条件下,各类型草原土壤细菌脂肪酸与总PLFA含量均显著高于放牧草地,真菌脂肪酸含量则因草原类型不同各有差异;放牧导致各类型草原革兰氏阳性细菌PLFAs/革兰氏阴性细菌PLFAs（GPPLFAs/GNPLFAs）比值显著降低,而除了克氏针茅草原,细菌PLFAs/真菌PLFAs比值则显著升高。PLFAs主成分分析表明,放牧和围栏处理对内蒙古针茅草原土壤微生物群落结构产生影响,且围栏处理的影响程度大于放牧处理。经相关分析表明,氯仿熏蒸法和PLFA分析方法之间有很好的一致性,且土壤微生物PLFAs与土壤有机质、全磷、硝态氮显著相关。     

土壤微生物生物量熏蒸提取法中转换系数K_(EC)的测定研究

本文综述了熏蒸提取法中用于计算土壤微生物生物量时所需的转换系数KEC的测定方法 ,并对现有的各种方法的应用性作了简要的评价 ;同时指出了目前常采用的KEC值在使用上应注意的事项     

坝上地区退耕还林还草措施对土壤细菌的影响

以河北坝上地区实施退耕还林还草18年后的耕地、林地、灌丛和草地为研究对象,通过测定土壤理化性质并结合氯仿熏蒸浸提法和高通量测序技术,分析了坝上地区退耕还林还草措施对土壤微生物生物量、细菌多样性和群落结构特征的影响.结果发现:林地的土壤含水量有微弱下降趋势,但不同植被恢复类型下土壤中的碳、氮、磷、钾等元素含量无显著差异....     

土壤微生物生物量抽提检定法的研究--土壤微生物生物-C的烘杀抽提容量法检定

本文是在前人提出的氯仿熏蒸分解法测定土壤微生物生物-C的基础上，经过对比试验研究的实践，作了适应性的修改。这种新方法可直接应用于酸性土壤微生物生物-C的测定。现暂称之为烘杀抽提容量分析法。该法较之前者更为方便，有效，而且能够大大缩短测定周期。     

土壤微生物生物量碳的表观周转时间测定方法

土壤微生物生物量碳周转对土壤有机质和养分循环起着决定作用。本研究建立了土壤微生物生物量碳周转时间的测定方法。培养条件下 (2 5℃、10 0 %湿度 ) ,加入14 C标记葡萄糖标记土壤微生物生物量碳 ,在 10 0d培养期内 ,每隔 2 0d测定一次14 C标记微生物生物量碳 (14 C BC) ,采用一级热力学方程拟合测定期内 (2 0～ 10 0d) 14 C BC 的周转速率常数 (k) ,由此计算土壤微生物生物量碳的表观周转时间。测定的 5个土壤在培养条件下微生物生物量碳的周转时间为 93～ 4 0 0d ,根据培养温度和实际田间年平均温度推算得到田间条件下土壤微生物生物量碳的周转时间为 1 0～ 4 1a。其主要影响因子为土壤质地 ,土壤利用方式的影响较小。土壤微生物生物量碳的周转时间能较好地反映土壤微生物生物量的周转状况及其与土壤有机质的周转和积累的关系。     

The influence of tillage on the proportion of organic carbon and nitrogen in the microbial biomass of medium-textured soils in a humid climate

Summary Microbial biomass C and N respond rapidly to changes in tillage and soil management. The ratio of biomass C to total organic C and the ratio of mineral N flush to total N were determined in the surface layer (0–5 cm) of low-clay (8–10%), fine sandy loam, Podzolic soils subjected to a range of reduced tillage (direct drilling, chisel ploughing, shallow tillage) experiments of 3–5 years' duration. Organic matter dynamics in the tillage experiments were compared to long-term conditions in several grassland sites established on the same soil type for 10–40 years. Microbial biomass C levels in the grassland soils, reduced tillage, and mouldboard ploughing treatments were 561, 250, and 155 g g^-1 soil, respectively. In all the systems, microbial biomass C was related to organic C (r=0.86), while the mineral N flush was related to total N (r=0.84). The average proportion of organic C in the biomass of the reduced tillage soils (1.2) was higher than in the ploughed soils (0.8) but similar to that in the grassland soils (1.3). Reduced tillage increased the average ratio of mineral N flush to total soil N to 1.9, compared to 1.3 in the ploughed soils. The same ratio was 1.8 in the grassland soils. Regression analysis of microbial biomass C and percent organic C in the microbial biomass showed a steeper slope for the tillage soils than the grassland sites, indicating that reduced tillage increased the microbial biomass level per unit soil organic C. The proportion of organic matter in the microbial biomass suggests a shift in organic matter equilibrium in the reduced tillage soils towards a rapid, tillage-induced, accumulation of organic matter in the surface layer.     

