两株绿脓杆菌对石油污染土壤的修复作用

本文旨在研究环境条件下微生物对石油污染土壤的修复情况。从矿井周边土样定向筛选出两株绿脓杆菌,摇瓶降解实验发现,两菌混合培养10 d原油降解率达到95.67%,比单菌培养提高至少32%,即两菌对原油降解具有协同作用。根据降解实验结果制备了混合修复菌剂,并且人工构建石油污染场地,展开中试场地修复试验,模拟不同的操作条件下土壤中原油的降解情况。经60 d修复发现,添加了菌剂的场地,石油烃含量下降趋势明显,每克土壤中石油烃含量从初始的0.8%降至0.1%–0.3%,其中额外添加有机肥作为补充碳氮源的场地,总石油烃降解率最高,达到85.28%。而未添加菌剂的对照组石油烃含量仅减少25.85%。     

Bioremediation of polycyclic aromatic hydrocarbons by fungal laccases engineered by directed evolution

Fungal laccases are useful for several remarkable transformations, such as bioremediation of polycyclic aromatic hydrocarbons (PAHs), synthesis of phenolic-based resins, oxidation of lignin derivatives and others. Most of these substrates are barely water-soluble, and although polar organic co-solvents may be added to enhance their solubility, transformation rates dramatically decrease due to the negative effect of organic solvents on the protein structure. Laccase from Myceliophthora thermophila variant T2 (MtLT2) has been submitted to laboratory evolution in Saccharomyces cerevisiae with the aim of improving activity and stability in organic co-solvents. Some 4500 clones created by random mutagenesis were screened in two rounds of directed evolution. Libraries were explored under increasing concentrations of acetonitrile and ethanol, and several mutants with improved features were purified and further characterised. Turnover rates of MtLT2 in 30% (v/v) acetonitrile and 50% (v/v) ethanol were increased up to 6.5- and 7.5-fold, respectively. The best variants showed similar rates in 20% (v/v) acetonitrile or 30% (v/v) ethanol as the parent type in aqueous media. Mutant laccases were also tested for the oxidation of anthracene in the presence of 20% (v/v) acetonitrile.     

Bioremediation of polycyclic aromatic hydrocarbons by fungal laccases engineered by directed evolution

Fungal laccases are useful for several remarkable transformations, such as bioremediation of polycyclic aromatic hydrocarbons (PAHs), synthesis of phenolic-based resins, oxidation of lignin derivatives and others. Most of these substrates are barely water-soluble, and although polar organic co-solvents may be added to enhance their solubility, transformation rates dramatically decrease due to the negative effect of organic solvents on the protein structure. Laccase from Myceliophthora thermophila variant T2 (MtLT2) has been submitted to laboratory evolution in Saccharomyces cerevisiae with the aim of improving activity and stability in organic co-solvents. Some 4500 clones created by random mutagenesis were screened in two rounds of directed evolution. Libraries were explored under increasing concentrations of acetonitrile and ethanol, and several mutants with improved features were purified and further characterised. Turnover rates of MtLT2 in 30% (v/v) acetonitrile and 50% (v/v) ethanol were increased up to 6.5- and 7.5-fold, respectively. The best variants showed similar rates in 20% (v/v) acetonitrile or 30% (v/v) ethanol as the parent type in aqueous media. Mutant laccases were also tested for the oxidation of anthracene in the presence of 20% (v/v) acetonitrile.     

Preliminary screening of some fungal strains for protein upgrading of sugar-beet pulp

Summary Fourteen fungal strains were grown on sugar-beet pulp (SBP) in a semisolid state with the view of evaluating their capabilities in increasing the protein content of SBP. The highest increases were obtained withSporotrichum thermophile no 89 Paprican andThielavia terrestric ATCC 38088, which gave a protein content of 14.6% and 13.4% respectively after a 3-day culture or a net increase of 48% and 36% respectively over that of the unfermented SBP.     

Action of phosphine on production of aflatoxins by various Aspergillus strains isolated from foodstuffs.

