Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 21
Issue 1
DOI:10.30825/5.ejpau.15.2018.21.1, EJPAU 21(1), #03.
Available Online: http://www.ejpau.media.pl/volume21/issue1/art-03.html


Hanna Miszkiewicz, Olga Marchut-Mikołajczyk, Lidia Mielcarz, Stanisław Bielecki
Institute of Technical Biochemistry, Lodz University of Technology, Poland



The effect of concentration of lignite, derived from the Polish mine in Bełchatów, pretreated with 8M nitric acid, on the yield of its biosolubilization by Fusarium oxysporum 1101 strain was studied. Observed decrease in pH during lignite biosolubilization by the F. oxysporum 1101 was correlated with amounts of humic and fulvic acids released from the substrate. Conversely, in the lignite-free medium the strain synthesized alkaline compounds that caused an increase in pH. The highest biosolubilization yield was achieved when the culture medium was supplemented with 5% of  lignite (3-times higher than in the medium supplemented with 10% of coal). In this case, for the first 8 days, the products with gradually decreasing molecular masses were efficiently released (the E4/E6 ratio was growing) and then polymerized (the E4/E6 ratio was reduced), and the content of condensed aromatic rings was decreased. The FTIR analysis showed that the relative intensity of the peaks reflected the concentration of functional groups in the tested products. Only the spectrum of products released from 10% coal contained the additional peak, corresponding to -CH(Ar) groups.

Key words: Fusarium oxysporum 1101, lignite solubilization, coal loading.


Lignite plays an important role in many countries as the primary medium of electricity and heat. Due to oil and gas reserve depletion, it is becoming necessary do develop an effective technology for the conversion of lignite. Current technology of electric energy and heat production is based on the combustion of lignite. However, these processes causes harmful emissions of carbon dioxide, sulfur and nitrogen oxides into the atmosphere. Furthermore, classic gasification and liquefaction of lignite require high temperature, high pressure and high energy consumption. Therefore, new technologies for lignite conversion, which will reduce the devastation of the environment are required.

Biosolubilization of lignite yields products, which can be used for chemical or biotechnological synthesis. The process involves the use of various types of microorganisms capable of converting the raw material into another energy form. Different bacterial species [6, 11, 14, 20], molds [1, 4, 12, 23] and white and brown rot fungi [2, 3, 16], can change the coal structure by its solubilization or depolymerization.

Depending on the coal properties, various factors influence the process of its bioconversion. The solubilization of coal with a high content of carboxyl groups is positively affected by alkali metabolites, while in case of lignite with a high content of ester bonds the presence of esterases and in the lignite with high content of specific metal ion the presence of chelators plays an important role. Given the more aromatic and lignin-like structure ligninolytic enzymes positively affect the lignite solubilization [5, 15]. High lignite biosolubility also depends on the interaction between the coal, surfactants, microorganisms and their enzymes [18].

Lignite solubilization is a non-enzymatic process that occurs at alkaline pH (7–10). It is mediated by various specific substances, like alkaline metabolites, surfactants and chelating agents [19].

Depolymerization of lignite occurs at low pH (3–6) and is mediated by oxidoreductases (laccase, lignin peroxidase and Mn-peroxidase), and some hydrolases. The process generates yellow, low molecular weights substances structurally similar to fulvic acids [7].

Changing the coal structure due to the initial oxidation with  nitric acid (HNO3), hydrogen peroxide (H2O2), ozone (O3) or radiation makes it more susceptible to microbial degradation. Bioconversion of coal occurs at an ambient temperature and pressure, is one phase process, and the product retain 97.5% of the raw lignite heating value [10, 13, 17].

In the last two decades bioconversion of various lignites by microorganisms was described in the literature. Most authors described the bioconversion of coal using fungal strains such as Trichoderma atroviride [1, 8, 12], Hypocrea lixii [22], Penicillium decumbens P6 isolated from mining environments [9].

