A simple screening procedure for selecting fungi with potential for use in the bioremediation of contaminated land

A simple initial screening procedure for selecting strains of white-rot fungi with potential for use in bioremediation of contaminated sites is described. Besides the ability to degrade low molecular weight PAHs, isolates were screened for their growth rate on straw-based agar media, their potential to tolerate high concentrations of phenanthrene and their ability to out-grow the cellulolytic fungus Trichoderma harzianum on straw agar plates. Results from simple in vitro tests were correlated with the ability of the different strains to degrade PAHs in sand microcosms. It was found that fungal growth rate on straw-based agar media in the presence of phenanthrene correlated well with the ability of the different fungi to degrade PAHs in sand microcosms. Whereas growth rate on straw-based agar plates per se was indicative of the ability of white-rot fungi to establish in the presence of a competing fungus, it was a poor indicator of the fungus’ ability to degrade PAHs.     

Characterization of main sulfur source of wood-degrading basidiomycetes by S K-edge X-ray absorption near edge spectroscopy (XANES)

The main wood degraders in aerobic terrestrial ecosystems belong to the white- and brown-rot fungi, where their biomass can be created on wood decay only. However, total sulfur (S) concentration in wood is very low and only little is known about the different sulfur compounds in wood today. Sulfur-starved brown-rot fungi Gloeophyllum trabeum and Oligoporus placenta were incubated on sterilized pine wood blocks whereas Lentinus cyathiformis and the white-rot fungi Trametes versicolor were incubated on sterilized beech wood blocks. After 19 weeks of incubation, the S oxidation status was analyzed in wood, in degraded wood, and in biomass of wood-degrading fungi by synchrotron based S K-edge XANES, and total S and sulfate were quantified. Total sulfur and sulfate content in pine wood blocks were approximately 50 and 1 ??g g⁻¹, respectively, while in beech wood approximately 100 and 20 ??g g⁻¹ were found, respectively. Sulfur in beech was dominated by sulfate-esters. In contrast, pine wood also contained larger amounts of reduced S. Three out of four selected fungi caused a reduction of the S oxidation state in wood from oxidized S (sulfate-ester, sulfate) to intermediate S (sulfonate, sulfoxide) or reduced S (thiols, e.g., proteins, peptides, enzyme cofactors). Only O. placenta shifted thiol to sulfonate. Growth experiments of these fungi on selective minimal media showed that in particular cysteine (thiol), sulfonates, and sulfate enhanced total mycelium growth. Consequently, wood-degrading fungi were able to utilize a large variety of different wood S sources for growth but preferentially transformed in vivo sulfate-esters and thiol into biomass structures.     

Studies of growth conditions in wood for three white-rot fungi and their cellulaseless mutants

White-rot fungi, which have the ability to degrade all the wood components including lignin, are of great interest in biotechnological processes based on wood and other lignocellulosic materials. It was demonstrated earlier that enough lignin can be degraded to cause a decrease in the energy demand for production of thermomechanical pulp if wood chips are pretreated by cellulaseless mutants of white-rot fungi. This paper concerns the growth conditions in wood for three white-rot fungi and their cellulaseless mutants in order to determine optimal conditions for such pretreatment processes. The pH and temperature optima have been determined as well as the growth rate in wood. The results show that the growth rate in wood. at least for Cel 44 (a cellulaseless mutant of Sporotrichum pulverulentum), is not the rate-limiting step in delignification. From different mixtures of urea and NH₄H₂PO₄ the optimal nitrogen source was determined for the mutants. The optimal C/N ratio was found to vary between 160/1 and 400/1. It is suggested that the lower the C/N ratio, the faster the growth. It was also demonstrated that both water- and acetone-extractable substances in wood supported the growth of cellulaseless mutants. When some glucose was added to the wood, the weight loss caused by Cel 44 increased. All these observations support earlier findings that lignin in wood cannot be degraded by white-rot fungi unless a more easily metabolizable carbon source is used simultaneously.     

