STRUCTURAL STUDIES ON SOIL POLYSACCHARIDE

The polysaccharide extracted by alkali from a Countesswells series soil has been fully methylated and the hydrolysis products identified by GC-MS. The parent neutral sugars are galactose, glucose, mannose, arabinose, xylose, fucose and rhamnose and these constitute about 40 per cent of the polysaccharide. The analysis shows that hexose components are predominantly present in 1 → 3 and 1 → 4 linkages and pentose sugar in 1 → 4 linkages. About 20 per cent of the residues were in branching positions. From the number of non-reducing terminal groups present the average molecular weight of the methylated material has been calculated to be about 1460 compared with a value of 2700 obtained by vapour pressure osmometry. This contrasts with much higher values reported for unmethylated soil polysaccharides. The mixture of derivatives obtained supports the concept that soil polysaccharide originates in both plants and microorganisms.     

Effect of various procedures on the yield of methylated carbohydrate substances obtained from soil

Abstract

The molecular shape of a polysaccharide, which determines its cohesive and water retention properties, depends on the types of bonding present between sugar residues. These may be determined by permethylation and hydrolysis. Neither acetylation, nor reduction of soil prior to methylation, increased the overall yield of methylated product, although acetylation resulted in a greater proportion being released in the first methylation which may avoid some ß‐elimination reactions on remethylation. Reduction by NaBH₄ before each methylation step did not result in any large differences in the proportions of different sugar residues, suggesting that ß‐elimination reactions did not have a prominent effect in the remethylation of the soil residue. Grinding and freeze‐milling of the soil prior to methylation gave a greater release of methylated product in the first three methylations and the yield was about 15% greater overall.     

Nature of soil carbohydrate and its association with soil humic substances

The fulvic acid and alkali-soluble polysaccharide fractions of a sandy loam arable soil of the Countesswells series have been subjected to acid hydrolysis or methylation and the products examined by infra-red and NMR spectroscopy, pyrolysis mass spectrometry and chemical analysis. Infra-red and NMR spectroscopy of the polysaccharide fraction indicated that the substance was predominantly carbohydrate, although sugars accounted for less than one-third of the weight by chemical analysis. Pyrolysis mass spectrometry con-firmed the presence of sugars by sugar anhydride formation, but also showed the presence of ‘secondary’ or ‘pseudo’ polysaccharide. The fulvic acid contained only 2–3% sugars by chemical analysis, whereas a much larger carbohydrate component was suggested by physicochemical analyses. Infra-red and pyrolysis mass spectrometry difference spectra for the residues after acid hydrolysis indicated the release of material with some of the characteristics of glycoprotein. Most of the carbohydrate present in the fulvic acid was of the secondary or pseudo polysaccharide type. The hypothesis that the secondary polysaccharide could be a degraded polysaccharide structure in which some sugar residues have been partly transformed to melanoidins by Maillard reaction is explored.     

Components of soil organic matter under grass and arable cropping

Components of the organic matter have been studied in three soils from adjacent sites with different long-term treatments: soil I, prolonged arable cultivation; soil II, 17 years under grass after prolonged arable cultivation; and soil III, old pasture. Contents of total organic C in the top 15cm were 0.9% in soil I. 1.7% in soil II and 4.8% in soil III. The light fraction, comprising partially decomposed materials with a specific gravity < 2.06, represented greater proportions of the organic C in soils II and III (20–23 per cent) than in soil I (8.5 per cent). The light fraction of soil III had a relatively high N content.The proportions of the soil organic C released, by hydrolysis, as neutral sugars, uronic acids, amino sugars, amino acids and phenolic acids were generally similar in the three soils, although uronic acids and phenolic acids constituted somewhat greater proportions in soils II and III than in I.The light fractions contained greater proportions of neutral sugars and phenolic acids, and smaller proportions of amino sugars and amino acids than the whole soils.     

