Effects of humic substances on fluorometric DNA quantification and DNA hybridization

DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities. Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic substances and vascular plant extract interfered with the fluorometric measurement of DNA concentration using Hoechst dye H33258 and PicoGreen reagent. Quantification of DNA amended with humic substances (20-80 ng/microl) using the Hoechst dye assay was more reliable than with PicoGreen reagent. A simple procedure was developed to improve the accuracy for determining the DNA concentration in the presence of humic substances. In samples containing up to 80 ng/microl of humic acids, the fluorescence of the samples were measured twice: first without Hoechst dye to ascertain any fluorescence from impurities in the DNA sample, followed with Hoechst dye addition to obtain the total sample fluorescence. The fluorescence of the Hoechst dye-DNA complex was calculated by subtracting the fluorescence of the impurities from the fluorescence of the sample. Vascular plant extract and humic substances reduced the binding of DNA onto the nylon membrane. Low amounts (<2.0 microg) of humic substances derived from estuarine waters did not affect the binding of 100 ng of target DNA to nylon membranes. DNA samples containing 1.0 microg of humic substances performed well in DNA hybridizations with DIG-labeled oliogonucleotide and chromosomal probes. Therefore, we suggest that DNA samples containing low concentrations of humic substances (<20 ng/microl) could be used in quantitative membrane hybridization without further purification.     

Influence of soluble humic substances on the growth of Algæ and blue-green Algæ

A stimulatory effect of soluble humic acid and sodium humate of soft coal origin on the growth of green algæChlorella andScenedesmus was established. On the contrary, the growth of blue-green algæ was not affected by humic substances. For green microorganisms soluble humic substances act more as a fertilizer than a bacteriostatic substance. The possibility of application of soluble humic substances as selective fertilizer of algæ at the expense of blue-green algæ at concentrations up to 300 mg/L is discussed.     

Size distribution of dissolved (<200 nm) organic carbon and aluminium in alkaline and humic lakes

Chemical equilibrium calculations treating humic substances as the simple diprotic phtalic acid, predicted no Al-humus at pH>6.5 in humic lake water. However, dissolved (<200 nm) organic carbon (DOC) and dissolved Al appeared to be linearly (r=0.597, P<0.001) correlated in samples from five different humic surface waters in The Netherlands with a DOC range of 10–36 mg.l^–1 and a (mean) pH range of 6.85–8.47. Yet, organic carbon (C_org) and Al did no exhibit similar size distributions between 5 and 200 nm revealed by ultrafiltration. Averaged 25% of the C_org and >50% of the Al occurred in the fraction <5 nm. Only in this fraction the C_org and Al were linearly correlated (r=0.515, P<0.001). This result suggests the presence of organic ligands in the DOC pool of humic waters having smaller molecular sizes and higher Al stability constants than the humic substances used to model aquatic Al speciation.     

Responses of the toxic cyanobacterium Microcystis aeruginosa to iron and humic substances.

Iron is an essential element to marine biota. Different types of dissolved organic matter (DOM), such as humic substances have impacts on the marine coastal waters iron chemistry. The aim of the study was to examine how the presence of humic substances (both aquatic and sedimentary) may affect iron bioavailability to the bloom-forming cyanobacterium Microcystis aeruginosa Kutzing incubated on standard and modified mineral BG-11 media. The final iron concentrations in the growth media ranged from 0.1 to 100microM. The results demonstrate that both the growth rate and the concentration of chlorophyll a in cultures of M. aeruginosa are limited by insufficient (<10microM) Fe concentrations. The addition of aquatic humic substances in the presence of iron in concentrations <0.1microM increased the optical density 25-fold, and the production of chlorophyll a 15-fold as compared with the cultures exposed to iron only at the same concentration. Sedimentary humic acids in the presence of iron at a concentration of 10microM reduced the growth and production of chlorophyll a by 50% as compared to the cultures exposed to iron only at the same concentration. Possible mechanisms of humic substances - metal ion - alga interactions are discussed. It is suggested that aquatic humic substances could be of great importance in the formation of cyanobacteria blooms.     