Degradation of petroleum hydrocarbons (C6-C40) and crude oil by a novel Dietzia strain

A novel bacterial strain, DQ12-45-1b, was isolated from the production water of a deep subterranean oil-reservoir. Morphological, physiological and phylogenetic analyses indicated that the strain belonged to the genus Dietzia with both alkB (coding for alkane monooxygenase) and CYP153 (coding for P450 alkane hydroxylase of the cytochrome CYP153 family) genes and their induction detected. It was capable of utilizing a wide range of n-alkanes (C6-C40), aromatic compounds and crude oil as the sole carbon sources for growth. In addition, it preferentially degraded short-chain hydrocarbons (?C25) in the early cultivation phase and accumulated hydrocarbons with chain-lengths from C23 to C27 during later cultivation stage with crude oil as the sole carbon source. This is the first study to report the different behaviors of a bacterial species toward crude oil degradation as well as a species of Dietzia degrading a wide range of hydrocarbons.     

Effects of Nitrogen Supplements on Degradation of Aspen Wood Lignin and Carbohydrate Components by Phanerochaete chrysosporium

A supplement of KH₂PO₄, MgSO₄, CaCl₂, trace elements, and thiamine accelerated the initial rate of aspen wood decay by Phanerochaete chrysosporium but did not increase the extent of lignin degradation. Asparagine, casein hydrolysate, and urea supplements (1% added N) strongly inhibited lignin degradation and weight loss. The complex nitrogen sources peptone and yeast extract stimulated lignin degradation and weight loss. Albumen and NH₄Cl had intermediate effects. Conversion of [¹⁴C]lignin to ¹⁴CO₂ and water-soluble materials underestimated lignin degradation in the presence of the complex N sources. The highest ratio of lignin degradation to total weight loss and the largest increase in cellulase digestibility occurred during the decay of unsupplemented wood. Rotting of aspen wood by P. chrysosporium gives smaller digestibility increases than have been found with some other white-rot fungi.     

Beh?et's: A Disease or a Syndrome? Answer from an Expression Profiling Study

Behçet’s disease (BD) is a chronic, relapsing, multisystemic inflammatory disorder with unanswered questions regarding its etiology/pathogenesis and classification. Distinct manifestation based subsets, pronounced geographical variations in expression, and discrepant immunological abnormalities raised the question whether Behçet’s is “a disease or a syndrome”. To answer the preceding question we aimed to display and compare the molecular mechanisms underlying distinct subsets of BD. For this purpose, the expression data of the gene expression profiling and association study on BD by Xavier et al (2013) was retrieved from GEO database and reanalysed by gene expression data analysis/visualization and bioinformatics enrichment tools. There were 15 BD patients (B) and 14 controls (C). Three subsets of BD patients were generated: MB (isolated mucocutaneous manifestations, n = 7), OB (ocular involvement, n = 4), and VB (large vein thrombosis, n = 4). Class comparison analyses yielded the following numbers of differentially expressed genes (DEGs); B vs C: 4, MB vs C: 5, OB vs C: 151, VB vs C: 274, MB vs OB: 215, MB vs VB: 760, OB vs VB: 984. Venn diagram analysis showed that there were no common DEGs in the intersection “MB vs C” ∩ “OB vs C” ∩ “VB vs C”. Cluster analyses successfully clustered distinct expressions of BD. During gene ontology term enrichment analyses, categories with relevance to IL-8 production (MB vs C) and immune response to microorganisms (OB vs C) were differentially enriched. Distinct subsets of BD display distinct expression profiles and different disease associated pathways. Based on these clear discrepancies, the designation as “Behçet’s syndrome” (BS) should be encouraged and future research should take into consideration the immunogenetic heterogeneity of BS subsets. Four gene groups, namely, negative regulators of inflammation (CD69, CLEC12A, CLEC12B, TNFAIP3), neutrophil granule proteins (LTF, OLFM4, AZU1, MMP8, DEFA4, CAMP), antigen processing and presentation proteins (CTSS, ERAP1), and regulators of immune response (LGALS2, BCL10, ITCH, CEACAM8, CD36, IL8, CCL4, EREG, NFKBIZ, CCR2, CD180, KLRC4, NFAT5) appear to be instrumental in BS immunopathogenesis.     

