Rhizodegradation of PAHs differentially altered by C3 and C4 plants.

Pyrosequencing of 16S ribosomal RNA (rRNA) was employed to characterize bacterial communities colonizing the rhizosphere of plants with C3 and C4 photosynthetic pathways grown in soil contaminated with polycyclic aromatic hydrocarbons (PAHs) after 60 and 120 days. The results of this study exhibited a clear difference in bacterial diversity between the rhizosphere and non-rhizosphere samples and between the rhizospheres of the C3 and C4 plants after 120 days. In both C3 and C4 rhizospheres, an incremental change in PAHs degrading bacterial genera was observed in the 120th day samples compared to the 60th day ones. Among the PAHs degrading bacterial genera, Pseudomonas showed good resistance to PAHs in the 120th day rhizosphere of both C3 and C4 plants. Conversely, the genus Sphingomonas showed sensitivity to PAHs in the 120th day rhizosphere soils of C3 plants only. Also, a significant increase in the PAHs degrading genera was observed at 120th day in the C4 rhizosphere in comparison to the C3 rhizosphere, which was reflected in a reduced PAHs concentration measured in the soil remediated with C4 plants rather than C3 plants. These results suggest that the rhizoremediation of PAHs was primarily governed by the plant photosystems, which led to differences in root secretions that caused the variation in bacterial diversity seen in the rhizospheres. This study is the first report to demonstrate the greater effectiveness of C4 plants in enhancing the PAHs degrading bacterial community than C3 plants.

Phytoremediation efficacy assessment of polycyclic aromatic hydrocarbons contaminated soils using garden pea (Pisum sativum) and earthworms (Eisenia fetida).

Endpoint assessment using biological systems in combination with the chemical analysis is important for evaluating the residual effect of contaminants following remediation. In this study, the level of residual toxicity of polycyclic aromatic hydrocarbons (PAHs) after 120 days of phytoremediation with five different plant species:- maize (Zea mays), Sudan grass (Sorghum sudanense), vetiver (Vetiveria zizanioides), sunflower (Helianthus annuus) and wallaby grass (Austrodanthonia sp.) has been evaluated by ecotoxicological tests such as root nodulation and leghaemoglobin assay using garden pea (Pisum sativum) and acute, chronic and genotoxicity assays using earthworm (Eisenia fetida). The phytoremediated soil exhibited lesser toxicity supporting improved root nodulation and leghaemoglobin content in P. sativum and reducing DNA damage in E. fetida when compared to contaminated soil before remediation. Also, the results of the ecotoxicological assays with the legume and earthworm performed in this study complemented the results obtained by the chemical analysis of PAHs in phytoremediated soil. Therefore, these findings provide a basis for a framework in which remediation efficacy of PAHs-contaminated sites can be evaluated effectively with simple ecotoxicological bioassays using legumes and earthworms.

Low molecular weight organic acids enhance the high molecular weight polycyclic aromatic hydrocarbons degradation by bacteria.

The biodegradation potential of three bacterial cultures isolated from the rhizosphere of maize (Zea mays) and Sudan grass (Sorghum sudanense) grown in PAHs contaminated soils to degrade benzo[a]pyrene (BaP) and pyrene (PYR) was assessed. Of the three bacterial cultures isolated, two belonged to Gram-positive bacteria of phylum Actinobacteria namely Arthrobacter sp. MAL3 and Microbacterium sp. MAL2. The Gram-negative bacterial culture was Stenotrophomonas sp. MAL1, from the phylum Proteobacteria. The cultures were grown in the presence of BaP and PYR as sole carbon sources and with the addition of low molecular weight organic acids (LMWOAs) mixture. After 10-14 days of exposure, all the bacterial isolates exhibited a complete degradation of PYR with the addition of LMWOAs mixture, whereas only 38.7% of BaP was degraded by Stenotrophomonas sp. MAL1 with the addition of LMWOAs mixture. In addition, enhanced PAHs biodegradation by bacterial culture was observed when the PAHs present as mixture (BaP + PYR) with the addition of LMWOAs. Dioxygenase genes were detected in Stenotrophomonas sp. MAL1 (phnAC), and Arthrobacter sp. MAL3 (nidA and PAH-RHDα). Therefore, this study provides new insights on the influence of LMWOAs in enhancing the degradation of high molecular weight (HMW) PAHs in soil by rhizosphere bacterial cultures.

