Purification,characterization, and coal depolymerizing activity of lignin peroxidase from <Emphasis Type="Italic">Gloeophyllum sepiarium</Emphasis> MTCC-1170

Lignin peroxidase from the liquid culture filtrate of Gloeophyllum sepiarium MTCC-1170 has been purified to homogeneity. The molecular weight of the purified enzyme was 42 kDa as determined by SDS-PAGE. The K _m values were 54 and 76 μM for veratryl alcohol and H₂O₂, respectively. The pH and temperature optima were 2.5 and 25°C, respectively. Depolymerization of coal by the fungal strain has been demonstrated using humic acid as a model of coal. Depolymerization of humic acid by the purified lignin peroxidase has been shown by the decrease in absorbance at 450 nm and increase in absorbance at 360 nm in presence of H₂O₂. Depolymerization of humic acid by the purified enzyme has also been demonstrated by the decrease in the viscosity with time of the reaction solution containing humic acid, H₂O₂, and the purified lignin peroxidase. The influence of NaCl and NaN₃ and inhibitory effects of various metal chelating agents on the lignin peroxidase activity were studied.     

Evaluation of extractants for soil humic substances

Summary An evaluation of alkaline extractants was done by isotachophoresis and compared with infrared, ultraviolet and visible spectroscopic analysis. The electrophoretograms show that the humic acid extracted. This result is partially corroborated by the E₄/E₆ ratio. Differences in the carboxyl content and TRIS. In the pyrophosphate humic acid three fractions of high molecular weight were not extracted. This resultis partially corroborated by the E₄/E₆ ratio. Differences in the carboxyl content found by ir spectra of the TRIS humic acid and the NaOH humic acid were not shown in the isotachophoretic separation.     

On Comparison Between Fungal and Bacterial Pretreatment of Coal for Enhanced Biogenic Methane Generation

Owing to better understanding of subsurface geochemical carbon recycling and real-time active methanogenesis in major coal basins around the globe, substantial share of subsurface methane generation is attributed to biogenic origin. Since coal, being complex geopolymer, does not appear to be a favorable microbial substrate, enhancement in biogenic methane yield depends on its degradation into simpler organic substrates. This review puts forward a comparative analysis of fungal and bacterial pretreatment for determining the extent of facilitation in initial degradation of coal, which is still rate limiting step in overall conversion of coal into methane. Primarily, the initial fungal degradation of coal differs from bacterial pretreatment of coal in terms of the nature of released organics. On the basis of previous reports, fungal pretreatment of coal yields, majorly, polyaromatic hydrocarbons, however, bacterial pretreatment results in the generation of mixed organics pool of aromatics and aliphatics. The presence of aliphatics may be prospected for achieving greater conversion rates of coal conversion into methane. Considering the criticality of preliminary degradation of coal and associated issues, the fate of commercial biogenic methane generation would be dictated by the factors pertaining to geological considerations and reservoir geology, chemistry of coal and associated water tables, geomicrobial considerations and economic viability.     

Fungal biosolubilization of Rhenish brown coal monitored by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide

Residues and coal fractions that remained after the biosolubilization of Rhenish brown coal by strains of Lentinula edodes and Trametes versicolor have been studied by Curie-point pyrolysis/gas chromatography/mass spectrometry using tetraethylammonium hydroxide (NEt₄OH) at 610 °C. To differentiate methyl derivatives of esters and ethers from free or bound hydroxyl and carboxyl groups NEt₄OH was used in the thermochemolysis experiments instead the commonly used tetramethylammonium hydroxide. A comparison of humic acid fractions before and after fungal attack shows considerable alteration of the soluble macromolecules of coal. Depending on the coal fraction studied and the fungi used, the assortment of fatty acid esters released during the pyrolysis varies significantly. Furthermore, dicarbonic acid ethyl diesters as well as ethyl derivatives of aromatic ethers and acids yield information about humic acid structure and the biosolubilization of brown coal. Variations in the mixture produced are possibly caused by differences in the pattern of extracellular enzymes secreted that attack the macromolecular structural elements of brown coal. Therefore pyrolysis of native and microbiologically altered geomacromolecules using NEt₄OH allows one to differentiate between free hydroxyl groups as well as substances that are attached to humic substances via ester or ether bridges, and their methylated counterparts. Received: 13 July 1998 / Received revision: 12 October 1998 / Accepted: 16 October 1998     

