Isolation of keratinolytic fungi from a coal mine dump

During the study of fungal succesion in the coal mine dump in Brzezinka (Poland), soil samples were examined for keratinolytic fungi. These micro-organisms were rather poorly represented in the area studied. Out of 300 soil samples examined, only 48 (16%) were positive for keratinolytic fungi.Trichophyton ajelloi andArthroderma curreyi were the prevailing species. These species occurred practically at two locations, i.e. on the naked carbon rocks inhabited by algae crops (chiefly byCyanophyta) and in the pine litter. It can be supposed that the occurrence of keratinolytic fungi was more dependent on the favourable general conditions such as increasing organic matter content, microflora, and humidity than on the presence of keratin remains in the soil. Because of the lack of potentially pathogenic fungi, the coal mine dump examined cannot be considered as an important source of fungal infection.     

New lipid‐producing,cold‐tolerant yellow‐green alga isolated from the rocky mountains of colorado

A new strain of yellow‐green algae (Xanthophyceae, Heterokonta), tentatively named Heterococcus sp. DN1 (UTEX accession number UTEX ZZ885), was discovered among snow fields in the Rocky Mountains. Axenic cultures of H. sp. DN1 were isolated and their cellular morphology, growth, and composition of lipids were characterized. H. sp. DN1 was found to grow at temperatures approaching freezing to accumulate large intracellular stores of lipids. H. sp. DN1 produces the highest quantity of lipids when grown undisturbed with high light in low temperatures_. Of particular interest was the accumulation of eicosapentaenoic acid, known to be important for human nutrition, and palmitoleic acid, known to improve biodiesel feedstock properties. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:853–861, 2013     

Identification and characterization of a novel salt‐tolerant esterase from a Tibetan glacier metagenomic library

A salt‐tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p‐nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three‐dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat‐resistant features. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:890–899, 2015     

Isolation of fungi from soil using the keratin-baiting technique

Of 236 soil samples baited with wool, some 71.2% (168) produced fungal growth. Gliocladium (25.0%), Paecilomyces (14.8%), Trichophyton (11.9%) species were the most prevalent in soil moistened with sterile distilled water (SDW) containing antibiotics. On the other hand, in soil moistened with SDW only, Trichophyton (32.6%), Paecilomyces (27.5%), Diheterospora (16.5%), Gliocladium (13.6%) and Fusarium (13.1%) species were more common. Of the known potential pathogens, the Paecilomyces and Fusarium species were frequently isolated from soil collected in parks, cleared areas, paddocks, rivers and roadsides. All five sites are areas of human and animal activity. The keratinolytic species were Microsporum cookei, M. gypseum complex, Trichophyton ajelloi and T. terrestre, which are regarded as nonpathogens with the exception of M. cookei and M. gypseum complex.     

Local Adaptation of Metallicolous and Non‐Metallicolous Anthyllis vulneraria Populations: Their Utilization in Soil Restoration

Restoration of metalliferous mine soils requires using plant species tolerant to high metal concentrations and adapted to nutrient‐poor soil. Legumes can increase plant productivity through N₂‐fixation, but they are often scarce in metalliferous sites. We examined survival, growth, and tolerance of four populations of a legume, Anthyllis vulneraria, from two metalliferous (MET) Zn‐Pb mine sites, Avinières (AV) ([Zn‐EDTA] = 26,000 mg/kg) and Eylie (EY) ([Zn‐EDTA] = 4,632 mg/kg), and two non‐metalliferous (NMET) sites located in the south of France with the aim to select the most appropriate populations for restoration of mined soils. In a common garden experiment, plants from each population were reciprocally grown in soil from the provenance of each population. The two NMET populations exhibited high mortality and low growth rates in soil from the mined sites. The AV MET exhibited a high growth rate in metalliferous soils, but showed high mortality in non‐metalliferous soils. The growth of the EY MET was very low in the AV‐contaminated soil, but was the highest of all populations in moderately and non‐metalliferous soils. Plants from the AV MET population showed a high growth and survival in metalliferous soil and would be appropriate in the restoration of metal‐contaminated sites (>30,000 mg Zn kg^?1). The EY MET population would be adapted to the restoration of moderate metal‐contaminated soils (<30,000 mg Zn kg^?1). Taking into account the broad distribution of A. vulneraria, these two populations could be suitable for the restoration of derelict mine sites in mediterranean and temperate regions of Europe and North America.     