Progressive Degradation of Crude Oil n-Alkanes Coupled to Methane Production under Mesophilic and Thermophilic Conditions

Although methanogenic degradation of hydrocarbons has become a well-known process, little is known about which crude oil tend to be degraded at different temperatures and how the microbial community is responded. In this study, we assessed the methanogenic crude oil degradation capacity of oily sludge microbes enriched from the Shengli oilfield under mesophilic and thermophilic conditions. The microbial communities were investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes combined with cloning and sequencing. Enrichment incubation demonstrated the microbial oxidation of crude oil coupled to methane production at 35 and 55°C, which generated 3.7±0.3 and 2.8±0.3 mmol of methane per gram oil, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that crude oil n-alkanes were obviously degraded, and high molecular weight n-alkanes were preferentially removed over relatively shorter-chain n-alkanes. Phylogenetic analysis revealed the concurrence of acetoclastic Methanosaeta and hydrogenotrophic methanogens but different methanogenic community structures under the two temperature conditions. Candidate divisions of JS1 and WWE 1, Proteobacteria (mainly consisting of Syntrophaceae, Desulfobacteraceae and Syntrophorhabdus) and Firmicutes (mainly consisting of Desulfotomaculum) were supposed to be involved with n-alkane degradation in the mesophilic conditions. By contrast, the different bacterial phylotypes affiliated with Caldisericales, “Shengli Cluster” and Synergistetes dominated the thermophilic consortium, which was most likely to be associated with thermophilic crude oil degradation. This study revealed that the oily sludge in Shengli oilfield harbors diverse uncultured microbes with great potential in methanogenic crude oil degradation over a wide temperature range, which extend our previous understanding of methanogenic degradation of crude oil alkanes.     

Importance of phosphoinositide-dependent signaling pathways in the control of gene expression in resting cells and in response to phytohormones

“Phosphoinositide” refers to phosphorylated forms of phosphatidylinositol, including phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. Both of these molecules could be in vivo substrates of plant phospholipase C. These phosphoinositides can also be biologically active “per se,” by directly binding to proteins and thus altering their location and/or activity. The use of pharmacological agents in Arabidopsis suspension cells allowed us to identify genes whose expression was positively or negatively controlled, in the basal state, by products of phosphoinositide-dependent phospholipase C. In this basal state, it seems that no genes exhibit a phosphoinositide-dependent expression “per se.” However, many genes whose expression is altered in the presence of phospholipase C inhibitors appeared to be responsive to salicylic acid. This allowed us to show that salicylic acid acts both by increasing the phosphoinositide pool and by inhibiting the phospholipase C. In response to salicylic acid it is possible to identify genes whose expression is controlled by products of PI-PLC, but also genes whose expression is controlled by phosphoinositides “per se.” Our data highlight the importance of phosphoinositide-dependent pathways in gene expression in resting cells and in response to phytohormones.     

Growth and Mycorrhizal Community Structure of Pinus sylvestris Seedlings following the Addition of Forest Litter

We report the effects of pine and oak litter on species composition and diversity of mycorrhizal fungi colonizing 2-year-old Pinus sylvestris L. seedlings grown in a bare-root nursery in Lithuania. A layer of pine or oak litter was placed on the surface of the nursery bed soil to mimic natural litter cover. Oak litter amendment appeared to be most favorable for seedling survival, with a 73% survival rate, in contrast to the untreated mineral bed soil (44%). The concentrations of total N, P, K, Ca, and Mg were higher in oak growth medium than in pine growth medium. Relative to the control (pH 6.1), the pH was lower in pine growth medium (5.8) and higher in oak growth medium (6.3). There were also twofold and threefold increases in the C content of growth medium with the addition of pine and oak litter, respectively. Among seven mycorrhizal morphotypes, eight different mycorrhizal taxa were identified: Suillus luteus, Suillus variegatus, Wilcoxina mikolae, a Tuber sp., a Tomentella sp., Cenococcum geophilum, Amphinema byssoides, and one unidentified ectomycorrhizal symbiont. Forest litter addition affected the relative abundance of mycorrhizal symbionts more than their overall representation. This was more pronounced for pine litter than for oak litter, with 40% and 25% increases in the abundance of suilloid mycorrhizae, respectively. Our findings provide preliminary evidence that changes in the supply of organic matter through litter manipulation may have far-reaching effects on the chemistry of soil, thus influencing the growth and survival of Scots pine seedlings and their mycorrhizal communities.     

