Bacterial Outer Membrane Vesicles as Antibiotic Delivery Vehicles.

Bacterial outer membrane vesicles (OMVs) are nanometer-scale, spherical vehicles released by Gram-negative bacteria into their surroundings throughout growth. These OMVs have been demonstrated to play key roles in pathogenesis by delivering certain biomolecules to host cells, including toxins and other virulence factors. In addition, this biomolecular delivery function enables OMVs to facilitate intra-bacterial communication processes, such as quorum sensing and horizontal gene transfer. The unique ability of OMVs to deliver large biomolecules across the complex Gram-negative cell envelope has inspired the use of OMVs as antibiotic delivery vehicles to overcome transport limitations. In this review, we describe the advantages, applications, and biotechnological challenges of using OMVs as antibiotic delivery vehicles, studying both natural and engineered antibiotic applications of OMVs. We argue that OMVs hold great promise as antibiotic delivery vehicles, an urgently needed application to combat the growing threat of antibiotic resistance.

Heterozygous, Polyploid, Giant Bacterium, Achromatium, Possesses an Identical Functional Inventory Worldwide across Drastically Different Ecosystems.

Achromatium is large, hyperpolyploid and the only known heterozygous bacterium. Single cells contain approximately 300 different chromosomes with allelic diversity far exceeding that typically harbored by single bacteria genera. Surveying all publicly available sediment sequence archives, we show that Achromatium is common worldwide, spanning temperature, salinity, pH, and depth ranges normally resulting in bacterial speciation. Although saline and freshwater Achromatium spp. appear phylogenetically separated, the genus Achromatium contains a globally identical, complete functional inventory regardless of habitat. Achromatium spp. cells from differing ecosystems (e.g., from freshwater to saline) are, unexpectedly, equally functionally equipped but differ in gene expression patterns by transcribing only relevant genes. We suggest that environmental adaptation occurs by increasing the copy number of relevant genes across the cell's hundreds of chromosomes, without losing irrelevant ones, thus maintaining the ability to survive in any ecosystem type. The functional versatility of Achromatium and its genomic features reveal alternative genetic and evolutionary mechanisms, expanding our understanding of the role and evolution of polyploidy in bacteria while challenging the bacterial species concept and drivers of bacterial speciation.

The Structure of the Lipid A of Gram-Negative Cold-Adapted Bacteria Isolated from Antarctic Environments.

Gram-negative Antarctic bacteria adopt survival strategies to live and proliferate in an extremely cold environment. Unusual chemical modifications of the lipopolysaccharide (LPS) and the main component of their outer membrane are among the tricks adopted to allow the maintenance of an optimum membrane fluidity even at particularly low temperatures. In particular, the LPS' glycolipid moiety, the lipid A, typically undergoes several structural modifications comprising desaturation of the acyl chains, reduction in their length and increase in their branching. The investigation of the structure of the lipid A from cold-adapted bacteria is, therefore, crucial to understand the mechanisms underlying the cold adaptation phenomenon. Here we describe the structural elucidation of the highly heterogenous lipid A from three psychrophiles isolated from Terra Nova Bay, Antarctica. All the lipid A structures have been determined by merging data that was attained from the compositional analysis with information from a matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS) and MS2 investigation. As lipid A is also involved in a structure-dependent elicitation of innate immune response in mammals, the structural characterization of lipid A from such extremophile bacteria is also of great interest from the perspective of drug synthesis and development inspired by natural sources.

Cell Architecture of the Giant Sulfur Bacterium Achromatium oxaliferum: Extra-cytoplasmic Localization of Calcium Carbonate Bodies.

Achromatium oxaliferum is a large sulfur bacterium easily recognized by large intracellular calcium carbonate bodies. Although these bodies often fill major parts of the cells' volume, their role and specific intracellular location are unclear. In this study, we used various microscopy and staining techniques to identify the cell compartment harboring the calcium carbonate bodies. We observed that Achromatium cells often lost their calcium carbonate bodies, either naturally or induced by treatments with diluted acids, ethanol, sodium bicarbonate and UV radiation which did not visibly affect the overall shape and motility of the cells (except for UV radiation). The water-soluble fluorescent dye fluorescein easily diffused into empty cavities remaining after calcium carbonate loss. Membranes (stained with Nile Red) formed a network stretching throughout the cell and surrounding empty or filled calcium carbonate cavities. The cytoplasm (stained with FITC and SYBR Green for nucleic acids) appeared highly condensed and showed spots of dissolved Ca2+ (stained with Fura-2). From our observations, we conclude that the calcium carbonate bodies are located in the periplasm, in extra-cytoplasmic pockets of the cytoplasmic membrane and are thus kept separate from the cell's cytoplasm. This periplasmic localization of the carbonate bodies might explain their dynamic formation and release upon environmental changes.