Phosphine is a food fumigant, used until now as an insecticide and rodenticide. The present work researches the action of phosphine treatment on growth and aflatoxin production of 23 Aspergillus strains. Production of aflatoxins B1, B2, G1, and G2 decreased in almost all cases by a ratio of 10 to 100. Phosphine treatment therefore seems favorable to prevent growth of various Aspergillus strains, in the context of keeping food safe.     

不同菌株固态发酵玉米秸秆生产饲料蛋白的比较研究

利用24株能够降解纤维素和木质素的菌种对玉米秸秆粉进行了单菌株发酵、多菌株组合发酵以及不同氮源发酵生产饲料蛋白的比较研究.结果表明24株单菌株发酵中F-21的发酵产物真蛋白含量最高(平均为7.64%);以F-5,F-17,F-21和F-24组成的多菌株发酵体系,经3d发酵后,发酵产物粗蛋白含量由2.80%提高到10.07%,比原料本身的粗蛋白含量高259.6%;粗纤维含量由38.17%降低到36.07%;氨基酸总量由2.1%增加到5.7%,比原料本身高171.4%,且氨基酸种类齐全;尿素和(NH4)2SO4的添加量与发酵产物真蛋白含量的关系呈抛物线,对相同添加量以尿素效果较好,而在尿素中,2%的添加量为最好.聚类分析将24株单菌株发酵后真蛋白含量和对照分为4组,其中G3{F-1,F-21}发酵效果最好.G1{F-3,F-5,F-6,F-7,F-8,F-12,F-13,F-15,F-17,F-19,F-20,F-22}次之,G2{F-2,F-4,F-9,F-10,F-11,F-14,F-18,F-23,F-24}较差,G4{对照,F-16}最差.试验结果表明,由F-5,F-17,F-21和F-24组成的多菌株发酵体系为发酵秸秆生产饲料蛋白的优良菌株.     

Production of extracellular enzymes by Antarctic fungal strains

 Thirty-three fungal strains, isolated from different sites on Victoria Land (continental Antarctica), were plate-screened for their ability to produce twelve extracellular enzymes. Lipases were generally present and in high quantities in almost all the strains. Polygalacturonase, as well as amylase and phosphatase, was common. Glucose oxidase, protease and DNAase appeared to be generally low or absent. Many strains, producing a limited number of enzymes, appeared to have a low eco-nutritional versatility while a few, such as Verticillium cfr. lecanii no. 1, V. cfr. lecanii no. 3, Aspergillus versicolor and Phoma sp. no. 2, showing a diversified enzymatic competence, are probably advantaged in extreme terrestrial environments characterized by low competition. The possibility of utilizing the enzyme-producing ability of these fungi in applied research is also discussed. Received: 3 November 1995/Accepted: 29 May 1996     

Beta-amylases from various fungal strains. A review

An overview presentation is made on the current global status of fungal beta3-amylases, their characteristics and applications in various industries. Among the few available report on beta-amylase producing fungal strains, many showed a preference for a cultivation temperature of 28 degrees C, acidic pH and soluble starch as an inducer of enzyme synthesis. In some fungal strains, alpha-amylase and alpha-glucosidases were found to be present as major contaminating enzymes. Although the existence of a few starch digesting and raw starch adsorbing fungal strains were reported, detailed study on molecular biology of corresponding fungal genes was not available.     

Biomodification and biodeterioration of carbon coatings by fungal strains

Over the past years, surface protection by hard carbon coatings has been increasingly applied in many fields. This study aimed to characterize changes in the properties of carbon coatings brought about by the growth of fungi (the first report about these processes was published by Kaczorowska et al., Nanodiam, PWN, 2006, 99–116). Alterations in the structure of diamond-like carbon (DLC) and nanocrystalline diamond (NCD) coatings, examined by Raman spectroscopy, XRD, XPS, and FTIR, were found to be caused by the processes of oxidation (incorporation of oxygen atoms) and reduction (hydrogenation) occurring within graphite carbon and amorphous carbon domains (components of the surface layer of the tested coatings along with the diamond domain). Determination of the activity of selected enzymes synthesized by the studied strains of filamentous fungi revealed that the observed biomodification and biodeterioration processes involved, among others, laccase, Mn-dependent peroxidase, two catechol dioxygenases, and esterases. The biosynthesis of these enzymes by fungi was enhanced when graphite was added to their culture media. Because of the high risk of fungal infections, the quality of carbon coatings should be routinely controlled by standard microbiological tests employing suitable strains of filamentous fungi (e.g., Aspergillus niger) and yeasts synthesizing enzymes that attack carbon materials.     

