Combined iron and sulfate reduction biostimulation as a novel approach to enhance BTEX and PAH source-zone biodegradation in biodiesel blend-contaminated groundwater.

The use of biodiesel as a transportation fuel and its growing mandatory blending percentage in diesel increase the likelihood of contaminating groundwater with diesel/biodiesel blends. A 100L-field experiment with B20 (20% biodiesel and 80% diesel, v/v) was conducted to assess the potential for the combined biostimulation of iron and sulfate reducing bacteria to enhance BTEX and PAH biodegradation in a diesel/biodiesel blend-contaminated groundwater. A B20 field experiment under monitored natural attenuation (MNA) was used as a baseline control. Ammonium acetate and a low-cost and sustainable product recovered from acid mine drainage treatment were used to stimulate iron and sulfate-reducing conditions. As a result, benzene and naphthalene concentrations (maximum concentrations were 28.1µgL-1 and 10.0µgL-1, respectively) remained lower than the MNA experiment (maximum concentrations were 974.7µgL-1 and 121.3µgL-1, respectively) over the whole experiment. Geochemical changes were chronologically consistent with the temporal change of the predominance of Geobacter and GOUTA19 which might be the key players responsible for the rapid attenuation of benzene and naphthalene. To the best of our knowledge, this is the first field experiment to demonstrate the potential for the combined iron and sulfate biostimulation to enhance B20 source-zone biodegradation.

Functional Basis of Microorganism Classification.

Correctly identifying nearest "neighbors" of a given microorganism is important in industrial and clinical applications where close relationships imply similar treatment. Microbial classification based on similarity of physiological and genetic organism traits (polyphasic similarity) is experimentally difficult and, arguably, subjective. Evolutionary relatedness, inferred from phylogenetic markers, facilitates classification but does not guarantee functional identity between members of the same taxon or lack of similarity between different taxa. Using over thirteen hundred sequenced bacterial genomes, we built a novel function-based microorganism classification scheme, functional-repertoire similarity-based organism network (FuSiON; flattened to fusion). Our scheme is phenetic, based on a network of quantitatively defined organism relationships across the known prokaryotic space. It correlates significantly with the current taxonomy, but the observed discrepancies reveal both (1) the inconsistency of functional diversity levels among different taxa and (2) an (unsurprising) bias towards prioritizing, for classification purposes, relatively minor traits of particular interest to humans. Our dynamic network-based organism classification is independent of the arbitrary pairwise organism similarity cut-offs traditionally applied to establish taxonomic identity. Instead, it reveals natural, functionally defined organism groupings and is thus robust in handling organism diversity. Additionally, fusion can use organism meta-data to highlight the specific environmental factors that drive microbial diversification. Our approach provides a complementary view to cladistic assignments and holds important clues for further exploration of microbial lifestyles. Fusion is a more practical fit for biomedical, industrial, and ecological applications, as many of these rely on understanding the functional capabilities of the microbes in their environment and are less concerned with phylogenetic descent.

Ethyl tert-butyl ether (ETBE)-degrading microbial communities in enrichments from polluted environments.

The ethyl tert-butyl ether (ETBE) degradation capacity and phylogenetic composition of five aerobic enrichment cultures with ETBE as the sole carbon and energy source were studied. In all cases, ETBE was entirely degraded to biomass and CO2. Clone libraries of the 16S rRNA gene were prepared from each enrichment. The analyses of the DNA sequences obtained showed different taxonomic compositions with a majority of Proteobacteria in three cases. The two other enrichments have different microbiota with an abundance of Acidobacteria in one case, whereas the microbiota in the second was more diverse (majority of Actinobacteria, Chlorobi and Gemmatimonadetes). Actinobacteria were detected in all five enrichments. Several bacterial strains were isolated from the enrichments and five were capable of degrading ETBE and/or tert-butyl alcohol (TBA), a degradation intermediate. The five included three Rhodococcus sp. (IFP 2040, IFP 2041, IFP 2043), one Betaproteobacteria (IFP 2047) belonging to the Rubrivivax/Leptothrix/Ideonella branch, and one Pseudonocardia sp. (IFP 2050). Quantification of these five strains and two other strains, Rhodococcus sp. IFP 2042 and Bradyrhizobium sp. IFP2049, which had been previously isolated from one of the enrichments was carried out on the different enrichments based on quantitative PCR with specific 16S rRNA gene primers and the results were consistent with the hypothesized role of Actinobacteria and Betaproteobacteria in the degradation of ETBE and the possible role of Bradyrhizobium strains in the degradation of TBA.