An automated anion-exchange chromatographic procedure for the estimation of saccharides in acid hydrolysates of soil

It is well known that the major neutral monosaccharide components released from soil by acid hydrolysis are glucose, galactose, mannose, arabinose, xylose, ribose, rhamnose and fucose. A colorimetric determination of the saccharide mixture released from soil is unsatisfactory for determining an accurate figure for soil saccharides. More precise information can be obtained by the determination of monosaccharides after separation by chromatography. Paper and thin-layer chromatography for quantitative analysis are rather time consuming and laborious. The gas chromatographic procedure was applied successfully for the analysis of sugars in soil hydrolysates by OADES et at. (7). Preparation of the various derivatives for gas chromatography still requires many steps, much handling of the sample, and considerable time, although final analysis of the product derivative is accomplished in an hour or two.     

Structure of soil carbohydrates resistant to periodate oxidation

Permethylation has been applied to the polysaccharide in soil, which has been oxidized with 0.2 m NaIO₄ and treated with 0.1 m Na₂B₄O₇. The amounts and types of methylated sugar obtained on hydrolysis have been compared with those from unoxidized soil.
As well as a number of derivatives corresponding to 1 -3 linked glucose, arabinose and xylose, considerable proportions of 1-4 linked glucose and xylose also persisted. The polysaccharides appear to persist for both chemical and physical reasons.     

外源氮添加对土壤微生物残体积累动态的影响－基于Meta分析

微生物残体是土壤有机碳库的重要贡献者。为明确外源氮添加对土壤微生物残体积累动态的影响,本文收集整理了1980—2020年已发表的文献,共选取122组试验观测数据,利用整合分析方法（Meta-analysis）,以微生物残体标识物－氨基糖为目标组分,定量分析了不同种类和数量的外源氮添加对土壤中微生物来源细胞残体积累数量和组成比例的影响,并系统解析其主要影响因素。结果表明:外源氮添加（0 ~ 6000 kg hm?1）对微生物细胞残体的积累有显著的促进作用,并能引起土壤中真菌和细菌来源细胞残体相对比例发生明显变化。与不加氮对照相比,氮添加使土壤氨基糖总量增加27%,其中氨基葡萄糖、氨基半乳糖和胞壁酸含量分别增加22.5%、29.8%和19.0%。同时,不同种类外源氮素添加对氨基糖积累特征的影响也有所不同,表现为有机氮（如动物厩肥）比无机氮添加对氨基糖积累的促进作用更大。此外,氮添加对氨基糖的影响程度还与土壤自身的碳氮比、土地利用类型和自然降雨量等环境因子密切相关。其中是否添加碳源对微生物残体的响应有较大影响,表现为:无碳源添加会降低土壤氨基糖葡萄糖和胞壁酸对氮添加的响应,削弱了微生物残体对土壤有机质的贡献比例;而氮源同时配合碳源添加条件下,土壤氨基糖积累量显著高于单一氮源添加的处理,说明氮添加对微生物残体积累的影响存在着碳氮耦合效应。     

Determination of neutral sugars in soil by capillary gas chromatography after derivatization to aldononitrile acetates

We have quantified ribose, rhamnose, arabinose, xylose, fucose, mannose, glucose, and galactose in soil by gas chromatography (GC) simultaneously after converting to aldononitrile acetate derivatives. A recommended single-hydrolytic step by 4 M trifluoroacetic acid (TFA) at 105 °C for 4 h was more effective for releasing soil neutral sugars from non-cellulosic carbohydrates and better suited to our purification procedure compared with the sulphuric acid hydrolysis. Linearity of the GC detection for each neutral sugar was in the range of 10-640 μg ml⁻¹ and the recovery of neutral sugars from the spiked soil samples ranged from 76% to 109.7%. The coefficients of variation of the neutral sugars in four soils were lower than 2.0% for the instrument and 4.6-7.6% for the whole determination procedures. Compared with the trimethylsilyl (TMS) derivatization, the recovery of our newly modified method was more satisfactory and the reproducibility of ribose was improved significantly. Moreover, the aldononitrile acetate derivative was more stable than TMS derivative. Therefore, it is a promising approach suitable for a routine use in the quantitative analysis of soil neutral sugars, since it is fast, sensitive, and reproducible.     