Active organic matter distribution in the surface 15 cm of undisturbed and cultivated soil

Summary Organic-matter dynamics near the soil surface influence plant nutrient supplies. To evaluate the effects of cultivation on active soil organic-matter distribution in the surface 15 cm, we measured total organic C, Kjeldahl N, microbial biomass C and N (chloroform fumigation), respirable C and mineralizable N (aerobic incubations) in 1-cm layers from 0–10 cm and in 2.5-cm layers from 10–15 cm in adjacent areas of undisturbed (shortgrass steppe) and cultivated (four years wheat-fallow rotation) Ascalon sandy loam soil (Aridic Argiustoll). The active organic matter was highly concentrated in the surface centimeter of undisturbed soil. In undisturbed soil, microbial biomass C and N concentrations were more than five times greater at 0–1 cm than at 2–15 cm. Respirable-C and mineralizable-N concentrations (20-day incubations) were 8 and 18 times greater at 0–1 cm than at 2–15 cm. Below 3 cm, the concentrations were equal in both cultivated and undisturbed soil. Cultivation reduced the average concentrations at 0-15 cm of microbial biomass C and N by 62% and 32%, and of respirable C and mineralizable N by 71% and 46%, by reducing the concentrations at 0-1 cm to levels comparable to the 2- to 15-cm layers of the undisturbed soil. Decreases after cultivation ensued from disrupting the surface 1-cm layer.     

Effect of CO<Subscript>2</Subscript> on nitrification and immobilization of NH<Subscript>4</Subscript><Superscript>+</Superscript>-N

A laboratory incubation experiment was conducted to demonstrate that reduced availability of CO₂ may be an important factor limiting nitrification. Soil samples amended with wheat straw (0%, 0.1% and 0.2%) and (¹⁵NH₄)₂SO₄ (200 mg N kg^–1 soil, 2.213 atom% ¹⁵N excess) were incubated at 30±2°C for 20 days with or without the arrangement for trapping CO₂ resulting from the decomposition of organic matter. Nitrification (as determined by the disappearance of NH₄⁺ and accumulation of NO₃^–) was found to be highly sensitive to available CO₂ decreasing significantly when CO₂ was trapped in alkali solution and increasing substantially when the amount of CO₂ in the soil atmosphere increased due to the decomposition of added wheat straw. The co-efficient of correlation between NH₄⁺-N and NO₃^–-N content of soil was highly significant (r =0.99). During incubation, 0.1–78% of the applied NH₄⁺ was recovered as NO₃^– at different incubation intervals. Amendment of soil with wheat straw significantly increased NH₄⁺ immobilization. From 1.6% to 4.5% of the applied N was unaccounted for and was due to N losses. The results of the study suggest that decreased availability of CO₂ will limit the process of nitrification during soil incubations involving trapping of CO₂ (in closed vessels) or its removal from the stream of air passing over the incubated soil (in open-ended systems).     