Isolation and Characterization of Coal Solubilizing Aerobic Microorganisms from Salt Range Coal Mines,Pakistan

Microbial solubilization of coal has been considered as a promising technology to convert raw coal into valuable products. In the present study, initially a total of 50 different aerobic bacterial and fungal isolates have been isolated from soil, coal and water samples of Dulmial Coal Mines, Chakwal, Pakistan, but on the basis of solubilization potential, only four isolates were selected for further study. The intensity of biosolubilization was measured by determining the weight loss of the coal pieces, which was observed to be about 25.93% by Pseudomonas sp. AY2, 36.36% by Bacillus sp. AY3 and 50% by Trichoderma sp. AY6, while Phanerochaete sp. AY5 showed maximum coal solubilization potential i.e. 66.67% in 30 days. UV/Vis spectrum revealed an increase in the pattern of absorbance of all treated samples compared to control referring to solubilization. Fourier transform infrared spectroscopy indicated alterations in the structure of treated coal in comparison to control coal suggesting breakdown in the complex structure of coal. The major absorbance bands in infrared spectroscopy for solubilization product were attributed to carbonyl (1,600 cm^?1), hydroxyl (3,450 cm^?1), cyclane (2,925 cm^?1), ether linkage (1,000–1,300 cm^?1), carboxyl (3,300–2,500 cm^?1) and side chains of aromatic ring (1,000–500 cm^?1). The presence of microorganisms and surface erosion of coal residues compared to control samples were observed by scanning electron microscopy, which suggested that isolated microorganisms were able to survive in coal for a longer period of time. Therefore, the present study concluded that microorganisms isolated from coal mines have excellent potential for coal solubilization which is considered as a crucial step in coal methanogenesis allowing them to be used successfully for in situ methane production to meet future energy demands.     

Phyto-bioconversion of hard coal in the Cynodon dactylon/coal rhizosphere

Fundamental processes involved in the microbial degradation of coal and its derivatives have been well documented. A mutualistic interaction between plant roots and certain microorganisms to aid growth of plants such as Cynodon dactylon (Bermuda grass) on hard coal dumps has recently been suggested. In the present study coal bioconversion activity of nonmycorrhizal fungi was investigated in the C. dactylon/coal rhizosphere. Fungal growth on 2% Duff-agar, gutation formation on nitric acid treated coal and submerged culture activity in nitrogen-rich and -deficient broth formed part of the screening and selection of the fungi. The selected fungal isolates were confirmed to be found in pristine C. dactylon/coal rhizosphere. To simulate bioconversion, a fungal aliquot of this rhizosphere was used as inoculum for a Perfusate fixed bed bioreactor, packed with coal. The results demonstrate an enhanced coal bioconversion facilitated by low molecular weight organics and the bioconversion of coal may be initiated by an introduction of nitrogen moieties to the coal substrate. These findings suggest a phyto-bioconversion of hard coal involving plant and microbes occurring in the rhizosphere to promote the growth of C. dactylon. An understanding of this relationship can serve as a benchmark for coal dumps rehabilitation as well as for the industrial scale bioprocessing of hard coal.     