In this study, we report biosolubilization  of Polish lignite from the mine in Bełchatów,  pre-oxidized with nitric acid, by fungus Fusarium oxysporum 1101 in a submerge culture. The study quantifies the degree of lignite solubilization and humification and emulsion index under these culture conditions, as well as the effect of lignite concentration on microbial solubilization.


Coal and pre-treatment
Lignite used in the study was sampled from Lignite Mine in Bełchatów (Poland) and grounded to small particles with diameters of 1–2 mm in a grinding mill. Subsequently, the coal was pre-oxidized with 8M nitric acid (6g carbon / 10 ml of nitric acid) and left for 48 hours at room temperature. After the pretreatment the coal was washed with distilled water until a neutral pH. Then pre-oxidized carbon was dried at 60°C for 72 hours before use and autoclaved (121°C, 20 min.).

The fungal strain Fusarium oxysporum 1101,  isolated in the Institute of Technical Biochemistry from surface water in the place sort of lignite mine in Bełchatów was used in the study. Strain genetic identification was made by amplifying the ITS1-ITS2 region including 5.8S r DNA region, using ITS1 and ITS4 primers. The strain was deposited in the pure cultures collection of the Institute of Technical Biochemistry, Lodz University of Technology. Preliminary studies have shown that Fusarium oxysporum 1101 has the ability to lignite biosolubilization. For activation (30°C, 7 days) and storage (4°C) of the strain petri dishes of Czapek-Dox medium 3% agar-solidified was used.

Inoculum preparation
The inoculum was prepared by culturing the fungus in 500 mL flasks with 100 ml of sterile Czapek-Dox medium containing 0.2% (w / v) sodium glutamate and 0.1% (w / v) of fine coal with a grain size of about 150–300 microns. The growth medium was inoculated with 1 cm disk cut from the lined plate. The culture was incubated for 3 days at 30°C on an orbital shaker at 300 rpm.

Growth media and culture conditions
The growth medium used for inoculum preparation and experiments contained (per liter): 10 g glucose, 2 g of sodium glutamate, 3 g NaNO3, 2 g K2HPO4, 0.5 g MgSO4 × 7H2O, 0.5 g KCl, 1 g of fine coal. The culture was incubated at 30°C on an orbital shaker at 300 rpm.

Lignite biosolubilization in submerge cultures
The strain was cultivated  in 500 ml flasks containing 100 ml of growth medium and 10% (v / v) inoculum at 30°C on an orbital shaker at 300 rpm for 3 days. Then the culture broth was supplemented with 2.5, 5, 7.5 and 10% (w / v) of sterile, pre-oxidized with nitric acid lignite and the culture was continued for 12 days under the same conditions.

Supernatants of the culture broths were obtained by centrifuging  for 15 minutes at 10 000 rpm and an ambient temperature.

pH measurement
The pH of the supernatant was determined using a pH meter.

Determination of humic and fulvic acids
The released amounts of humic acid (A450nm) and fulvic acids (A650nm) were determined using the indirect method by measuring the absorbance of suitably diluted supernatant.

Emulsification index E24
Emulsifying ability of the fungal strain was determined by adding 2 ml of a hydrocarbon (diesel oil) to the same amount of  supernatant,  mixing  with  a  vortex  for  2  min,  and  leaving  to  stand  for  24  hours.  The  emulsification index (E24)  is  given  as   percentage of height of emulsified zone (mm) divided by total height of the liquid column (mm) [17].

E24 = (EZ / TH) × 100%

EZ – emulsified zone
TH – total height (5 cm)

Chemical analysis of biosolubilization products
Bio-liquefaction products were characterized by using ultraviolet-visible (UV-VIS) spectroscopy and infrared spectroscopy (FTIR).

In the UV-VIS spectroscopy analysis, products were scanned with light of wave lengths from 200 nm to 650 nm using a spectrophotometer (PG T80 UV/VIS).

In order to determine the specific organic chemical groups in the bio-liquefaction products the pellets were analyzed using an infrared spectrum analyzer (Thermo Nicolet Avatar 330) with light wave lengths from 4000 cm-1 to 450 cm-1 [19].