Multiple factors drive the abundance and diversity of the diazotrophic community in typical farmland soils of China.

Biological nitrogen fixation plays an important role in nitrogen cycling by transferring atmospheric N2 to plant-available N in the soil. However, the diazotrophic activity and distribution in different types of soils remain to be further explored. In this study, 152 upland soils were sampled to examine the diazotrophic abundance, nitrogenase activity, diversity and community composition by quantitative polymerase chain reaction, acetylene reduction assay and the MiSeq sequencing of nifH genes, respectively. The results showed that diazotrophic abundance and nitrogenase activity varied among the three soil types. The diazotrophic community was mainly dominated by Bradyrhizobium, Azospirillum, Myxobacter, Desulfovibrio and Methylobacterium. The symbiotic diazotroph Bradyrhizobium was widely distributed among soils, while the distribution of free-living diazotrophs showed large variation and was greatly affected by multiple factors. Crop type and soil properties directly affected the diazotrophic ɑ-diversity, while soil properties, climatic factors and spatial distance together influenced the diazotrophic community. Network structures were completely different among all three types of soils, with most complex interactions observed in the Red soil. These findings suggest that diazotrophs have various activities and distributions in the three soil types, which played different roles in nitrogen input in agricultural soil in China, being driven by multiple environmental factors.

Intracellular calcite and sulfur dynamics of Achromatium cells observed in a lab-based enrichment and aerobic incubation experiment.

We investigated the intracellular dynamics of calcite and sulfur in the large sulfur-oxidizing, calcite-accumulating bacterium Achromatium, with an emphasis on oxygen exposure as a physiological control. For this purpose, morphological changes and possible accretion mechanisms of calcite granules in cells that were freshly collected from natural Achromatium-containing sediment were compared to cells from the same source after prolonged exposure to atmospheric oxygen. Intracellular sulfur is oxidized and removed in response to oxygen exposure. Calcite granules also undergo distinct oxygen-related dynamics; they alternate between tightly packaged, smooth granules with narrow but sharply defined interstitial spaces in atmospheric oxygen-exposed cells, and more loosely packaged granules with irregular, bumpy surface texture and larger interstitial spaces in cells that were not artificially exposed to oxygen. These results suggest that morphological changes of the calcite granules reflect their changing physiological role inside the cell. Sulfur oxidation and calcite dissolution appear to be linked in that proton generation during sulfur oxidation is buffered by gradual calcite erosion, visible in the smooth, rounded surface morphology observed after oxygen exposure. Our results support the hypothesis that calcite dynamics buffer the intracellular pH fluctuations linked to electron acceptor limitation during proton-consuming sulfide oxidation to sulfur, and electron acceptor abundance during proton-generating sulfur oxidation to sulfate.

How bacteria hack the matrix and dodge the bullets of immunity.

Haemophilus influenzae, Moraxella catarrhalis and Pseudomonas aeruginosa are common Gram-negative pathogens associated with an array of pulmonary diseases. All three species have multiple adhesins in their outer membrane, i.e. surface structures that confer the ability to bind to surrounding cells, proteins or tissues. This mini-review focuses on proteins with high affinity for the components of the extracellular matrix such as collagen, laminin, fibronectin and vitronectin. Adhesins are not structurally related and may be lipoproteins, transmembrane porins or large protruding trimeric auto-transporters. They enable bacteria to avoid being cleared together with mucus by attaching to patches of exposed extracellular matrix, or indirectly adhering to epithelial cells using matrix proteins as bridging molecules. As more adhesins are being unravelled, it is apparent that bacterial adhesion is a highly conserved mechanism, and that most adhesins target the same regions on the proteins of the extracellular matrix. The surface exposed adhesins are prime targets for new vaccines and the interactions between proteins are often possible to inhibit with interfering molecules, e.g heparin. In conclusion, this highly interesting research field of microbiology has unravelled host-pathogen interactions with high therapeutic potential.

Analysis of the Anti-Cancer Effects of Cincau Extract (Premna oblongifolia Merr) and Other Types of Non-Digestible Fibre Using Faecal Fermentation Supernatants and Caco-2 Cells as a Model of the Human Colon.