Metabolomics reveals defensive mechanisms adapted by maize on exposure to high molecular weight polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons are an important group of persistent organic pollutants. Using plants to remediate PAHs has been recognized as a cost-effective and environmentally friendly technique. However, the overall impact of PAHs on the regulation of plant metabolism has not yet been explored. In this study, we analyzed the alteration in the maize (Zea mays L.) metabolome on exposure to high molecular weight PAHs such as benzo[a]pyrene (BaP) and pyrene (PYR) in a hydroponic medium, individually and as a mixture (BaP + PYR) using GC-MS. The differences in the metabolites were analyzed using XCMS (an acronym for various forms (X) of chromatography-mass spectrometry), an online-based data analysis tool. A significant variation in metabolites was observed between treatment groups and the unspiked control group. The univariate, multivariate and pathway impact analysis showed there were more significant alterations in metabolic profiles between individual PAHs and the mixture of BaP and PYR. The marked changes in the metabolites of galactose metabolism and aminoacyl tRNA biosynthesis in PAHs treated maize leaves exhibit the adaptive defensive mechanisms for individual and PAHs mixture. Therefore, the metabolomics approach is essential for an understanding of the complex biochemical responses of plants to PAHs contaminants. This knowledge will shed new light in the field of phytoremediation, bio-monitoring, and environmental risk assessment.

Comparison of plants with C3 and C4 carbon fixation pathways for remediation of polycyclic aromatic hydrocarbon contaminated soils.

The phytoremediation technique has been demonstrated to be a viable option for the remediation of polycyclic aromatic hydrocarbons (PAHs) contaminated sites. This study evaluated the potential applicability of plants with C3 and C4 carbon fixation pathways for the phytoremediation of recalcitrant high molecular weight (HMW) PAHs contaminated soil. A 60 and 120-day greenhouse study was conducted which showed higher degradation of HMW PAHs in soil grown with C4 plants when compared to C3 plants. Also, no PAHs were detected in the maize cobs, sunflower, wallaby, and Sudan grass seeds at the end of the experiment. The effect of plants in modifying the microbial community and dynamics in the rhizosphere was also examined by measuring soil biochemical properties such as dehydrogenase activity and water-soluble phenols. The results demonstrate a substantial difference in the microbial populations between planted and unplanted soils, which in turn facilitate the degradation of PAHs. To the best of our knowledge, this study for the first time evaluated the phytoremediation efficacy through the A. cepa cyto- and genotoxicity assay which should be considered as an integral part of all remediation experiments.

Rhodococcus wratislaviensis strain 9: An efficient p-nitrophenol degrader with a great potential for bioremediation.

A Gram-positive bacterium, Rhodococcus wratislaviensis strain 9, was isolated from groundwater contaminated with nitrophenolics and trichloroethene following enrichment culture technique. The cells of strain 9 grown on LB broth (uninduced) degraded 720 µM p-nitrophenol (PNP) within 12 h, and utilized as a source of carbon and energy. Orthogonal experimental design analysis to determine optimal conditions for biodegradation of PNP showed that pH had a significant positive effect (P ≤ .05) on bacterial degradation of PNP, while glucose, di- and tri-nitrophenols exhibited significant negative effect. Cell-free extracts obtained from PNP-grown culture that contained 20 µg mL-1 protein degraded 90% of 720 µM PNP within 5 h of incubation. Two-dimensional protein analysis revealed differential expression of the oxygenase component of PNP monooxygenase and an elongation factor Tu in PNP-grown cells, but not in those grown on glucose. The strain 9 remediated laboratory wastewater containing 900 µM PNP efficiently within 14 h, indicating its great potential in bioremediation of PNP-contaminated waters.

Impact of plant photosystems in the remediation of benzo[a]pyrene and pyrene spiked soils.