Use of antagonistic bacilli for biocontrol of fungi degrading fresh wood

The species composition of micromycete complexes colonizing aspen, birch, and pine wood was studied. Calculation of the Sorens-Tchekanovsky similarity coefficients showed that these complexes shared a high degree of similarity. They were dominated by representatives of the genera Penicillium, Paecilomyces, Trichoderma, and Rhizopus. Some antagonistic bacilli inhibited growth and development of wood-decay fungi in vitro and led to the formation of spheroplasts on growing hyphae. A study of possible involvement of bacillary mycolytic enzymes in the inhibition of wood-decay fungi demonstrated selectivity of their lytic effect, which was determined by the genus and species of micromycetes and did not correlate with their relative resistance to antagonists.     

The Genome of the Moderate Halophile Amycolicicoccus subflavus DQS3-9A1T Reveals Four Alkane Hydroxylation Systems and Provides Some Clues on the Genetic Basis for Its Adaptation to a Petroleum Environment

The moderate halophile Amycolicicoccus subflavus DQS3-9A1^T is the type strain of a novel species in the recently described novel genus Amycolicicoccus, which was isolated from oil mud precipitated from oil produced water. The complete genome of A. subflavus DQS3-9A1^T has been sequenced and is characteristic of harboring the genes for adaption to the harsh petroleum environment with salinity, high osmotic pressure, and poor nutrient levels. Firstly, it characteristically contains four types of alkane hydroxylases, including the integral-membrane non-heme iron monooxygenase (AlkB) and cytochrome P450 CYP153, a long-chain alkane monooxygenase (LadA) and propane monooxygenase. It also accommodates complete pathways for the response to osmotic pressure. Physiological tests proved that the strain could grow on n-alkanes ranging from C10 to C36 and propane as the sole carbon sources, with the differential induction of four kinds of alkane hydroxylase coding genes. In addition, the strain could grow in 1–12% NaCl with the putative genes responsible for osmotic stresses induced as expected. These results reveal the effective adaptation of the strain DQS3-9A1^T to harsh oil environment and provide a genome platform to investigate the global regulation of different alkane metabolisms in bacteria that are crucially important for petroleum degradation. To our knowledge, this is the first report to describe the co-existence of such four types of alkane hydroxylases in a bacterial strain.     

Oxalate production by Basidiomycetes,including the white-rot species Coriolus versicolor and Phanerochaete chrysosporium

Oxalate was found to accumulate in liquid culture media from the growth of the white-rot basidiomycetes Coriolus versicolor, Heterobasidion annosum, Pleurotus florida and Phanerochaete chrysosporium. Whereas little oxalate accumulated during active growth, millimolar concentrations of oxalate were detected in culture media during the stationary phase. The basidiomycete Agaricus bisporus, the cultivated mushroom, also accumulated oxalate in its culture medium in the stationary phase. In comparison, the brown-rot fungi Amyloporia xantha, Coniophora marmorata, C. puteana and Poria vaporaria accumulated oxalate in the primary metabolic phase and throughout growth up to 35 days. Oxalate accumulation (0.04–10.0 mm) in white-rot cultures did not lower the pH of the medium during growth, whereas in brown-rot cultures oxalate (2.0–20.0 mm) reduced the media pH during growth. Cultures of Agaricus bisporus, C. puteana and Coriolus versicolor grown on solid media containing high levels of calcium (50 or 100 mm calcium chloride) produced calcium oxalate crystals to varying extents on the surface of the hyphae. Correspondence to: C. S. Evans     

