Biological activity of humus acids isolated from spruce wood decomposed by fungi

Biological activity of humic acid, hymatomelanic acid, lignofulvic acid, and fulvic acids isolated from peat, from spruce wood decomposed by the fungusFomes marginatus (Fr.) Gillet, and from spruce wood decomposed by the fungusPoria cinerascens Bres. was tested on cress seedlings (Lepidium sativum L.). The results evaluated by the analysis of variance indicate that humus acids show a high degree of affinity in their biological activity which is a condition for the applicability of the decomposition of wood by fungi as a model of the humus formation. Humic and hymatomelanic acids, products of an advanced step of humification, have a predominantly stimulatory effect especially on the hypocotyl. Lignofulvic acids and fulvic acids exert a rather inhibitory effect with the root being more affected.     

Humic-like substances of bacterial origin. I. Some aspects of the formation and nature of humic-like substances produced by Pseudomonas.

Two bacterial strains Pseudomonas acidovorans No 26 and Pseudomonas sp. No 4 grown in Conn and yeast extract-glucose media, or in the media enriched with tyrosine, were found to produce dark brown pigment. It was shown that in the bacterial cultures numerous phenolic and quinone-type compounds were formed and transformed to humic-like polymers. Formation of humic-like substances started in the bacterial cells and was accompanied by the presence of phenyloxidases in the bacterial cultures. The bacterial "humic acids" were obtained from the supernatants in amounts varing from 0.05 to 0.865 mg/1 mg of dry weight of cells and from the cells in amounts of 0.02 to 0.165 mg/1 mg of dry weight of cells, depending on the medium used and time of incubation. The IR spectra of the bacterial "humic acids" appeared to be very similar to IR spectrum of the synthetic humic acids (Fluka A.G.) and contained the same chemical groups as the soil humic acids. The culture medium after growth of the strain No 26 was fractionated into "fulvic, hymatomelanic and humic acid" fractions. The hydrolysates from the obtained fractions contained amino acids and uronic acids. The amino acid composition appeared to be very similar to that of soil humic acids.     

Activity of humus acids from peat as studied by means of some growth regulator bioassays

Biologioal activities of humic acid (Na salt), hymatomelanic acid (Na salt), lignofulvic acid and of fulvic acids isolated from peat were studied by means of the auxin-, gibberellin-, and cytokinin- bioassays. All the four tested fractions showed higher or lower stimulating activity in these bioassays. However, the stimulating effect is considerably lower and cannot be interpreted as phytohormone activity. Some fractions in some concentrations also showed inhibitory effects. The manner of biological action of the studied fractions might bo the result of interaction of their respective components.     

Effects of humic materials on virus recovery from water.

Humic and fulvic acids were tested for their ability to interfere with virus recovery by microporous filters. Two electropositively charged types of filter (Seitz S and Zeta Plus 60S) were used to concentrate poliovirus in the presence of humic materials. Humic acid inhibited virus adsorption, but even at the highest humic acid concentrations tested (200 mg/liter), 30 to 40% of the virus was recovered by the filters. Fulvic acid, tested with Zeta Plus filters, did not affect virus recovery. For comparison, two electronegatively charged filter types were tested (Cox and Balston). These two types of filter were more sensitive to interference at lower concentrations of humic acid than the more positively charged filters. With Balston filters, at humic acid concentrations above 10 mg/liter, most of the virus was recovered in the filtrate. Fulvic acid, tested with Balston filters, did not interfere with virus recovery. With the electropositively charged filters, the humic materials adsorbed efficiently, even at high input concentrations. Interference with virus adsorption occurred at humic acid concentrations which were below the level of saturation of the filters. In addition, in high-volume experiments, humic acid led to premature blockage of the filters. The efficiency of virus recovery by a second concentration step, organic flocculation of the filter eluate, was tested. For all the filter types tested, this procedure was not affected by the presence of humic or fulvic acid in the input water.     