The phytoremediation potential of 14 different plant species belonging to C3 and C4 carbon fixation pathway for soils spiked with polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) and pyrene (PYR) was investigated. A glasshouse experiment was conducted to measure the changes in morphological, physiological, biochemical parameters and the bioaccumulation and biodegradation ability of the plants in soils spiked with 48 and 194 mg kg-1 of B[a]P and PYR, respectively. The per cent removal efficacy of B[a]P and PYR by the tested plant species over a period of 50 days was from 6 to 26% and 14 to 40% respectively. The maximum removal of both B[a]P and PYR was observed in Sudan grass (C4), vetiver (C4), maize (C4), and sunflower (C3). In terms of accumulation in root and shoot, the concentration of PYR was higher in both C3 and C4 plant species when compared to B[a]P. Overall the results indicated that C4 plants were more efficient than their C3 counterparts in terms of morphological, physiological, biochemical and degradation ability of PAHs.

Identification of a new operon involved in desulfurization of dibenzothiophenes using a metagenomic study and cloning and functional analysis of the genes.

The presence of sulphur-substituted hydrocarbons in fossil fuels are one of main reasons for the release of sulfur oxides into the environment. Dibenzothiophenes (DBT) are organic sulfur-containing molecules in crude oil, which have the potential for biological oxidation, with the sulphur being removed through an enzymatic cleavage of the CS bonds. Therefore, finding new strains that can desulfurize this compound has recently become a point of interest. In this study, three new genes involved in the bacterial desulfurization of Dibenzothiophene, which were sequenced in the course of a metagenomic study, were isolated by PCR amplification in the laboratory. The activities of these genes were then analysed following insertion into an expression vector and cloning in Escherichia coli DH5α cells. Based on the results, all three genes were actively expressed and their products could act on their corresponding substrates.

Microbial diversity and hydrocarbon degrading gene capacity of a crude oil field soil as determined by metagenomics analysis.

Soils contaminated with crude oil are rich sources of enzymes suitable for both degradation of hydrocarbons through bioremediation processes and improvement of crude oil during its refining steps. Due to the long term selection, crude oil fields are unique environments for the identification of microorganisms with the ability to produce these enzymes. In this metagenomic study, based on Hiseq Illumina sequencing of samples obtained from a crude oil field and analysis of data on MG-RAST, Actinomycetales (9.8%) were found to be the dominant microorganisms, followed by Rhizobiales (3.3%). Furthermore, several functional genes were found in this study, mostly belong to Actinobacteria (12.35%), which have a role in the metabolism of aliphatic and aromatic hydrocarbons (2.51%), desulfurization (0.03%), element shortage (5.6%), and resistance to heavy metals (1.1%). This information will be useful for assisting in the application of microorganisms in the removal of hydrocarbon contamination and/or for improving the quality of crude oil. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:638-648, 2016.

The Biodiversity Changes in the Microbial Population of Soils Contaminated with Crude Oil.

Crude oil spills resulting from excavation, transportation and downstream processes can cause intensive damage to living organisms and result in changes in the microbial population of that environment. In this study, we used a pyrosequencing analysis to investigate changes in the microbial population of soils contaminated with crude oil. Crude oil contamination in soil resulted in the creation of a more homogenous population of microorganisms dominated by members of the Actinomycetales, Clostridiales and Bacillales (all belonging to Gram-positive bacteria) as well as Flavobacteriales, Pseudomonadales, Burkholderiales, Rhizobiales and Sphingomonadales (all belonging to Gram-negative bacteria). These changes in the biodiversity decreased the ratios of chemoheterotrophic bacteria at higher concentrations of crude oil contamination, with these being replaced by photoheterotrophic bacteria, mainly Rhodospirillales. Several of the dominant microbial orders in the crude oil contaminated soils are able to degrade crude oil hydrocarbons and therefore are potentially useful for remediation of crude oil in contaminated sites.

A Review on the Genetics of Aliphatic and Aromatic Hydrocarbon Degradation.

