Effect of biodelignification of rice straw on humification and humus quality by Phanerochaete chrysosporium and Streptomyces badius

The effect of biodelignification of rice straw by two different ligninolytic organisms, Phanerochaete chrysosporium (white-rot fungus) and Streptomyces badius (actinomycetes), on humus quality was investigated during a 56-day incubation at 30 °C. Lignin degradation, the release of humic extract (HE), humic acid (HA) and fulvic acid (FA), E4/E6 ratio of HA, and humification index (HI, HA/FA) were measured during the incubation. Lignin was degraded by both organisms, but to different extents. Lignin was degraded to 41% and 31% by P. chrysosporium and S. badius, respectively. HE released by P. chrysosporium and S. badius were, respectively, 2.10 and 2.13 times larger than that in the control at the maximum values. A significant correlation between lignin degradation and humus-related parameters involving HA fraction showed that both organisms are converting lignin to humic substances.     

肥料类型对盐碱地蒙古黄芪生长与光合特征及药材产量和品质的影响

以一年生蒙古黄芪种苗为材料,在盐碱地探究二年生蒙古黄芪根系产量和药效成分对不同肥料(海藻酸水溶肥、腐殖酸水溶肥、微生物菌剂生物肥、复合肥和有机肥)的响应以及其光合生理机制,为盐碱地蒙古黄芪的推广种植提供参考。结果显示:在盐碱地,微生物菌剂生物肥的施用对蒙古黄芪的抗逆性、光合作用效率、生长特性、根系产量及质量均无显著性影响;复合肥的施用显著促进了蒙古黄芪地上部分的生长,但对根系产量无显著影响,反而显著降低了其抗逆性、光合作用效率和药效成分;海藻酸水溶肥、腐殖酸水溶肥和有机肥的施用均显著提高了蒙古黄芪抗逆性、光合作用效率、地下部分的生长及产量、质量;施用有机肥的蒙古黄芪药材产量最高(7 046.2 kg·hm^-2),并与施用除腐殖酸水溶肥以外的其余处理差异显著,施用腐殖酸水溶肥和有机肥的蒙古黄芪药效成分含量最高并与其余施肥处理差异显著,它们的黄芪甲苷含量均达到0.13%以上,毛蕊异黄酮葡萄糖苷含量均达到0.06%以上。研究表明,在盐碱地蒙古黄芪栽培过程中,施用有机肥在保证蒙古黄芪药效成分较高的同时,还具有最高的药材产量,可认为是最佳肥料。     

Isolation of humic acid from the brown algaPilayella littoralis

A standard humic acid extraction procedure has been used to isolate dark brown organic residues from samples of the macroscopic marine brown algaPilayella littoralis. The residues are insoluble in water, but soluble at high pH, and are similar in elemental composition, ash content, UV-visible, IR, PMR and X-Ray fluorescence spectra, X-Ray diffractograms and scanning electron micrographs to residues of a humic acid isolated from municipal compost. These results indicate thatPilayella produces humic acids.Author for correspondence     

Contribution of bacterial cell nitrogen to soil humic fractions

Summary Living cells ofSerratia marcescens, uniformly labelled with¹⁵N, were added to samples of maple (Acer saccharum) and black spruce (Picea mariana) forest soils. After different periods of incubation from zero time to 100 days, the soils were subjected to alkali-acid and phenol extraction to provide humic acid, fulvic acid, humin and humoprotein fractions. Significant amounts of the cell nitrogen were recovered in the humic and fulvic acids immediately after addition. After incubation, less cell, nitrogen appeared in the humic acid and more in the fulvic acid. The amount of cell nitrogen recovered in the humin fraction increased with incubation. Roughly 5 to 10 per cent of the added cell nitrogen was found as amino acid nitrogen from humoprotein in a phenol extract of the humic acid. The data are consistent with the occurrence of co-precipitation of biologically labile biomass nitrogen compounds with humic polymers during the alkaline extraction procedure involved in the humic-fulvic fractionation.     

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.     