Action of phosphine on production of aflatoxins by various Aspergillus strains isolated from foodstuffs

Phosphine is a food fumigant, used until now as an insecticide and rodenticide. The present work researches the action of phosphine treatment on growth and aflatoxin production of 23 Aspergillus strains. Production of aflatoxins B1, B2, G1, and G2 decreased in almost all cases by a ratio of 10 to 100. Phosphine treatment therefore seems favorable to prevent growth of various Aspergillus strains, in the context of keeping food safe.     

Xylanase production by fungal strains on spent sulphite liquor

Xylanase production by seven fungal strains was investigated using concentrated spent sulphite liquor (SSLc), xylan and d-xylose as carbon substrates. An SSLc-based medium induced xylanase production at varying levels in all of these strains, with Aspergillus oryzae NRRL 3485 and Aspergillus phoenicis ATCC 13157 yielding activities of 164 and 146 U ml⁻¹, respectively; these values were higher than those obtained on xylan or d-xylose with the same fungal strains. The highest xylanase activity of 322 U ml⁻¹ was obtained with Aspergillus foetidus ATCC 14916 on xylan. Electrophoretic and zymogram analysis indicated three xylanases from A. oryzae with molecular weights of approximately 32, 22 and 19 kDa, whereas A. phoenicis produced two xylanases with molecular weights of about 25 and 21 kDa. Crude xylanase preparations from these A. oryzae and A. phoenicis strains exhibited optimal activities at pH 6.5 and 5.0 and at 65 and 55°C, respectively. The A. oryzae xylanolytic activity was stable at 50°C over the pH range 4.5–10. The crude xylanase preparations from these A. oryzae and A. phoenicis strains had negligible cellulase activity, and their application in the biobleaching of hardwood pulp reduced chlorine dioxide consumption by 20–30% without sacrificing brightness.     

Bioremediation of DDT in soil by genetically improved strains of soil fungus Fusarium solani

Bioremediation of DDT in soil by genetically improved recombinants of the soil fungus Fusarium solani was studied. The parent strains were isolated from soil enriched with DDD or DDE (immediate anaerobic and aerobic degradation products of DDT), as further degradation of these products are slow processes compared to the parent compound. These naturally occurring strains isolated from soil, however, are poor degraders of DDT and differed in their capability to degrade its metabolites such as DDD, DDE, DDOH and DBP and other organochlorine pesticides viz. kelthane and lindane. Synergistic effect was shown by some of these strains, when grown together in the medium containing DDD and kelthane under mixed culture condition. No synergism in DDE degradation was observed with the strains isolated from enriched soil. DDD-induced proteins extracted from individual culture filtrate (exo-enzyme) when subjected to SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed complementary polypeptide bands in these strains i.e., each strain produced distinct DDD degrading polypeptide bands and the recombinant or hybrid strains produced all of the bands of the two parents and degraded DDD better than the parental strains. Recombinant hybrid strains with improved dehalogenase activity were raised by parasexual hybridisation of two such complementary isolates viz. isolate 1(P-1) and 4(P-2) showing highest complementation and are compatible for hyphal fusion inducing heterokaryosis. These strains are genetically characterised as Kel⁺Ben^RDBP^-Lin^- and Kel^-Ben^rDBP⁺Lin⁺ respectively.Recombinants with mixed genotype, i.e., Kel⁺Ben^RDBP⁺Lin⁺ showing superior degradation quality for DDT were selected for bioremediation study. Recombination was confirmed by polypeptide band analysis of DDD induced exo-proteins from culture filtrate usingSDS-Polyacrylamide Gel Electrophoresis (PAGE) and RAPD (Random Amplified Polymorphic DNA) of genomic DNA using PCR (Polymerase Chain Reaction) technique. SDS-PAGE showed combination of DDD induced polypeptide bands characteristic of both the parents in the recombinants or the hybrids. PCR study showed the parent specific bands in the recombinant strains confirming gene transformation.     