Comparison of Acridine Orange, Acriflavine, and Bisbenzimide Stains for Enumeration of Bacteria in Clear and Humic Waters

In highly humic water, acridine orange precipitated with dissolved humic matter, resulting in such bright background fluorescence that no bacteria could be seen. With bisbenzimide staining, a similar precipitate was nonfluorescent but obscured many cells. An acriflavine staining method proved useful and reproducible both in clear and in humic waters. Fading of fluorescence was not a problem, and stained samples could be stored after preparation. The fluorescence of cells stained with acriflavine was weaker than that with acridine orange, making counting extremely small cells slightly more difficult with the former stain.     

陆地生态系统中水溶性有机质的环境效应

目前水溶性有机质（Dissolved Organic Matter)已逐步成为陆地生态系统中的一个研究热点。系统地评述了陆地生态系统中DOM的组成特点及其环境效应。尽管关于陆地生态系统中DOM的研究还不完善,至今对其性质,组成和分类方法等问题看法不一,但现有结果已经表明DOM是一种十分活跃的重要化学组分,它对陆地生态系统中污染物质的溶解,吸附,解吸,吸收,迁移和生物毒性,微生物活动以及土壤形成过程等均有显著的影响。影响DOM在地生态系统中的环境效应的主要因素包括：DOM与污染物的络合作用,污染物溶解／沉淀作用,土壤对DOM的吸附作用,土壤质地,酸碱缓冲作用等。     

Speciation of iodine in high iodine groundwater in china associated with goitre and hypothyroidism

Iodine intake affects the occurrence of disease in a population. Excessive iodine intake may be caused by a high iodine content of drinking water. Tap water in few locations in Europe contains up to 139 μg/L mostly bound to humic substances, probably leaching from marine sediments in the aquifers. Even higher iodine contents have been found in Chinese waters, previously shown to associate with goitre and hypothyroidism. The aims were to elucidate speciation of high iodine groundwater from deep wells in China and to compare with high iodine waters from Europe. Water was sampled from eight wells in five villages along Bohai Bay, China. Macro-molecules and low molecular weight (MW) substances were separated by size exclusion chromatography (high performance liquid chromatography, Superose 12 HR 10/30, buffer 0.1 M Tris, pH 7.0). Organic material was evaluated by A280 and iodine in fractions measured by the Ce/As method after alkaline incineration. Iodine content of well water varied from 135 to 880 μg/L (median 287 μg/L). The amount of organic material in water was low with A280, <1–5 mAU. The chromatographic traces were similar between samples: One peak of iodine eluted around K _AV 0.65 corresponding to MW 5 kDa (humic substances) and one peak at V _total (iodide/low MW substances). The fraction of iodine in macro-molecules, suggested to be humic substances, varied from 8% to 70% (median 27%). Iodine and peak absorbance were associated (p = 0.006). In conclusion, iodine in iodine-rich deep well water in northern China may have marine origin and may associate with humic substances, comparable to shallow well iodine-rich water in Europe. High iodine intake from iodine-rich water suggests the cause of endemic goitre and hypothyroidism in some areas in China being iodine.     

Inhibition of phosphatase activity by dissolved humic substances and hydrolytic reactivation by natural ultraviolet light

1. Phosphatases released extracellularly by aquatic micro-organisms often complex with humic compounds that are released from decomposing tissues of plants and imported in dissolved and colloidal forms to lakes and rivers. 2. Dissolved humic substances from several natural sources formed complexes with phosphatases of bacterial and algal origin and reduced hydrolytic activity by non-competitive inhibition. Restoration of the hydrolytic enzyme activities from the humic substances–enzyme complexes increased progressively over time when exposed experimentally to natural and artificial ultraviolet (UV) irradiance. 3. Greater phosphatase restoration occurred from humic acid–phosphatase complexes when humic acids were extracted from dissolved organic matter (DOM) of mixed natural plant sources, than when humic acids were isolated from a decomposing single plant species. 4. The data support a previously suggested hypothesis that phosphatases and other enzymes in aquatic ecosystems can complex with humic substances that dominate the DOM pool. These humic substances–enzyme complexes, in which the enzyme is temporarily inactivated, can be transported with water movements and displaced to other sites within the ecosystem. Upon exposure to UV irradiance in the photic zone, functional enzymes can be released. The potential for inactivation and storage of enzyme activity, relocation within the ecosystem, and subsequent reactivation holds important implications for regulation of nutrient cycling in fresh waters.     