Long-term recovery from mine drainage disturbance of a macroinvertebrate community in the Ichi-kawa River,Japan

The effects of mine drainage on a benthic macroinvertebrate community and its recovery have been investigated for 25 years in the Ichi-kawa River in western Japan, focusing especially on change in community structure. Concentrations of arsenic, copper and zinc in the water were distinctly higher at sites just below the drainage than an upstream reference site before mine closure in 1973. Benthic communities there were severely damaged, as evidenced by reductions in the number of families and biomass. Chironomidae and a mayfly, Epeorus latifolium, predominated at the impacted sites, whereas stenopsychid caddisflies were dominant at the reference site. After mine closure, zinc concentrations significantly decreased downstream, although they remained higher than at the reference site. Following this, family richness and biomass of benthic communities clearly increased. In addition, the percentage of Trichoptera increased and, finally, Stenopsychidae became the dominant family in 1996 at all sites except just below the drainage. Therefore, the dominance of Stenopsychidae at the impacted sites is expected to be an indicator of complete recovery of the benthic community from the effect of mine drainage.     

自然演替过程中铜尾矿土壤酶活性的变化

铜陵矿区堆放了大量不同时期的铜尾矿,形成大量铜尾矿废弃地,其中的大部分铜尾矿废弃地处于自然生态恢复状态。并形成典型的原生演替系列。为探讨废弃地自然生态恢复中不同演替阶段植物群落变化与土壤酶活性变化的关系,对铜陵3处不同时期弃置的尾矿废弃地进行了调查。随着自然生态系统演替的进行,尾矿中土壤酶活性逐步提高,维管植物群落下尾矿中土壤酶活性〉隐花植物结皮下尾矿中土壤酶活性〉裸地处土壤酶活性;位于植物群落下的尾矿废弃地中脲酶、碱性磷酸酶、过氧化氢酶的活性从表层至下层逐渐降低。不同植物群落对尾矿中土壤酶活性影响有很大差异,位于木贼群落下的尾矿中几种土壤酶的活性高于位于白茅群落和中华结缕草群落下的尾矿中酶的活性。相关分析表明,土壤酶活性与有机质、总N表现出显著的正相关。     

湖北铜绿山古铜矿遗址区木本植物对重金属富集能力的分析

对湖北铜绿山古铜矿遗址区的木本植物种类组成及生长概况进行了调查,并采用原子吸收光谱法测定了木本植物地上部和地下部及其根际土壤中重金属含量;在此基础上,分析了各树种对Cu、Cd和Pb的富集作用,并对木本植物的生态修复意义进行了初步评价。结果表明:在该古铜矿遗址区共有木本植物13科14属14种,其中乔木13种、灌木1种;木本植物的树龄4~25 a、株高3.7~22.6 m、胸径5.3~29.0 cm,长势均较好。木本植物根际土壤中Cu、Cd、Pb、Cr、Mn和Zn的平均含量分别为3 166.73、3.66、137.06、31.32、1 774.43和208.32 mg·kg-1,其中,土壤中Cu、Cd和Pb的含量达到重度污染等级。各树种地上部和地下部的Cu、Cd和Pb的含量差异明显,且均为Cu含量最高、Cd含量最低;各树种的地上部和地下部对Cu、Cd和Pb的富集系数及综合富集系数也均有明显差异,但对Cd的富集系数均最大。法国冬青〔Viburnum odoratissimum var.awabuki(K.Koch)Zabel ex Rumpl.〕、梧桐〔Firmiana platanifolia(Linn.f.)Marsili〕、桂花〔Osmanthus fragrans(Thunb.)Lour.〕和刺槐(Robinia pseudoacacia Linn.)地上部的综合富集系数较高,苦楝(Melia azedarach Linn.)、女贞(Ligustrum lucidum Ait.)、法国冬青和樟树〔Cinnamomum camphora(Linn.)Presl〕地下部的综合富集系数较高;总体上看,法国冬青、苦楝、女贞、梧桐和桂花对Cu、Cd和Pb的平均综合富集系数均大于1,具有较高的重金属富集能力。根据研究结果,建议在Pb污染区域可选择栽植二球悬铃木〔Platanus×acerifolia(Ait.)Willd.〕和构树〔Broussonetia papyrifera(Linn.)L’Hert.ex Vent.〕等树种,在Cd污染区域可选择栽植法国冬青、梧桐、刺槐和苦楝等树种,而在Cu-Cd-Pb复合污染区域应栽植法国冬青、苦楝、女贞、梧桐、桂花和刺槐等树种。     

