Effect and potential ecological significance of the interaction of humic acids with two aquatic extracellular proteases

1. The effects of humic acids and fulvic acids on an extracellular serine and metalloprotease purified from a strain of Aeromonas hydrophila isolated from a freshwater wetland were examined. The serine protease was inhibited by humic acids at pH and humic acid concentrations found naturally in the wetland where this strain of A. hydrophila was isolated. The metalloprotease was not inhibited by humic acids at any pH investigated. Fulvic acids had no effect upon either protease. 2. Inhibition of serine protease activity by humic acids was reversed by increasing the pH to 9, as well as increasing the ionic strength by addition of CaCl₂- It was concluded that the interaction between humic acids and the serine protease was primarily ionic. 3. The formation of a humic acid-serine protease complex increased the half-life of enzymatic activity in dilute solutions. Humic acids had no effect on the stability of the metalloprotease. 4. There was no clear effect of humic acids on the growth of A. hydrophila, indicating that either the serine protease is not involved in the rate-limiting step of growth or that sufficient activity exists even when the serine protease is inhibited by humic acids. 5. Increasing the enzymatic lifetime through association with humic acids may be an adaptive mechanism which could result in energy conservation due to a decreased requirement for protein synthesis.     

Production of humic acids from oil palm empty fruit bunch by submerged fermentation with Trichoderma viride: Cellulosic substrates and nitrogen sources

The novelty of this study was to produce humic acids by submerged fermentation of empty fruit bunch (EFB) with Trichoderma viride and to investigate the effects of the cellulosic substrates and the organic sources of nitrogen on the biotechnological production of these acids. The results obtained indicate the potential application of EFB, a waste of oil palm processing, for humic acids production. Because EFB contains cellulose, hemicellulose and lignin, fermentations were also performed using these polymers as carbon sources, separately or in combination. After 120 h of fermentation, significant production of humic acids was observed only in cultures containing either EFB or a mixture of the three polymers. Use of either potato peptone or yeast extract as a nitrogen source yielded nearly identical patterns of fungal growth and production of humic acids. The data obtained from microscopic imaging of T. viride growth and sporulation in EFB, coupled with the determined rates of production of humic acids indicated that the production of these acids is related to T. viride sporulation. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:631–637, 2013     

Extracellular enzyme activities of Bjerkandera adusta R59 soil strain, capable of daunomycin and humic acids degradation

Geotrichum-like strain R59, the anamorphic form of the white-rot fungus, Bjerkandera adusta, was isolated from soil. It was found to completely decolorize and degrade 10% daunomycin post-production effluent during 10 days of incubation at 26°C. Strain R59 produced only low levels of ligninolytic enzymes when grown on wheat straw- or beech sawdust-containing media, but in the presence of humic acids derived from brown coal it synthesized significant amounts of laccase and lipase. This phenomenon was coupled with the fungus entering the idiophase and the appearance of aerial mycelium. B. adusta strain R59 was found to completely decolorize 0.03% humic acids from brown coal and lessive soil and to partially decolorize humic acids isolated from a chernozem during 14 days of growth. This ability of strain R59 could be useful in constructing a new generation of biologically active filters for the purification of humic acids-contaminated drinking waters.     

Shoot initiation by humic acids of selected tropical crops grown in tissue culture

Summary The effect of humic acid on shoot development from nodal segments in tissue culture was tested. The species wereGnetum gnemon, Elletaria cardamomum, andPogostemon cablin. Humic acids were extracted from a peat soil sampled from South Sumatra, Indonesia. Alkaline extraction was conducted on two series of 12-hour agitations followed by centrifugation and sedimentation at pH below 2. Explants were 1-cm long single nodes ofG. gnemon andP. cablin, and lateral buds ofE. cardamomum on MS medium. Benzyladenine was added at 0.1 mg/L for the first two and at 0.3 mg/L for the latter. The treatments included six, seven, and eight levels of humic acid concentrations for the respective species in a completely randomized design with 10 replicates. Effect of the treatment was evaluated on the basis of the initiation period of shoots and/or roots, and the number and height of the shoots. The initiation period of the shoots was significantly shortened in the presence of humic acids. Root initiation was significantly induced especially when humic acids were used in liquid medium. In combinations with BA, the addition of humic acids at 400 mg/L, 40 mg/L, and 300 mg/L yielded the fastest growth ofG. gnemon, E. cardamomum, andP. cablin, respectively.Abbreviations HA humic acids - BA benzyladenine - MS Murashige & Skoog     