腐殖质在环境污染物生物降解中的作用研究进展

腐殖质物质在地球的生态环境中大量存在,它不仅可以在有毒化合物的生物降解和生物转化过程中起到氧化还原中间体的作用,加速有毒物质的降解和转化。也可以作为唯一末端电子受体,接受来自一些有机酸或者甲苯等环境中有毒物质提供的电子,偶联能量的产生,支持菌体的生长,形成一种新的细菌厌氧呼吸形式——腐殖质呼吸。因此,对腐殖质在环境有毒物质的生物降解和生物转化过程中的作用进行研究,不仪对于深入理解细菌呼吸的本质具有重要的理论意义,而且对于环境有毒物质的降解和转化以及元素的生物地球化学循环具有重要的生态学意义,同时对地球表面的有毒物质进行更有效的生物降解具有重要的现实意义。     

Artificial humification of lignin architecture: Top-down and bottom-up approaches

Humic substances readily identifiable in the environment are involved in several biotic and abiotic reactions affecting carbon turnover, soil fertility, plant nutrition and stimulation, xenobiotic transformation and microbial respiration. Inspired by natural roles of humic substances, several applications of these substances, including crop stimulants, redox mediators, anti-oxidants, human medicines, environmental remediation and fish feeding, have been developed. The annual market for humic substances has grown rapidly for these reasons and due to eco-conscious features, but there is a limited supply of natural coal-related resources such as lignite and leonardite from which humic substances are extracted in bulk. The structural similarity between humic substances and lignin suggests that lignocellulosic refinery resulting in lignin residues as a by-product could be a potential candidate for a bulk source of humic-like substances, but structural differences between the two polymeric materials indicate that additional transformation procedures allowing lignin architecture to fully mimic commercial humic substances are required. In this review, we introduce the emerging concept of artificial humification of lignin-related materials as a promising strategy for lignin valorization. First, the core structural features of humic substances and the relationship between these features and the physicochemical properties, natural functions and versatile applications of the substances are described. In particular, the mechanism by which humic substances stimulate the growth of plants and hence can improve crop productivity is highlighted. Second, top-down and bottom-up transformation pathways for scalable humification of small lignin-derived phenols, technical lignins and lignin-containing plant residues are described in detail. Finally, future directions are suggested for research and development of artificial lignin humification to achieve alternative ways of producing customized analogues of humic substances.     

Mechanisms of alkaline buffering by peat and quantitative estimation of its buffering capacity

The alkaline buffering capacity of humic substances in persistent organic materials such as peats has been utilized for remediation of alkaline soils. The purposes of the present study were to reveal the mechanism of alkaline buffering and to quantify the alkaline buffering capacity of humic substances contained in three peat samples obtained from Hokkaido, Canada, and Sri Lanka. Acidic functional groups in humic acid and fulvic acid extracted from the three natural peats were quantified by alkalimetric titration and pK distribution analyses of the titration curves. The results indicate that the inherent degree of humification of the organic matter can be identified from the pK distribution, which showed that the humic substances in peats contained diprotic acids. The present study also showed that the ratios of amounts of deprotonation to organic carbon content, as well as the organic carbon content, were needed for quantification of the pH buffering capacity. Moreover, the study showed that the composition of the peat from Sri Lanka was different from those of other peats and that it is a effective organic material for buffering under alkaline conditions.     

Removal of Humic Substances from Aqueous Solution by Fungal Pellets

The removal of humic substances from aqueous solution at pH=4.8 and 6.0 and 25 °C by pure culture of Aspergillus niger has been examined. The removal of humic substances from aqueous phase was monitored by following the decrease in absorbance at 370 nm. COD and fungal growth were also measured. The initial concentration of humic and fulvic acids, pH and diameter of fungal pellets were found to be the most important parameters. The results suggested that decolorization of batch culture was a consequence of several processes. In the first 5 hours for pellets with diameter 1-3 mm and 10 hours for pellets with diameter 2-5 mm adsorption of humic substances was dominant. Afterwards, desorption of slightly bound humic substances and their degradation, as well as degradation of remaining humic substances in the solution, continued simultaneously through the period of 10 days.     