A comparison ofin vitro andin vivo effect of clay minerals,humic acid and micronutrients on the activity of fungicides againstRhizoctonia solani

Activity of nine fungicides against mycelial growth ofRhizoctonia solani in potato dextrose broth and in pot tests as seed treatment against cowpea seedling rot in infested soil was differentially in fluenced by clay minerals, humic acid and micronutrients. Humic acid, extracted from farmyard manure, considerably lowered the activity, bothin vitro andin vivo, of all fungicides except chloroneb. Montmorillonite caused substantial decrease in disease control by fungicides but enhanced the toxicity of 2-methoxyethyl mercury chloride (MEMC), and quintozene in culture. Kaolinite inactivated carbendazim, benomyl and thiophanatemethyl in nutrient broth but had little effect on disease control by these fugicides. The six micronutrients altered the activity of fungicides to varying levels often without definite correlation betweenin vitro andin vivo results. The results ofin vitro growth inhibition tests are largely inapplicable to dieseas control tests in infested soil mainly due to the differences in the ambient conditions of the two systems.     

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.     

Mass development of the flagellate Gonyostomum semen (Raphidophyta) in Swedish forest lakes - an effect of acidification?

During the last 20–30 years the flagellate Gonyostomum semen has become more abundant in lakes, especially small humic lakes, in Scandinavia. Mass development of the alga has been reported from areas affected by anthropogenic acid deposition, and reports from bathers of health problems are becoming frequent. Although there is an apparent connection between the appearance of Gonyostomum and acidification, it seems not to be the low pH per se that is the cause, but rather interlinked factors. The present tendency towards a wider distribution of the alga in non-humic lakes, emphasizes the importance of a better understanding of small humic lakes.     

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     

Isolation of humic acid from the brown algaeAscophyllum nodosum,Fucus vesiculosus,Laminaria saccharina and the marine angiospermZostera marina

Recent discovery of humic acid (HA) in the free-living, brown algaPilayella littoralis has prompted a search for HA in other live plants. Marine algaeCodium fragile andMonostoma oxyspermum (greens),Chondrus crispus,Palmaria palmata andPolysiphonia lanosa (reds),Ascophyllum nodosum, Fucus vesiculosus andLaminaria saccharina (browns) andZostera marina (marine angiosperm) were investigated for their HA content. Only the brown algae and the marine angiosperm contained HA, which was extracted by a standard procedure augmented with necessary removal of alginic acid (where applicable). The isolated products were identified as HA by comparison of their analytical data, uv-visible, FTIR,¹H NMR spectra and morphologies with those of authentic HA isolated from municipal compost.Authors for correspondence     

Soil humic substances affect transport properties of tonoplast vesicles isolated from oat roots

The effect of a low molecular size (<5 KDa) humic fraction, essentially fulvic acids, on microsomal and tonoplast ion-stimulated ATPase activity was studied. After 20 min of pre-incubation with microsomal vesicles from oat roots, humic substances at organic C concentration of up to 0.5 μg cm^-3 increased KCl-stimulated ATPase activity, while they inhibited enzyme activity at higher concentrations. Cl^--stimulated ATPase activity of tightly sealed tonoplast-enriched vesicles was similarly affected by <5 KDa humic substances. This behaviour was not observed when gramicidin D was added to the assay medium. Proton transport by vesicles incubated up to 5 min with <5 KDa humic molecules was affected in a concentration-dependent manner, strongly resembling that observed for ATP hydrolysis, whereas it was severely reduced when the assay conditions were close to those used for measuring ATP hydrolysis (20 min pre-incubation of vesicles with humic substances). The transmembrane electrical potential was negatively affected, irrespective of the concentration of humic molecules. Furthermore, a 15-min pre-incubation strongly reduced the formation of a potential gradient. The size and concentrations of humic substances employed make an interaction with the vacuolar membrane of root cells plausible. The results show that the main target of humic molecules is the electrical membrane potential and suggest a possible way of interference of these naturally occurring substances with the biochemical mechanisms involved in plant mineral nutrition.     

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.     

Influence of humic, fulvic and hydrophilic acids on the growth, photosynthesis and respiration of the dinoflagellate Prorocentrum minimum (Pavillard) Schiller

