Angel E. Absalón

Isolation and Selection of a Highly Tolerant Microbial Consortium with Potential for PAH Biodegradation from Heavy Crude Oil-Contaminated Soils

A degrading microbial consortium highly tolerant to three-, four- and five-ring polycyclic aromatic hydrocarbons (PAHs) was selected from 50 fungal and bacterial isolates obtained from crude oil-contaminated soils. Morphological and molecular studies indicated that isolated fungi belonged to genera Aspergillus, Penicillium, Fusarium, Trichoderma, Scedosporium, and Acremonium and bacteria to Pseudomonas, Klebsiella, Bacillus, Enterobacter, Streptomyces, Stenotrophomonas, Kocuria, and Delftia genera. Individual fungal and bacterial isolates were evaluated for their potential to tolerate high concentrations of different molecular weight PAHs, as phenantrene (Phe), pyrene (Pyr), and benzo[a]pyrene (BaP) by surface plate assays, showing significant differences in extension rates for fungi and inhibition ratios for bacteria when both were exposed to 0–6,000xa0mg of PAHs per liter. Trichoderma asperellum H15, Aspergillus nomius H7, Aspergillus flavus H6, Pseudomonas aeruginosa B7, Klebsiella sp. B10, and Stenotrophomonas maltophilia B14 grew using PAHs as sole carbon source and presented a remarkably high tolerance to PAHs, up to 6,000xa0mgxa0l−1. The consortium composed of 12 fungal and bacterial PAH-tolerant isolates for the bioremediation of a PAH-contaminated soiled to a removal of 87.76xa0% Phe, 48.18xa0% Pyr, and 56.55xa0% BaP after 14xa0days. The degrading microbial consortium presented high potential for bioremediation and may be useful for the treatment of sites polluted with PAHs due to their elevated tolerance to high molecular weight (HMW) PAHs and their capacity to utilize them as energy source. This is the first study which evaluated the microbial tolerance to extreme concentrations of PAHs, resulting in a degrading consortium and highly tolerant consortium compared with those reported in other studies, where the concentrations tested are low.

Degradation of polycyclic aromatic hydrocarbons in soil by a tolerant strain of Trichoderma asperellum.

Trichoderma asperellum H15, a previously isolated strain characterized by its high tolerance to low (LMW) and high molecular weight (HMW) PAHs, was tested for its ability to degrade 3–5 ring PAHs (phenanthrene, pyrene, and benzo[a]pyrene) in soil microcosms along with a biostimulation treatment with sugarcane bagasse. T. asperellum H15 rapidly adapted to PAH-contaminated soils, producing more CO2 than uncontaminated microcosms and achieving up to 78xa0% of phenanthrene degradation in soils contaminated with 1,000xa0mg Kg−1 after 14xa0days. In soils contaminated with 1,000xa0mg Kg−1 of a three-PAH mixture, strain H15 was shown to degrade 74xa0% phenanthrene, 63xa0% pyrene, and 81xa0% of benzo[a]pyrene. Fungal catechol 1,2 dioxygenase, laccase, and peroxidase enzyme activities were found to be involved in the degradation of PAHs by T. asperellum. The results demonstrated the potential of T. asperellum H15 to be used in a bioremediation process. This is the first report describing the involvement of T. asperellum in LMW and HMW-PAH degradation in soils. These findings, along with the ability to remove large amounts of PAHs in soil found in the present work provide enough evidence to consider T. asperellum as a promising and efficient PAH-degrading microorganism.

Biodegradation of a mixture of PAHs by non-ligninolytic fungal strains isolated from crude oil-contaminated soil.