All chemicals used in the studies were analytical grade and purchased from Sigma-Aldrich (USA) and Chempur (Poland).


The study investigated the effect of lignite (pre-oxidized with nitric acid) concentration in the range of 2.5 to 10% on its biosolubilization process performed by Fusarium oxysporum 1101. Figure 1 shows the dynamics of pH changes in F. oxysporum 1101 submerge cultures.

Fig. 1. The dynamics of changes in culture pH of the supernatants containing different amounts of brown coal

The pH of F. oxysporum 1101 culture without the addition of coal, after three days of cultivation increased from 7.0 to 8.6, and after fifteen days reached 8.9. The rise in pH during the first phase of the process is thought to be a consequence of biosynthesis of alkaline substances by fungal strain. In the culture with the lowest coal concentration (2.5%) changes in the culture pH ranged from 8.6 to 7.1 and, for the highest coal concentration (10%) from 8.6 to 4.79.

The dynamics of pH changes observed in the experiment was related to the amount of humic and fulvic acids released into the culture and the acidic character of the lignite pre-oxidized with nitric acid. Under the applied culture conditions (intensive mixing, temperature), the elution of residual nitric acid adsorbed deep into the pores of coal during its pre-treatment appeared. After 12 days the biosolubilization, the lowest pH (4.8) was observed in the sample containing 10% of coal.

Obtained results confirmed that the F. oxysporum 1101 strain used in the experiment produced alkaline compounds, which were evolved to the culture and which participated in the solubilization of lignite.

Holker et al. [8] observed an increase in pH of the culture broth (after 7 days reached a value of 9) during  Fusarium oxysporum 1101 growth in medium containing glutamate. Under these conditions, fungal strain showed the capacity to lignite solubilization.

The effect of the coal concentration on the dynamics of humic acids release in submerge culture of F. oxysporum 1101 is shown in Figure 2.

Fig. 2. The effect of brown coal concentration in F. oxysporum 1101 submerge cultures on the dynamics of humic acids release

In the study we found that when the lignite was added to a 72 h F. oxysporum 1101 culture, after the first three days an increase in humic acid dissolution rate for the tested lignite concentrations was observed. For the next 4 days growth of the parameter has been retarded. From the 10th day of biosolubilization to the end of the process a marked increase in the release of humic acids was observed. The highest amount of humic acids (A450nm – 86) was obtained after  12 days of the process for variants with 5% of lignite. In the presence of 10% of lignite the lowest humic acids concentration (A450nm – 31) was observed. The release of humic acids from lignite in a sterile medium occurred in a very small extent and the absorbance (A450nm) averaged 1.9 (data not shown).

According to Tripathi et al. [21] the rate of coal solubilization decreases with increasing dose of coal. This phenomenon may be related to a lack of nutrients, or no acceptable concentration of the product, followed by the product inhibition. This may be also related to the unavailability to the surface of coal particles due to pore clogging by debris of dead cells and their metabolites. Oboirien et al. [12] found that the optimal concentration of coal range from 5 to 10% (w / v). Rising above the optimum inhibited the growth of microorganisms that are involved in lignite biosolubilization. In the presence of 10% (w / v) of lignite fragmentation of the mycelium was observed.

The effect of coal concentration on the dynamics of fulvic acids release in submerge culture of Fusarium oxysporum 1101 is shown in Figure 3.

Fig. 3. The effect of brown coal concentration in the F. oxysporum 1101 culture on the dynamics of fulvic acids release

The nature of the dynamics of fulvic acids release was similar to this obtained for humic acids. The highest amount of fulvic acids (A650nm – 12.8 and 13.5) was observed in cultures containing 2.5 and 5% of lignite respectively, while the lowest (A650nm – 4.9) in culture with 10% of the substrate. During the solubilization of lignite in sterile medium, under the influence of mixing and temperature, a trace amounts of fulvic acids were observed (data not shown).