Blooms of the dinoflagellate Prorocentrum minimum often occur in coastal regions characterized by variable salinity and elevated concentrations of terrestrially derived dissolved organic carbon (DOC). Humic, fulvic and hydrophilic acid fractions of DOC were isolated from runoff entering lower Narragansett Bay immediately after a rainfall event and the influence of these fractions upon P. minimum growth, cell yield, photosynthesis and respiration was examined. All organic fractions stimulated growth rates and cell yields compared with controls (no organic additions), but the extent of stimulation varied with the fraction and its molecular weight. Greatest stimulations were observed with humic and fulvic acids additions; cell yields were more than 2.5 and 3.5 times higher than with hydrophilic acid additions while growth rates were 21 and 44% higher, respectively. Responses to additions of different molecular weight fractions of each DOC fraction suggest that growth rate effects were attributable to specific molecular weight fractions: the >10,000 fraction of humic acids, both the >10,000 and <500 fractions of fulvic acids and the <10,000 fraction of hydrophilic acids. The form and concentration of nitrogen (as NO₃⁻ or NH₄⁺) present also influenced P. minimum response to DOC; 10–20 μg ml⁻¹ additions of fulvic acid had no effect upon growth rates in the presence of NH₄⁺ but significantly increased growth rates in the presence of NO₃⁻, a relationship probably related to fulvic acid effects upon trace metal bioavailability and subsequent regulation of the biosynthesis of enzymes required for NO₃⁻ assimilation. The influence of DOC additions on P. minimum respiration and production rates also varied with the organic fraction and its concentration. Production rates ranged from 1.1 to 3.4 pg O₂ cell⁻¹ h⁻¹, with highest rates observed upon exposure to fulvic and hydrophilic acid concentrations of >10 μm ml⁻¹. Low concentrations (5–10 μg ml⁻¹) of humic acid had no statistically significant effect upon production, but exposure to concentrations >25 μg ml⁻¹ resulted in a 30% decrease in O₂ evolution, probably due to light attenuation by the highly colored humic acid fraction. Respiration rates ranged from 1.2 to 2.7 pg O₂ cell⁻¹ h⁻¹ and were elevated upon exposure to both fulvic and hydrophilic acids, but not to humic acid. These results demonstrate that terrestrially derived DOC fractions play an active role in stimulation of P. minimum growth via direct effects upon growth, yield and photosynthesis as well as via indirect influences such as interactions with nitrogen and effects upon light attenuation.     

Degradation of humic acid of a forest soil by some fungal isolates

Summary In a laboratory incubation study the humic acid isolated from a forest soil of Palamau (Bihar) was subjected to biodegradation for a period of six weeks by using nine cultures of fungi. These fungi were tested earlier for their cellulose decomposing ability. The humic acid was used as sole source of carbon, nitrogen and carbon plus nitrogen in Czapek-Dox broth. Of the nine culturesAspergillus awamori (IARI),Penicillium sp. (Ranchi),Humicola insolense (Hissar) were found to be very effective in decomposing humic acid. The humic acid used as sole source of carbon was most efficiently degraded followed by that used as carbon+nitrogen source. When it was used as sole source of nitrogen, it could not be degraded so efficiently. This may be due to unavailability of its nitrogen to these microorganisms.     

Endoglucanase Activity of Compost-Dwelling Fungus Paecilomyces inflatus is Stimulated by Humic Acids and Other Low Molecular Mass Aromatics

Summary Paecilomyces inflatus isolated from municipal waste compost was found to have cellulolytic activity in several solid and liquid media. This study was done to reveal the multifarious effects of municipal waste compost on endoglucanase activity of P. inflatus. The highest enzyme activities under the conditions of solid-state fermentation were measured in authentic compost samples compared with wood, straw and bran substrates. In surface liquid cultures glucose, cellobiose, xylan, Avicel cellulose, carboxymethylcellulose (CM-cellulose), starch and citrus pectin were used as carbon sources. All carbon sources supported the growth of P. inflatus. However, only CM-cellulose, cellobiose and pectin noticeably stimulated endoglucanase (EG) activity. Further stimulation of EG activity was obtained in cultures containing 1% CM-cellulose as a carbon source by supplementation with low-molecular mass aromatic compounds vanillin, veratric acid and benzoic acid, and with soil humic acid (SHA). SHA and veratric acid were found to be the most efficient elicitors of the cellulolytic activity. P. inflatus was able to utilize nitrate and ammonium as pure nitrogen sources in media containing cellulose.     