Microbial residues as indicators of soil restoration in South African secondary pastures

Prolonged intensive arable cropping of semiarid grassland soils in the South African Highveld resulted in a significant loss of C, N and associated living and dead microbial biomass. To regenerate their soils, farmers converted degraded arable sites back into secondary pastures. The objective of this study was to clarify the contribution of microorganisms to the sequestration of C and N in soil during this regeneration phase. Composite samples were taken from the topsoils of former arable land, namely Plinthustalfs, which had been converted to pastures 1-31 years ago. Amino sugars were determined as markers for microbial residues in the bulk soil and in selected particle-size fractions. The results showed that when C and N contents increased during the secondary pasture usage, the amino sugar concentration in the bulk soil (0-5 cm) recovered at similar magnitude and reached a new steady-state level after approximately 90 years, which corresponded only to 90% of the amino sugar level in the primary grassland. The amino sugar concentration in the clay-sized fraction recovered to a higher end level than in the bulk soil, and also at a faster annual rate. This confirms that especially the finer particles contained a high amount of amino sugars and were responsible, thus, for the restoration of microbially derived C and N. The incomplete recovery of amino sugars in bulk soil can only in parts be attributed to a slightly coarser texture of secondary grassland that had lost silt through wind erosion. The soils particularly had also lost the ability to restore microbial residues below 5 cm soil depth. Overall, the ratios of glucosamine to muramic acid also increased with increasing duration of pasture usage, suggesting that fungi dominated the microbial sequestration of C and N whereas the re-accumulation of bacterial cell wall residues was less pronounced. However, the glucosamine-to-muramic acid ratios finally even exceeded those of the primary grassland, indicating that there remained some irreversible changes of the soil microbial community by former intensive crop management.     

Saccharides of ectomycorrhizal fungal sclerotia as sources of forest soil polysaccharides

ABSTRACT

The neutral monosaccharide composition of forest soils differs from that of non-forest soils suggesting there is an accumulation of microbial saccharides. Ectomycorrhizal (ECM) fungi can be responsible as the fungi are typical in forest soils. We investigated neutral saccharides of ECM fungal sclerotia to determine what part it might play in the origin of forest soil polysaccarides. Sclerotial grain (SG) was collected from the O, A1 and A2 horizons of a soil of subalpine forest of Mt. Ontake, central Japan. Neutral saccharides in soil and SG were analyzed by two step hydrolysis with sulfuric acid and gas-chromatography of alditol acetate derivatives. Saccharides accounted for 6.0?16% of the SG by carbon content. The SG contained predominantly easily hydrolysable (EH)-glucose, which accounted for 75–85% of the composition depending on grain size and the soil horizon, followed by mannose (7.7?15%), galactose (2.2?4.8%) and non-easily hydrolysable (NEH)-glucose (1.7?6.1%). The SG contained all of these sugars irrespective of its size. The SG collected from the A1 and A2 horizons contained all sugar components found in that from the O horizon, except for fucose in that from A2 horizon. The monosaccharide composition of SG indicates that accumulation of ECM fungal sclerotial polysaccharides might have been responsible for enlarging the molar ratios of (galactose + mannose) /(arabinose + xylose) and EH-glucose/NEH-glucose of forest soils. The proportions of SG saccharides relative to soil saccharides were 3.6, 1.2, and 0.83% for the O, A1 and A2 horizons, respectively. These levels of the proportion are considerable as ECM fugal sclerotia are the products of a limited species among hundreds and thousands of microbial species inhabiting forest soils. The sclerotia forming ECM fungal species such as Cenococcum geophilum may be key sources of forest soil polysaccharides.     