Effect of soil CO2 concentration on microbial biomass

The effect of increasing soil CO₂ concentration was studied in six different soils. The soils were incubated in ambient air (0.05 vol.% CO₂) or in air enriched with CO₂ (up to 5.0 vol.% CO₂). Carbon dioxide evolution, microbial biomass, growth or death rate quotients and glucose decay rate were measured at 6, 12 and 24 h of CO₂ exposure. The decrease in soil respiration ranged from 7% to 78% and was followed by a decrease in microbial biomass by 10–60% in most cases. High CO₂ treatments did not affect glucose decay rate but the portion of C_gluc mineralized to CO₂ was lowered and a larger portion of C_gluc remained in soils. This carbon was not utilized by soil microorganisms. Received: 30 August 1996     

Comparison of methods for extraction of organic N monomers from soil microbial biomass

One way of investigating the function of soil is via the pool of low molecular weight organic compounds in the soil microbial biomass. This is because low molecular weight organic compounds have key roles in metabolism of soil microbes, can function in osmotic adjustment and other stress responses, and are intermediates in the breakdown of polymers to inorganic nutrients. Methods for measuring low molecular weight microbial metabolites in soil rely upon extracting total metabolites and then subtracting the contribution from metabolites in the soil extracellular matrix (i.e. microbial = total − extracellular). Recent studies have tested methods for extracting organic N monomers from the extracellular matrix of soil, but there has not been similar testing of methods for extracting total organic N monomers. The aims of this study were to examine methods for extracting total organic N monomers by a) contrasting chloroform gas fumigation with chloroform direct extraction, and b) examining whether it is possible to extract soil with two methods that combine quenching of metabolic activity with extraction, namely cold methanol/chloroform/water and hot aqueous ethanol. To evaluate methods, organic N compounds were extracted from soil and then capillary electrophoresis–mass spectrometry identified and quantified 42 organic N monomers including amino acids, quaternary ammonium compounds, nucleobases and nucleosides, amines and polyamines. Absolute concentrations of 32 out of the 42 quantified organic N monomers were significantly different between soil extracted by chloroform gas fumigation and chloroform direct extraction. These differences were probably a function of gains and losses of compounds due to oxidation, hydrolysis and deamidation during the two-day chloroform gas fumigation. Cold methanol/chloroform/water yielded large amounts of the extremely labile compound ergothioneine, probably because the extraction method rapidly quenched metabolic activity. The primary limitation of extraction with methanol/chloroform/water is that it was ineffective at extracting strongly cationic compounds (e.g. polyamines). Extraction with hot aqueous ethanol was unsuccessful with soil presumably because soil microbes are difficult to lyse. It is recommended that future studies examining organic N monomers in soil microbial biomass use chloroform direct extraction or cold methanol/chloroform/water rather than chloroform gas fumigation.     

Urease activity of microbial biomass in soils as affected by cropping systems

 The impacts of crop rotations and N fertilization on different pools of urease activity were studied in soils of two long-term field experiments in Iowa; at the Northeast Research Center (NERC) and the Clarion-Webster Research Center (CWRC). Surface soil samples (0–15 cm) were taken in 1996 and 1997 in corn, soybeans, oats, or meadow (alfalfa) plots that received 0 or 180 kg N ha^–1, applied as urea before corn and an annual application of 20 kg P and 56 kg K ha^–1. The urease activity in the soils was assayed at optimal pH (THAM buffer, pH 9.0), with and without toluene treatment, in a chloroform-fumigated sample and its nonfumigated counterpart. The microbial biomass C (C_mic) and N (N_mic) were determined by chloroform fumigation methods. The total, intracellular, extracellular and specific urease activities in the soils of the NERC site were significantly affected by crop rotation, but not by N fertilization. Generally, the highest total urease activities were obtained in soils under 4-year oats–meadow rotations and the lowest under continuous corn. The higher total activities under multicropping systems were caused by a higher activity of both the intracellular and extracellular urease fractions. In contrast, the highest values for the specific urease activity, i.e. of urease activity of the microbial biomass, were found in soils under continuous soybean and the least under the 4-year rotations. Total and extracellular urease activities were significantly correlated with C_mic (r>0.30* and >0.40**) and N_mic (r>0.39** and >0.44**) in soils of the NERC and CWRC sites, respectively. Total urease activity was significantly correlated with the intracellular activity (r>0.73***). About 46% of the total urease activity of the soils was associated with the microbial biomass, and 54% was extracellular in nature. Received: 25 May 1999     

Loss of labile organic carbon from subsoil due to land-use changes in subtropical China