Effects of humic materials on virus recovery from water

Humic and fulvic acids were tested for their ability to interfere with virus recovery by microporous filters. Two electropositively charged types of filter (Seitz S and Zeta Plus 60S) were used to concentrate poliovirus in the presence of humic materials. Humic acid inhibited virus adsorption, but even at the highest humic acid concentrations tested (200 mg/liter), 30 to 40% of the virus was recovered by the filters. Fulvic acid, tested with Zeta Plus filters, did not affect virus recovery. For comparison, two electronegatively charged filter types were tested (Cox and Balston). These two types of filter were more sensitive to interference at lower concentrations of humic acid than the more positively charged filters. With Balston filters, at humic acid concentrations above 10 mg/liter, most of the virus was recovered in the filtrate. Fulvic acid, tested with Balston filters, did not interfere with virus recovery. With the electropositively charged filters, the humic materials adsorbed efficiently, even at high input concentrations. Interference with virus adsorption occurred at humic acid concentrations which were below the level of saturation of the filters. In addition, in high-volume experiments, humic acid led to premature blockage of the filters. The efficiency of virus recovery by a second concentration step, organic flocculation of the filter eluate, was tested. For all the filter types tested, this procedure was not affected by the presence of humic or fulvic acid in the input water.     

Share of carbohydrates in the humus substances composition

The effect of different concentrations of hydrochloric acid on the qualitative an quantitative content of carbohydrates in humus fraction hydrolyzates was investigated. Three humic acid preparations were subjected to the usual procedure for obtaining fractions of humic and hymatomelanic acids. The carbohydrate content of the hydrolyzates was determined after their Chromatographic separation on Whatman 1, using the Partridge system and detection with acid anilin phtalate. The results showed that the qualitative carbohydrate content in the hydrolyzates of most humus fractions of samples used was identical. With increasing acid concentrations the amount of hydrolyzable carbohydrates usually increased, the total amount of carbohydrate in the hydrolyzate not exceeding 4% . The difference in the carbohydrate content as between hydrolysis with 0-9N and 5-3N HC1 was sometimes fourfold. A higher content of hexoses than of pentoses was generally found.     

Purification and crystallization of glycogen phosphorylase from Saccharomyces cerevisiae.

Glycogen phosphorylase from Saccharomyces cerevisiae is activated by the covalent phosphorylation of a single threonine residue in the N terminus of the protein. We have hypothesized that the structural features that effect activation must be distinct from those characterized in rabbit muscle phosphorylase because the two enzymes have unrelated phosphorylation sites located in dissimilar protein contexts. To understand this potentially novel mechanism of activation by phosphorylation, we require information at atomic resolution of the phosphorylated and unphosphorylated forms of the enzyme. To this end, we have purified, characterized and crystallized glycogen phosphorylase from S. cerevisiae. The enzyme was isolated from a phosphorylase-deficient strain harboring a multicopy plasmid containing the phosphorylase gene under the control of its own promoter. One liter of cultured cells yields 12 mg of crystallizable material. The purified protein was not phosphorylated and had an activity of 4.7 units/mg in the presence of saturating amounts of substrate. Yeast phosphorylase was crystallized in four different crystal forms, only one of which is suitable for diffraction studies at high resolution. The latter belongs to space group P4(1)2(1)2 with unit cell constants of a = 161.1 A and c = 175.5 A Based on the density of the crystals, the solvent content is 49.7%, indicating that the asymmetric unit contains the functional dimer of yeast phosphorylase.     

Quantitation of glycogen synthase and phosphorylase protein in mouse liver: correlation between enzymatic protein and enzyme activity

Phosphorylase and glycogen synthase protein were measured in normal and genetically diabetic (C57BL/KsJ db/db) mice liver extracts using rocket immunoelectrophoresis, and these data correlated with measurements of total phosphorylase and total glycogen synthase activities, respectively. Phosphorylase protein in 5-week-old normal mice was about 5 micrograms/mg protein and reached 8 micrograms/mg protein by 9 weeks. In comparison, the diabetic mice had elevated levels of phosphorylase protein (11-13 micrograms/mg protein) which correlated with an increased total phosphorylase activity compared to normals. The correlation coefficient for the phosphorylase activity vs protein plot was highly significant (r = 0.73, P less than 0.001). The molar concentration of phosphorylase subunit in normal mouse liver was calculated to be 11 microM and up to 23 microM in the diabetic mice. The liver concentration of glycogen synthase was relatively constant in normal mice at 400 ng/mg protein (corresponding to approximately 1.4 microM) but varied from 230 to 441 ng/mg protein (0.9 to 1.8 microM) in diabetic mice. There was little correlation between glycogen synthase activity and enzymatic protein (r = 0.15). These results indicate (1) that phosphorylase is present at concentrations approximately 10 times that of glycogen synthase, and (2) that glycogen synthase activity is relatively more dependent upon factors other than the amount of enzymatic protein.     