The degree of lignite humification  (E4 / E6) was calculated on the base of absorbance measured at 450 and 650 nm. The value of this ratio doesn’t dependent on the concentration of humic substances in examined solution but is related to their structure and origin. The values  of E4 / E6 ranged from 5.8 to 7.2 (Fig. 4). Essentially the E4 / E6 ratio increases with lowering of molecular weight and the content of condensed aromatic rings and with the rise in the oxygen content [4].

Fig. 4. The effect of brown coal concentration on the humification degree (changes on E4/E6 ratio) in brown coal biosolubilization

Character of changes of the E4 / E6 rate for supernatants containing 2.5 or 5% of coal differed from those obtained for samples with 7.5 or 10% of the substrate. After three days of lignite biosolubilization the values of E4 / E6 reached appropriately 6.37 (for 2.5% of coal), 6.9 (for 5% of coal) and 7.1 (for the 7.5 and the 10% of coal). For the variants of the culture containing 2.5 and 5% of coal at first (8 days) an increase in the examined rate was observed. This indicated an effective release of products with lower molecular weight and lower content of fused aromatic rings and an increase in oxygen content. By the end of the process a decrease in the E4 / E6 value was observed (5.8 in variants containing 2.5% of lignite up to 6.2 for variants with 5% of lignite), what suggested the polymerization of released compounds.

However for biosolubilization variants containing 7.5 and 10% of coal a significant decrease in the E4 / E6 coefficient value was observed between 3rd and 8th day of the process. After this time an appreciable increase until the 10th day of biosolubilization and subsequent fall in the E4/E6 ratio in the end of the process was observed.  This indicated that in the first phase of biosolubilization a decrease in a content of condensed aromatic rings and an increase in the oxygen content  occurred.  In consequence humic acids of more complex structure and a higher molecular weight were released. These compounds were degraded to fulvic acids – the compounds of lower molecular weight, and  next their polymerization was observed.

These results indicate that the course of biosolubilization  of lignite used in the concentration lower than 5 % differ from this obtained for higher coal concentration.

It is known that biosurfactants have a positive effect on lignite biosolubilization [18]. F.oxysporum 1101 strain, used in the study has the ability for biosurfactants production which resulted in the ability to lignite solubilization [17]. The presence of these compounds was confirmed by measuring the emulsification index (E24) in post-culture supernatant fluids. It has been found that the value of E24 increased with the increasing concentration of lignite added to the medium (Tab. 1). The highest value of the emulsification index – E24 (36%) was observed in the 13th day of the process, in variant supplemented with 10% of lignite.

Table 1. The effect of brown coal concentration on the emulsification index (E24)
Cultivation time [day]
Coal loading [% w/v]
Emulsification index E24

After 15 days of biosolubilization of pre-treated with nitric acid lignite received black, liquid products were analyzed with UV-VIS spectrophotometer in the range of 200–600 nm. The results of UV-VIS analysis are shown in Figure 5.

Fig. 5. UV- VIS analysis of brown coal biosolubilization products

Absorbance maximum for all the tested products (2.5, 5, 7.5 and 10% of lignite initially oxidized with nitric acid) was found at about 240 nm, which means that in the analyzed biosolubilization products, alkyl substitutes of unsaturated aldehydes and ketones were present. Small differences in the absorbance range of 210–280 nm indicating a large variety of compounds. The spectrum of 10% lignite biosolubilization products differed from the others in the range above 300 nm, which indicates a higher content of azo groups and conjugated olefins. Absorption in the range of 260–300 nm indicates the presence of aromatic structures in the solubilization products.

Since many aromatic rings in the lignite structure are connected by a complex bonds, lignite is present as a solid. After biosolubilization, aromatic structures were found in the fluid, which means that the microorganisms can degrade certain bonds in the lignite, so that some single aromatic rings were released from the lignite and formed a fluid [19].