Contamination by uranium mine drainages affects fungal growth and interactions between fungal species and strains

The presence of aquatic hyphomycetes has been reported for several heavy metal-contaminated waters. Tolerance probably is one adaptation to coping with heavy metals. To help clarify this issue strains of two species of aquatic hyphomycetes (Tricladium splendens Ingold and Varicosporium elodeae Kegel) were isolated from a reference stream and a stream contaminated with heavy metals and grown on malt extract agar prepared with reference and contaminated water to characterize colony morphology, growth rate, growth inhibition and interaction among species and strains. In V. elodeae the morphology of colonies differed between strains. Colony diameter increased linearly over time with growth rates being lower for strains isolated from contaminated than from reference streams (mostly for V. elodeae). Strains from the contaminated stream grew faster in medium prepared with contaminated water than in medium prepared with reference water, while for strains from the reference stream there was no significant difference in growth rates on the two media. In interacting isolates radial growth toward the opposing colony was generally lower than toward the dish edge. Percentage growth inhibition was higher for isolates in intraspecific interactions (13-37%) than in interspecific interactions (3-27%). However differences in growth inhibition experienced by interacting isolates were observed only in three cases out of 16. The difference between the percentage inhibition caused and experienced by a given isolate was highest in interactions involving isolates with distinct growth rates. Our results suggest that strains from the reference stream tolerate heavy metals while strains from the contaminated stream seem to be adapted to contaminated waters. We hypothesize that in natural environments fungal species-specific limits of tolerance to metal contamination might determine an abrupt or gradual response of the original fungal community to mine pollution giving origin to a poorer fungal community dominated by adapted strains with distinct functional efficiency.     

Defective pyocin particles produced by some mutant strains of Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa, defective in the production of active R-type pyocins, were isolated from pyocinogenic strains and their products were characterized. Polysheath-like structures were found in induced lysates of 29 out of 42 mutants. Two mutants (strain P15-16 and M189) were found to produce special defective particles, which were characterized in detail. The other 11 mutants did not produce significant amounts of any structure visible under an electron microscope. Serum blocking powers were found in lysates from P15-16 and M189 to significant amounts. Defective particle produced by strain P15-16 lacked the sheath component, whereas M189 had morphological defects at the junction between sheath and baseplate, and also in the architecture of baseplate. Both defective particles could adsorb to the surface of bacteria, that were sensitive to pyocin, at the tip of their fibers without killing cells. All M189 particles attached to the bacteria had the extended sheaths. Therefore, attachment to the bacteria by fibers is not sufficient to kill cells, and contraction of sheath must occur after the initial adsorption by fibers for pyocin to express its biological activity. Defective particles of strain P15-16, which was derived from strain P15 (a pyocin R1 producer), could be converted to active forms by an in vitro complementation reaction with extracts from certain mutants originated from strain PAO (a pyocin R2 producer). This result indicated the exchangeability of components between R-type pyocins belonging to the different groups.     

Bioremediation of pulp and paper mill effluent by a novel fungal consortium isolated from polluted soil

Two basidiomycetous fungi (Merulius aureus syn. Phlebia sp. and an unidentified genus) and a deuteromycetous fungus (Fusarium sambucinum Fuckel MTCC 3788) were isolated from soils affected with effluents of a pulp and paper mill over several years. These isolates were immobilized on nylon mesh and the consortium was used for bioremediation of pulp and paper mill effluent in a continuously aerated bench-top bioreactor. The treatment resulted in the reduction of color, lignin and COD of the effluent in the order of 78.6%, 79.0% and 89.4% in 4 days. A major part of reductions in these parameters occurred within first 24h of the treatment, which was also characterized by a steep decline in the pH of the effluent. During this period, total dissolved solids, electrical conductivity and salinity of the effluent also registered marked decline. It is pertinent to note that this is the first report of bioremediation of pulp and paper mill effluent by an immobilized fungal consortium.