INFLUENCE OF FE, CU, MN, ZN, CO CHELATORS ON THE BIOTRANSFORMATION OF HUMIC SUBSTANCES OF LIGNITE

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1 INFLUENCE OF FE, CU, MN, ZN, CO CHELATORS ON THE BIOTRANSFORMATION OF HUMIC SUBSTANCES OF LIGNITE G. Angelova 1, K. Chakalov 1, T. Popova 1 and V. Savov 1 ROMB ltd, 1 Aboba Str. 166 Sofia, Bulgaria Sofia University St Kliment Ohridski, Faculty of Biology Correspondence to: Gergana Angelova romb_ltd@abv.bg ABSTRACT Preparations, containing humic substances become more and more popular in modern technologies for organic farming during the past years. The purpose of the study is to determine both the influence of the supporting agent modified zeolites during biodestruction of lignite by Т.harzianum, and the possibility for using the formed humic substances as a matrix for immobilization of enzyme systems. The influence of chelate form of Fe, Cu, Mn, Zn, Co, Ca, Mg in composition with natural zeolite (clinoptilolite Eastern Rhodopes, Bulgaria ammonia form) upon the biotransformation of humic substances from lignite is studied. Lignite from Stanyantsy Mine is inoculated with Т.harzianum 1 5 CFU/g; modified zeolites with concentration g/1g lignite are added to the medium. Research data show that N-zeolites, modified with Cu, Zn, Mn and a combination of them, and used as a supporting agent during the biotransformation of lignite by T.harzianum, affect the humification and the formation of enzyme systems depending on the concentration of the saturating micro element. Zinc ions have a lower influence, and manganese ions slow down the humificaion to some extent. Zeolite components, modified with Mn increase the activity of polyphenol oxidase and decrease the activity of peroxidase. Zeolite enrichment with Zn increases the activity of polyphenol oxidase and peroxidase, and the increased concentration of Cu ions in zeolites lead to an increased activity of polyphenol oxidase and peroxidase. Keywords: biotransformation, humic substances, Trichoderma harzianum Introduction Humic substances have been an object of scientific research for more than years. A significant feature of HA is the saturation of the functional groups of molecules: carboxyl, phenol, alcohol, hydroxyl, chinoid, amide and amine groups. Кононова considers HA as a product of condensation of aromatic compounds of phenolic type with amino acids. The reactivity of HA is explained with the presence of the mentioned functional groups (1, ). HA of lignite are no soluble in water and their extraction is realized through alkaline solutions and the formed salts with univalent ions are humates physiologically and chemically active coal humates preparations. It is well known that micro fungi take a great part in the degradation of carbon complexes such as starch, cellulose, 796 pectin, lignin, lignocellulose, xylane and so on. Trichoderma reesei is well known with its ability to finish the required enzyme reactions and to lead to the degradation of cellulose to glucose. Degradation of lignocellulose is typical for some Basidiomycetes. Lignin degradation however is a secondary metabolic process that is not obligatory for the growth of most micro fungi. Mycelium granules of Trichoderma viride are used for biodegradation of natural humic substances in water solutions. Some ligninolytic cultures of Phanerochaete chrysosporium have a relatively good ability to degrade the macromolecules of coal. Pokorny R, et al (3) report about biodegradation of lignite by strains isolated from coal mines Trichoderma atroviride, T. viride, T. Pseudokoningi. The practical application of degraded lignite is connected with its usage as a substrate for cultivation of other microorganisms. The prevailing opinion is that microorganisms suitable for biodegradation of these substrates in a bioreactor are: micro