Heathland restoration in The Netherlands: Effects of turf cutting depth on germination of Arnica montana

Abstract. Germination experiments were conducted in a heathland after turf cutting and in a climate chamber to investigate the effects of turf cutting depth, aluminium toxicity and aluminium detoxification by humic acids and base cations on the germination and establishment of Arnica montana. Turfs were cut at three different depths, creating a gradient from organic to mineral soils. Germination and establishment of A. montana were negatively correlated with turf cutting depth. The removal of organic matter resulted in a major decrease in organic fraction of the soil and its nutrients. It also resulted in a considerable decrease in moisture content and humic acids. Additional liming after turf cutting increased germination and establishment in all plots and at all depths. Germination experiments under controlled conditions in a climate chamber revealed a significantly higher germination at low aluminium/calcium (Al:Ca) ratios. High Al:Ca ratios resulted in poor germination, suggesting Al toxicity. Addition of humic acids increased germination, even at high Al:Ca ratios, suggesting immobilization of Al by humic acids. It is concluded that turf cutting can have a marked effect on the success of heathland restoration. It results in the intended removal of the eutrophic layer but also in the unintentional removal of much of the buffering mechanisms and/or Al immobilizing compounds. Additional buffering and/or less deep turf cutting may be necessary to allow germination and establishment of rare herbaceous species such as A. montana.     

Effects of humic and fulvic acids on growth of tobacco

Summary Organic matter was extracted with a mixture of 0.1M Na₄P₂O₇ and 0.1M NaOH from soil and a commercial organic matter product, Aqua Humus, and separated into humic and fulvic acids according to their solubility in acid and alkaline solutions. They were purified by passage through a cation exchange resin, freeze-dried, and used for characterization and seedling growth studies. The CEC increased with pH for both humic and fulvic acids. Data from tobacco seedling studies show that humic and fulvic acids from both sources increased root number and length at low concentrations while at high concentrations, humic acids inhibited length and number of roots but fulvic acids had no effect. The results support the proposition that fractions of organic matter referred to as humic and fulvic acids affect root development by means other than as a source of nutrients.     

STIMULATION OF GROWTH IN SCENEDESMUS OBLIQUUS (CHLOROPHYCEAE) BY HUMIC ACIDS UNDER IRON LIMITED CONDITIONS1,2

Stimulatory affects of humic acids of molecular weight 30,000 or greater on iron-starved Scenedesmus obliquus (Turp.) Kütz. in association with bacteria were studied by growth and Fe uptake experiments. Humic acids stimulated growth of Fe-starved cells by decreasing the lag phase and extending the growth phase. Humic acids stimulated increased algal growth in medium containing EDTA as well as in medium containing no EDTA, indicating humic acids are not stimulating algal growth under Fe limiting conditions by creating a soluble Fe pool. Humic acids decreased Fe availability to Fe-starved S. obliquus. Iron bound to humic acids is unavailable for uptake by Fe-starved cells indicating growth stimulation is not due to chelation effects alone. Stimulation of growth is not a membrane phenomenon as humic acids show the same stimulatory effect when in contact with cells or separated by dialysis membrane. Humic acids also stimulate growth in the dark, with and without aeration, indicating use as a heterotrophic substrate. A photoheterotrophic mechanism is indicated by increased algal growth caused by illuminating cultures, containing humic acids but excluding CO₂.     

Effect of<Emphasis Type="Italic">Chalara longipes</Emphasis> on decomposition of humic acids from<Emphasis Type="Italic">Picea abies</Emphasis> needle litter

The effect of the saprotrophic ascomycete Chalara longipes on the decomposition of humic acids was determined in a cultivation experiment. The fungus was incubated in liquid cultures in a full-strength (F system) and an organic nitrogen-free medium (F-N system), both amended with pure humic acids isolated from spruce forest litter. Fungal biomass production was highest in the F system with humic acids and lowest in the F-N system, the effect of organic nitrogen and humic acids being significant (p < 0.05). The presence of organic nitrogen seems to be essential for growth. The fungus utilized humic acids; molar mass distribution in media obtained by gel-permeation chromatography showed decrease in the humic acids fraction. Decolorization of the media reached 75 and 64 % in the F and F-N systems, respectively. The molar mass of humic acids probably also decreased as estimated from the increase in the absorbance A465/A665 (A4/A6) ratio. It is assumed that the utilization of humic acids may be mediated by the production of organic acids (as their components) and production of some oxidative enzymes.     