Humic substances biological activity at the plant-soil interface: From environmental aspects to molecular factors

Humic substances (HS) represent the organic material mainly widespread in nature. HS have positive effects on plant physiology by improving soil structure and fertility and by influencing nutrient uptake and root architecture. The biochemical and molecular mechanisms underlying these events are only partially known. HS have been shown to contain auxin and an “auxin-like” activity of humic substances has been proposed, but support to this hypothesis is fragmentary. In this review article, we are giving an overview of available data concerning molecular structures and biological activities of humic substances, with special emphasis on their hormone-like activities.Key words: auxin, humic substances, root, soil, sustainable agriculture     

水葫芦对藻类的克制效应

水葫芦对藻类生长有克制作用。其机制主要是由于水葫芦根系向水体分泌的有机物质能伤害和杀死藻类。用水葫芦种植水培养藻类,使藻类的光合作用速率显著降低,叶绿素a破坏,细胞还原TTC的能力下降。在荧光显微镜下可看到藻细胞从鲜红色变为淡蓝绿色。     

A Method for Studying Photosynthetic Capacities of Unicellular Algae Based on in vivo Chlorophyll Fluorescence

The correlation between photosynthesis and DCMU-induced fluorescence increase has been studied in four species of unicellular green algae from inoculation of the cultures to the stationary growth phase. The fluorescence increase induced by DCMU was high in exponentially growing cultures but decreased when the cultures approached the stationary phase of growth. Because of the good correlation between photosynthesis and DCMU-induced fluorescence increase, the fluorescence technique is a promising method for describing changes in photosynthetic capacities of algal populations in natural waters.     

Study of humic substance transformation in phosphatic soil in terms of nucleophilicity and electrophilicity

The applicability of the nucleophilicity/electrophilicity concept to the explanation of mechanisms of formation and transformation of humic substances was considered. Sequential time changes in the structure and properties of humic substances in phosphatic soil have been revealed. Different elemental composition of humic and fulvic acids at different stages of humus transformation was due to different patterns of electron density distribution in phosphorus-modified fragments of humic molecules. The important role of metals and phosphorus in realization of different pathways of humic substance transformation was demonstrated.     

Further Evidence that Humic Substances Have the Potential to Modulate the Reproduction of the Nematode Caenorhabditis elegans

Humic isolates have previously been shown to modulate the reproduction of the nematode, Caenorhabditis elegans Maupas . The aim of this study was to find out if a hormone‐like effect is an intrinsic feature of humic substances. Therefore, the same nematode bioassay was used with ten humic substances (XAD and/or reverse osmosis isolates). The results confirmed the hormone‐like effect of humic substances, nine isolates significantly increased the numbers of offspring per worm, one isolate did not modulate the reproduction of C. elegans. This effect occurred with XAD isolates and with reverse osmosis isolates as well. Thus, this effect is an intrinsic feature of humic substances. This reproduction modulating potential of humic substances may be due to alkylaromatics that are major structural and photo stable structural components, rather than to sterols that are only minor structural components. This is a new view about the direct interactions of humic with aquatic organisms. The effects haven been mathematically modeled by a simple approach, considering a hormone‐like effect (β) and a non‐hormone like effect, such as nutrient effect (α) on growth. The different effects observed can be interpreted in terms of trade‐offs between these two competing mode of actions.     

