Re-inoculation of autoclaved soil as a non-sterile treatment for xenobiotic sorption and biodegradation studies

Autoclaved soil is commonly used for the study of xenobiotic sorption and as an abiotic control in biodegradation experiments. Autoclaving has been reported to alter soil physico-chemical and xenobiotic sorption characteristics such that comparison of autoclaved with non-autoclaved treatments in soil aging and bioavailability studies may yield misleading results. Experiments could be improved by using autoclaved soil re-inoculated with indigenous microorganisms as an additional or alternative non-sterile treatment for comparison with the sterile, autoclaved control. We examined the effect of autoclaving (3 x 1 h, 121°C, 103.5 KPa) on the physico-chemical properties of a silt loam soil (pH 7.2, 2.3% organic carbon) and the establishment of indigenous microorganisms reintroduced after autoclaving. Sterilisation by autoclaving significantly (p ≤ 0.05) decreased pH (0.6 of a unit) and increased concentrations of water-soluble organic carbon (WSOC; nontreated = 75 mg kg^-1; autoclaved = 1526 mg kg^-1). The initial first-order rate of ¹⁴C-2,4-dichloro-UL-phenol (2,4-DCP) adsorption to non-treated, autoclaved and re-inoculated soil was rapid (K₁ = 16.8-24.4 h^-1) followed by a slower linear phase (K₂). In comparison with autoclaved soil (0.038% day^-1), K₂ values were higher for re-inoculated (0.095% day^-1) and nontreated (0.181% day^-1) soil. This was attributed to a biological process. The Freundlich adsorption coefficient (K(f)) for autoclaved soil was significantly (p ≤ 0.05) higher than for re-inoculated or non-treated soil. Increased adsorption was attributed to autoclaving-induced changes to soil pH and solution composition. Glucose-induced respiration of autoclaved soil after re-inoculation was initially twice that in the non-treated control, but it decreased to control levels by day 4. This reduction corresponded to a depletion of WSOC. 2,4-DCP mineralisation experiments revealed that the inoculum of nonsterile soil (0.5 g) contained 2,4-DCP-degrading microorganisms capable of survival in autoclaved soil. The lag phase before detection of significant 2,4-DCP mineralisation was reduced (from 7 days to ≤3 days) by pre-incubation of re-inoculated soils for 7 and 14 days before 2,4-DCP addition. This was attributed to the preferential utilisation of WSOC prior to the onset of 2,4-DCP mineralisation. Cumulative ¹⁴CO₂ evolved after 21 days was significantly lower (p ≤ 0.05) from non-treated soil (25.3%) than re-inoculated soils (ca 45%). Experiments investigating sorption-biodegradation interactions of xenobiotics in soil require the physico-chemical properties of sterile and non-sterile treatments to be as comparable as possible. For fundamental studies, we suggest using re-inoculated autoclaved soil as an additional or alternative non-sterile treatment.

Biosensing 2,4-dichlorophenol toxicity during biodegradation by Burkholderia sp. RASC c2 in soil

Biodegradation of the model pollutant, 2,4-dichlorophenol (2,4-DCP) by Burkholderia sp. RASC c2, in contaminated soil was assessed by combining chemical analysis with a toxicity test using Escherichia coli HB101 pUCD607. E. coli HB101 pUCD607 was previously marked with luxCDABE genes, encoding bacterial bioluminescence and was used as an alternative to Microtox. Mineralization of ¹⁴C-2,4-DCP (196.2 μg g^-1 dry wt) in soil occurred rapidly after a 24 h lag. Correspondingly, 2,4-DCP concentrations in soil and soil water extracts decreased with time and concentrations in the latter were at background levels (<0.12 μg mL^-1) after day 2. Toxicity of soil water extracts to the lux-based biosensor also decreased with time. Mean light output of E. coli was stimulated by ~1.5 X control values in soil water extracts when concentrations of 2,4-DCP were approaching the limit of detection by HPLC but returned to values equivalent to those of controls when soil water 2,4-DCP concentrations were below the detection limit. No mineralization or microbial growth was detected in noninoculated microcosms. 2,4-DCP concentration in sterile controls decreased significantly with time as did toxicity to E. coli Lux-based E. coli was a sensitive biosensor of 2,4-DCP toxicity during biodegradation and results complemented chemical analysis.