Hyphal growth and mycorrhiza formation by the arbuscular mycorrhizal fungus Glomus claroideum BEG 23 is stimulated by humic substances

Effects of humic substances (humic acid or fulvic soil extract) or saprophytic microorganisms (Paecilomyces lilacinus and an unidentified actinomycete) on growth of mycelium and mycorrhiza formation by Glomus claroideum BEG23 were studied in a hydroponic system. Humic substances stimulated root colonization and production of extraradical mycelium by the mycorrhizal fungus. Both humic and fulvic acids tended to decrease populations of culturable bacteria and fungi in the cultivation system, indicating a moderately antibiotic activity. The addition of saprophytic microorganisms able to use humic substances to the cultivation system further stimulated the development of the mycorrhizal fungus. However, stimulation of G. claroideum was also observed when the saprophytic microorganisms were heat-killed, suggesting that their effect was not linked to a specific action on humic substances. The results indicate that humic substances may represent a stimulatory component of the soil environment with respect to arbuscular mycorrhizal fungi.     

Humic Materials Offer Photoprotective Effect to Escherichia coli Exposed to Damaging Luminous Radiation

The behavior ofEscherichia coli immersed in aqueous systems amended with humic acids, under PAR, UV-A, UV-B, and simulated solar radiation was examined. Culturability, ability to elongate, functioning of the electron transport systems, and glucose uptake were assessed. Humic substances in the range from 1 to 50 mg L⁻¹ protected cells from photoinactivation. Decrease in culturability and cellular activities was significantly (p<0.05) less in the presence of humic material. However, humic acid were not used as nutrients. Neither irradiated nor nonirradiated humic solutions (50 mg L⁻¹) supported the growth of 10⁵ cells ml⁻¹. However, humic acids dissolved in 0.9% NaCl efficiently absorbed light over wavelengths from 270 to 500 nm. Also, a photoprotective effect against simulated sunlight was observed when humic acid were not in contact with but rather enveloped the cellular suspensions in double-wall microcosms. The protection afforded by humic acids against luminous radiation likely derives from their ability to absorb these radiations and hence reduces the amount of energy reaching the cells.     

Quinone-respiration improves dechlorination of carbon tetrachloride by anaerobic sludge

The impact of humic acids and the humic model compound, anthraquinone-2,6-disulfonate (AQDS), on the biodegradation of carbon tetrachloride (CT) by anaerobic granular sludge was studied. Addition of both humic acids and AQDS at sub-stoichiometric levels increased the first-order rate of conversion of CT up to 6-fold, leading to an increased production of inorganic chloride, which accounted for 40–50% of the CT initially added. Considerably less dechlorination occurred in sludge incubations lacking humic substances. By comparison, very limited dechlorination occurred in sterile controls with autoclaved sludge. Accumulation of chloroform (1–10%) and dichloromethane (traces) also accounted for the CT converted. The accumulation of a chlorinated ethene, perchloroethylene (up to 9% of added CT), is also reported for the first time as an end-product of CT degradation. A humus-respiring enrichment culture (composed primarily of a Geobacter sp.) derived from the granular sludge also dechlorinated CT, yielding products similar to the AQDS-supplemented granular sludge consortium. The dechlorination of CT by the Geobacter enrichment was dependent on the presence of AQDS or humic acids, which were reduced during the assays. The reduced form of AQDS, anthrahydroquinone-2,6-disulfonate, was shown to cause the chemical reduction of CT when incubated in sterile medium. The results taken as a whole indicate that the formation of reduced humic substances by quinone-respiring microorganisms can contribute to the reductive dechlorination of CT.     