Effects of humic acid-herbicide interactions on the growth of Pisum sativum in nutrient solution

Slight increases in root length and dry matter production were measured in roots and shoots of Pisum sativum seedlings, when germinated seeds were grown for eight days in a controlled environment chamber, on Nitch nutrient solution to which 10 mg L^-1 soil humic acid (HA) had been added. A concentration of 100 mg L^-1 of HA produced a small reduction in dry matter production of shoots. In the presence of herbicides, 10 mg L^-1 cycluron or 100 mg L^-1 prometone, root elongation and shoot growth was reduced by varying degrees with respect to the control. Nutrient solutions containing 100 mg L^-1 cycluron, 10 or 100 mg L^-1 alachlor, or as little as 0.1 mg L^-1 of 2,4-D resulted in an almost complete suppression of the growth of pea seedlings. The addition of 10 or 100 mg L^-1 of HA to nutrient solution containing the herbicide was found to be either ineffective in enhancing growth, or even further reduced growth, compared to samples grown in the presence of the herbicide alone. The results of this preliminary study suggest that the generally accepted view that humic substances exert a positive effect on plant growth may not occur when humic substances interact with herbicides.     

Combined effects of an oxidative enzyme and dissolved humic substances on 13C-labelled 2,4-D herbicide as revealed by high-resolution 13C NMR spectroscopy

Phenoxyalkanoic acids are a widely used class of herbicides. This work employed high-resolution ¹³C NMR to study the structural changes induced by humic substances and horseradish perodixase on 2,4-dichorophenoxyacetic acid (2,4-D) ¹³C-labelled in the side chain. NMR spectra showed that humic substances chemically catalyze abiotic splitting of [¹³C]2,4-D into 2,4-dichlorophenol and [¹³C]acetic acid at pH 7 but not at pH 4.7. Peroxidase did not catalyze the oxidative degradation of [¹³C]2,4-D at any pH tested and inhibited the effect of humic substances. Catalytic degradation by humic substances was attributed to free-radical reactions enhanced by the stereochemical contribution of large conformational structures formed by heterogeneous humic molecules at neutral pHs. Inhibition of 2,4-D degradation when humic substances were combined with peroxidase was explained by modification of both chemical and conformational humic structure due to peroxidase-promoted oxidative cross-coupling among humic molecules. Our findings show for the first time that the abiotic degradation of 2,4-D is catalyzed by dissolved humic substances at neutral pH. Journal of Industrial Microbiology & Biotechnology (2001) 26, 70–76. Received 09 February 2000/ Accepted in revised form 22 May 2000     

The zooplankton and other limnological features of a humic coastal lagoon (Lagoa Comprida,Macé, R.J.) in Brazil

Comprida lagoon is a shallow lagoon separated from the sea by a sand barrier. It has a brown-coloured freshwater with a high concentration of humic compounds. Its zooplankton community and limnological features were studied from March 1992 to December 1995. The lagoon was characterized by low transparency, acid water and relative constant physical–chemical features, except during sporadic marine entrances. The zooplankton, composed of holoplanktonic and meroplanktonic forms, consisted of 60 taxa. Eleven were permanent elements of the community: e.g. Bosminopsis deitersi, nauplii and copepodites of `Diaptomus' azureus. B. deitersi correlated positively with the lowest pH values and with the highest total dissolved nitrogen. Only four taxa correlated with chlorophyll-a concentration. Pigmentation in `D.' azureus is suggested to be as an energy reserve in a system where phytoplankton is probably light-limited.     

Effect of electron mediators on current generation and fermentation in a microbial fuel cell

Effects of select electron mediators [9,10-anthraquinone-2,6-disulfonic acid disodium salt (AQDS), safranine O, resazurin, methylene blue, and humic acids] on metabolic end-products and current production from cellulose digestion by Clostridium cellulolyticum in microbial fuel cells (MFCs) were studied using capillary electrophoresis and traditional electrochemical techniques. Addition of the mediator resazurin greatly enhanced current production but did not appear to alter the examined fermentation end-products compared to MFCs with no mediator. Assays for lactate, acetate, and ethanol indicate that the presence of safranine O, methylene blue, and humic acids alters metabolite production in the MFC: safranine O decreased the examined metabolites, methylene blue increased lactate formation, and humic acids increased the examined metabolites. Mediator standard redox potentials (E ₀) reported in the literature do not coincide with redox potentials in MFCs due presumably to the electrolytic complexity of media that supports bacterial survival and growth. Current production in MFCs: (1) can be effected by the mediator redox potential while in the media, which may be significantly shifted from E ₀, and (2) depended on the ability of the mediator to access the bacterial electron source, which may be cytoplasmic. In addition, some electron mediators had significant effects on metabolic end-products and therefore the metabolism of the organism itself. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.