2 fungi T. atroviride and Penicillium sp., bacteria (Pseudomonas sp.), and yeast Candida (3,,5,6,7 and 8). The purpose of the examination is to determine the influence of chelated forms of the micro elements Fe, Mn, Zn, Cu, Co and the macro elements Ca and Mg on the biotransformation of lignite and the related immobilization of enzymes on humic substances. Materials and methods Experiments are realized for determining the influence of chelated forms of micro and macro elements, put on the mineral matrix, on the biocatalitical activity of Trichoderma harzianum in the case of degradation of low rank coal from Stanyantsy Mine. with this solution. % solution of each chelated form is used; 1 ml are taken from the solution and are used for the moistening of 1g modified zeolite. Zeolites are introduced as.5 weight %. Microfungal inoculation is realized with conidia - 5,х1 7 CFU. Except for the described proteinates no carbon source is added to the medium. The samples are put in geotextyle envelopes and are cultivated in a thermostat at 33 о С during the first three days and at о С during the rest 11 days. The samples are dried at temperature of 8 о С, grinded and analyzed. The organic content by Method of Kononova (9), and enzyme activity of the samples are determined. Procedure Chelated forms of micro and macro elements are solved in distillated water and ammonia form of zeolites is moistened Cf relative,% 1 8 6,7,7 6,7 8,7 1,7 Ch relative, % ,7,7 6,7 8,7 1,7 a) Сf = exp(, ,6916*); r=-.58 b) Сh = 91,5136 +,58598*; r=.58 Fig. 1 a) and 1 b) Influence of on relative content of fulvic(a) and humic (b) acids in bio-transformed lignite Scheme of the experiment and Content of micro- and macroelements in the zeolite supporting agent Chelated proteinate form Content of the element in the zeolite composition,% рн 1. Fe,6,75. Mn,96 5,3 3. Zn 1,19,6. Cu,96,9 5. Co,6,9 6. Ca,96 1,7 7. Mg,6 9, ml alcohol extract from tuja cones/75 ml coal n.d. n.d. 9. 8Х +Fe+Mn+Zn+Cu+Co+Ca+Mg Х + 3g scots pine pollen /75ml coal n.d. n.d horse-radish n.d. n.d. TABLE 1 Results and Discussion The data presented in Tables 1 and show that HA formation is рн determined with relatively weak influence of SPECIAL EDITION/ON-LINE YEARS OF ACADEMIC EDUCATION IN BIOLOGY

3 this factor. The magnesium form of chelates transforms coal to almost 1 % humic acids at relatively lower than that of Са chelate. Cupric and zinc forms of chelates increase to the biggest extent the formation of fulvic acids.data presented in Table 3 and Figures -5 show the enzyme activity of preparations and the influence of formed humic substances upon this index. Results show that enzyme activity of products is higher than that of the Control; the activity is especially high when extract from tuja cones is added to the medium. Со and Са chelates increase the activity of peroxidase almost twice; Са, Mg and Fe chelators increase the activity of polyphenol oxidase with more than 5 %. Results show that introduction of chelated forms of micro and macro elements increase the enzyme activity of preparations in comparison to the Control. The increase of fulvic acids content in the medium leads to a decrease of the enzyme activity of preparations. Total and relative content of humic acids has a positive influence on the activity of polyphenol oxidase and leads to a drastic decrease of peroxidase activity. Organic carbon and humic composition of biotransform lignite Total organic Treatment Carbon Control Х,95a 39,97 6,b 1. Fe,9 39,6 63,8. Mn,33 39,11 6,1 3. Zn,5 39,9 61,91. Cu,18 39,5 61,15 5. Co 3,7 39,86 58,89 6. Ca 3,57 39,73 59,33 7. Mg 3,55 39,83 59, ml Tuja extract/75 ml lignite,8 38,55 6,6 9. 8Х +Fe+Mn+Zn+Cu+Co+Ca+Mg,7 38,1 59, Х + 3g scots pine pollen /75ml 3,75 38,75 lignite 61,9 9, extract of horse-radish,5 69,9 a - % of the sample; b-% of the total org C; c-% of the total humic substances This contradicts to the results about the influence of on the peroxidase activity. The results from the analysis of the formed humic-like substances and of the enzyme activity of preparations after introducing of extract from tuja cones show that relatively high amount of humic acids are concentrated in the medium, but the relative part of fulvic acids is bigger than that in the case of biotransformation with zeolites, modified with mono chelators forms Mn, Ca and Mg as a supporting agent. Scots pine pollen and tuja extract have a stronger effect when used on the background of all chelated forms. Tuja extract leads to a drastic increase of peroxidase activity. Horse-radish extract increases significantly polyphenol oxidase and peroxidase activity of bio transformed lignite. Data from the regression analysis in this group of products, presented on Figures a), b), c) and d) show that the higher the total content of Сh is, the lower is the enzyme activity of preparations. In the cases when the relative part of Сh is higher peroxidase activity increases. The activity of polyphenol oxidase is better when is higher. TABLE Total organic C in the extract С h % 3,37a 93,67c,8 97,3 3, 9,7,11 9,3,9 9,87 1,5 91,39,87 97,3 3,5 99,79 3,6 95,76,5 98,9,7 95,75,78 8,1 С f 1,58a 6,33c,6,57 1,9 5,3,3 9,57 1,17 8,61, 8,61,7,97,5,1 1,,,5 1,1 1,1,5,67 15,86 Е:Е6 Optical density 7,7 7,3 7,3 6,7 7,5 7,8 6,58 6,8 6,98 6,7 6,76 5,9 798

