Biostimulant activity of humic substances extracted from leonardites

Background and aims

Biostimulants are natural compounds that enhance plant growth and plant nutrient use efficiency. In this study, biostimulant effects of humic substances (HS) extracted from leonardites were analysed on the metabolism of maize plants grown in hydroponic conditions.

Methods

HS extracted from four leonardites were tested for their auxin-like and gibberellin-like activities. Then, 11 day old maize seedlings were treated for 48 h with five concentrations (0, 0.1, 0.5, 1, and 10 mg C L^?1) of HS. After sampling, root growth and morphology, glutamine synthetase (GS) activity, glutamate synthase (GOGAT) activity, total protein content, soluble sugars content, phenylalanine ammonia-lyase (PAL) activity, soluble phenols, and free phenolic acids were analysed.

Results

HS from leonardites had similar spectroscopic pattern, with small differences. The HS from the South Dakota lignite (HS_USA) had more carboxylic groups, whereas the three from Turkish mines had more aromatic and aliphatic structures. HS_USA best enhanced total root growth, root surface area, and proliferation of secondary roots. Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production. HS stimulated also PAL enzyme activity, followed by a higher production of total soluble phenols, p-hydroxybenzoic acid, p-coumarilic acid, and chlorogenic acid.

Conclusion

This study found that, although the activity of the HS depended on the origin of the leonardite, these compounds can be attributed to the biostimulant products, eliciting plant growth, nitrogen metabolism, and accumulation of phenolic substances.

     

Chemical characterization of humic substances extracted from organic-waste-amended soils

Humic substances were extracted from a soil treated, in a 4-year experiment, at different rates with a sludge from anaerobic treatment of combined civil and industrial wastes, and with agricultural manure. Elemental and chemical analyses, molecular weight (MW) distribution and infrared (IR) spectroscopy were performed on the purified humic acids (HA). Organic wastes significantly increased the HA content of the treated soils and improved CEC. The C/N, C/H and C/O ratios of HA extracted from the original wastes showed a higher degree of humification for sludge than for manure. This difference was also noticed for the C/N ratio of soil humic extracts, indicating a faster humification process for the sludges in soil. The content of oxygen-containing functional groups was lower than the ‘model’ HA reported in the literature, and even more so for HA from sludges, reflecting their anaerobic formation. MW distribution and E₄/E₆ ratios showed that the sludge material had a higher molecular complexity than manure, supporting the high degree of humification attributed to the former. HA extracted from sludge-treated soils revealed a molecular dimension increasing with the application doses of waste material. Infrared spectra showed that HA formed in soils after waste additions reflected the chemical composition of the original organic material, which was rich in aliphatic groups and peptides for sludge and in carbohydrates for manure. On the basis of this study, it is concluded that not only are organic waste additions able to build up the HA content in soils but the HA formed assume the chemical characteristics and the degree of humification of the original material.     

Description of characteristics of humic substances from different waste materials

Humic acids (HAs) extracted from different organic wastes have been characterised by chemical methods. The chemical properties of HAs showed differences depending on the source from which they were obtained. The C content in HAs from organic wastes (41.1-63.2%) fluctuated around the C value in soil HA with the exception of composted bark and tobacco dust. Compared with soil HA, the N contents of HAs from sewage sludge and brewery sludge were found much higher than the others. E4:E6 ratios for HAs in organic wastes were generally greater than that for soil HA, which indicated a low degree of condensation and humification. The carboxyl and phenolic-OH group contents ranged 0.51-2.23 and 11.1-20.7 meq g(-1), respectively. High values of carboxyl and phenolic-OH contents indicated that these materials were still within early stages of humification.     