Because of the high diversity of hydrocarbons, degradation of each class of these compounds is activated by a specific enzyme. However, most of other downstream enzymes necessary for complete degradation of hydrocarbons maybe common between different hydrocarbons. The genes encoding proteins for degradation of hydrocarbons, including the proteins required for the uptake of these molecules, the specific enzyme used for the initial activation of the molecules and other necessary degrading enzymes are usually arranged as an operon. Although the corresponding genes in many phylogenetic groups of microbial species show different levels of diversity in terms of the gene sequence, the organisation of the genes in the genome or on plasmids and the activation mode (inductive or constitutive), some organisms show identical hydrocarbon-degrading genes, probably as a result of horizontal gene transfer between microorganisms.

Enhanced removal of petroleum hydrocarbons using a bioelectrochemical remediation system with pre-cultured anodes.

Bioelectrochemical remediation (BER) systems such as microbial fuel cells (MFCs) have recently emerged as a green technology for the effective remediation of petroleum hydrocarbon contaminants (PH) coupled with simultaneous energy recovery. Recent research has shown that biofilms previously enriched for substrate degrading bacteria resulted in excellent performance in terms of substrate removal and electricity generation but the effects on hydrocarbon contaminant degradation were not examined. Here we investigate the differences between enriched biofilm anodes and freshly inoculated new anodes in diesel fed single chamber mediatorless microbial fuel cells (DMFC) using various techniques for the enhancement of PH contaminant remediation with concomitant electricity generation. An anodophilic microbial consortium previously selected for over a year through continuous culturing with a diesel concentration of about 800mgl(-1) and which now showed complete removal of this concentration of diesel within 30days was compared to that of a freshly inoculated new anode MFC (showing 83.4% removal of diesel) with a simultaneous power generation of 90.81mW/m(2) and 15.04mW/m(2) respectively. The behaviour of pre-cultured anodes at a higher concentration of PH (8000mgl(-1)) was also investigated. Scanning electron microscopy observation revealed a thick biofilm covering the pre-cultured anodic electrode but not the anode from the freshly inoculated MFC. High resolution imaging showed the presence of thin 60nm diametre pilus-like projections emanating from the cells. Anodic microbial community profiling confirmed that the selection for diesel degrading exoelectrogenic bacteria had occurred. Identification of a biodegradative gene (alkB) provided strong evidence of the catabolic pathway used for diesel degradation in the DMFCs.

Multiwall carbon nanotubes increase the microbial community in crude oil contaminated fresh water sediments.

Since crude oil contamination is one of the biggest environmental concerns, its removal from contaminated sites is of interest for both researchers and industries. In situ bioremediation is a promising technique for decreasing or even eliminating crude oil and hydrocarbon contamination. However, since these compounds are potentially toxic for many microorganisms, high loads of contamination can inhibit the microbial community and therefore reduce the removal rate. Therefore, any strategy with the ability to increase the microbial population in such circumstances can be of promise in improving the remediation process. In this study, multiwall carbon nanotubes were employed to support microbial growth in sediments contaminated with crude oil. Following spiking of fresh water sediments with different concentrations of crude oil alone and in a mixture with carbon nanotubes for 30days, the microbial profiles in these sediments were obtained using FLX-pyrosequencing. Next, the ratios of each member of the microbial population in these sediments were compared with those values in the untreated control sediment. This study showed that combination of crude oil and carbon nanotubes can increase the diversity of the total microbial population. Furthermore, these treatments could increase the ratios of several microorganisms that are known to be effective in the degradation of hydrocarbons.

A second component of the SltA-dependent cation tolerance pathway in Aspergillus nidulans.