Ethyl tert-butyl ether (ETBE) biodegradation by a syntrophic association of Rhodococcus sp. IFP 2042 and Bradyrhizobium sp. IFP 2049 isolated from a polluted aquifer.

Ethyl tert-butyl ether (ETBE) enrichment was obtained by adding contaminated groundwater to a mineral medium containing ETBE as the sole carbon and energy source. ETBE was completely degraded to biomass and CO2 with a transient production of tert-butanol (TBA) and a final biomass yield of 0.37 ± 0.08 mg biomass (dry weight).mg(-1) ETBE. Two bacterial strains, IFP 2042 and IFP 2049, were isolated from the enrichment, and their 16S rRNA genes (rrs) were similar to Rhodococcus sp. (99 % similarity to Rhodococcus erythropolis) and Bradyrhizobium sp. (99 % similarity to Bradyrhizobium japonicum), respectively. Rhodococcus sp. IFP 2042 degraded ETBE to TBA, and Bradyrhizobium sp. IFP 2049 degraded TBA to biomass and CO2. A mixed culture of IFP 2042 and IFP 2049 degraded ETBE to CO2 with a biomass yield similar to the original ETBE enrichment (0.31 ± 0.02 mg biomass.mg(-1) ETBE). Among the genes previously described to be involved in ETBE, MTBE, and TBA degradation, only alkB was detected in Rhodococcus sp. IFP 2042 by PCR, and none were detected in Bradyrhizobium sp. IFP 2049.

Monitoring of bacterial communities during low temperature thermal treatment of activated sludge combining DNA phylochip and respirometry techniques.

Sludge reduction is one of the major challenges in biological wastewater treatment. One approach is to increase the sludge degradation yield together with the biodegradation kinetics. Among the various sludge pretreatment strategies proposed, thermal pretreatment at around 65 °C was described as promising. The enhancement in the biodegradation activity due to the selection of thermophilic hydrolytic bacteria was proposed, but further experiments are needed to demonstrate the specific role of these bacteria. In this study, concentrated activated sludge grown at 20 °C was subjected to thermal treatment at 65 °C for different periods. The originality of the work relied on a polyphasic approach based on the correlation between kinetics (chemical oxygen demand, COD; mixed liquor suspended solids, MLSS), bacterial activity (respirometry) and bacterial community structure (phylochip monitoring) in order to characterize the mechanisms involved in the thermal reduction of sludge. The bacterial activity in the aeration basin decreased to a very low level when recycling sludge was treated at 65 °C from 13 to 60 h, but then, started to increase after 60 h. In parallel to these fluctuations in activity, a drastic shift occurred in the bacterial community structure with the selection of thermophilic bacteria (mainly related to genera Paenibacillus and Bacillus), which are known for their specific hydrolases.

Long-term persistence and bacterial transformation potential of transplastomic plant DNA in soil.

The long-term physical persistence and biological activity of transplastomic plant DNA (transgenes contained in the chloroplast genome) either purified and added to soil or naturally released by decaying tobacco leaves in soil was determined. Soil microcosms were amended with transplastomic tobacco leaves or purified plant DNA and incubated for up to 4 years. Total DNA was extracted from soil and the number of transgenes (aadA, which confers resistance to both spectinomycin and streptomycin) was quantified by quantitative PCR. The biological activity of these transgenes was assessed by transformation in the bacterial strain Acinetobacter sp. BD413(pBAB2) in vitro. While the proportion of transgenes recovered increased with the increasing amount of transplastomic DNA added, plant DNA was rapidly degraded over time. The number of transgenes recovered decreased about 10,000 fold within 2 weeks. Data reveal, however, that a small fraction of the plant DNA escaped degradation. Transgene sequences were still detected after 4 years and transformation assays showed that extracted DNA remained biologically active and could still transform competent cells of Acinetobacter sp. BD413(pBAB2). The approach presented here quantified the number of transgenes (based on quantitative PCR of 50% of the gene) released and persisting in the environment over time and provided new insights into the fate of transgenic plant DNA in soil.

