Max M. Häggblom

Embryonic exposure to tetrabromobisphenol A and its metabolites, bisphenol A and tetrabromobisphenol A dimethyl ether disrupts normal zebrafish (Danio rerio) development and matrix metalloproteinase expression.

Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant that is persistent in the environment and detected in human serum and breast milk. TBBPA is microbiologically transformed in anaerobic environments to bisphenol A (BPA) and in aerobic environments to TBBPA dimethyl ether (TBBPA DME). Despite the detection of TBBPA DME in the environment, the resulting toxicity is not known. The relative toxicity of TBBPA, BPA and TBBPA DME was determined using embryonic exposure of zebrafish, with BPA and TBBPA DME exhibiting lower potency than TBBPA. TBBPA exposure resulted in 100% mortality at 3 (1.6mg/L) and 1.5μM (0.8mg/L), whereas BPA and TBBPA DME did not result in significant embryonic mortality in comparison to controls. While all three caused edema and hemorrhage, only TBBPA specifically caused decreased heart rate, edema of the trunk, and tail malformations. Matrix metalloproteinase (MMP) expression was measured due to the role of these enzymes in the remodeling of the extracellular matrix during tissue morphogenesis, wound healing and cell migration. MMP-2, -9 and -13 expression increased (2-8-fold) after TBBPA exposure followed by an increase in the degradation of collagen I and gelatin. TBBPA DME exposure resulted in only a slight increase (less than 2-fold) in MMP expression and did not significantly increase enzymatic activity. These data suggest that TBBPA is more potent than BPA or TBBPA DME and indicate that the trunk and tail phenotypes seen after TBBPA exposure could be due in part to alteration of proper MMP expression and activity.

EXOTIC PLANT SPECIES ALTER THE MICROBIAL COMMUNITY STRUCTURE AND FUNCTION IN THE SOIL

Exotic plant species are increasingly becoming the focus of research and have been identified as a component of human-induced global change. Successful invaders may alter soil conditions, but the effect of exotic species on soil microbial communities has not been studied. We studied two exotic understory plant species (Japanese barberry [Berberis thunbergii] and Japanese stilt grass [Microstegium vimineum]) in hardwood forests in northern New Jersey, USA. We sampled bulk and rhizosphere soils under the two exotic species, as well as under a co-occurring native species (blueberry [Vaccinium spp.]). We indexed the structure (by measuring phospholipid fatty acid [PLFA] profiles) and function (by measuring enzyme activities and substrate-induced respiration [SIR] profiles) of microbial communities in the sampled soils. Soils under the three species differed in microbial community structure and function. These differences were observed in both the rhizosphere and bulk soil samples. Differences in the structural variables were correlated to differences in the functional variables as demonstrated by canonical correlation analysis. These results indicate that successful exotic invasive species can have profound effects on the microbial community of the soil.

Experimental analysis of the effect of exotic and native plant species on the structure and function of soil microbial communities

Invasions of exotic plant species are among the most pervasive and important threats to natural ecosystems, however, the effects of plant invasions on soil processes and the soil biota have rarely been investigated. We grew two exotic and a native under-story plant species in the same mineral soil from a non-invaded forest stand in order to test whether observed differences in the field could be experimentally produced in the greenhouse. We characterized changes in the soil microbial community structure (as indexed by PLFAs) and function (as indexed by enzyme activities and SIR), as well as changes in potential nitrogen mineralization rates. We found that the invasion of two very dissimilar exotic species into the under-story of deciduous forests in eastern North America can rapidly cause changes in most of the studied soil properties. At the end of the three-month incubation, soils under the exotic species had significantly different PLFA, enzyme and SIR profiles than both initial soils and soils where native shrubs had been grown. We also observed a significant increase in pH and nitrification rates under one of the exotic plants. Such changes in the soil are potentially long-term (e.g. changes in soil pH) and are therefore likely to promote the re-invasion of these and other exotics. Both management of exotic plant invasions and the restoration of native communities must take into account exotic species effects on the soil.

Isolation and characterization of polycyclic aromatic hydrocarbon-degrading bacteria associated with the rhizosphere of salt marsh plants.

