Associations of pseudomonas species and forage grasses enhance degradation of chlorinated benzoic acids in soil

Microorganisms and plants can be used as bioremediation agents to clean up contaminated soil sites in a cost effective and environmentally friendly manner. Furthermore, associations of plants and bacteria can act synergistically and thereby, eliminate difficulties encountered when using a single agent. The purpose of my thesis research was to develop and characterize associations of plants and bacteria that degraded chlorinated benzoic acids (CBA) in soil. The contaminants were used as model compounds because they are present in soils contaminated with polychlorinated biphenyls (PCB) or chlorinated pesticides. Sixteen forage grasses in combination with 12 bacterial inoculants were screened for their ability to promote the degradation of CBA in soil. The CBAs were added to soil as single isomers, or in tertiary mixtures. The effect of inoculants on the root associated microbial community was assessed by fatty acid methyl ester (FAME) profiles as well as carbon substrate utilization as determined by the Biolog system. In addition, inoculant stimulation of the CBA degradative activity of roots and/or rhizosphere soil was determined by in vitro and hydroponic systems. Degradative enzymes were isolated from root exudates through the use of ultrafiltration and chromatography. Five associations of plants and bacteria degraded CBA to a greater extent than plants without bacterial inoculants. Plant-bacterial associations that increased 2-chlorobenzoic acid (2CBA) degradation had little effect on di-chlorinated benzoic acid degradation. Furthermore, the effective inoculants altered the root-associated microbial community of Bromus biebersteinii and simultaneously increased the CBA degradative activity of roots. Although these inoculants had little effect on the microbial community composition of Elymus dauricus, they stimulated a plant enzyme capable of degrading 2CBA in the rhizosphere. Bacterial inoculants stimulated CBA degradation by altering the microbial community present on the root surface and thereby increasing the ability of this community to degrade CBA. Alternatively, inoculants stimulated the production of a plant enzyme(s) that degraded 2CBA. My research has demonstrated that specific interactions between plants and bacteria promote contaminant degradation in soil, and suggests that new remediation strategies can be developed based on such interactions

Strain-Specific Ureolytic Microbial Calcium Carbonate Precipitation

During a study of ureolytic microbial calcium carbonate (CaCO3) precipitation by bacterial isolates collected from different environmental samples, morphological differences were observed in the large CaCO3 crystal aggregates precipitated within bacterial colonies grown on agar. Based on these differences, 12 isolates were selected for further study. We hypothesized that the striking differences in crystal morphology were the result of different microbial species or, alternatively, differences in the functional attributes of the isolates selected. Sequencing of 16S rRNA genes showed that all of the isolates were phylogenetically closely related to the Bacillus sphaericus group. Urease gene diversity among the isolates was examined by using a novel application of PCR-denaturing gradient gel electrophoresis (DGGE). This approach revealed significant differences between the isolates. Moreover, for several isolates, multiple bands appeared on the DGGE gels, suggesting the apparent presence of different urease genes in these isolates. The substrate affinities (Km) and maximum hydrolysis rates (Vmax) of crude enzyme extracts differed considerably for the different strains. For certain isolates, the urease activity increased up to 10-fold in the presence of 30 mM calcium, and apparently this contributed to the characteristic crystal formation by these isolates. We show that strain-specific calcification occurred during ureolytic microbial carbonate precipitation. The specificity was mainly due to differences in urease expression and the response to calcium

Polycyclic Aromatic Hydrocarbon Release from a Soil Matrix in the In Vitro Gastrointestinal Tract

Soil ingestion is an important exposure route by which immobile soil contaminants enter the human body. We assessed polycyclic aromatic hydrocarbon (PAH) release from a contaminated soil, containing 49 mg PAH kg–1, using a SHIME (Simulator of the Human Intestinal Microbial Ecosystem) reactor comprising the stomach, duodenal, and colon compartments. Polycyclic aromatic hydrocarbon release was defined as that fraction remaining in the digest supernatant after centrifugation for 5 min at 1500 x g. The PAH release in the stomach digest was only 0.44% of the total PAH present in soil, resulting in PAH concentrations of 23 µg PAH L–1 chyme. The lower PAH releases in duodenum (0.13%) and colon (0.30%) digests, compared with the stomach digest, were thought to be attributed to combined complexation and precipitation with bile salts, dissolved organic matter, or colon microbiota. We studied these complexation processes in an intestinal suspension more in depth by preparing mixtures of 9-anthracenepropionic acid, a Bacillus subtilis culture, and cholin as model compounds for PAHs, organic matter, and bile salts, respectively. Bile salts or organic matter in the aqueous phase initially enhance PAH desorption from soil. However, desorbed PAHs may form large aggregates with bile and organic matter, lowering the freely dissolved PAH fraction in the supernatant. Using the model compounds, mathematical equations were developed and validated to predict PAH complexation processes in the gastrointestinal tract. Contaminant release and subsequent complexation in the gut is an important prerequisite to intestinal absorption and thus bioavailability of that contaminant. The data from this research may help in understanding the processes to which PAHs are subjected in the gastrointestinal tract, before intestinal absorption

Metal oxides and annealed metals as alternatives to metal salts for fixed-ratio metal mixture ecotoxicity tests in soil.

In soil metal ecotoxicology research, dosing is usually performed with metal salts, followed by leaching to remove excess salinity. This process also removes some metals, affecting metal mixture ratios as different metals are removed by leaching at different rates. Consequently, alternative dosing methods must be considered for fixed ratio metal mixture research. In this study three different metal mixture dosing methods (nitrate, oxide and annealed metal dosing) were examined for metal concentrations and toxicity. In the nitrate metal dosing method leaching reduced total metal retention and was affected by soil pH and cation exchange capacity (CEC). Acidic soils 3.22 (pH 3.4, CEC 8 meq/100g) and WTRS (pH 4.6, CEC 16 meq/100g) lost more than 75 and 64% of their total metals to leaching respectively while Elora (6.7 pH, CEC 21 meq/100g) and KUBC (pH 5.6, CEC 28 meq/100g) with higher pH and CEC only lost 13.6% and 12.2% total metals respectively. Metal losses were highest for Ni, Zn and Co (46.0%, 63.7% and 48.4% metal loss respectively) whereas Pb and Cu (5.6% and 20.0% metal loss respectively) were mostly retained, affecting mixture ratios. Comparatively, oxide and annealed metal dosing which do not require leaching had higher total metal concentrations, closer to nominal doses and maintained better mixture ratios (percent of nominal concentrations for the oxide metal dosing were Pb = 109.9%, Cu = 84.6%, Ni = 101.9%, Zn = 82.3% and Co = 97.8% and for the annealed metal dosing were Pb = 81.7%, Cu = 80.3%, Ni = 100.5%, Zn = 89.2% and Co = 101.3%). Relative to their total metal concentrations, nitrate metal dosing (lowest metal concentrations) was the most toxic followed by metal oxides dosing while the annealed dosing method was generally non-toxic. Due to the lack of toxicity of the annealed metals and their higher dosing effort, metal oxides, are the most appropriate of the tested dosing methods, for fixed-ratio metal mixtures studies with soil invertebrates