Human pathogens abundant in the bacterial metagenome of cigarettes.

BACKGROUND: Many studies have evaluated chemical, heavy metal, and other abiotic substances present in cigarettes and their roles in the development of lung cancer and other diseases, yet no studies have comprehensively evaluated bacterial diversity of cigarettes and the possible impacts of these microbes on respiratory illnesses in smokers and exposed nonsmokers. OBJECTIVES: The goal of this study was to explore the bacterial metagenomes of commercially available cigarettes. METHODS: A 16S rRNA-based taxonomic microarray and cloning and sequencing were used to evaluate total bacterial diversity of four brands of cigarettes. Normalized microarray data were compared using principal component analysis and hierarchical cluster analysis to evaluate potential differences in microbial diversity across cigarette brands. RESULTS: Fifteen different classes of bacteria and a broad range of potentially pathogenic organisms were detected in all cigarette samples. Most notably, we detected Acinetobacter, Bacillus, Burkholderia, Clostridium, Klebsiella, Pseudomonas aeruginosa, and Serratia in > or = 90% of all cigarette samples. Other pathogenic bacteria detected included Campylobacter, Enterococcus, Proteus, and Staphylococcus. No significant variability in bacterial diversity was observed across the four different cigarette brands. CONCLUSIONS: Previous studies have shown that smoking is associated with colonization by pathogenic bacteria and an increased risk of lung infections. However, this is the first study to show that cigarettes themselves could be the direct source of exposure to a wide array of potentially pathogenic microbes among smokers and other people exposed to secondhand smoke. The overall public health implications of these findings are unclear at this time, and future studies are necessary to determine whether bacteria in cigarettes could play important roles in the development of both infectious and chronic respiratory diseases.

Leaching and transformability of transgenic DNA in unsaturated soil columns.

Unsaturated soil columns were used to examine the transport of the plasmid pLEPO1 and plant DNA (transplastomic tobacco DNA), both carrying an antibiotic resistance gene (aadA gene), and the capacity of bacteria to incorporate the gene in their genome after its passage through the soil. Soil columns containing a top leaf layer had sterile water percolated through them at a rate of 0.5mLh(-1). DNA from column leachate water was extracted and analyzed. Quantitative measurements included total DNA concentrations in the water and the transformation frequencies of Acinetobacter sp. BD413 by DNA in the column effluent. Qualitative measurements included the relative degradation of DNA after passage in the columns by agarose gel electrophoresis and the potential of effluent DNA to transform bacteria, leading to the production of antibiotic-resistant bacteria. The presence of aadA gene in the leachate water of soil columns suggests the mobility of DNA in unsaturated soil medium. The extent of DNA degradation was found to be proportional to its residence time in the soil column while a fraction of DNA was always able to incorporate into the Acinetobacter genome under all conditions studied. These results suggest that biologically active transgenic DNA might be transported downward by rain in unsaturated soils.

Visual evidence of horizontal gene transfer between plants and bacteria in the phytosphere of transplastomic tobacco.

Plant surfaces, colonized by numerous and diverse bacterial species, are often considered hot spots for horizontal gene transfer (HGT) between plants and bacteria. Plant DNA released during the degradation of plant tissues can persist and remain biologically active for significant periods of time, suggesting that soil or plant-associated bacteria could be in direct contact with plant DNA. In addition, nutrients released during the decaying process may provide a copiotrophic environment conducive for opportunistic microbial growth. Using Acinetobacter baylyi strain BD413 and transplastomic tobacco plants harboring the aadA gene as models, the objective of this study was to determine whether specific niches could be shown to foster bacterial growth on intact or decaying plant tissues, to develop a competence state, and to possibly acquire exogenous plant DNA by natural transformation. Visualization of HGT in situ was performed using A. baylyi strain BD413(rbcL-DeltaPaadA::gfp) carrying a promoterless aadA::gfp fusion. Both antibiotic resistance and green fluorescence phenotypes were restored in recombinant bacterial cells after homologous recombination with transgenic plant DNA. Opportunistic growth occurred on decaying plant tissues, and a significant proportion of the bacteria developed a competence state. Quantification of transformants clearly supported the idea that the phytosphere constitutes a hot spot for HGT between plants and bacteria. The nondisruptive approach used to visualize transformants in situ provides new insights into environmental factors influencing HGT for plant tissues.