ABSTRACT Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from contaminated estuarine sediment and salt marsh rhizosphere by enrichment using either naphthalene, phenanthrene, or biphenyl as the sole source of carbon and energy. Pasteurization of samples prior to enrichment resulted in isolation of gram-positive, spore-forming bacteria. The isolates were characterized using a variety of phenotypic, morphologic, and molecular properties. Identification of the isolates based on their fatty acid profiles and partial 16S rRNA gene sequences assigned them to three main bacterial groups: gram-negative pseudomonads; gram-positive, non-spore-forming nocardioforms; and the gram-positive, spore-forming group,Paenibacillus. Genomic digest patterns of all isolates were used to determine unique isolates, and representatives from each bacterial group were chosen for further investigation. Southern hybridization was performed using genes for PAH degradation fromPseudomonas putida NCIB 9816-4, Comamonas testosteroni GZ42, Sphingomonas yanoikuyae B1, andMycobacterium sp. strain PY01. None of the isolates from the three groups showed homology to the B1 genes, only two nocardioform isolates showed homology to the PY01 genes, and only members of the pseudomonad group showed homology to the NCIB 9816-4 or GZ42 probes. The Paenibacillus isolates showed no homology to any of the tested gene probes, indicating the possibility of novel genes for PAH degradation. Pure culture substrate utilization experiments using several selected isolates from each of the three groups showed that the phenanthrene-enriched isolates are able to utilize a greater number of PAHs than are the naphthalene-enriched isolates. Inoculating two of the gram-positive isolates to a marine sediment slurry spiked with a mixture of PAHs (naphthalene, fluorene, phenanthrene, and pyrene) and biphenyl resulted in rapid transformation of pyrene, in addition to the two- and three-ringed PAHs and biphenyl. This study indicates that the rhizosphere of salt marsh plants contains a diverse population of PAH-degrading bacteria, and the use of plant-associated microorganisms has the potential for bioremediation of contaminated sediments.

Microbial community structure and activity in arsenic‐, chromium‐ and copper‐contaminated soils

Microbial community structure, potential microbial activity and As resistance were affected by arsenic (As), chromium (Cr) and copper (Cu) contamination in soils of abandoned wood impregnating plants. Contaminated soils differed in the concentrations of soil acid-soluble and total water-soluble As, Cr and Cu, and in the concentration of bioavailable As analyzed with a bacterial sensor. Phospholipid fatty acid (PLFA) and 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) profiles indicated that exposure to high metal contamination or subsequent effects of this exposure permanently changed microbial community structure. The total number of colony forming units (CFU) was not affected by metal contamination and the As(V)-resistant bacterial ratio to total heterotrophic plate counts was high (0.5-1.1) and relatively independent of the concentration of As. In contrast, the proportion of As(III)-resistant bacteria was dependent on the concentration of As in the soils and a significant positive relationship was found between the bioavailability of As and the proportion of As(III)-resistant bacteria. Dominant As-resistant isolates from contaminated soils were identified by their fatty acid methyl ester (FAME) profiles as Acinetobacter, Edwardsiella, Enterobacter, Pseudomonas, Salmonella and Serratia species. No differences were noted in glucose mineralization among contaminated and control soil samples within sites. Based on [(14)C]glucose mineralization the community was able to compensate for the reduced diversity. According to t-RFLP results, this was not due to a reversion towards the unexposed community, but mainly due to the appearance of new dominating species. This study, combining complementary culture-dependent and -independent methods, suggests that microbes are able to respond to soil metal contamination and maintain metabolic activity apparently through changes in microbial community structure and selection for resistance.

Isolation and Characterization of Diverse Halobenzoate-Degrading Denitrifying Bacteria from Soils and Sediments

ABSTRACT Denitrifying bacteria capable of degrading halobenzoates were isolated from various geographical and ecological sites. The strains were isolated after initial enrichment on one of the monofluoro-, monochloro-, or monobromo-benzoate isomers with nitrate as an electron acceptor, yielding a total of 33 strains isolated from the different halobenzoate-utilizing enrichment cultures. Each isolate could grow on the selected halobenzoate with nitrate as the terminal electron acceptor. The isolates obtained on 2-fluorobenzoate could use 2-fluorobenzoate under both aerobic and denitrifying conditions, but did not degrade other halobenzoates. In contrast, the 4-fluorobenzoate isolates degraded 4-fluorobenzoate under denitrifying conditions only, but utilized 2-fluorobenzoate under both aerobic and denitrifying conditions. The strains isolated on either 3-chlorobenzoate or 3-bromobenzoate could use 3-chlorobenzoate, 3-bromobenzoate, and 2- and 4-fluorobenzoates under denitrifying conditions. The isolates were identified and classified on the basis of 16S rRNA gene sequence analysis and their cellular fatty acid profiles. They were placed in nine genera belonging to either the α-, β-, or γ-branch of theProteobacteria, namely, Acidovorax,Azoarcus, Bradyrhizobium,Ochrobactrum, Paracoccus,Pseudomonas, Mesorhizobium,Ensifer, and Thauera. These results indicate that the ability to utilize different halobenzoates under denitrifying conditions is ubiquitously distributed in theProteobacteria and that these bacteria are widely distributed in soils and sediments.

Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus, Produced under Various Conditions

ABSTRACT This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 μg ml−1. The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 μg of cereulide g−1 (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 μg of cereulide ml−1 in liquid medium at room temperature (21 ± 1°C) in 1 to 3 days, during the stationary growth phase when cell density was 2 × 108 to 6 × 108 CFU ml−1. Cereulide production at temperatures at and below 8°C or at 40°C was minimal.

Fatty acids of fungi and nematodes: possible biomarkers in the soil food chain?

Abstract The fatty acid composition of 16 different soil fungi (ascomycetes, basidiomycetes, mitosporic fungi) and a fungal-feeding nematode Aphelenchoides sp. reared on seven fungal species was investigated. Additionally, fatty acid profiles of Aphelenchoides sp. and A. saprophilus grown on the same fungal food source,Agrocybe gibberosa, were compared. Thirteen predominant fatty acids were detected in the fungi. Most of them occurred in each of the 16 species, but relative quantities of individual fatty acids differed, in particular those of unsaturated ones. Most fungal species could be differentiated from each other on this basis. Our study revealed convergence, but also, differences, in the fatty acid composition of systematically related fungi, i.e. a taxonomic or phylogenetic relationship was not necessarily accompanied by similarity in fatty acid profiles. Nematodes comprised a wider fatty acid spectrum than fungi, with 17 predominant fatty acids, and a higher amount of long-chain, polyunsaturated acids than their fungal diet. Fungal host tissue may have supplied palmitic, oleic and linoleic acid present, whereas most of the long-chain unsaturated fatty acids were synthesized by the nematodes. Unsaturated fatty acids mainly belonged to the ω6 and 9 family, indicating a carboxyl-directed desaturation as a major metabolic pathway. The fungal host significantly affected the fatty acid profile of the nematodes. However, we could not assign individual fatty acids as biomarkers reflecting the dietary source, likely due to the considerable convergence within tested fungal species. Of the basidiomycetes analyzed Laccaria laccata, was distinctly separated from the others and this difference could also be detected in the nematodes showing the influence of food type. We conclude that the lipid composition of nematodes is controlled by both the nematode and its diet and that monitoring fatty acid patterns of soil animals may therefore provide a way to detect trophic interactions in belowground food webs.

INFLUENCE OF MICROBES ON THE MOBILIZATION, TOXICITY AND BIOMETHYLATION OF ARSENIC IN SOIL

To understand the effects of microbial activity on the mobilization and speciation of arsenic in soil, the cycling of arsenic was studied in microcosm experiments under laboratory conditions. Particular attention was paid to the biomethylation of arsenic and to the toxicity of inorganic and organic arsenic species for microbes. Microbes enhanced mobilization of arsenic from soil by 19-24% compared to formaldehyde inhibited controls. Formation of dissolved methylated arsenic species by microbes was low (< 0.1%) during the 5-day incubation. Even though methylation may function as a detoxification method, it was of minor importance in the soil tested.

PAH-degradation by Paenibacillus spp. and description of Paenibacillus naphthalenovorans sp. nov., a naphthalene-degrading bacterium from the rhizosphere of salt marsh plants.

Bacteria belonging to the genus Paenibacillus were isolated by enrichment from petroleum-hydrocarbon-contaminated sediment and salt marsh rhizosphere using either naphthalene or phenanthrene as the sole carbon source, and were characterized using phenotypic, morphological and molecular techniques. The isolates were grouped by their colony morphologies and polyaromatic hydrocarbon-degradation patterns. Phenanthrene-degrading isolates produced mottled colonies on solid media and were identified as P. validus by fatty acid methyl ester and 16S rRNA gene sequence analyses. In contrast, the naphthalene-degrading isolates with mucoid colony morphology were distantly related to Paenibacillus validus, according to fatty acid methyl ester and 16S rRNA gene sequence analyses. The predominant fatty acids of the mucoid isolates were 15:0 anteiso, 16:1omega11c, 16:0 and 17:0 anteiso, constituting, on average, 50.5, 12.0, 11.2 and 6.5% of the total, respectively. The G+C contents of their DNA ranged from 47 to 52 mol%. The 16S rDNA sequence analysis revealed the highest (< or = 94%) similarity to P. validus. In addition, phylogenetic analyses based on 16S rDNA sequences showed that the mucoid isolates formed a distinct cluster within Paenibacillus. DNA-DNA hybridization experiments showed only a 6% DNA similarity between the type strain of P. validus and mucoid strain PR-N1. On the basis of the morphological, phenotypic and molecular data, the naphthalene-degrading isolates merit classification as a new Paenibacillus species, for which the name Paenibacillus naphthalenovorans sp. nov. is proposed, with PR-N1T (= ATCC BAA-206T = DSM 14203T) as the type strain.