Green cincau (Premna oblongifolia Merr) is an Indonesian food plant with a high dietary fibre content. Research has shown that dietary fibre mixtures may be more beneficial for colorectal cancer prevention than a single dietary fibre type. The aim of this study was to investigate the effects of green cincau extract on short chain fatty acid (SCFA) production in anaerobic batch cultures inoculated with human faecal slurries and to compare these to results obtained using different dietary fibre types (pectin, inulin, and cellulose), singly and in combination. Furthermore, fermentation supernatants (FSs) were evaluated in Caco-2 cells for their effect on cell viability, differentiation, and apoptosis. Cincau increased total SCFA concentration by increasing acetate and propionate, but not butyrate concentration. FSs from all dietary fibre sources, including cincau, reduced Caco-2 cell viability. However, the effects of all FSs on cell viability, cell differentiation, and apoptosis were not simply explainable by their butyrate content. In conclusion, products of fermentation of cincau extracts induced cell death, but further work is required to understand the mechanism of action. This study demonstrates for the first time that this Indonesian traditional source of dietary fibre may be protective against colorectal cancer.

Proteins with CHADs (Conserved Histidine α-Helical Domains) Are Attached to Polyphosphate Granules In Vivo and Constitute a Novel Family of Polyphosphate-Associated Proteins (Phosins).

On the basis of bioinformatic evidence, we suspected that proteins with a CYTH (CyaB thiamine triphosphatase) domain and/or a CHAD (conserved histidine α-helical domain) motif might represent polyphosphate (polyP) granule-associated proteins. We found no evidence of polyP targeting by proteins with CYTH domains. In contrast, two CHAD motif-containing proteins from Ralstonia eutropha H16 (A0104 and B1017) that were expressed as fusions with enhanced yellow fluorescent protein (eYFP) colocalized with polyP granules. While the expression of B1017 was not detectable, the A0104 protein was specifically identified in an isolated polyP granule fraction by proteome analysis. Moreover, eYFP fusions with the CHAD motif-containing proteins MGMSRV2-1987 from Magnetospirillum gryphiswaldense and PP2307 from Pseudomonas putida also colocalized with polyP granules in a transspecies-specific manner. These data indicated that CHAD-containing proteins are generally attached to polyP granules. Together with the findings from four previously polyP-attached proteins (polyP kinases), the results of this study raised the number of polyP-associated proteins in R. eutropha to six. We suggest designating polyP granule-bound proteins with CHAD motifs as phosins (phosphate), analogous to phasins and oleosins that are specifically bound to the surface of polyhydroxyalkanoate (PHA) granules in PHA-accumulating bacteria and to oil droplets in oil seed plants, respectively.IMPORTANCE The importance of polyphosphate (polyP) for life is evident from the ubiquitous presence of polyP in all species on earth. In unicellular eukaryotic microorganisms, polyP is located in specific membrane-enclosed organelles, called acidocalcisomes. However, in most prokaryotes, polyP is present as insoluble granules that have been designated previously as volutin granules. Almost nothing is known regarding the macromolecular composition of polyP granules. Particularly, the absence or presence of cellular compounds on the surface of polyP granules has not yet been investigated. In this study, we identified a novel class of proteins that are attached to the surface of polyP granules in three model species of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria These proteins are characterized by the presence of a CHAD (conserved histidine α-helical domain) motif that functions as a polyP granule-targeting signal. We suggest designating CHAD motif-containing proteins as phosins [analogous to phasins for poly(3-hydroxybutyrate)-associated proteins and to oleosins for oil droplet-associated proteins in oil seed plants]. The expression of phosins in different species confirmed their polyP-targeting function in a transspecies-specific manner. We postulate that polyP granules in prokaryotic species generally have a complex surface structure that consists of one to several polyP kinases and phosin proteins. We suggest differentiating polyP granules from acidocalcisomes by designating them as polyphosphatosomes.

Pacificimonas aurantium sp. nov., Isolated from the Seawater of the Pacific Ocean.