Influence of mineral and organic fertilization on soil fungi,enzyme activities and humic substances in a long-term field experiment

Changes in microfungal communities, fungal activities and humic substances (HS) in agricultural soils kept under different fertilization regimes were observed and their causal relationships were investigated in a long-term field experiment. Fertilization did not change the abundance of HS-utilizing microfungi and, except for organic amendment alone, total culturable microfungi were also unaffected by this factor. Organic fertilization increased activities of manganese peroxidase (MnP) and proteinase, but decreased endo-1,4-beta-glucanase activity compared to the corresponding control without organic fertilization. In soils treated with mineral fertilizers, the activities of MnP, endo-1,4-beta-glucanase and proteinase were higher than in control without any mineral treatment. Both the aromaticity of fulvic acid and the molar mass of humic acid was lower in soil with organic fertilization, which may be a result of oxidative degradation mediated by higher MnP activity observed in treatments with organic fertilization.     

Molecular-sieve chromatography of humic substances extracted with chelating resin from chernozemic Ah horizons

Summary Humic substances extracted with chelating resin from a Dark Brown Chernozem (Typic Haploboroll) Ah, a Black Chernozem (Udic Haploboroll) Ah, and a Dark Gray Luvisol (Typic Cryoboralf) Ahe were fractionated by Bio-Gel P. The eluted material was combined to form three or four fractions for further characterization.Most of the extracted polydisperse humic substances of the three soils were eluted near V₀ and distributed around a single mean value. The ash content of this material varied from 1.36 to 1.78 per cent. Carboxyl acidity may thus be obtained without interference from any cations.The humic substances from the Dark Brown and Black Chernozem Ah gave similar elution profiles, whereas those from the Dark Gray Luvisol Ahe were less aliphatic and more aromatic in nature. The materials of low molecular weight had the highest content of ash, C, and carboxyl groups. They represented only 3.6 to 14.1 per cent of the C extracted and were considered, on the basis of increased C content, decreased optical density, and infrared spectral configurations, to consist of polynuclear, fused-ring, aromatic structures.     

Extracellular enzyme activities during humic acid degradation by the white rot fungi Phanerochaete chrysosporium and Trametes versicolor

Abstract The relationship between humic acid biodegradation and extracellular lignin peroxidase and Mn-dependent peroxidase activities of two white rot fungi, Phanerochaete chrysosporium and Tranetes versicolor , reported to be lignin degraders, was examined. In experimental conditions promoting culture aeration, particularly with T. versicolor no extracellular peroxidase activity could be detected unless humic acids were included in the culture medium. In the presence of humic acids, appreciable enzymatic activities were determined in the culture filtrate of the two fungi. However, T. versicolor was a more effective degrader than P. chrysosporium , and mineralization assays on synthetic humic acids with culture filtrates showed the important role played by Mn²⁺. The surfactant properties of humic acids are suggested to be responsible for the increase of enzymatic activities.     

Microbial dynamics and enzyme activities during rapid composting of municipal solid waste - a compost maturity analysis perspective

An investigation was carried out in the laboratory to find out the microbial dynamics and enzyme activities during rapid composting of municipal solid waste (MSW). Various treatments such as aeration (A), addition of chemical agents (glucose (G) and acetic acid (AA) and application of cellulolytic microbial (M) inoculum (Phanerochaete chrysosporium and Trichoderma reesei) were used to facilitate the decomposition of MSW. The result of the present investigation revealed that the degradation of organic substrates were quick (within 9-12 days) in case of rapid composting as indicated by the reduction (below 20) in C/N ratio. Whereas, normal composting took more than 20 days to attain C/N ratio of below 20. Estimation of selected enzymes (amylase, protease, phosphatase and cellulase) provided information on the substrate specific degradation profiles of various labile substrates contained in organic waste.     

Chemical and spectroscopic analysis of olive mill waste water during a biological treatment

The treatment of olive mill waste water was studied on the laboratory scale. Physico-chemical analyses showed the final products had a mean pH of 5.4 without neutralisation and 5.7 when lime was added to the process. Raising the pH by adding lime had a positive outcome on the degradation of phenols, whose levels were reduced by over 76%. The lime also changed the structure of the organic matter, as seen in the infra-red spectra. Combining the FT-IR and 13C NMR data showed that with addition of lime, the density of aliphatic groups decreased to the benefit of aromatic groups, indicating that polymerisation of the organic matter occurred during the bioprocess. Under our experimental conditions, the biotransformation of olive mill waste water appears to favour the stabilisation of the organic matter through mechanisms analogous to those that lead to the formation of humus in the soil.     