农田栽参土壤改良中肥料对土壤元素及酶活性的影响

为探究农田栽参土壤改良中肥料对土壤元素与土壤酶活性的影响, 于2019年5月21日采集经混合肥、土地乐、腐殖酸、木醋液、益生源处理过的土壤样品, 对其土壤养分与土壤酶活性进行研究。结果表明: 不同肥料在土壤中发挥出不同的作用, 施用混合肥的土壤SOM、ACa含量最高分别为139.51 g·kg-1、848.9 mg·kg-1。施用土地乐生物有机肥后土壤中AP和AMn含量最高分别为36.7 mg·kg-1、126.12 mg·kg-1。施用腐殖酸后土壤中AFe、电导率、AK数值最高分别为737.36 mg·kg-1、59.67 mg·kg-1、794.87 mg·kg-1。施用木醋液与益生源菌剂后各养分均有不同程度增加; 过氧化氢酶活性在混合肥处理后最低, 对照处理最高。漆酶活性在腐殖酸处理后最低, 木醋液处理最高。蔗糖酶活性在腐殖酸处理最低, 混合肥处理最高。脲酶活性在腐殖酸处理最低, 益生源处理后最高; 相关性分析表明, 土壤养分与土壤酶活性有密切关系, 存在不同程度的相关性。五种肥料在土壤改良中都有显著效果     

Rat liver glycogen metabolism in the perinatal period.

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen synthase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximum on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose or AMP on the respective phosphatases was considered. It was found that the Km of phosphorylase phosphatase for glucose in the prenatal period was 5--7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6--15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time. Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver. It is suggested that the rapid changes observed immediately after birth are due to an effect of gypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

Formation of mutagens following chlorination of humic acid. A model for mutagen formation during drinking water treatment

Aqueous chlorination of humic acids results in the formation of compounds with direct-acting mutagenic activity in the Ames/Salmonella plate assay for tester strains TA98, TA100, TA1535, TA1537 and TA1538. The addition of a rat-liver microsomal fraction (S9) plus cofactors causes a substantial decrease of activity, the extent of which is tester strain dependent. The non-chlorinated humic acids are not mutagenic either in the presence or absence of S9. Formation of mutagenic activity and of total organic halogen (TOX) is linearly related to humic concentration in the range of 0.2-1.6 mg/ml total organic carbon (TOC), and to chlorine concentration in the range of 0.1-1.0 chlorine equivalents per mole of carbon. The mutagenic activity is due predominantly to non-volatile compounds. Mutagenic activity is also detectable, after sample concentration by lyophilization, upon chlorination at a humic acid level of 0.02 mg/ml TOC. The specific mutagenic activities (per mg TOX), and also the degree of chlorine incorporation into humic acid, at 0.02 mg/ml TOC are similar to those present after chlorination at 1 mg/ml TOC. Production of mutagens is greatly dependent on the chlorination pH, with a pattern of decreasing mutagenic activity with increasing pH. This order of activity can be at least partially explained by the alkali liability of the compounds. Chlorination of commercial humic acids is proposed as a model for examination of mutagen formation during water chlorination.     

SOS Chromotest study concerning some appreciation criteria of humic substances' genotoxic potency

The genotoxicity of 5 compounds: 2 fulvic acids, a trade humic acid, a synthetic humic material (SHM), and 2,5-dihydroxybenzoic acid, was assessed after chlorination, by means of the SOS Chromotest for tester strain E. coli PQ 37 without metabolic activation. Chlorination was carried out for humic material concentration of 0.5 mg total organic carbon per liter, and chlorine concentrations in the range of 0.1-2.0 chlorine equivalents per mole of carbon. Among all the considered criteria that can account for potent toxicity: chemical degradation determined by the UV absorption decrease, chlorine consumption, average molecular weight, only the polymerization index (O.D. 665 nm/O.D. 465 nm) can be related to the genotoxicity of humic samples. This latter criterion appears a possible predictor of genotoxic potency, revealed subsequent to the aqueous chlorination of humic materials. Looking at the various genotoxic activities of the tested compounds, SHM can be considered a better model of natural humic materials than the trade humic acid.     