A Gram-negative bacterium, denoted JLT2012(T), was isolated from the surface water of the Pacific Ocean. This aerobic bacterium was rod shaped and devoid of flagella, displayed gliding motility, and grew in characteristic orange colonies. The bacterium contained ubiquinone Q-10 as the major respiratory quinone, and spermidine and spermine as the major polyamine compounds. The dominant fatty acids were C18:1ω7c and/or C18:1ω6c (34.7 %), C16:0 (21.3 %), and C18:0 (15.9 %), whereas the polar lipids consisted mainly of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four sphingoglycolipids, and several unknown glycolipids. The G + C content DNA was found to be 65.5 mol%. Comparative 16S rRNA gene sequence analysis revealed that strain JLT2012(T) formed a distinct lineage within the genus Pacificimonas (formerly known as Pacificamonas) and shared the highest sequence similarity with the type strain of Pacificimonas flava JLT2015(T) (96.0 %). Data combined from different studies on the phenotypic, phylogenetic, and genomic characteristics indicated that strain JLT2012(T) is a representative of a novel species within Pacificimonas for which the name Pacificimonas aurantium sp. nov. (type strain JLT2012(T)=LMG 27361(T)=CGMCC 1.12399(T)) is proposed.

Traqueobronquitis por Elizabethkingia meningoseptica en una paciente portadora de traqueostomía / Tracheobronchitis by Elizabethkingia meningoseptica in a patient with tracheostomy

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Gram-negative enteric rods associated to early implant failure and peri-implantitis: case report and systematic literature review / Bacilos entéricos gram-negativos asociados a fracaso temprano de implantes y peri-implantitis: reporte de un caso y revisión sistemática de literatura

The microbiota associated with failed implants includes Pseudomonas and Gram-negative enteric rods. The present study reports a case of Escherichia coli associated to early implant failed that was resistant in vitro to doxycycline, amoxicillin, metronidazole, and clindamycin, but was susceptible in vitro to ciprofloxacin and aminoglycosides. The literature concerning the prevalence of the opportunistic microorganisms in early implant failure and peri-implantitis patients, and the usual treatment of these patients harboring Pseudomonas and enteric rods was also revised.

La microbiota asociada con los implantes fallidos incluye Pseudomonas y bacilos entéricos Gram-negativos. En el presente estudio se informa acerca de un caso de Escherichia coli asociada a un fallo temprano del implante resistente in vitro a la doxiciclina, amoxicilina, metronidazol y clindamicina, pero susceptible in vitro a ciprofloxacina y aminoglucósidos. Se realizó una revisión de la literatura sobre la prevalencia de los microorganismos oportunistas en pacientes con insuficiencia temprana del implante y periimplantitis, y el tratamiento habitual de estos pacientes portadores de Pseudomonas y bacilos entéricos.

[Selection of a community of acidochemolithotrophic microorganisms with a high oxidation rate of pyrrhotite- containing sulphide ore flotatation concentrate].

A community of acidochemolithotrophic microorganisms with a high oxidation rate of pyrrhotite-containing sulphide ore flotation concentrate was selected. The Acidithiobacillus caldus OP-1 and Ferroplasma acidiphilum OP-2 cultures were identified to be dominating members. The presence of the Acidithio- bacillusferrooxidans OP-3, Leptospirillumferriphilum OP-4, and Sulfobacillus thermosulfidooxidans OP-5 cultures in the community's composition was also mentioned. The analysis results of solid residues of the process showed a greater elemental sulfur oxidation level and gold recovery when the initial pH value in tank I was maintained at a level of 1.8-2.0 (90.5%) rather than 1.6-1.8 (86.3%).

Microbial degradation of furanic compounds: biochemistry, genetics, and impact.

Microbial metabolism of furanic compounds, especially furfural and 5-hydroxymethylfurfural (HMF), is rapidly gaining interest in the scientific community. This interest can largely be attributed to the occurrence of toxic furanic aldehydes in lignocellulosic hydrolysates. However, these compounds are also widespread in nature and in human processed foods, and are produced in industry. Although several microorganisms are known to degrade furanic compounds, the variety of species is limited mostly to Gram-negative aerobic bacteria, with a few notable exceptions. Furanic aldehydes are highly toxic to microorganisms, which have evolved a wide variety of defense mechanisms, such as the oxidation and/or reduction to the furanic alcohol and acid forms. These oxidation/reduction reactions constitute the initial steps of the biological pathways for furfural and HMF degradation. Furfural degradation proceeds via 2-furoic acid, which is metabolized to the primary intermediate 2-oxoglutarate. HMF is converted, via 2,5-furandicarboxylic acid, into 2-furoic acid. The enzymes in these HMF/furfural degradation pathways are encoded by eight hmf genes, organized in two distinct clusters in Cupriavidus basilensis HMF14. The organization of the five genes of the furfural degradation cluster is highly conserved among microorganisms capable of degrading furfural, while the three genes constituting the initial HMF degradation route are organized in a highly diverse manner. The genetic and biochemical characterization of the microbial metabolism of furanic compounds holds great promises for industrial applications such as the biodetoxifcation of lignocellulosic hydrolysates and the production of value-added compounds such as 2,5-furandicarboxylic acid.