Topsoil carbon (C) stocks are known to decrease as a consequence of the conversion of natural ecosystems to plantations or croplands; however, the effect of land use change on subsoil C remains unknown. Here, we hypothesized that the effect of land use change on labile subsoil organic C may be even stronger than for topsoil due to upward concentration of plantations and crops root systems. We evaluated soil labile organic C fractions, including particulate organic carbon (POC) and its components [coarse POC and fine POC], light fraction organic carbon (LFOC), readily oxidizable organic carbon, dissolved organic carbon (DOC) and microbial biomass down to 100 cm soil depth from four typical land use systems in subtropical China. Decrease in fine root biomass was more pronounced below 20 cm than in the overlying topsoil (70% vs. 56% for plantation and 62% vs. 37% for orchard. respectively) driving a reduction in subsoil labile organic C stocks. Land use changes from natural forest to Chinese fir plantation, Chinese chestnut orchard, or sloping tillage reduced soil organic C stocks and that of its labile fractions both in top and subsoil (20–100 cm). POC reduction was mainly driven by a decrease in fine POC in topsoil, while DOC was mainly reduced in subsoil. Fine POC, LFOC and microbial biomass can be useful early indicators of changes in topsoil organic C. In contrast, LFOC and DOC are useful indicators for subsoil. Reduced proportions of fine POC, LFOC, DOC and microbial biomass to soil organic C reflected the decline in soil organic C quality caused by land use changes. We conclude that land use changes decrease C sequestration both in topsoil and subsoil, which is initially indicated by the labile soil organic C fractions.     

Freeze-dried soil extraction method for the measurement of microbial biomass C

A new method for the measurement of microbial biomass C by direct extraction of freeze-dried soil with either 0.5M K₂SO₄ or 0.5M NaHCO₃ was evaluated. The underlying principle of the method is that rehydrating a freeze-dried soil releases cytoplasmic organic compounds from desiccated and disrupted microbial cells. Nineteen soils under various management regimes were sampled to test the proposed method, in which each soil sample was split into two subsamples. One subsample was kept in the field-moist condition at 4°C. The other subsample was brought to 100% water-holding capacity and frozen at –20°C for 24h. The frozen soil was then freezedried. Both subsamples were extracted with 0.5M K₂SO₄ or 0.5M NaHCO₃ at a soil-to-extractant ratio of 1-to-4 (w/v) and organic C determined in the extract (C_{K2 SO4} or C_NaHCO3). The net freeze-dry stimulated increase in extracted C was correlated (r ²=0.98 for C_{K2 SO4} or 0.93 for <$>\rm C_{NaHCO_3})<$> more closely with microbial biomass C (C_MB) measured as net evolution of CO₂–C by chloroform fumigation incubation (CFI) than with total C (r ²=0.42 for C_{K2 SO4} or 0.47 for C_NaHCO3). Based on linear regression equations, extraction efficiency coefficients (K _EC) were used to calculate C_MB from C_{K2 SO4} or C_NaHCO3 as follows: C_MB=C_{K2 SO4}/0.152±0.004 C_MB=C_NaHCO3/0.257±0.01 The relationship between the C_MB and the flushes of C extracted after rehydration of freeze-dried soil showed that the K _EC values were more consistent for C_{K2 SO4} than C_NaHCO3. The freeze-dried soil extraction was a fast, precise, reliable and safe method for measuring microbial biomass C in soil. Received: 27 May 1996     

Soil CO2 evolution: Response from arginine additions

Short-term response of soil C mineralization following drying/rewetting has been proposed as an indicator of soil microbial activity. Houston Black clay was amended with four rates of arginine to vary microbial responses and keep other soil properties constant. The evolution of CO₂ during 1 and 3 days following rewetting of dried soil was highly related to CO₂ evolution during 10 days following chloroform fumigation (r² = 0.92 and 0.93, respectively) which is a widely used method for soil microbial biomass C, which disrupts cellular membranes. This study suggest that the release of CO₂ following rewetting of dried soil with no amendments other than heat and water can be highly indicative of soil microbial activity and possibly be used as a quantitative measurement of soil biological quality in Houston Black soils.