Effects of N-1-naphthylphthalamic acid on the growth and bud formation of tobacco callus grown in vitro

The effect of N-1 -naphthylphthalamic acid (NPA), indole-3-aceticacid (IAA) and kinetin on callus growth and bud formation wasstudied mainly by a tobacco callus culture method. Callus producedfrom Nicotiana tabacum var. Wisconsin 38 was used as the testplant material. Callus growth on nutrient agar containing 2mg/liter of IAA was promoted by NPA added at a concentrationof 0.5 mg/liter with 0.4 mg/liter of kinetin or by NPA addedat 5 mg/liter in the absence of kinetin. At a high concentrationof 50 mg/liter, however, NPA inhibited growth on the mediumcontaining 2 mg/liter IAA and no kinetin. Kinetin reduced thisNPA inhibition. In the presence of 0.4 mg/liter kinetin and2 mg/liter IAA, when the concentration of NPA was 50 mg/liter,buds were initiated after calluses were grown on the test mediumfor 7 weeks in dim light, but no buds formed when NPA was omittedfrom the above medium. The control of callus growth and bud initiation is based onthe active ratio of auxin (IAA) to cytokinin (kinetin) in themedium and NPA added to the medium can promote or inhibit callusgrowth and induce bud formation. Therefore, it is proposed thatNPA can itself reduce auxin activity or enhance cytokinin activityand hence change the active ratio of the two regulators. NPAmay enhance the activity of cytokinin (here supplied as kinetin)but cannot substitute for it. ¹Present address: Department of Biology, Wisconsin State University,Oshkosh, Wisconsin 54901, U. S. A. (Received March 10, 1969; )     

Rat liver glycogen metabolism in the perinatal period

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen sythase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximun on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose of AMP on the respective phosphatases was considered. It was found that the Km of phosphatase for glucose in the prenatal period was 5–7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6–15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time.Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver.It is suggested that the rapid changes observed immediately after birth are due to an effect of hypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

Copper dissociation as a mechanism of fungal laccase denaturation by humic acid

Effects of humic acid on removal of hydroxy polychlorobiphenyls (PCBs) with laccase from Trametes versicolor were studied. In the absence of humic acid, hydroxy PCBs were rapidly degraded by laccase. However, the rate constants decreased with increasing humic acid concentration, the reactions being completely inhibited at 150 mg l^-1 of humic acid. Peroxidase from Arthromyces ramosus was not inhibited by the same treatment. The activity of humic acid-deactivated laccase was completely restored by copper ions (500 M of Cu²⁺ in 150 mg l^-1 of humic acid), but not by other metal ions (Zn²⁺, Fe²⁺ and Hg²⁺). Humic acid-deactivated laccase purified by gel permeation chromatography (GPC) showed no activity against 2,2-azino-bis(3-ethylbenzthiazoline sulfonic acid) diammonium salt and 3,5-dichloro-4-hydroxybiphenyl, but its activity was restored by copper ion treatment. Humic acid-deactivated laccase showed similar properties, such as GPC retention time and copper ion requirements for activity, to those of laccase deactivated by nitrilotriacetic acid. The extent of humic acid inhibition, expressed as activity non-recoverable by copper ion treatment, increased over time more rapidly than that of the humic acid-free control. These results suggest that short-term inactivation of laccase, i.e., less than 1 day, is attributable to a depletion of copper ion.     

Influence of Stabilizer Concentration on Effectiveness of Chlorine as an Algicide

Cyanuric acid, used as chlorine stabilizer in swimming pool waters, has a relatively minor effect on the algicidal efficiency of free chlorine. The toxicity of free chlorine to three swimming pool algae was reduced slightly by 25 mg of cyanuric acid per liter if inhibiting, but less than algicidal, concentrations of chlorine were employed. Higher stabilizer concentrations (50, 100, and 200 mg/liter) generally resulted in no further reduction in the algicidal efficiency of free chlorine beyond that observed at 25 mg/liter.     

Improved laminaribiose phosphorylase production by <Emphasis Type="Italic">Euglena gracilis</Emphasis> in a bioreactor: A comparative study of different cultivation methods

Laminaribiose phosphorylase (EC 2.4.1.31) catalyzes a reversible phosphorolysis reaction in which laminaribiose, a very high value sugar is produced. This enzyme is not being produced commercially therefore, to realize the most effective method for producing laminaribiose phosphorylase and obtaining as much activity units as possible per liter of culture, different cultivation methods of Euglena gracilis were compared. Heterotrophic and mixotrophic cultivations of Euglena gracilis in two different pHs, in flask and bioreactor were performed. The reverse phosphorolysis activity of laminaribiose phosphorylase produced under different cultivation methods was measured. The heterotrophic approach showed to be the more effective cultivation method as 47.6 IU/L was obtained compared to 27 IU/L in the mixotrophic one. The heterotrophic cultivation then was further investigated under two different pH values of the culture media. The culture at pH 6.8 resulted in 7.94 IU/L/day whereas only 4.06 was obtained for the culture at pH 4. Cultivation in a bioreactor resulted in a distinctive amount of 191.5 IU/L and an activity yield of 9.7 IU/g glucose compared to 5.4 in flask cultivation. Heterotrophic cultivation of Euglena gracilis in a bioreactor containing a culture media at pH 6.8 and controlled operation conditions showed enhanced laminaribiose phosphorylase activity production per liter and day of cultivation.     