4 Enzyme activity of preparations No of sample Kind of sample mg Purpugaline/1g /3` Х Control Zeolite,5 % Measurement to control 7,7 1,8 Х1 +Fe chelate 1g/75 ml lignite +5,73 - Х +Mn chelate1g/75 ml lignite +3,6 +,6 Х3 +Zn chelate1g/75 ml lignite +,57 +,17 Х +Cu chelate1g/75 ml lignite +,3 +3, Х5 +Co chelate1g/75 ml lignite +,37 +,97 Х6 +Ca chelate1g/75 ml lignite +5,57 +,1 Х7 +Mg chelate1g/75 ml lignite +,83 +3,93 Х8 +5 ml tuja extract +8,5 +,75 Х9 No 8+3,5 g from 1 to 7 (,5 g from each one) +31,75 +1,5 Х1 No 9+3 g scots pine pollen +7,5 +1,5 Х11 No1+horse-radish extract +19,5 +1,5 Note the chelates are calculated as g/75 ml lignite, but they are introduced together with the zaolites. TABLE 3 6,5 5 5,5,5 3 3,5 1,5,,8 1, 1,6, Cf t Ch t Fig. a) Fig. 3 b) 6,5 5 5,5,5 3 3,5 1, Ch,7,7 6,7 8,7 1,7 Fig. c) Fig.5 d) Fig. a) Influence of the total content of fulvic acids in bio-transformed lignite on the polyphenol oxidase activity of preparations: = exp(1, ,6368*Cf t);r= -,75 Fig. 3 b) Influence of the total content of humic acids in bio-transformed lignite on the peroxidase activity of preparations: = exp(7,618-3,91*ch t); r = -,63 Fig. c) Influence of the relative content of humic acids in bio-transformed lignite on the polyphenol oxidase activity of preparations: = 1/(-1, ,68/Ch) ;r =,737 Fig. 5 d) Influence of pн on the peroxidase activity of preparations: =,1977*^7,976; r =,71 SPECIAL EDITION/ON-LINE YEARS OF ACADEMIC EDUCATION IN BIOLOGY

5 Conclusions Chelated forms of Cu and Zn support the microfungal biotransformation of lignite, leading to an increase of the fulvic acids content; Ca and Mg increase Ch content up to 97 and 99,8%. Chelated forms of Fe, Mn, Zn, Cu, Co, Ca, Mg, when introduced with the modified zeolites NH -form, have a positive effect on the enzyme activity of the preparations from the Trihoderma harzianum metabolic activity, especially on the background of plant extracts. REFERENCES 1. Гулько А.Е. (1993) Гуминовые вещества в биосфере. М., Орлов Д.С. (993) Гуминовые вещества в биосфере. М., Fakoussa R.M. (199) Fuel Process. Technol.,, Fakoussa R.M. and M. Hofrichter. (1999) Appl. Microbiol. Biotechnol., 5, Hofrichter M. and W. Fritsche. (1997) Appl. Microbiol. Biotechnol., 7, Hofrichter M., K. Scheibner, I. Schneegaß, D. Ziegenhagen and W. Fritsche.(1998) Appl. Microbiol. Biotechnol., 9, Hofrichter M., D. Ziegenhagen, T. Vares, M. Friedrich, M. G. Jäger, W. Fritsche and A. Hatakka. (1998) FEBS Lett., 3, Hofrichter M., T. Lundell, A. Hatakka. (1 Appl. Environ. Microbiol., 67, Kononova M.M. (1966) Soil Organic Matter. (e). Pergamon, N.Y., pp. 5. 8

Characterization of Humic System in Fertilizer Raw Materials

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