Fatty Acids of Chthonomonas calidirosea, of a novel class Chthonomonadetes from a recently described phylum Armatimonadetes.

A Gram-negative, aerobic, pink-pigmented, rod-shaped bacterium Chthonomonas calidirosea (strain T49(T)) with an optimal temperature for growth of 68 °C, isolated from soil samples from Hell's Gate in the Tikitere geothermal system (New Zealand), was the first cultivated bacterium of the novel phylum Armatimonadetes (formerly candidate division OP10). The lipid composition of C. calidirosea presents a number of unusual features both in the fatty acids and polar lipids. This contribution reports on the fatty acid profile of C. calidirosea. Transmethylation of bacterial biomass yielded fatty acid methyl esters and hydrocarbons, including squalene, partially hydrogenated squalenes, and diploptene. The only type of unsaturation found in C. calidirosea fatty acids was cis-Δ5, as revealed by GCMS of dimethyl disulfide (DMDS) adducts, and the lack of trans-unsaturation absorbance at 960-980 cm(-1) in the IR spectrum of fatty acids methyl esters. An unidentified component X with ECL 16.86 (BP1) and ECL 17.27 (BP20) was also observed, with molecular ion at m/z 282 ("17:1"). X did not form DMDS adducts, nor was affected by mild hydrogenation conditions, indicating the likely presence of a ring rather than unsaturation. The presence of a cyclopropane ring with cis-stereochemistry was confirmed by the (1)H-NMR spectrum. Hydrogenation of X in acetic acid resulted in formation of straight chain 17:0, 5-methyl- and 6-methyl-16:0 fatty acid methyl esters, thus confirming the structure of a novel 5,6-methylene hexadecanoic acid. The major fatty acids of a solid media-grown C. calidirosea were as follows (in weight % of total fatty acids): 16:0 (25.8), i17:0 (19.3), ai17:0 (13.5), 16:1∆5 (8.8), i17:1∆5 (6.8), 5,6-methylene 16:0 (5.2), i16:0 (4.4), 18:0 (3.6), 18:1∆5 (3.2).

[Identification and denitrification characteristics of a thermophilic aerobic denitrifier].

A bacterial strain TAD1 with high nitrogen removal efficiency was isolated from biofilm of the biotrickling filter of a coal-fired power plant by thermophilic domestication. This bacterium was Gram negative, short rod, with the size of (0.67-0.89) microm x (1.03-1.41) microm. It was identified as Chelatococcus sp. according to its physiological properties and the analysis of its 16S rDNA gene. Studied on its function of aerobic denitrification at the temperature of 50 degrees C, the results showed that nitrate in the culture media was efficiently removed from 63.79 mg/L to 0.46 mg/L, and the nitrogen removal efficiency was up to 99.12% in 24 hours, and no nitrite was observed during the incubation, the major end product of the denitrification was nitrogen. During the denitrification of TAD1, the pH in the culture medium gradually increases, while the oxidation-reduction potential gradually decreases. The factors affecting aerobic denitrification by strain TAD1 were also discussed, indicating that the most suitable pH value for aerobic denitrification was 7.0-9.0, and the DO was 2.1-7.2 mg/L.

Cadherin domains in the polysaccharide-degrading marine bacterium Saccharophagus degradans 2-40 are carbohydrate-binding modules.

The complex polysaccharide-degrading marine bacterium Saccharophagus degradans strain 2-40 produces putative proteins that contain numerous cadherin and cadherin-like domains involved in intercellular contact interactions. The current study reveals that both domain types exhibit reversible calcium-dependent binding to different complex polysaccharides which serve as growth substrates for the bacterium.

Haloferula luteola sp. nov., an endophytic bacterium isolated from the root of a halophyte, Rosa rugosa, and emended description of the genus Haloferula.