龙南钾矿区常见蕨类植物可培养内生真菌的多样性

植物内生真菌作为一类特殊的微生物资源,与宿主在长期的生态系统演化过程中形成了互惠共生关系,通过多样化途径来增强植物体的营养生理和抗性机能,对宿主植物产生多种有益生物学作用,在植物演替过程中具有重要的生态学意义。这种特殊微生物资源近年来倍受关注,而利用植物-真菌共生体强化植物在矿区逆境中生长,提高矿区生物修复效率是一个新的研究热点。为探明钾矿区不同蕨类植物内生真菌的物种多样性、群落组成以及生态分布规律,该研究以芒萁、狗脊、禾秆蹄盖蕨、海金沙、华中铁角蕨、井栏边草和乌蕨等植物为材料,采用组织分离、形态学鉴定等方法,对其内生真菌多样性进行分析。结果表明:从7种植物中共分离获得377株内生真菌,总分离率在2.50%~4.52%之间。经鉴定377个菌株隶属于链格孢属、曲霉属、枝孢菌属和轮枝孢属等25个分类单元,其中链格孢属、曲霉属、枝孢菌属和轮枝孢属等在所有被调查蕨类植物中都有分布,为优势属,共计为185株,占总株数的49.07%,但它们在每种植物的分布存在明显差异;7种蕨类植物内生真菌总定植率为叶高于根状茎(P0.05),多样性指数在0.502~0.867之间,但每种植物及其不同组织部位的内生真菌定植率和多样性指数存在一定的差异;从相似性分析来看,同一个钾矿区不同蕨类植物内生真菌菌群之间的相似性程度较低,相似性系数在0.189~0.587之间。该研究结果不仅丰富了植物内生真菌种质资源,而且为进一步开展植物内生真菌强化宿主植物在钾矿区生长适应机制的研究奠定了基础。     

狮子山铜尾矿植物对铜的吸收及土壤特的影响

我国铜矿储藏丰富,铜矿开采带来巨大经济利益的同时,也对生态环境造成极大的破坏,这种恶劣的环境严重阻碍了植物的定居,但是自然界物种繁多,总有一些植物能适应这种环境而生存下来.本文通过对狮子山优势植物吸收和积累铜的分析,发现这些植物均能富集较多的铜,在土壤铜含量很高的情况下,依然生长旺盛,没有出现受害症状,成为尾矿上的优势种,并形成了单优群落或多优小群落.这些植物的存在改变了土壤的理化特性,降低了土壤中的重金属的含量,提高了土壤的全N、全P、全K和有机质含量,一定程度上改善了土壤的不良环境,在尾矿的植被恢复和土壤修复中起着非常重要的作用.     