硼砂对灰棕紫泥土尿素转化与脲酶活性的影响

采用室内培养,连续取样测定培养期内0%、0.5%、1%、1.5%和2%的硼砂对灰棕紫泥土中尿素、铵态氮、硝态氮和脲酶活性变化的影响。结果表明,硼砂能抑制脲酶活性,进而减缓尿素水解速度与铵态氮释放速度,0.5%的硼砂效果较弱,随硼砂用量的增大,效果增强;硼砂对硝态氮的积累影响不大。由于硼本身就是作物必需营养元素,因此作为尿素缓释剂有着良好的应用前景。     

氮肥和羊粪对内蒙古典型草原土壤多糖含量及组成的影响

采用三氟乙酸(TFA)水解、糖醇乙酸酯衍生、气相色谱法测定土壤多糖含量,研究了内蒙古羊草草原围栏封育并连续5年施用氮肥和羊粪的表层土壤的多糖含量和组成特征。结果表明:长期施用氮肥显著降低土壤多糖含量6%～19%;施用羊粪显著提高土壤多糖含量20%。施氮肥或羊粪都降低了(半乳糖+甘露糖)(/阿拉伯糖+木糖)(GM/AX)和甘露糖/木糖(M/X)的比例,表明施肥降低了微生物多糖对土壤多糖的贡献,但是施氮肥的土壤降低幅度大于施羊粪的土壤。这表明,长期施氮肥和羊粪都将改变土壤多糖含量和组成。     

Effect of periodate treatment of soil on carbohydrate constituents and soil aggregation

Soil from a field under long-term grass was treated with 0.02m sodium periodate for various periods up to 1176 h, followed by 0.1 d sodium tetraborate for 6 h. This destroyed an increasing proportion of microaggregates >45 μm and carbohydrate. After periodate treatment for 6 h about 70% of the soil sugars remained in the residue as measured by reducing sugar content and about 67% as individual sugars measured by gas-liquid chromatography. After 48 h the reducing sugar content was about 45%. An inverse linear relationship was established between the proportion by weight of microaggregates >45 μm and residual carbohydrate. The residual carbohydrate showed an enrichment in sugars commonly found in plant materials; glucose, arabinose and xylose, suggesting that the microbial carbohydrate had been preferentially destroyed. When the concentration of the periodate was increased to 0.05 m the residue contained about 50% of the original carbohydrate after 6 h treatment, and 25% after 48 h and an additional increase of about 10% in the proportion by weight of particles in the <45 μm range. These results throw doubt on the validity of assumptions made in a number of studies about the limited extent to which soil polysaccharide is involved in aggregation.     

土壤碳水化合物的转化与累积研究进展

碳水化合物虽然仅占土壤有机质的10%~20%,却是土壤中最活跃的有机碳库,是微生物的主要能源和碳源,并参与土壤团聚体的形成,是土壤有机质和土壤性质研究中的重要指标和对象。本文综述了土壤碳水化合物种类、来源和在团聚体中的分布特征、微生物群落结构对碳水化合物的转化和积聚的影响,同时讨论了耕作、施肥等农业措施对碳水化合物含量和分布的影响。从现有文献资料来看,碳水化合物的含量因土壤粒级不同而异;外源碳水化合物在土壤中的转化和累积与微生物群落结构特征密切相关,细菌在单糖等简单碳水化合物的转化中起主要作用,而复杂碳水化合物则首先在真菌的作用下水解成单糖;真菌和革兰氏阳性菌丰富的土壤更有利于碳水化合物来源碳的累积,但是详细的转化过程和存在形态认识有限,有待深入研究。     