Quality evaluation of co-composted wheat straw, poultry droppings and oil seed cakes

Poultry droppings, neem cake, castor cake, jatropha cake and grass clippings were used separately as organic nitrogen additives to decrease the high C:N ratio of wheat straw. Composting was carried out aerobically in presence of fungal consortium developed by including Aspergillus awamori, Aspergillus nidulans, Trichoderma viride and Phanerochaete chrysosporium. The degraded product was characterized to assess the technical viability of organic nitrogen supplements as well as fungal consortium in improving the quality of compost and hastening the process of decomposition of high lignocellulolytic waste. Evaluation of maturity showed that mixture of wheat straw, poultry dropping and jatropha cake had the lowest C:N ratio of 10:1, the highest humic acid fraction of 3.15%, the lowest dehydrogenase activity and a germination index exceeding 80% in 60 days of decomposition. Inoculated and grass clipping amended wheat straw–poultry dropping mixture resulted in compost with highest humus content of 11.8% and C:N ratio of 13.5, humic acid fraction of 2.84% and germination index of 59.66%. Fungal consortium was effective in improving the humus content of all the composted mixtures. In some treatments, germination index could not be correlated with C:N ratio. Non edible oil seed cake supplemented substrate mixtures did not respond to fungal inoculation as far as C:N ratio was concerned.     

Nitrous Acid Reactivation of Ultraviolet-Irradiated Transforming DNA from Hemophilus influenzae

Partial recovery of ultraviolet-damaged denatured or native transforming DNA from Hemophilus influenzae, has been obtained by exposing the irradiated DNA in the denatured form to nitrous acid. Some factors that affect this recovery are described. An erythromycin marker (E₂₀) was not reactivated. The UV damage reactivable by nitrous acid is different from that repaired by the photoreactivating enzyme from bakers' yeast. The pretreatment with nitrous acid affords a slight protection for denatured C₂₅ DNA and Sm₂₅₀ DNA against ultraviolet irradiation, but this pretreatment sensitized the E₂₀ DNA to this irradiation.     

Purification, characterisation and coal depolymerisation activity of lignin peroxidase from Lenzitus betulina MTCC-1183

Lignin peroxidase from the culture filtrate of Lenzitus betulina MTCC-1183 has been purified to homogeneity using concentration by ultrafiltration and anion exchange chromatography on DEAE cellulose. The molecular weight of the purified lignin peroxidase using SDS-PAGE analysis was 43 kDa. Specific activity of the enzyme was 29.58 IU/mg. The K _m values for veratryl alcohol and H₂O₂ for the purified enzyme were 54 and 81 μM, respectively. The k _cat value of the purified enzyme was 2.3 s^?1 using 3,4-dimethoxybenzyl alcohol as the substrate. The optimal conditions for the lignin peroxidase assay were detected at pH 2.4 and 22°C. Thermal stability of the purified enzyme has also been studied and its activation energy for deactivation was 287 kJ/mol. The purified lignin peroxidase depolymerised humic acid in presence of H₂O₂. Depolymerisation of coal by the L. betulina MTCC-1183 has been demonstrated using humic acid as a model of coal.     

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     

Biological Coal Conversions

Abstract

The discovery in 1982 that microorganisms can interact with, and presumably modify, the chemical structure of coal has focused attention on the potential for the use of microbial cultures or isolated microbial enzymes in coal conversion. Biological processes based on this activity will promote the effective utilization of lowrank coal, which is poorly suited to direct combustion. Research in this area may also support the development of improved biological technologies for the cleaning of highrank coal and/or the treatment of coalderived waste.     

Degradation of Soil Humic Extract by Wood- and Soil-Associated Fungi, Bacteria, and Commercial Enzymes

Abstract An alkaline humic extract (HE) of a black calcareous forest mull was exposed to 36 fungal and 9 eubacterial isolates in liquid standing culture. At 21 d in fungi, and 4 d in bacteria, the groups of wood-degrading basidiomycetes, terricolous basidiomycetes, ectomycorrhizal fungi, soil-borne microfungi, and eubacteria had reduced the absorbance (A ₃₄₀) of HE media by 57, 28, 19, 26 and 5%, respectively. Gel permeation chromatography revealed that the large humic acid molecules were more readily degraded than the smaller fulvic acid molecules and served as a sole source of carbon and energy. The more active HE degraders reduced the overall molecular weight of humic and fulvic acids by 0.25 to 0.47 kDa. They also reduced the chemical reactivity of HE to tetrazotized o-dianisidine, indicating the degradation of hydroxylated aromatic molecules (which are responsible for this reaction). Decreases in absorbance, molecular weight, and reactivity were caused by fungal manganese peroxidase, horseradish peroxidase, β-glucosidase, and abiotic oxidants such as H₂O₂ and Mn(III) acetate. It is concluded that fungi, some of which are propagated in contaminated soils to control xenobiotics, metabolize HE compounds enzymatically. They use enzymes which are also involved in the degradation of soil xenobiotics. Because of reductions in the molecular weight of HE, which is a potential carrier of heavy metal ions and xenobiotics, solubility and motility of humic substances in soil and surface waters are increased. Received: 4 March 1998; Accepted: 1 June 1998     