铜尾矿废弃地土壤动物多样性特征

土壤动物的恢复、群落演替及其多样性对于铜尾矿废弃地的生态重建具有重要意义.为了解铜尾矿废弃地土壤动物群落多样性特征,在铜陵市铜尾矿区设置4个样地23采样点,共捕获土壤动物4622只个体,隶属5门10纲18目29类.结果表明,铜尾矿自然废弃地与其对照组土壤动物多样性差异明显,自然废弃地土壤动物丰富度指数d、大型土壤动物密度、中小型土壤动物密度、多样性指数H'和D·G指数均小于其对照组且存在显著差异.而复垦废弃地中小型土壤动物密度和D·G指数小于其对照组且存在显著差异,多样性指数H'大于其对照组且差异极显著,其他土壤动物群落指标与其对照组均无显著差异.相似系数q表明,外围林地间土壤动物群落相似性最大,其次是复垦废弃地与外围林地间,自然废弃地与其它3个样地间相似性最小.从垂直分层来看,复垦废弃地土壤动物表聚性强于自然废弃地,尾矿废弃地表聚性强于外围林地.灰色关联分析表明,铜尾矿区土壤理化性质与土壤动物群落结构指标关系密切,其中最重要的因素是土壤含水量和总钾含量,重金属全镉含量也不容忽视,植被因素和土壤全铜含量对土壤动物的影响相对较小.由此可见,土壤含水量、土壤基质的优劣、土壤有机质与营养元素的含量等因素限制了铜尾矿废弃地土壤动物的恢复与重建,而这些因素的改善主要归功于尾矿废弃地的土地复垦和作物种植.所以,尾矿废弃地的复垦与利用有利于土壤动物的恢复与重建.     

Single‐cell sequencing unveils the lifestyle and CRISPR‐based population history of Hydrotalea sp. in acid mine drainage

Acid mine drainage (AMD) is characterized by an acid and metal‐rich run‐off that originates from mining systems. Despite having been studied for many decades, much remains unknown about the microbial community dynamics in AMD sites, especially during their early development, when the acidity is moderate. Here, we describe draft genome assemblies from single cells retrieved from an early‐stage AMD sample. These cells belong to the genus Hydrotalea and are closely related to Hydrotalea flava. The phylogeny and average nucleotide identity analysis suggest that all single amplified genomes (SAGs) form two clades that may represent different strains. These cells have the genomic potential for denitrification, copper and other metal resistance. Two coexisting CRISPR‐Cas loci were recovered across SAGs, and we observed heterogeneity in the population with regard to the spacer sequences, together with the loss of trailer‐end spacers. Our results suggest that the genomes of Hydrotalea sp. strains studied here are adjusting to a quickly changing selective pressure at the microhabitat scale, and an important form of this selective pressure is infection by foreign DNA.     

Characterization of an ethanol‐tolerant 1,4‐β‐xylosidase produced by Pichia membranifaciens

Aims: The purification and biochemical properties of the 1,4‐β‐xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol‐tolerant 1,4‐β‐xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G‐100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS‐PAGE. The activity of 1,4‐β‐xylosidase was optimum at pH 6·0 and at 35°C. The activity had a K_m of 0·48 ± 0·06 mmol l^?1 and a V_max of 7·4 ± 0·1 μmol min^?1 mg^?1 protein for p‐nitrophenyl‐β‐d ‐xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4‐β‐xylosidase from P. membranifaciens to be used in the bioethanol production process.     

不同植物群落下铜尾矿废弃地生物结皮中真菌群落结构的比较

分布于铜尾矿废弃地的裸地表面及维管植物群落中的生物土壤结皮在尾矿废弃地生态恢复过程中扮演重要角色。利用分子生物学技术探讨了不同维管植物下以及不同演替阶段的生物土壤结皮中真菌的多样性及其群落结构的变化。结果表明:生物土壤结皮中的真菌主要包括子囊菌门(Ascomycota)、担子菌门(Basidiomycota)和壶菌门(Chytridiomycota),其中子囊菌门占绝对优势,其相对丰度为55.12%—87.73%,其次为担子菌门相对丰度为12.27%—43.86%;不同样本真菌群落结构在门、纲、目以及属的水平存在显著差异;生物土壤结皮中真菌群落结构和多样性的差异与维管植物群落类型以及演替阶段不同的生物土壤结皮的类型有关,与基质化学性质之间无显著的相关性。     

Isolation and characterization of a novel copper‐inducible metallothionein gene of a ciliate,Tetrahymena tropicalis lahorensis