Effects of humic and fulvic acids on poliovirus concentration from water by microporous filtration

Because naturally occurring organic matter is thought to interfere with virus adsorption to microporous filters, humic and fulvic acids isolated from a highly colored, soft surface water were used as model organics in studies on poliovirus adsorption to and recovery from electropositive Virosorb 1MDS and electronegative Filterite filters. Solutions of activated carbon-treated tap water containing 3, 10, and 30-mg/liter concentrations of humic or fulvic acid were seeded with known amounts of poliovirus and processed with Virosorb 1MDS filters at pH 7.5 or Filterite filters at pH 3.5 (with and without 5 mM MgCl2). Organic acids caused appreciable reductions in virus adsorption and recovery efficiencies with both types of filter. Fulvic acid caused greater reductions in poliovirus recovery with Virosorb 1MDS filters than with Filterite filters. Fulvic acid interference with poliovirus recovery by Filterite filters was overcome by the presence of 5 mM MgCl2. Although humic acid reduced poliovirus recoveries by both types of filter, its greatest effect was on virus elution and recovery from Filterite filters. Single-particle analyses demonstrated MgCl2 enhancement of poliovirus association with both organic acids at pH 3.5. The mechanisms by which each organic acid reduced virus adsorption and recovery appeared to be different for each type of filter.     

Kinetin-induced tuberization of potato in vitro: on the mode of action of kinetin.

The tuberization percentage of etiolated potato (Solatium luberosumL.) sprout sections cultured in vitro in the absence of kinetinwas 61% (endogenous tuberization potential). The presence of5 mg/liter kinetin in the medium increased tuberization to 94%. In a time course experiment soluble ADP glucose: -l,4-glucan-4-glucosyl transferase (starch synthetase) activity extractedfrom etiolated sprout sections cultured in vitro remained low(one twenty-fifth of the activity present in the mother tubersat time 0), and was almost constant throughout 21 days of culture.No difference in activity was found in sections grown in kinetin-containingmedium in comparison with those grown in the absence of kinetin. During tuber initiation of etiolated potato stolons culturedin vitro, kinetin caused stimulation of phosphorylase activitywhich continued until the late stages of tuberization when italso stimulated ADP glucose pyrophosphorylase activity. Kinetindid not affect amylase activity. Addition of 11 mg/liter kinetinto the enzyme reaction mixture did not change the activity ofsolublestarch synthetase in vitro. The net accumulation of starch caused by kinetin was partiallythe result of the stimulation of starch biosynthesis throughphosphorylase at an early stage, and both phosphorylase andpyrophosphorylase at a later stage. ¹ Present address: Department of Horticulture, Washington StateUniversity, Pullman, WA 99163, USA. (Received January 6, 1976; )     

Effects of humic and fulvic acids on poliovirus concentration from water by microporous filtration.

Because naturally occurring organic matter is thought to interfere with virus adsorption to microporous filters, humic and fulvic acids isolated from a highly colored, soft surface water were used as model organics in studies on poliovirus adsorption to and recovery from electropositive Virosorb 1MDS and electronegative Filterite filters. Solutions of activated carbon-treated tap water containing 3, 10, and 30-mg/liter concentrations of humic or fulvic acid were seeded with known amounts of poliovirus and processed with Virosorb 1MDS filters at pH 7.5 or Filterite filters at pH 3.5 (with and without 5 mM MgCl2). Organic acids caused appreciable reductions in virus adsorption and recovery efficiencies with both types of filter. Fulvic acid caused greater reductions in poliovirus recovery with Virosorb 1MDS filters than with Filterite filters. Fulvic acid interference with poliovirus recovery by Filterite filters was overcome by the presence of 5 mM MgCl2. Although humic acid reduced poliovirus recoveries by both types of filter, its greatest effect was on virus elution and recovery from Filterite filters. Single-particle analyses demonstrated MgCl2 enhancement of poliovirus association with both organic acids at pH 3.5. The mechanisms by which each organic acid reduced virus adsorption and recovery appeared to be different for each type of filter.