Cell-Free Solubilization of Coal by Polyporus versicolor

Solubilization of coal was demonstrated with filtrates (0.45-μm-pore-size filters) obtained from the broth in which Polyporus versicolor had grown. The rate and extent of solubilization were dependent on the age of the fungal cultures, the particle size of the coal, the pH of the filtrates, and the presence of proteins in the filtrates. The rate of solubilization of coal was significantly reduced after proteins in the filtrates were denatured by acid hydrolysis.     

Degradation of lignite (low-rank coal) by ligninolytic basidiomycetes and their manganese peroxidase system

Ligninolytic basidiomycetes (wood and leaf-litter-decaying fungi) have the ability to degrade low-rank coal (lignite). Extracellular manganese peroxidase is the crucial enzyme in the depolymerization process of both coal-derived humic substances and native coal. The depolymerization of coal by Mn peroxidase is catalysed via chelated Mn(III) acting as a diffusible mediator with a high redox potential and can be enhanced in the presence of additional mediating agents (e.g. glutathione). The depolymerization process results in the formation of a complex mixture of lower-molecular-mass fulvic-acid-like compounds. Experiments using a synthetic ¹⁴C-labeled humic acid demonstrated that the Mn peroxidase-catalyzed depolymerization of humic substances was accompanied by a substantial release of carbon dioxide (17%–50% of the initially added radioactivity was released as ¹⁴CO₂). Mn peroxidase was found to be a highly stable enzyme that remained active for several weeks under reaction conditions in a liquid reaction mixture and even persisted in sterile and native soil from an opencast mining area for some days. Received: 31 July 1998 / Received revision: 29 September 1998 / Accepted: 2 October 1998     

Spectroscopic and elemental investigation of microbial decomposition of aquatic fulvic acid in biological process of drinking water treatment

As humic substances left in treated water tend to form trihalomethans during chlorination, their removal in water treatment processes is a significant concern for drinking water supplies. One of the removal technologies, the biofilm reactor is studied for the microbial decomposition of aquatic fulvic acid (AFA). The AFA is characterized by elemental analysis, UV-Vis, ¹³C-NMR, and IR spectroscopic methods. The spectroscopic and elemental investigation was capable of characterizing the microbial decomposition of AFA. Biologically treated fulvic acid was in a more oxidized state; its spectra displayed a higher degree of condensation of aromatic constituents than influent fulvic acid. Microbial degradation of AFA was more active in the low molecular weight fractions and intensively occurred in the aliphatic fraction.Abbreviations A the absorbance at wavelength - specific absorptivities - AFAs aquatic fulvic acids - AHS aquatic humic substances - COD chemical oxygen demand - Da dalton - DO dissolved oxygen - E₄/E₆ratio a ratio between absorbance at 465 and 665 nm - FA fulvic acid - IR infrared - NMR nuclear magnetic resonance - TOC total organic carbon - UV-Vis ultraviolet-visible     

Molecule-size distribution of soluble hiunic compounds from different natural waters

It this study the use of Sephadex G-25, Sephadex LH-20 and CPG-10-75 for the separation of soluble humic compoutids frotn fresh water is tested. It is shown by Sephadex G-25 gel filtration that differences in molecule-size distribution of soluble humic compounds in one lake at different titnes and between lakes can be predicted by E₂₅₀ and E₃₆₅ measurements.     

