Biodegradation of humic substances by microscopic filamentous fungi: chromatographic and spectroscopic proxies
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The study of interactions between humic substances (HSs) and soil filamentous fungi is the key to understanding the sustainable soil functioning. The present work aims to examine the decomposition of HSs by filamentous dark-pigmented fungus Alternaria alternatа under the laboratory conditions and to determine the effect of easily assimilable organic carbon on this process. Analyzing such polydisperse substances like HSs by a complex integrated methodology makes it possible to explore the data on their decomposition by microorganisms.
Materials and methods
To achieve the aforementioned goals, we used chromatographic and spectroscopic approaches: low-pressure size-exclusion and hydrophobic interaction chromatography accompanied by absorption and fluorescence spectroscopy. To determine the effect cometabolism conditions produced on HS decomposition, two types of carbon substrates were added to the nutrient media: easily assimilable organic carbon (standard 0.3% or reduced 0.03% sucrose content) and hardly assimilable organic carbon (HSs), as well as their combinations. Five HS samples of different organic matter origin have been inspected: potassium humates (HPs) and humic acids (HAs) from coal, peat, and lignosulfonate. Correlation matrix and principal component analysis (PCA) were calculated for comprehensive data analysis.
Results and discussion
Transformations of the investigated HSs under fungal cultivation lead to the increase in the low molecular weight fraction, rise of hydrophilic fraction, enlargement of absorbance ratio A250/A365, shortening of the emission wavelength of the humic-type fluorescence, and growth in the fluorescence quantum yield measured with excitation at 355 nm. A positive correlation was observed between the accumulation of fungal biomass and the degree of HS decomposition. PCA analysis confirms that the difference in the results of HS decomposition largely depends on the sucrose content and the nature of HSs. We divided all the HS samples into four groups according to the degree of HS decomposition: original HS solutions, HPs altered using fungal cultivation at 0.03% sucrose, HAs after fungal cultivation at 0.03% sucrose, and finally, HSs (both HPs and HAs) after fungal cultivation at 0.3% sucrose.
In the laboratory experiments, we showed that (1) the isolated HAs were more effectively degraded than the parent HPs, and this process was more pronounced at a reduced sucrose content, and (2) the decomposition of stable organic compounds (HSs) was activated by the easily assimilable carbon sources (especially 0.3% sucrose) being present. We assume that it is the easily assimilable organic carbon that most likely triggers the HS degradation working as the priming effect in natural environments.
KeywordsAbsorption and fluorescence spectroscopy Decomposition of humic substances Easily assimilable organic carbon Filamentous fungi Hydrophobic interaction chromatography Size-exclusion chromatography
Absorbance ratio at 250 nm and 365 nm
Chromophoric dissolved organic matter
Humic acid isolated from HPcoal
Humic acid isolated from HPpeat
Hydrophobic interaction chromatography
Potassium humates produced from coal
Potassium humates produced from lignosulfonate
Potassium humates produced from peat
Low-pressure size-exclusion chromatography
Principal component analysis
Fluorescence quantum yield at 355 nm
Reversed-phase hydrophobic interaction chromatography
The study was funded by the RFBR research project no. 18-016-00078.
- Bingeman CW, Varner JE, Martin WP (1953) The effect of the addition of organic materials on the decomposition of an organic soil. Soil Sci Soc Am Proc 29:692–696Google Scholar
- Fedoseeva E, Khundzhua D, Terekhova V, Patsaeva S (2018) Use of absorption spectra and their second-order derivative to quantify degradation of lignohumate by filamentous fungi. Proc SPIE 106142B:1–7Google Scholar
- Kirk PM, Cannon PF, Minter DW, Stalpers JA (2008) Dictionary of the fungi (10th ed). CABI, WallingfordGoogle Scholar
- Milanovskii EYu (2006) Humus substances as a system of hydrophobic-hydrophilic compounds. Dissertation, Lomonosov Moscow State UniversityGoogle Scholar
- Osterman LA (1985) Chromatography of proteins and nucleic acids, Russia, MoscowGoogle Scholar
- Patsaeva S, Khundzhua D, Trubetskoj OA, Trubetskaya OE (2018) Excitation-dependent fluorescence quantum yield for freshwater chromophoric dissolved organic matter from northern Russian lakes. J Spectroscopy 3168320:3168320–1–3168320–7Google Scholar
- Perminova IV, Frimmel FH, Kudryavtsev AV, Kulikova NA, Abbt-Braun G, Hesse S, Petrosyan VS (2003) Molecular weight characteristics of humic substances from different environments as determined by size exclusion chromatography and their statistical evaluation. Environ Sci Technol 37(11):2477–2485CrossRefGoogle Scholar
- Shubina D, Fedoseeva E, Gorshkova O, Patsaeva S, Terekhova V, Timofeev M, Yuzhakov V (2010) The “blue shift” of emission maximum and the fluorescence quantum yield as quantitative spectral characteristics of dissolved humic substances. EARSeL eProceedings 9(1):13–21Google Scholar
- Stepanov AA (2005) Structural features of amphiphilic humic acid fractions from a southern chernozem. Eurasian Soil Sci 38(8):843–847Google Scholar
- Trubetskoj OA, Richard C, Voyard G, Marchenkov VV, Trubetskaya OE (2018) Molecular size distribution of fluorophores in aquatic natural organic matter: application of HPSEC with multi-wavelength absorption and fluorescence detection following LPSEC-PAGE fractionation. Environ Sci Technol 52(9):5287–5295CrossRefGoogle Scholar
- Zavarzina AG, Lisov AA, Zavarzin AA, Leontievsky AA (2011) Fungal oxidoreductases and humification in forest soils. In: Shukla G, Varma A (eds) Soil enzymology, (Vol. 22 of Soil Biology), pp 207–229Google Scholar