沼液替代化学氮肥对滨海稻田土壤有机氮和细菌群落的影响

  【目的】  明确沼液替代化肥对稻田土壤有机氮(SON)矿化的影响及其与土壤微生物群落变化的关系。  【方法】  田间试验于2017年在江苏滨海稻田上进行,在总氮投入量225 kg/hm2的前提下,设置4个沼液替代化肥氮比例:0%、33%、66%、100% (即,BS0、BS33、BS66、BS100处理)。试验连续进行3年后(2019年),取土测定了有机氮的矿化特征,分析有机氮中酸解氮、非酸解氮、酸解铵态氮、氨基酸态氮、氨基糖态氮、酸解未知态氮的含量及细菌组成结构。  【结果】  与BS0处理相比,BS66处理的土壤氮矿化势(N₀)增幅最大,达39.7%。氨基酸态氮和非酸解氮含量沼液施用处理有显著升高(P<0.05),BS66处理的氨基酸态氮较BS0增加了39.2%,BS100处理的非酸解氮含量增加了73.9%。沼液替代化肥提高了土壤Chloroflexi (绿弯菌门)与Actinobacteria (放线菌门)的相对丰度,显著降低了Nitrospirae (硝化螺旋菌门)的相对丰度(P<0.05)。不同细菌属对沼液替代化肥水平的响应差异较大,随着沼液替代化肥比例的上升Subgroup_6_unclassified的相对丰度增加;而Subgroup_17_unclassified的相对丰度则呈下降趋势;KD4-96_unclassified的相对丰度在BS33处理下降低而在BS66处理及BS100处理下升高;沼液替代化肥模式下新增了相对丰度≥1%的细菌属Subgroup_7_unclassified。冗余分析结果显示,土壤N₀与Thermodesulfovibrionia_unclassified呈极显著负相关(P<0.01),非酸解氮与Subgroup_6_unclassified呈显著正相关(P<0.05),而与Betaproteobacteria_unclassified呈显著负相关(P<0.05),氨基酸态氮与Proteobacteria_unclassified、氨基糖态氮与KD4-96_unclassified呈显著正相关(P<0.05),酸解未知态氮和Desulfarculaceae_unclassified呈显著负相关(P<0.05)。  【结论】  沼液替代化肥改变了土壤微生物群落结构,增加了土壤氮库容量,显著提高了土壤氮矿化势,增强了土壤供氮能力。     

Land-use effects on the composition of organic matter in particle-size separates of soil: I. Lignin and carbohydrate signature

Soil from Eutrochrept A horizons under long-term spruce forest (Sf), mixed deciduous forest (Df), permanent grassland (Gp) and arable rotation (Ar) was fractionated according to particle size and analysed for contents of C, N, lignin-derived phenols and carbohydrates. Whole soil from Sf, Df, Gp and Ar contained 84, 59, 73 and 25 g C kg^?1 soil, respectively. For all sites, the C content declined and C/N ratio increased in the order: clay (<2 μm), silt (2–20 μm), sand (20–2000 μm). Clay and silt were significantly lower in C in Ar than in Sf, Df and Gp, C associated with sand being substantially lower under arable rotation. The yield of lignin-derived phenols decreased and carboxyl functionality and methoxyl demethylation of lignin derivatives increased with decreasing particle size, indicating a progressive lignin alteration. Whole soil from Sf and Gp was substantially higher in vanillyl (V), syringyl (S) and cinnamyl (C) units (VSC) than soil from Df and Ar. Compared to whole soil, clay was depleted and sand enriched in VSC. Only sand appeared to be affected significantly by land use. Sand from Ar and Df was more enriched in VSC than sand from Gp and Sf. Whole soil carbohydrates decreased in the order: Gp>Ar>Df>Sf. Sand- and clay-sized separates were enriched in carbohydrates compared to silt. Carbohydrates in sand were mainly of plant origin whereas microbially-derived sugars accounted for a larger proportion in the clay. Compared to Sf, Df and Gp, clay from Ar was enriched and sand depleted in microbial sugars. Lignin and carbohydrate distribution patterns indicate that organic matter was in a more advanced stage of decomposition in the sand separates from forest than from agricultural A horizons. The forest soils also show a higher degree of oxidative changes in lignin associated with clay. In contrast, differences between silt from the four A horizons were small.     