Hydrolytic enzyme activities and degradation of storage components in cotyledons of germinatingPhaseolus mungo seeds

Phaseolus mungo seeds were allowed to germinate in the dark, and time-course changes in contents of protein fractions, starch, soluble α-amino nitrogen and reducing sugars and in hydrolytic enzyme activities in cotyledons were investigated. In cotyledons of germinated seeds, marked increases in proteolytic (caseolytic, globulytic and gelatin-hydrolyzing) activities and amylolytic activity occurred with concurrent mobilization of storage proteins and starch. Removal of axis organs from seeds at very early stages of germination caused the deteriorated breakdown of storage components and decreased development of proteolytic enzymes in the cotyledons, but this treatment did not significantly affect the appearance of amylolytic activity. The experimental results are discussed in comparison with the hydrolytic enzyme activities of germinating seeds of other leguminous species.     

Effects of humic substances derived from organic waste enhancement on the growth and mineral nutrition of maize

A physico-chemical process has been developed to transform and enhance lignocellulosic waste in liquid humic extracts: humic-like substances (HLS). The aim of this study was to determine the effects of HLS on plant physiology in order to consider their agricultural use as organic fertilizers. The effects of HLS were evaluated on maize seed germination, and their impact on growth, development and mineral nutrition was studied on maize plants cultivated under hydroponic conditions. The experimental results showed that HLS do not increase the percentage and rate of germination but enhance the root elongation of seeds thus treated. Positive effects were also observed on the whole plant growth as well as on root, shoot and leaf biomass. These effects can be related to the high water and mineral consumption of plants undergoing this treatment. The high water efficiency indicated that such plants produce more biomass than non-treated plants for the same consumption of the nutrient solution. Furthermore, the use of HLS induced a flowering precocity and modified root development suggesting a possible interaction of HLS with developmental processes. Considering the beneficial effect of HLS on different stages of plant growth, their use may present various scientific and economic advantages. The physico-chemical transformation of sawdust is an interesting way of enhancing organic waste materials.     

Effect of cereal brans on Lentinula edodes growth and enzyme activities during cultivation on forestry waste

AIMS: To develop strategies for increasing the growth of Lentinula edodes in eucalyptus residues. To this end, we have examined the effects of cereal brans additions on production of mycelial biomass and enzymes. METHODS AND RESULTS: Three isolates of the mushroom shiitake, L. edodes (Berk. Pegler), were evaluated for enzyme and ergosterol production on eucalyptus residue supplemented with 5, 10, 15 and 20% (w/w) of soya, wheat or rice brans. Nitrogen imput on eucalyptus residues accelerated mycelial growth by supplying the L. edodes with this limiting nutrient. High levels of enzymes activities were produced in eucalyptus residues supplemented by soya bran. Comparison of cellulose and xylanase production with manganese peroxidase (MnP) at 20% soya bran indicated that hydrolytic enzymes, but oxidative enzymes were reduced. CONCLUSIONS: Mycelial growth measurements revealed that eucalyptus residues supplemented with cereal brans supported fast growth of L. edodes, indicating that mycelium extension is related to the bioavailability of nitrogen. The type and concentration of nutrient supplement has a considerable effect both on substrate colonization and on the type of hydrolytic and oxidative enzymes produced. These characteristics may be useful for mushroom growing. SIGNIFICANCE AND IMPACT OF THE STUDY: Lentinula edodes is commercially important for edible mushroom production and supplements which enhance growth and enzymes production might also be beneficial for mushroom yields.     