Depolymerization of low-rank coal by extracellular fungal enzyme systems. III.In vitro depolymerization of coal humic acids by a crude preparation of manganese peroxidase from the white-rot fungus Nematoloma frowardii b19

The in vitro depolymerization of humic acids derived from German lignite (low-rank coal, brown coal) was studied using a manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii b19. The H₂O₂ required was continuously generated by glucose oxidase. Mn peroxidase depolymerized high-molecular-mass humic acids by forming fulvic-acid-like compounds. The depolymerization process was accompanied by the decolorization of the dark-brown humic acid fraction soluble in alkaline solutions (decrease in absorbance at 450 nm) and by the yellowish coloring of the fraction of acid-soluble fulvic-acid-like compounds (increase in absorbance at 360 nm). The Mn peroxidase of N. frowardii b19 has been proved to be highly stable; even after an in vitro reaction time of 7 days in the presence of humic acids, less than 10% loss in total oxidizing activity was detectable. Received: 16 September 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

Comparative effects of soil organic matter fractions on phosphatase activities in wheat roots

Summary Soil organic matter was obtained from two agricultural soils using alkali extraction followed by acidification to produce humic and fulvic acids which were further fractionated by adsorption and gel chromatography.All the products inhibited the activity of phosphatase prepared from wheat roots, but to different extents. Humic acids produced a greater inhibition of enzyme activity than either the fulvic acids or water extracts of soil. Aspergillin, fromAspergillus niger, had a similar C, H and N content to humic acid and produced a similar inhibition of phosphatase activity.The inhibitions produced by corresponding fractions derived from the two soils were slightly different, but the trends between similar fractions from different soils were comparable. The lower mol. wt. components of humic acid inhibited phosphatase activity to a greater extent than higher mot. wt. fractions. Although fulvic acid comprised only low mol. wt. components it was less effective in inhibiting enzyme activity than those components of comparable mol. wt. present in the corresponding humic acid. Synthetic polymaleic acid, produced an inhibition of phosphatase activity similar to that caused by fulvic acid.     

Technical note: Examining the use of ethylenediaminetetraacetic acid for humic extraction of ancient bone

We examined the efficacy of ethylenediaminetetraacetic acid (EDTA) for removing humic contaminants from collagen extracted from ancient bone. Humic contaminants must be removed to obtain reliable stable isotope values from ancient bone collagen, given that humic acids have consistently lower δ¹³C values than collagen. The purpose of our research was to examine if EDTA treatment could effectively remove humic contaminants from bone collagen and thus serve as an alternative to the commonly implemented sodium hydroxide (NaOH) treatment, which may be associated with large collagen losses in poorly preserved samples. We compared the isotopic and elemental composition of ancient samples treated with EDTA alone, samples demineralized in hydrochloric acid (HCl) and rinsed in EDTA, samples treated with HCl alone, and samples demineralized in HCl and rinsed with NaOH. The samples used in the analyses were selected because they presented evidence of substantial humic contamination. We found that NaOH was the most effective agent for reducing humic contaminants as evidenced by the samples treated with this agent having higher δ¹³C values and lower C:N ratios relative to other treatments. The results from samples treated with EDTA suggest that this chemical cannot effectively remove humic contaminants given that these samples had significantly higher C:N ratios and lower δ¹³C and δ¹⁵N values relative to the HCl/NaOH treatment. Our results demonstrate that when performing stable isotope analysis of ancient bone collagen suspected to be contaminated with humic acids, NaOH is the most effective chemical agent to remove humic contaminants, while EDTA cannot perform this task.     

Depolymerization of low-rank coal by extracellular fungal enzyme systems.

 A miniscale screening system was developed to detect depolymerizing activities of fungi toward low-rank coals. This system was suitable for the determination of changes in molecular masses as well as for the measurement of the enzymes responsible. A total of 486 fungal strains of different ecophysiological and taxonomic groups were tested for their ability to decolorize agar media containing coal-derived humic acids; 38 wood- and litter-decaying basidiomycetes caused a strong bleaching effect and 49 a weak effect. In contrast, micromycetes were proved to be unable to decolorize the coal substances. The wood-decaying fungus Nematoloma frowardii b19 most effectively bleached the medium. It could be demonstrated by gel-permeation chromatography that the strain really depolymerizes the high molecular-mass fractions of coal humic acids by forming fulvic-acid-like compounds. Extracellular enzyme activities of oxidases and peroxidases towards 2,2′-azinobis(3-ethylbenzthiazolinesulphonate) were extractable from the agar media. Received: 5 February 1996/Received revision: 15 April 1996/Accepted: 22 April 1996     