Nine native non-ligninolytic fungal strains were isolated from Maya crude oil-contaminated soil and selected based on their ability to grow and use crude oil and several polycyclic aromatic hydrocarbons (PAHs) as carbon source, for their application to PAH removal in soil. The fungi were identified by PCR amplification of intergenic transcribed sequences regions and microbiological techniques, and results showed them to be part of the genera Fusarium, Neurospora, Aspergillus, Scedosporium, Penicillium, Neosartorya and Talaromyces. A primary selection of fungi was made in minimal medium plates, considering the tolerance to different concentrations of PAHs for each strain. The radial extension rate exhibited significant differences (pxa0<xa00.05) from 200 to 1,000xa0mg of PAHs mixture l−1. A secondary selection of Aspergillus terreus, Talaromyces spectabilis, and Fusarium sp. was achieved based on their tolerance to 2,000xa0mg of a mixture of Phenanathrene and Pyrene kg−1 of soil in a solid-state microcosm system for 2 weeks. The percentage of PAH removal obtained by the three strains was approximately 21xa0% of the mixture.

Clinicopathological characterization and genomic sequence differences observed in a highly virulent fowl Aviadenovirus serotype 4

ABSTRACT Highly virulent fowl aviadenoviruses (genus: Aviadenovirus) represent a significant risk in poultry farming that may contribute to increased mortality rates and may adversely affect the growth performance of poultry flocks. In this study, we performed the clinicopathological characterization of a FAdV strain SHP95 isolated from a commercial farm and its whole genome sequencing. The study revealed that the isolated strain is a highly virulent serotype 4 FAdV that can cause 100% mortality in day-old specific pathogen free (SPF) chickens with a dose of 2.5u2009×u2009105 TCID50. At a lower viral dose (1.5u2009×u2009104 TCID50), the infection in day-old SPF chickens caused 40% mortality and lesions characteristic for Hepatitis-hydropericardium syndrome (HHS). The viral strain was detectable by real time PCR in chicken organs, including the lymphoid organs until day 28 after infection. The whole genome assembly of strain SHP95 revealed a size of 45,641u2005bp, which encodes for 42 viral open reading frame (ORF). The comparative analysis in the genome shows 98.1% similarity between strain SHP95 and other FAdV-4 genomes reported. The major differences in the genome sequence between pathogenic and non-pathogenic fowl Adenovirus were identified in the right arm of the genome.

Heterologous Expression of Manganese Peroxidase in Aspergillus niger and Its Effect on Phenanthrene Removal from Soil

A strain of Aspergillus niger, previously isolated from sugarcane bagasse because of its capacity to degrade phenanthrene in soil by solid culture, was used to express a manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium, aiming at increasing its polycyclic aromatic hydrocarbons degradation capacity. Transformants were selected based on their resistance to hygromycin B and the discoloration induced on Poly R-478 dye by the peroxidase activity. The recombinant A. niger SBC2-T3 strain developed MnP activity and was able to remove 95% of the initial phenanthrene (400 ppm) from a microcosm soil system after 17 days, whereas the wild strain removed 72% under the same conditions. Transformation success was confirmed by PCR amplification using gene-specific primers, and a single fragment (1,348 bp long, as expected) of the recombinant mnp1 was amplified in the DNA from transformants, which was absent from the parental strain.

Comparative metagenomic analysis of PAH degradation in soil by a mixed microbial consortium

In this study, we used a taxonomic and functional metagenomic approach to analyze some of the effects (e.g. displacement, permanence, disappearance) produced between native microbiota and a previously constructed Polycyclic Aromatic Hydrocarbon (PAH)-degrading microbial consortium during the bioremediation process of a soil polluted with PAHs. Bioaugmentation with a fungal-bacterial consortium and biostimulation of native microbiota using corn stover as texturizer produced appreciable changes in the microbial diversity of polluted soils, shifting native microbial communities in favor of degrading specific populations. Functional metagenomics showed changes in gene abundance suggesting a bias towards aromatic hydrocarbon and intermediary degradation pathways, which greatly favored PAH mineralization. In contrast, pathways favoring the formation of toxic intermediates such as cytochrome P450-mediated reactions were found to be significantly reduced in bioaugmented soils. PAH biodegradation in soil using the microbial consortium was faster and reached higher degradation values (84% after 30 d) as a result of an increased co-metabolic degradation when compared with other mixed microbial consortia. The main differences between inoculated and non-inoculated soils were observed in aromatic ring-hydroxylating dioxygenases, laccase, protocatechuate, salicylate and benzoate-degrading enzyme genes. Based on our results, we propose that several concurrent metabolic pathways are taking place in soils during PAH degradation.