Chemical and structural evolution of humic acids during organic waste composting

The effects of the composting process on the chemical and structural properties of humic acids have been studied in seven different organic waste mixtures from different origin. Only slight changes in elemental composition have been found in the humic acids after the composting process pointing to a more aromatic structure with higher proportions of oxygen and nitrogen. Functional groups were the most sensitive to the changes caused by the composting process showing a marked increase in the total acidity and phenolic, carboxyl and carbonyl groups. Gel permeation chromatography showed a slight increase in the average molecular weight of the humic acids. Infrared spectroscopy did not show important differences in humic acid structure but a clear decrease in the intensity of the bands in the region 3000–2850 cm^-1 corresponding to the aliphatic fractions. As a general result, the composting process yields humic acids in which the elemental and functional composition are closer to that of the more humified soil humic acids.     

Effects of different organic waste amendments on soil microaggregates stability and molecular sizes of humic substances

Three soils which had been amended for several years with pig slurry, cattle slurry, and sewage sludge were dry-sieved to obtain microaggregates in the size range of 250–125, 125–50, and <50 μm. With amendments, aggregate size distribution of whole soils was shifted to larger sizes, especially for the most fragile soil, whereas percent content of microaggregates decreased except for the lower size aggregates of the fragile soil. Particle size distribution of microaggregates revealed an increase in percent sand and a reduction of percent silt and clay in the <50 μg size fraction for all soils. These results showed the aggregation effect induced by the organic waste additions. Aggregate stability of microaggregates revealed significant correlation with humic substances content (humic acids alone and humic plus fulvic acids) and non significant with total organic matter substantiating the belief that humic substances are the predominant binding agents in this aggregation range. Molecular weight distribution of humic acids extracted from microaggregates of unamended soils demonstrated that the lower the soil aggregate size distribution, the larger the contribution of the high molecular weight fraction. All microaggregates from amended soils showed a progressive increase of the high molecular weight humic acids with decreasing size, reaching a maximum in the <50 μm fraction. In this aggregate size a parallel enhancement of the aggregate stability was also evident. It is concluded that a close relationship exists between aggregate stability and high molecular weight humic substances. Additions to soils of organic material containing high molecular weight constituents would represent a useful management practice to improve aggregate stability.     

Chemical properties and hydrolytic enzyme activities for the characterisation of two-phase olive mill wastes composting

Two-phase olive mill waste (TPOMW) is a semisolid sludge generated during the extraction of olive oil by the two-phase centrifugation system. Among all the available disposal options, composting is gaining interest as a sustainable strategy to recycle TPOMW for agricultural purposes. The quality of compost for agronomical use depends on the degree of organic matter stabilization, but despite several studies on the topic, there is not a single method available which alone can give a certain indication of compost stability. In addition, information on the biological and biochemical properties, including the enzymatic activity (EA) of compost, is rare. The aim of this work was to investigate the suitability of some enzymatic activities (beta-glucosidase, arylsulphatase, acid-phosphatase, alkaline-phosphatase, urease and fluorescein diacetate hydrolysis (FDA)) as parameters to evaluate organic matter stability during the composting of TPOMW. These enzymatic indices were also compared to conventional stability indices. For this purpose two composting piles were prepared by mixing TPOMW with sheep manure and grape stalks in different proportions, with forced aeration and occasional turnings. The composting of TPOMW followed the common pattern reported previously for this kind of material with a reduction of 40-50% of organic matter, a gradual increase in pH, disappearance of phytotoxicity and formation of humic-like C. All EA increased during composting except acid-phosphatase. Significant correlations were found between EA and some important conventional stability indices indicating that EA can be a simple and reliable tool to determine the degree of stability of TPOMW composts.     

Utilization of lignocellulosic waste by the edible mushroom,Pleurotus

Lignocellulosic waste represents huge amounts of unutilized renewable resource. The use of the polysaccharides in the lignocellulosic complex is limited due to their high lignin content. White rot fungi are capable of selectively degrading lignin, thereby upgrading it. The focus of this article is on the potential utilization of edible mushrooms of the genus Pleurotus, via solid state fermentation, using cotton plant stalks as a substrate. This material poses agrotechnical problems since the stalks have a fibrous structure similar to that of hardwood. Potential uses for this material are as a fuel in rural areas, a substrate for mushrooms, an animal feed and substrate for paper making. In this study, degradation of cotton stalks by Pleurotus is described using chemical analyses and scanning electron microscopy. During four weeks of solid state fermentation, lignin content significantly decreased and in vitro digestibility was increased. The fermentation product was consumed by ruminants at a level of up to 40% of their diet.     