Detection of potential transgenic plant DNA recipients among soil bacteria.

The likelihood of gene transfer from transgenic plants to bacteria is dependent on gene number and the presence of homologous sequences. The large number of transgene copies in transplastomic (transgenes contained in the chloroplast genome) plant cells as well as the prokaryotic origin of the transgene, may thus significantly increase the likelihood of gene transfer to bacteria that colonize plant tissues. In order to assess the probability of such transfer, the length of homologous DNA sequences required between the transgene and the genome of the bacterial host was assessed. In addition, the probability that bacteria, which co-infect diseased plants, are transformable and have sequences similar to the flanking regions of the transgene was evaluated. Using Acinetobacter baylyi strain BD143 and transplastomic tobacco plants harboring the aadA gene (streptomycin and spectinomycin resistance), we found that sequences identical to the flanking regions containing as few as 55 nucleotides were sufficient for recombination to occur. Consequently, a collection of bacterial isolates able to colonize tobacco plant tissue infected by Ralstonia solanacearum strain K60 was obtained, screened for DNA sequence similarity with the chloroplastic genes accD and rbcL flanking the transgene, and tested for their ability to uptake extracellular DNA (broad host-range pBBR1MCS plasmids) by natural or electro-transformation. Results showed that among the 288 bacterial isolates tested, 8% presented DNA sequence similarity with one or both chloroplastic regions flanking the transgene. Two isolates, identified as Pseudomonas sp. and Acinetobacter sp., were able to integrate exogenous plasmid DNA by electro-transformation and natural transformation, respectively. Our data suggest that transplastomic plant DNA recipients might be present in soil bacterial communities.

Effect of carbon and nitrogen input on the bacterial community structure of Neocaledonian nickel mine spoils.

Neocaledonian mine spoils are considered as an extreme environment because of their edaphic conditions, which are unfavourable for life. The principal characteristics of this soil are the high nickel content (20,000 ppm) and the very low carbon (0.2%) and nitrogen (0.01%) levels, which are certainly among the major limiting factors for heterotrophic bacterial growth. The aim of this work was to determine what changes could occur in the bacterial community structure of the mine spoils when a carbon and a nitrogen source were added. Soil bacterial response to nutrient addition was examined in both the mine spoils and an agricultural soil, which is characterized by normal levels of nutrients. 16S rRNA gene clone libraries constructed to characterize changes occurring in the different soil bacterial communities showed an important selection of Actinobacteria in the mine spoils as a consequence of nutrient amendment: Actinobacteria represented 75% and 96% of the bacterial community structure after succinate and glucose addition, respectively. This was observed only in the mine spoils and is probably a consequence of the extreme environmental conditions. Carbon amendment in the agricultural soil led to an increase in Firmicutes, mainly Bacillus sp.

Degradation and transformability of DNA from transgenic leaves.

The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the plant pathogen Ralstonia solanacearum. Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene. These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues. Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil.

In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria.

Interkingdom gene transfer is limited by a combination of physical, biological, and genetic barriers. The results of greenhouse experiments involving transplastomic plants (genetically engineered chloroplast genomes) cocolonized by pathogenic and opportunistic soil bacteria demonstrated that these barriers could be eliminated. The Acinetobacter sp. strain BD413, which is outfitted with homologous sequences to chloroplastic genes, coinfected a transplastomic tobacco plant with Ralstonia solanacearum and was transformed by the plant's transgene (aadA) containing resistance to spectinomycin and streptomycin. However, no transformants were observed when the homologous sequences were omitted from the Acinetobacter sp. strain. Detectable gene transfer from these transgenic plants to bacteria were dependent on gene copy number, bacterial competence, and the presence of homologous sequences. Our data suggest that by selecting plant transgene sequences that are nonhomologous to bacterial sequences, plant biotechnologists could restore the genetic barrier to transgene transfer to bacteria.