Amino acid distribution in some fungal melanins and of soil humic acids from Brazil

Summary Humic acids from four Brazilian topsoils of different origins and four soil fungal melanins, synthesized under two cultural conditions, were subjected to 6N HCl hydrolysis and their amino acid distribution patterns qualitatively and quantitatively determined. Both soil and fungal polymers showed similar patterns with aspartic acid, glutamic acid, glycine and alanine as the dominant amino acids. Some variations noted were more quantitative than qualitative, the similarities were more pronounced than differences, indicating that the fungal melanins may play a significant role in the formation of soil humic acid polymers. The humic acids of Brazilian soils had amino acid distribution patterns similar to those reported for humic acids of other tropical and temperate soils.     

Energy-driven uptake of humic acids by Aspergillus niger.

The uptake of humic acids by mycelia of Aspergillus niger was demonstrated to be energy-dependent with a sensitivity to sodium azide and to 2,4-dinitrophenol. Greater uptake of humic acids by submerged mycelium occurred at pH 3.0 and at 32 degrees C. The rate of uptake was influenced by the concentration of humic acids with an apparent Km of 0.2 grams/ml and with a Vmax of 0.13 mg humic acids per gram mycelial dry weight.10 min-1. In the absence of added energy source, Vmax of 0.05 mg humic acids per gram mycelial dry wt.10 min-1 was obtained; however, the affinity for humic acids by this uptake system was the same as for the energy-driven process. Apparent binding of humic acids to cell structures was indicated because only 41.8% of the humic acids taken up by the energy-dependent system could be recovered.     

In vivo-decolorization of coal-derived humic acids by laccase-excreting fungus Trametes versicolor

Lignite (brown coal) can be liquefied/solubilized with several fungi by different mechanisms. When applied industrially, only catalytic mechanisms can compete with chemical methods. The well-known fungal ligninolytic peroxidases are at a disadvantage, in that the relatively expensive hydrogen peroxide must be used as a cofactor. Comparing several fungal strains, we observed that the fungus Trametes versicolor is able to decolorize coal-derived humic acids, producing a considerable amount of laccase in the process. During this reaction the amount of humic acids decreases whilst that of fulvic acids increases; this was verified by optical density measurement and GPC after the two substance classes had been separated. Received: 27 August 1998 / Received revision: 4 November 1998 / Accepted: 7 November 1998     

Quenching of fluorescence of phenolic compounds and modified humic acids by cadmium ions

The interaction of a number of phenolic compounds, being ‘model fragments’ of humic acids, with cadmium ions was investigated. The fluorescence quenching method was used to determine the complexation constants of these compounds with cadmium ions. It was established that bonding of phenolic compounds by cadmium ions at рН 7 is weak and reaches a maximum value of 15% for interaction with resorcinol. It was demonstrated that modification of humic acids by the mechanoactivation method increases by three times bonding of cadmium ions, which is caused by strengthening the acid properties of carboxyl and hydroxyl groups at the aromatic ring. Copyright © 2016 John Wiley & Sons, Ltd.     

Influence of Naturally Occurring Humic Acids on Biodegradation of Monosubstituted Phenols by Aquatic Bacteria

Samples of the microbial community from Lake Michie, a mesotrophic reservoir in central North Carolina, were adapted to various levels (100 to 1,000 μg/liter) of natural humic acids in chemostats. The humic acids were extracted from water samples from Black Lake, a highly colored lake in the coastal plain of North Carolina. After adaptation, the microbial community was tested for its ability to degrade the monosubstituted phenols m-cresol, m-aminophenol, and p-chlorophenol. Adaptation to increasing levels of humic acids significantly reduced the ability of the microbial communities to degrade all three phenols. The decline in biodegradation was accompanied by a decrease in the number of specific compound degraders in the adapted communities. Short-term exposure of the community to increasing levels of humic acids had no significant effect on the ability of the community to degrade m-cresol. Thus the suppressive effect of humic acids on monosubstituted phenol metabolism was the result of long-term exposure to the humic materials. Increasing the levels of inorganic nutrients fed to the chemostats during the humic acid adaptation had little effect on the suppressive influence of the humic acids, indicating that nutrient limitation was probably not responsible for the metabolic suppression. The results of the study suggest that long-term exposure to humic acids can reduce the ability of microbial communities to respond to monosubstituted phenols.