Morphological changes and growth of filamentous fungi in the presence of high concentrations of PAHs

In this study, we evaluated the effect of low and high molecular weight polycyclic aromatic hydrocarbons (PAHs), i.e., Phenanthrene, Pyrene and Benzo[a]pyrene, on the radial growth and morphology of the PAH-degrading fungal strains Aspergillus nomius H7 and Trichoderma asperellum H15. The presence of PAHs in solid medium produced significant detrimental effects on the radial growth of A. nomius H7 at 4,000 and 6,000 mg L−1 and changes in mycelium pigmentation, abundance and sporulation ability at 1,000–6,000 mg L−1. In contrast, the radial growth of T. asperellum H15 was not affected at any of the doses tested, although sporulation was observed only up to 4,000 mg L−1 and as with the H7 strain, some visible changes in sporulation patterns and mycelium pigmentation were observed. Our results suggest that fungal strains exposed to high doses of PAHs significantly vary in their growth rates and sporulation characteristics in response to the physiological and defense mechanisms that affect both pigment production and conidiation processes. This finding is relevant for obtaining a better understanding of fungal adaptation in PAH-polluted environments and for developing and implementing adequate strategies for the remediation of contaminated soils.

Complete genome sequence of a non-pathogenic strain of Fowl Adenovirus serotype 11: Minimal genomic differences between pathogenic and non-pathogenic viruses

In this study, we conducted the clinicopathological characterization of a non-pathogenic FAdV-D serotype 11 strain MX95, isolated from healthy chickens, and its entire genome was sequenced. Experiments in SPF chickens revealed that the strain is a non-pathogenic virus that did not cause death at challenge doses of 1×106 TCID50. Additionally, the infection in SPF chickens caused no apparent damage in most of the organs analyzed by necropsy and histopathology, but it did cause inclusion body hepatitis; nevertheless it did not generate severe infectious clinical symptoms. The virus was detected in several chicken organs, including the lymphoid organs, by real-time polymerase chain reaction (PCR) until 42 days. The genome of FAdV-11 MX95 has a size of 44,326bp, and it encodes 36 open reading frames (ORFs). Comparative analysis of the genome indicated only 0.8% dissimilarity with a highly virulent serotype 11 that was previously reported.

Construction of PAH-degrading mixed microbial consortia by induced selection in soil

Bioremediation of polycyclic aromatic hydrocarbons (PAHs)-contaminated soils through the biostimulation and bioaugmentation processes can be a strategy for the clean-up of oil spills and environmental accidents. In this work, an induced microbial selection method using PAH-polluted soils was successfully used to construct two microbial consortia exhibiting high degradation levels of low and high molecular weight PAHs. Six fungal and seven bacterial native strains were used to construct mixed consortia with the ability to tolerate high amounts of phenanthrene (Phe), pyrene (Pyr) and benzo(a)pyrene (BaP) and utilize these compounds as a sole carbon source. In addition, we used two engineered PAH-degrading fungal strains producing heterologous ligninolytic enzymes. After a previous selection using microbial antagonism tests, the selection was performed in microcosm systems and monitored using PCR-DGGE, CO2 evolution and PAH quantitation. The resulting consortia (i.e., C1 and C2) were able to degrade up to 92% of Phe, 64% of Pyr and 65% of BaP out of 1000xa0mgxa0kg-1 of a mixture of Phe, Pyr and BaP (1:1:1) after a two-week incubation. The results indicate that constructed microbial consortia have high potential for soil bioremediation by bioaugmentation and biostimulation and may be effective for the treatment of sites polluted with PAHs due to their elevated tolerance to aromatic compounds, their capacity to utilize them as energy source.

Phenanthrene Removal from Soil by a Strain of Aspergillus niger Producing Manganese Peroxidase of Phanerochaete chrysosporium

© 2013 Cortes-Espinosa and Absalon, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Phenanthrene Removal from Soil by a Strain of Aspergillus niger Producing Manganese Peroxidase of Phanerochaete chrysosporium