冻融环境下亚高山森林凋落叶添加对土壤腐殖质动态的影响

亚高山森林土壤形成过程中,胡敏酸、富里酸等腐殖物质的累积是维持土壤肥力及物质循环的重要途径,它受到土壤基质质量、凋落叶和环境因素的调控.本研究以川西亚高山典型的针叶林、针阔混交林和阔叶林土壤为对象,采用室内培养控制冻融环境和凋落叶添加的方法,研究冻融环境下凋落叶添加对土壤腐殖物质累积的影响.结果表明:冻融循环环境下针叶林土壤腐殖质含量升高,而针阔混交林和阔叶林土壤腐殖质含量降低,且凋落叶对土壤腐殖质含量无显著影响.培养前期冻融环境下3种林型土壤胡敏酸净累积,净累积量大小为针阔混交林>针叶林>阔叶林,富里酸含量下降,下降程度为阔叶林>针阔混交林>针叶林,且凋落叶对土壤胡敏酸和富里酸含量无显著影响.随培养时间的延长,3种林型土壤胡敏酸及富里酸含量均下降.这表明,凋落叶对土壤腐殖质含量的影响与土壤基质质量存在密切关系,且受到冬季土壤冻融时间长短的影响.     

Ovarian steroidogenic enzyme activities during the rat estrous cycle

We have correlated the concentrations of serum LH, estradiol and progesterone with the activities of 2 ovarian steroid biosynthetic enzymes during the rat estrous cycle. Ovarian 3 β-hydroxysteroid dehydrogenase isomerase (3-βHSD) activity decreased from 29 ± 6 nmol/mg protein/ min (mean ± SEM) in diestrus, to 7 ± 0.4 nmol/mg protein/min in late proestrus (p < 0.005), and subsequently increased to 36 ± 9 nmol/mg protein/min in metestrus (p < 0.01). Ovarian 17-hydroxylase (17-OH) activity decreased from early to late proestrus (3.3 ± 0.2 vs 2.2 ± 0.2 nmol/mg protein/min, p <0.0025), and subsequently increased to 3.9 ± 0.2 in metestrus (p<0.001). Serum LH, estradiol and progesterone peaked during proestrus, and reached a nadir during estrus. We conclude that the activities of 3-βHSD and 17-OH in the rat ovary vary markedly during the estrous cycle. These changes may underlie the pattern of steroid secretion characteristic of this process.     

Improvement of olive oil phenolics content by means of enzyme formulations: Effect of different enzyme activities and levels

The present work deals with the development of an enzymatic treatment aiming at producing high-quality olive oil with increased phenolics content and antioxidant activity. Three different enzyme formulations, specifically pectinase, hemicellulase and cellulase (A), pectinase and hemicellulase (B), and only pectinase (C), were used either at three different level of each or in ternary and binary mixtures at a constant level. All of them were added to the olive paste (Italian cultivar Coratina) at the beginning of the malaxation step. Results demonstrated that an increased enzyme level led to higher phenolics content in the oils, and such an effect was found to be enzyme dependent, being greater when using formulation A, followed by formulations B and C. Two significant correlations were obtained between total polyphenols and o-diphenols contents versus antiradical power (R² = 0.8743 and R² = 0.8635, respectively), which pointed out the effectiveness of the proposed enzymatic treatment to produce olive oils characterized by low susceptibility to oxidation. A synergistic effect between the different enzymatic activities contained in the single formulations was observed by combining enzymes A and B. The ternary mixture was selected as the most efficient enzymatic system ensuring the highest phenolics content increase (40 and 37% for total polyphenols and o-diphenols contents, respectively) compared to the other enzymes when used at the same level.