Amino acid composition of soil peptides chromatographed by high performance liquid chromatography on C18 and C8 columns

Summary Low molecular weight fractions (LMW; <5000 daltons) of organic matter were isolated from three soils by a mild extraction procedure and gel-permeation chromatography. The peptides present in the LMW soil extracts were separated on a Whatman Partisphere C18 and a Beckman Ultrapore C8 column by reverse-phase high performance liquid chromatography (HPLC). The peptide fractions were collected, acid hydrolyzed, and analyzed for amino acid composition. The C8 bonded-phase column gave better separation of the LMW soil peptide material than the C18 column. The total quantities of amino acids released from LMW peptides by acid hydrolysis were greater than the quantities released by immobilized protease hydrolysis (Warman and Isnor 1990). Total soil N present in the form of LMW peptides in these three soils ranged from 4 to 15%. The total recovery of peptide amino acid-N showed little difference between the C18 and C8 columns for two of the soils tested.     

Distribution and partial characterisation of acid hydrolysable organic nitrogen in six New Zealand soils

Six New Zealand topsoils of widely different origins and properties were subjected to 6m HC1 hydrolysis and the distribution of N fractions and amino acids were determined qualitatively and quantitatively.Of the total-N in the soils studied 83–91%, was hydrolysable with 6m HCl. The largest proportion of the hydrolysable N was α-amino acid N (38– 42%). followed by hydrolysable-unknown N (HUN) (14–24%), and NH₄⁺-N (14–22%). A significant proportion (25–50%) of the HUN fraction was accounted for by the non α-amino acid-N. Oxidative (3% H₂O₂) hydrolysis released N-phenoxy amino acid-N and possibly N-compounds which were complexed with phenols and sugars. All soils had a similar amino-acid composition with a predominance of acidic amino-acids.     

Content and bioavailability of organic forms of nitrogen in the O horizon of a podzol

It is often thought that the most important source of nitrogen for plants and microorganisms comes from amino acids and amino sugars when they are hydrolysed in acid conditions. We did a microcosm experiment to test the hypothesis. In the experiment spruce seedlings (Picea abies L. Karst) were grown for 145 days in soil taken from a podzol Oa horizon under a long-term nitrogen fertilization experiment (control and N-treated soil). Net changes in different pools of organic N were determined using standard fractionation (acid hydrolysis and pyrophosphate extraction). During the experiment the amino acid and amino sugar pools decreased significantly (14% and 15% for the control and 10% and 17% for the N treatment), whereas no significant change was observed in the non-amino acid plus non-amino sugar fraction. On a per organic C basis there was even a significant increase in the non-amino acid plus non-amino sugar fraction of 11% for the control and 8% for the N treatment. Pyrophosphate extractions suggest that amino acids or amino sugars associated with the humin fraction were more accessible to microbes and plants than those associated with the humic acid, fulvic acid and hydrophilic substances. The long-term N fertilization (about 73 kg N ha⁻¹ was added annually as NH₄NO₃ during a 24-year period) resulted in an enrichment of all major fractions of organic N, i.e. amino acids, amino sugars and non-amino acids plus non-amino sugars. This enrichment was largely the result of small increases in all of the amino acids rather than large increases in just a few.     

The [13C]nuclear magnetic resonance spectrum of a methylated humic acid

A humic acid has been investigated by [¹³C]NMR spectroscopy using the methylated humic acid dissolved in chloroform which gave a relatively simple spectrum.Estimates of different types of C show that 21% of the C is aromatic, 35% consists of methylene chains and methyl groups, 3%, as the carbonyl C of methyl esters, and 13% as the methoxyl C of methylated phenols and alcohols. The rest, 28%, is not accounted for and may be mostly C bound to O as in polysaccharides or in peptides. No ketonic or quinonic groups were detected.The [¹³C]NMR spectrum of the methylated humic acid is entirely different from those of lignin and its derivatives thus suggesting that lignin residues play no significant role in the structure of the humic acid.