The transcriptional response to alkali metal cation stress is mediated by the zinc finger transcription factor SltA in Aspergillus nidulans and probably in other fungi of the pezizomycotina subphylum. A second component of this pathway has been identified and characterized. SltB is a 1272 amino acid protein with at least two putative functional domains, a pseudo-kinase and a serine-endoprotease, involved in signaling to the transcription factor SltA. Absence of SltB activity results in nearly identical phenotypes to those observed for a null sltA mutant. Hypersensitivity to a variety of monovalent and divalent cations, and to medium alkalinization are among the phenotypes exhibited by a null sltB mutant. Calcium homeostasis is an exception and this cation improves growth of sltΔ mutants. Moreover, loss of kinase HalA in conjunction with loss-of-function sltA or sltB mutations leads to pronounced calcium auxotrophy. sltA sltB double null mutants display a cation stress sensitive phenotype indistinguishable from that of single slt mutants showing the close functional relationship between these two proteins. This functional relationship is reinforced by the fact that numerous mutations in both slt loci can be isolated as suppressors of poor colonial growth resulting from certain null vps (vacuolar protein sorting) mutations. In addition to allowing identification of sltB, our sltB missense mutations enabled prediction of functional regions in the SltB protein. Although the relationship between the Slt and Vps pathways remains enigmatic, absence of SltB, like that of SltA, leads to vacuolar hypertrophy. Importantly, the phenotypes of selected sltA and sltB mutations demonstrate that suppression of null vps mutations is not dependent on the inability to tolerate cation stress. Thus a specific role for both SltA and SltB in the VPS pathway seems likely. Finally, it is noteworthy that SltA and SltB have a similar, limited phylogenetic distribution, being restricted to the pezizomycotina subphylum. The relevance of the Slt regulatory pathway to cell structure, intracellular trafficking and cation homeostasis and its restricted phylogenetic distribution makes this pathway of general interest for future investigation and as a source of targets for antifungal drugs.

A pyrosequencing-based analysis of microbial diversity governed by ecological conditions in the Winogradsky column.

The Winogradsky column is used as a microcosm to mimic both the microbial diversity and the ecological relationships between the organisms in lake sediments. In this study, a pyrosequencing approach was used to obtain a more complete list of the microbial organisms present in such columns and their ratios in different layers of this microcosm. Overall, 27 different phyla in these columns were detected in these columns, most (20 phyla) belonged to bacteria. Based on this study, Proteobacteria (mostly Sphingomonadales), Cyanobacteria (mostly Oscillatoriales) and Bacteroidetes (mostly Flavobacteriales) were the dominant microorganisms in the water, middle, and bottom layers of this column, respectively. Although the majority of organism in the water layer were photoautotrophic organisms, the ratio of the phototrophic organisms decreased in the lower layers, replaced by chemoheterotrophic bacteria. Furthermore, the proportion of aerobic chemoheterotrophic bacteria was greater in the higher layers of the column in comparison to the bottom. The green and purple sulfur phototrophic bacteria inhabited the bottom and middle of these columns, with none of them found in the water layer. Although the sulfur oxidizing bacteria were the dominant chemolithotrophic bacteria in the water layer, their ratio decreases in lower layers, being replaced with nitrogen oxidizing bacteria in the middle and bottom layers. Overall, the microbial population of these layers changes from a phototrophic and aerobic chemoheterotrophic organisms in the water layer to a mostly anaerobic chemoheterotrophic population of bacteria in the bottom layers.

A Comprehensive Review of Aliphatic Hydrocarbon Biodegradation by Bacteria.

Hydrocarbons are relatively recalcitrant compounds and are classified as high-priority pollutants. However, these compounds are slowly degraded by a large variety of microorganisms. Bacteria are able to degrade aliphatic saturated and unsaturated hydrocarbons via both aerobic and anaerobic pathways. Branched hydrocarbons and cyclic hydrocarbons are also degraded by bacteria. The aerobic bacteria use different types of oxygenases, including monooxygenase, cytochrome-dependent oxygenase and dioxygenase, to insert one or two atoms of oxygen into their targets. Anaerobic bacteria, on the other hand, employ a variety of simple organic and inorganic molecules, including sulphate, nitrate, carbonate and metals, for hydrocarbon oxidation.

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex in Aspergillus nidulans.