川西亚高山森林凋落物去除对土壤腐殖质动态的影响

森林凋落物作为森林土壤腐殖质的主要来源, 在土壤腐殖质的形成中发挥着重要作用, 但不同森林类型凋落物因其含量、组成等的不同, 对土壤腐殖质的影响也不同。该研究以川西亚高山针叶林、阔叶林和针阔混交林3种不同森林类型为对象, 采用凋落物原位控制实验, 对比研究不同关键期凋落物去除对土壤可提取腐殖质、胡敏酸和富里酸含量及胡敏酸/富里酸、胡敏酸/可提取腐殖质的影响。主要结果: (1)土壤可提取腐殖质、胡敏酸和富里酸含量在不同森林类型中差异显著。土壤可提取腐殖质含量总体表现为针叶林>针阔混交林>阔叶林, 胡敏酸含量总体表现为针阔混交林>针叶林>阔叶林, 而富里酸含量则表现为针叶林>阔叶林>针阔混交林, 其中3种林型中土壤腐殖质的主要成分为富里酸, 总体均表现为富里酸型。不同采样时期也显著影响了土壤可提取腐殖质、胡敏酸和富里酸含量, 总体均表现为先升高后下降的趋势。除个别采样时期外, 凋落物去除总体降低了土壤可提取腐殖质、胡敏酸和富里酸的含量。(2)胡敏酸/富里酸和胡敏酸/可提取腐殖质的结果显示3种林型土壤总体腐殖化程度均较低, 整体表现为针阔混交林>阔叶林>针叶林, 凋落物去除在一定程度上有利于提高阔叶林与针阔混交林的腐殖质品质。(3)相关分析表明不同凋落物处理间土壤可提取腐殖质与土壤有机碳含量、全氮含量和土壤含水量呈显著正相关关系, 与温度呈显著负相关关系。综上所述, 短期的凋落物去除会降低土壤腐殖物质的含量, 但不同林型间由于凋落物类型差异会导致土壤腐殖质的不同变化, 说明土壤腐殖质的动态变化受凋落物类型以及环境因素的综合调控。因此, 关于凋落物变化对土壤腐殖质的影响还需进一步的长期研究。     

Strong coupling between natural Planctomycetes and changes in the quality of dissolved organic matter in freshwater samples

Dilution-regrowth experiments coupled to fluorescence in situ hybridization were conducted with samples from two humic reservoirs in order to examine how inorganic nutrients (N, P) affect free-living bacterioplankton phylogenetic groups and subsequently the quality of dissolved organic matter (DOM). The experiments were complemented by analyses of the empirical relationships between the targeted bacteria, nutrients, DOM and grazers. The ratio of absorbance of waters (A) at 250 and 365 nm (A(250 nm):A(365 nm)), which has been found to increase with the proportion of small molecules in the DOM pool in other humic waters, was used as an index of DOM quality. When nutrient stimulated bacteria, both the responses of bacterial groups (in the absence of grazers) and the ratio A(250 nm):A(365 nm) were generally different between treatments (+N, +P, +NP), suggesting that in nutrient-poor systems, differences in the type of inorganic nutrient supply will ultimately cause differences in DOM quality. The ratio A(250 nm),:A(365 nm) peaked in the +N treatments where members of the Planctomycetes (PLA) were the most stimulated group, and across treatments, PLA best explained (positive relationship) variations in this ratio. Consistent with this, the in situ data showed that the removal of the negative effects of flagellates on PLA yielded the highest R(2) in attempts to use bacterial groups to explain variations in A(250 nm):A(365 nm). These findings provide lines of evidence, not previously demonstrated in natural waters, that Planctomycetes may be an important factor changing the DOM quality, particularly in nutrient-poor systems when supplied with inorganic N.     

腐殖质呼吸作用及其生态学意义

腐殖质呼吸是厌氧环境中普遍存在的一种微生物呼吸代谢模式.自1996年发现以来,日益成为生态学与环境科学领域的研究热点.在厌氧条件下,一些微生物能以腐殖质作为唯一电子受体,氧化环境中的有机质或者甲苯等环境有毒物质,产生CO2,参与碳循环;同时,腐殖质呼吸作用产生的还原态腐殖质可以还原环境中的一些氧化态物质,如Fe(III)、Mn(IV)、Cr(VI)、U(VI) 、硝基芳香化合物和多卤代污染物.因此,腐殖质呼吸能够影响环境中C、N、Fe、Mn以及一些痕量金属元素的生物地球化学循环,并且能够促进重金属以及有机污染物的脱毒,在水体自净、污染土壤原位修复、污水处理等方面具有积极作用.