A Gram-negative, non-spore-forming, endophytic bacterium, strain YC6886(T), was isolated from the root of a halophyte, Rosa rugosa, which inhabits coastal areas of Namhae Island off the southern coast of Korea. Cells were non-motile, obligately aerobic rods and formed pale-yellow colonies. The isolate grew at 4-32 °C (optimum 25-28 °C) and at pH 6.5-9.5 (optimum pH 7.5) and grew optimally with 2-3 % (w/v) NaCl, but NaCl was not an absolute requirement for growth. Strain YC6886(T) produced yellow carotenoid pigments. Strain YC6886(T) exhibited the highest 16S rRNA gene sequence similarity with Haloferula sargassicola MN1-1037(T) (97.4 %). Sequence similarities between strain YC6886(T) and other members of the genus Haloferula were 93.9-94.7 %. DNA-DNA relatedness between strain YC6886(T) and H. sargassicola KCTC 22202(T) and Haloferula rosea KCTC 22201(T) was 27 and 15 %, respectively. The major fatty acids were iso-C(14 : 0), C(16 : 0) and C(16 : 1)ω9c and minor components were C(14 : 0), C(18 : 0) and anteiso-C(15 : 0). The major respiratory quinone was menaquinone 9 and the DNA G+C content was 58.5 mol%. The polar lipid profile was composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unknown phospholipid and an unknown phosphoglycolipid. On the basis of phenotypic, chemotaxonomic, DNA-DNA hybridization and phylogenetic analysis, strain YC6886(T) represents a novel species in the genus Haloferula, for which the name Haloferula luteola sp. nov. is proposed. The type strain is YC6886(T) ( = KCTC 22447(T)  = DSM 21608(T)). An emended description of the genus Haloferula is also presented.

Microbial utilization of the industrial wastewater pollutants 2-ethylhexylthioglycolic acid and iso-octylthioglycolic acid by aerobic gram-negative bacteria.

Industrial wastewater from the production of sulfur containing esters and the resulting products of this synthesis, 2-ethylhexylthioglycolic acid (EHTG) and iso-octylthioglycolic acid (IOTG), were deployed in this study to enrich novel bacterial strains, since no wastewater and EHTG or IOTG degrading microorganisms were hitherto described or available. In addition, nothing is known about the biodegradation of these thiochemicals. The effect of this specific wastewater on the growth behaviour of microorganisms was investigated using three well-known Gram-negative bacteria (Escherichia coli, Pseudomonas putida, and Ralstonia eutropha). Concentrations of 5% (v/v) wastewater in complex media completely inhibited growth of these three bacterial strains. Six bacterial strains were successfully isolated, characterized and identified by sequencing their 16S rRNA genes. Two isolates referred to as Achromobacter sp. strain MT-E3 and Pseudomonas sp. strain MT-I1 used EHTG or IOTG, respectively, as well as the wastewater as sole source of carbon and energy for weak growth. More notably, both isolates removed these sulfur containing esters in remarkable amounts from the cultures supernatant. One further isolate was referred to as Klebsiella sp. strain 58 and exhibited an unusual high tolerance against the wastewater's toxicity without utilizing the contaminative compounds. If cultivated with gluconic acid as additional carbon source, the strain grew even in presence of more than 40% (v/v) wastewater. Three other isolates belonging to the genera Bordetella and Pseudomonas tolerated these organic sulfur compounds but showed no degradation abilities.

Phenanthrene metabolites bound to soil organic matter by birnessite following partial biodegradation.

The hypothesis that phenanthrene, an aromatic compound without a hydroxyl group, can form nonextractable residues in soil with the aid of phenanthrene-biodegrading bacteria and birnessite was tested. The mutant strain Sphingobium yanoikuyae B8/36 successfully accumulated cis-phenanthrene dihydrodiol, and the intermediate was readily radicalized and coupled into soil organic matter in the presence of birnessite. Phenanthrene and the intermediate disappeared from the soil in 96 h in the presence of birnessite, but the intermediate accumulation occurred without birnessite. By determining the total organic carbon contents before and after birnessite treatment, it could be seen that birnessite did not mineralize cis-phenanthrene dihydrodiol. Fourier transform infrared and ultraviolet analyses suggest instead that the intermediate was incorporated into the soil organic matter, forming nonextractable, bound residues. Increases in the aromaticity and pH in birnessite-treated soil also present more evidence for bound residue formation. The soil in which bound residue formed did not exhibit an acute toxicity of phenanthrene, but evidence indicated that such toxicity existed in the freshly spiked soil. In addition, a long-term column test revealed that the bound residues could not be eluted by the combination of water, 80% methanol, and U.S. Environmental Protection Agency Toxicity Characteristic Leaching Procedure solution (pH 2.88) for four months, implying stability of the nonextractable residues in the soil.