Fast biodegradation of long chain n-alkanes and crude oil at high concentrations with Rhodococcus sp. Moj-3449

Biodegradation of long chain n-alkanes and crude oil with fast rate and high concentration are desirable for bioremediation, especially in heavily oil-polluted areas, and enhanced oil recovery. We discovered Rhodococcus sp. Moj-3449 with such unique abilities by screening microorganisms for the growth on n-hexadecane at 30 mg/mL. The new strain grew very fast on 120 mg/mL of n-hexadecane giving a cell density of 14.7 g cdw/L after only 2 days’ incubation. During the growth with this strain, the oil–water phases were rapidly emulsified, giving rise to tolerance to high alkane concentration (250 mg/mL) and fast growth rate of 0.10–0.20 h^?1 for alkane concentration of 1–180 mg/mL. The degraded concentration of n-hexadecane increased linearly with the initial alkane concentration (1–250 mg/mL). Incubation on n-hexadecane at 250 mg/mL for 7 days gave a cell density of 13.5 g cdw/L and degraded 124 mg/mL of n-hexadecane. The strain grew also fast on n-dodecane (C12), n-tetradecane (C14), and n-octadecane (C18), with degradation preference of C14 (=C16) > C12 > C18. Different from many alkane-degrading strains, Rhodococcus sp. Moj-3449 was found to have subterminal oxidation pathway. Rhodococcus sp. Moj-3449 degraded also crude oil fast at 60–250 mg/mL, with a wide range of n-alkanes (C10–C35) as substrates in which C14–C19 are preferred. The degradation ability increased with initial oil concentration from 60 to 150 mg/mL and slightly decreased afterwards. Incubation on 150 mg/mL of crude oil for 7 days degraded 37% of n-alkanes. The outstanding ability of rapidly degrading long chain n-alkanes and crude oil at high concentration makes Rhodococcus sp. Moj-3449 potentially useful for bioremediation and microbial enhanced oil recovery.     

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)⁹ addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF6^8,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced^10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.     

KIAA1462, A Coronary Artery Disease Associated Gene,Is a Candidate Gene for Late Onset Alzheimer Disease in APOE Carriers

Alzheimer disease (AD) is a devastating neurodegenerative disease affecting more than five million Americans. In this study, we have used updated genetic linkage data from chromosome 10 in combination with expression data from serial analysis of gene expression to choose a new set of thirteen candidate genes for genetic analysis in late onset Alzheimer disease (LOAD). Results in this study identify the KIAA1462 locus as a candidate locus for LOAD in APOE4 carriers. Two genes exist at this locus, KIAA1462, a gene associated with coronary artery disease, and “rokimi”, encoding an untranslated spliced RNA The genetic architecture at this locus suggests that the gene product important in this association is either “rokimi”, or a different isoform of KIAA1462 than the isoform that is important in cardiovascular disease. Expression data suggests that isoform f of KIAA1462 is a more attractive candidate for association with LOAD in APOE4 carriers than “rokimi” which had no detectable expression in brain.     

Emulsification and degradation of "Bunker C" fuel oil by microorganisms

An enrichment culture procedure has been used to isolate mixed culture systems which grow upon “Bunker C” fuel oil. When inoculated into a mineral salts aqueous medium containing Bunker C oil, the mixed cultures initiate oil emulsification. Emulsification usually is observed in 24–48 hr. The role of microbes in this emulsification will be discussed. It appears that certain metabolic products produced by the microbe possess properties of surfactants. Bacteria and fungi have been isolated which possess the ability to cause emulsification. Freeze-dried biomass is also capable of emulsifying oil. Chromatographic analyses of biodegraded Bunker C fuel oil show that microorganisms selectively metabolize the n-paraffin fraction.