The unconventional adverse effects of fungal pretreatment on iturin A fermentation by Bacillus amyloliquefaciens CX-20

Fungal pretreatment is the most common strategy for improving the conversion of rapeseed meal (RSM) into value-added microbial products. It was demonstrated that Bacillus amyloliquefaciens CX-20 could directly use RSM as the sole source of all nutrients except the carbon source for iturin A fermentation with high productivity. However, whether fungal pretreatment has an impact on iturin A production is still unknown. In this study, the effects of fungal pretreatment and direct bio-utilization of RSM for iturin A fermentation were comparatively analysed through screening suitable fungal species, and evaluating the relationships between iturin A production and the composition of solid fermented RSM and liquid hydrolysates. Three main unconventional adverse effects were identified. (1) Solid-state fermentation by fungi resulted in a decrease of the total nitrogen for B. amyloliquefaciens CX-20 growth and metabolism, which caused nitrogen waste from RSM. (2) The released free ammonium nitrogen in liquid hydrolysates by fungal pretreatment led to the reduction of iturin A. (3) The insoluble precipitates of hydrolysates, which were mostly ignored and wasted in previous studies, were found to have beneficial effects on producing iturin A. In conclusion, our study verifies the unconventional adverse effects of fungal pretreatment on iturin A production by B. amyloliquefaciens CX-20 compared with direct bio-utilization of RSM.     

Degradation of low rank coal by Trichoderma atroviride ES11

A new isolate of Trichoderma atroviride has been shown to grow on low rank coal as the sole carbon source. T. atroviride ES11 degrades ∼82% of particulate coal (10 g l⁻¹) over a period of 21 days with 50% reduction in 6 days. Glucose (5 g l⁻¹) as a supplemented carbon source enhanced the coal solubilisation efficiency of T. atroviride ES11, while 10 and 20 g l⁻¹ glucose decrease coal solubilisation efficiency. Addition of nitrogen [1 g l⁻¹ (NH₄)₂SO₄] to the medium also increased the coal solubilisation efficiency of T. atroviride ES11. Assay results from coal-free and coal-supplemented cultures suggested that several intracellular enzymes are possibly involved in coal depolymerisation processes some of which are constitutive (phenol hydroxylase) and others that were activated or induced in the presence of coal (2,3-dihydrobiphenyl-2,3-diol dehydrogenase, 3,4-dihydro phenanthrene-3,4-diol dehydrogenase, 1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase, 1,2-dihydro-1,2-dihydroxyanthracene dehydrogenase). GC-MS analysis of chloroform extracts obtained from coal degrading T. atroviride ES11 cultures showed the formation of only a limited number of specific compounds (4-hydroxyphenylethanol, 1,2-benzenediol, 2-octenoic acid), strongly suggesting that the intimate association between coal particles and fungal mycelia results in rapid and near-quantitative transfer of coal depolymerisation products into the cell. An erratum to this article can be found at     

Humic substances change during the co-composting process of municipal solid wastes and sewage sludge

The change of the degree of stability of compost during the composting process was a kind of guideline for our study. This stability was estimated by monitoring the chemical fractionation (extraction of humic and fulvic acids, and humin) during two cycles of composting. Change of humin (H), humic-like acid carbon (CHA) and fulvic-like acid carbon (CFA) fractions during the composting process of municipal solid wastes were investigated using two windrows W1 (100% of municipal solid wastes) and W2 (60% of municipal solid wastes and 40% of dried sewage sludge). Humin and fulvic acid fractions in the two windrows decreased since the start of composting process and tend to stabilize. At the end of composting process, humic acid fraction is more important in the windrow without sludge (W1) than the one with sludge (W2). The humification indexes used in this study showed that the humic-like acid carbon fraction production takes place largely during the phase of temperature increase (thermophilic phase), and it appeared very active in the windrow W2. At the end of composting process, the E₄/E₆ ratio value indicated that the compost of W1 is more mature than the compost of W2. The humification ratio (HR) allowed a correct estimation of compost organic matter stabilization level.     

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.