The two isoforms of copper metallothionein (CuMT) gene of a copper resistant ciliate, Tetrahymena tropicalis lahorensis (Ttl), have been isolated and characterized. The molecular cloning and nucleotide sequencing of cDNAs coding for the two CuMT isoforms revealed that TtlCuMT1 gene has 300, while TtlCuMT2 has 327 nucleotides, both with ATG as the initiation codon and TGA as the translational termination codon. TAG codes for glutamine in TtlCuMT2 gene which is peculiar to Tetrahymena. The deduced or translated TtlCuMT1 and TtlCuMT2 peptide sequences contain 100 and 108 amino acid residues including 28 and 32 cysteine residues, respectively. The amino acid sequences of TtlCuMT1 and TtlCuMT2 have special features of two and three CXCXXCXCXXCXC intragenic tandem repeats with a conserved structural pattern of cysteine, respectively. The predicted tertiary structures of these two isoforms indicate two domains. Domain I and the initial part of domain II showed >98% homology with other Tetrahymena CuMT. On the basis of the differences in the domain II, the metallothionein subfamily 7b can be divided into two groups, one (TtlCuMT1) comprising >100 amino acids and the other (TtlCuMT2) comprising <100 amino acids. This is a novel finding of the present study as no such report on this type of classification exists at the moment. TtlCuMT1 has 95%, while TtlCuMT2 has 97% resemblance with the previously reported CuMT genes of Tetrahymena spp. SDS‐PAGE analysis using fluorescent probe as well as coomassie brilliant blue staining also confirmed the presence of metallothionein. J. Cell. Biochem. 110: 630–644, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of small increases in copper levels on culturable basidiomycetous yeasts in low‐nutrient soils

Aims: Investigating the effect of perturbations, with relatively low Cu concentrations, on yeast community composition in low‐nutrient virgin soil. Methods and Results: Culturable soil yeast populations were monitored at an experimental site treated with the fungicide copper oxychloride (10 mg Cu per kg soil). Yeast numbers were unaffected by additional Cu; however, a shift in yeast community composition from Hymenomycetes to Urediniomycetes species occurred. Subsequent growth experiments conducted with a synthetic liquid medium revealed that hymenomycetous and urediniomycetous yeasts were affected differently by 1 and 10 mg l^?1 Cu. Soil microcosm experiments then indicated that additional 10 mg kg^?1 Cu may improve the competitive ability of urediniomycetous yeasts in the presence of hymenomycetous yeasts. Conclusions: The shift from hymenomycetous to urediniomycetous yeasts, as a result of slightly increased soil Cu levels, may be because of hymenomycetous yeasts being more sensitive to elevated Cu levels and urediniomycetous yeasts having an improved competitive ability in the presence of elevated Cu levels. Significance and Impact of the Study: Yeast community composition of pristine low‐nutrient soils may change as a result of perturbations with relatively low concentrations of Cu. Urediniomycetous yeasts should be studied as potential bio‐indicators of Cu perturbations.     

Genotype‐by‐sequencing of the plant‐pathogenic fungi Pyrenophora teres and Sphaerulina musiva utilizing Ion Torrent sequence technology

Genetic and genomics tools to characterize host–pathogen interactions are disproportionately directed to the host because of the focus on resistance. However, understanding the genetics of pathogen virulence is equally important and has been limited by the high cost of de novo genotyping of species with limited marker data. Non‐resource‐prohibitive methods that overcome the limitation of genotyping are now available through genotype‐by‐sequencing (GBS). The use of a two‐enzyme restriction‐associated DNA (RAD)‐GBS method adapted for Ion Torrent sequencing technology provided robust and reproducible high‐density genotyping of several fungal species. A total of 5783 and 2373 unique loci, ‘sequence tags’, containing 16 441 and 9992 single nucleotide polymorphisms (SNPs) were identified and characterized from natural populations of Pyrenophora teres f. maculata and Sphaerulina musiva, respectively. The data generated from the P. teres f. maculata natural population were used in association mapping analysis to map the mating‐type gene to high resolution. To further validate the methodology, a biparental population of P. teres f. teres, previously used to develop a genetic map utilizing simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers, was re‐analysed using the SNP markers generated from this protocol. A robust genetic map containing 1393 SNPs on 997 sequence tags spread across 15 linkage groups with anchored reference markers was generated from the P. teres f. teres biparental population. The robust high‐density markers generated using this protocol will allow positional cloning in biparental fungal populations, association mapping of natural fungal populations and population genetics studies.