A mutation screen in Aspergillus nidulans uncovered mutations in the acdX gene that led to altered repression by acetate, but not by glucose. AcdX of A. nidulans is highly conserved with Spt8p of Saccharomyces cerevisiae, and since Spt8p is a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, the SAGA complex may have a role in acetate repression in A. nidulans. We used a bioinformatic approach to identify genes encoding most members of the SAGA complex in A. nidulans, and a proteomic analysis to confirm that most protein components identified indeed exist as a complex in A. nidulans. No apparent compositional differences were detected in mycelia cultured in acetate compared to glucose medium. The methods used revealed apparent differences between Yeast and A. nidulans in the deubiquitination (DUB) module of the complex, which in S. cerevisiae consists of Sgf11p, Sus1p, and Ubp8p. Although a convincing homologue of S. cerevisiae Ubp8p was identified in the A. nidulans genome, there were no apparent homologues for Sus1p and Sgf11p. In addition, when the SAGA complex was purified from A. nidulans, members of the DUB module were not co-purified with the complex, indicating that functional homologues of Sus1p and Sgf11p were not part of the complex. Thus, deubiquitination of H2B-Ub in stress conditions is likely to be regulated differently in A. nidulans compared to S. cerevisiae.

Influence of nutrient mixtures on p-nitrophenol degradation by Stenotrophomonas sp. isolated from groundwater.

We isolated strain CERAR5, a Stenotrophomonas sp., from an aquifer contaminated with chlorinated hydrocarbons that utilizes up to 1.0 mM PNP within 62 h in M9 medium as a source of carbon and nitrogen. To assess the potential of this strain for use in bioremediation, we investigated the influence of external sources of carbon and nitrogen on bacterial degradation of PNP following a full factorial design analysis. Glucose, sodium acetate, phenol, sodium nitrate and ammonium chloride were the factors chosen, while per cent removal of PNP, growth of the bacterial strain, and change in pH of the medium were the responses measured. Glucose and acetate had significant positive influence on the removal PNP. In particular, acetate exhibited a significant positive effect on all the three responses measured, clearly suggesting that the addition of acetate greatly contributes to an efficient bioremediation of habitats contaminated with PNP by Stenotrophomonas sp. CERAR5.

SAGA complex components and acetate repression in Aspergillus nidulans.

Alongside the well-established carbon catabolite repression by glucose and other sugars, acetate causes repression in Aspergillus nidulans. Mutations in creA, encoding the transcriptional repressor involved in glucose repression, also affect acetate repression, but mutations in creB or creC, encoding components of a deubiquitination system, do not. To understand the effects of acetate, we used a mutational screen that was similar to screens that uncovered mutations in creA, creB, and creC, except that glucose was replaced by acetate to identify mutations that were affected for repression by acetate but not by glucose. We uncovered mutations in acdX, homologous to the yeast SAGA component gene SPT8, which in growth tests showed derepression for acetate repression but not for glucose repression. We also made mutations in sptC, homologous to the yeast SAGA component gene SPT3, which showed a similar phenotype. We found that acetate repression is complex, and analysis of facA mutations (lacking acetyl CoA synthetase) indicates that acetate metabolism is required for repression of some systems (proline metabolism) but not for others (acetamide metabolism). Although plate tests indicated that acdX- and sptC-null mutations led to derepressed alcohol dehydrogenase activity, reverse-transcription quantitative real-time polymerase chain reaction showed no derepression of alcA or aldA but rather elevated induced levels. Our results indicate that acetate repression is due to repression via CreA together with metabolic changes rather than due to an independent regulatory control mechanism.

CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localisation or degradation of CreA.

The major regulatory protein in carbon repression in Aspergillus nidulans is CreA. Strains constitutively over-expressing creA show normal responses to carbon repression, indicating that auto-regulation of creA is not essential for CreA-mediated regulation. In these strains, high levels of CreA are present whether cells are grown in repressing or derepressing conditions, indicating large-scale degradation of CreA does not play a key role. CreA is located in the nucleus and cytoplasm in cells when grown in either repressing or derepressing conditions, and absence of CreB, CreD or AcrB does not affect either the localisation or amount of CreA. Therefore, CreA must require some modification or interaction to act as a repressor. Deletion analysis indicates that a region of CreA thought to be important for repression in Trichoderma reesei and Sclerotina sclerotiorum CreA homologues is not critical for function in Aspergillus nidulans.