FTIR spectra of lignite biosolubilization products are shown in Figure 6. The nature of FTIR spectra, regardless of the concentration of solubilized lignite were similar. The relative intensity of the peaks, reflecting the concentration of functional groups in the tested samples grew with the increasing coal concentration. Obtained main absorbance peaks corresponded to C-O bonds in phenols and alcohols (1132 cm-1), the ether and ester groups (1349 cm-1), the carbonyl and ketone groups (1582 cm-1) and O-H linkages (3159 cm-1). In the spectrum of biosolubilization products generated by F. oxysporum 1101 in samples containing 10% of lignite, a peak (850 cm-1) corresponding to the -CH(Ar) groups was found.

Fig. 6. FTIR spectra of biosolubilization products of brown coal used in different concentrations (2,5, 5 and 10%)

The major peaks from the FTIR spectrum of biosolubilization products of Chinese coal pre-oxidized with nitric acid are assigned to hydroxyl (3450 cm-1), carbonyl (1600 cm-1) and ether (1000–1300 cm-1) groups as well as the aromatic rings (1000–500 cm-1) in chains.

During the solubilization of pre-treated lignite used fungus degraded and broke the side chains of the aromatic rings and  destroyed  the bonds in the lignite. This resulted in the  release of single aromatic structures from the complex structure of the lignite to produce a solubilization product [19].


The study showed that the released in the lignite biosolubilization process humic and fulvic acids may cause the pH reduction, which is proportional to the amount of solubilized coal. In a culture without lignite pH value increased due to the secretion of alkaline compounds by  F. oxysporum 1101 strain.

The highest yield of biosolubilization (amount of released humic and fulvic acids) was obtained in the cultures containing 2.5 and 5% of lignite. For these variants until the 8th day of biosolubilization an efficient secretion of the lower molecular weight products (increase in the E4 / E6 ratio) and  decrease in a content of condensed aromatic followed by a polymerization of the released compounds (decrease of E4 / E6 to about 6) were observed.

The ability of F. oxysporum 1101strain for biosurfactants production grew up with the with the increasing concentration of lignite added to the medium. The highest value of the emulsification index – E24 (36%) was obtained in the 13th day of biosolubilization, in the variant containing 10% of lignite.

The nature of the UV-VIS spectra of the products of lignite biosolubilization used at different concentrations was similar. The only exception was sample with the presence of 10% of lignite, in which  higher content of azo groups and conjugated olefins  in the range of 300–400 nm was observed.

The concentration of biosolubilized lignite affected the nature of the infrared spectra. The relative intensity of the peaks, which reflect the concentration of functional groups in the tested samples, grew with the increasing lignite concentration. The spectrum of biosolubilization products generated by F. oxysporum 1101 in samples containing 10% differed from other samples. In this spectrum  a peak (850 cm-1) corresponding to the -CH(Ar) groups was found.

These studies show  that  the process of lignite biosolubilization performed by F. oxysporum 1101occurred most  favorably  in the submerged culture conditions, in the presence of 5% w/w of lignite. Biosolubilization is an environmentally friendly process which may be alternative to chemical methods for obtaining humic acids and other value-added products.


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Accepted for print: 22.02.2018

Hanna Miszkiewicz
Institute of Technical Biochemistry, Lodz University of Technology, Poland
B. Stefanowskiego 4/10
90-924 Łódź
email: hanna.miszkiewicz@p.lodz.pl

Olga Marchut-Mikołajczyk
Institute of Technical Biochemistry, Lodz University of Technology, Poland
B. Stefanowskiego 4/10
90-924 Łódź
email: olga.marchut-mikołajczyk@p.lodz.pl

Lidia Mielcarz
Institute of Technical Biochemistry, Lodz University of Technology, Poland
B. Stefanowskiego 4/10
90-924 Łódź

Stanisław Bielecki
Institute of Technical Biochemistry, Lodz University of Technology, Poland
B. Stefanowskiego 4/10
90-924 Łódź
email: stanisław.bielecki@p.lodz.pl

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