Assessment of Anaerobic Biodegradation of Aromatic Hydrocarbons: The Impact of Molecular Biology Approaches

One of the most cost effective methods of pollution remediation is through natural attenuation where the resident microorganisms are responsible for the breakdown of pollutants (Dou et al. 2008). Other forms of bioremediation - such as analogue enrichment, composting and bio-venting - also use the microbes already present in a contaminated site to enhance the remediation process. In order for these approaches to be successful, in an industrial setting, some form of monitoring needs to take place enabling conclusions to be drawn about the degradation processes occurring. In this review we look at some key molecular biology techniques that have the potential to act as a monitoring tool for industries dealing with contaminated land. 

Engineering passive bioreactive barriers: risk-managing the legacy of industrial soil and groundwater pollution

Permeable reactive barriers are a technology that is one decade old, with most full-scale applications based on abiotic mechanisms. Though there is extensive literature on engineered bioreactors, natural biodegradation potential, and in situ remediation, it is only recently that engineered passive bioreactive barrier technology is being considered at the commercial scale to manage contaminated soil and groundwater risks. Recent full-scale studies are providing the scientific confidence in our understanding of coupled microbial (and genetic), hydrogeologic, and geochemical processes in this approach and have highlighted the need to further integrate engineering and science tools.

A microbial fuel cell in contaminated ground delineated by electrical self-potential and normalized induced polarization data

There is a growing interest in the use of geophysical methods to aid investigation and monitoring of complex biogeochemical environments, for example delineation of contaminants and microbial activity related to land contamination. We combined geophysical monitoring with chemical and microbiological analysis to create a conceptual biogeochemical model of processes around a contaminant plume within a manufactured gas plant site. Self-potential, induced polarization and electrical resistivity techniques were used to monitor the plume. We propose that an exceptionally strong (>800 mV peak to peak) dipolar SP anomaly represents a microbial fuel cell operating in the subsurface. The electromagnetic and electrical geophysical data delineated a shallow aerobic perched water body containing conductive gasworks waste which acts as the abiotic cathode of microbial fuel cell. This is separated from the plume below by a thin clay layer across the site. Microbiological evidence suggests that degradation of organic contaminants in the plume is dominated by the presence of ammonium and its subsequent degradation. We propose that the degradation of contaminants by microbial communities at the edge of the plume provides a source of electrons and acts as the anode of the fuel cell. We hypothesize that ions and electrons are transferred through the clay layer that was punctured during the trial pitting phase of the investigation. This is inferred to act as an electronic conductor connecting the biologically mediated anode to the abiotic cathode. Integrated electrical geophysical techniques appear well suited to act as rapid, low cost sustainable tools to monitor biodegradation.

Resolving genetic functions within microbial populations: In situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization

Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope (C-13)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 mu M. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment.

Variation in stable carbon isotope fractionation during aerobic degradation of phenol and benzoate by contaminant degrading bacteria

Variation in the natural abundance stable carbon isotope composition of respired CO2 and biomass has been measured for two types of aerobic bacteria found in contaminated land sites. Pseudomonas putida strain NCIMB 10015 was cultured on phenol and benzoate and Rhodococcus sp. I-1 was cultured on phenol. Results indicate that aerobic isotope fractionations of differing magnitudes occur during aerobic biodegradation of these substrates with an isotopic depletion in the CO2 (Delta(13)C(phenol-CO2)) as much as 3.7 parts per thousand and 5.6 parts per thousand for Pseudomonas putida and Rhodococcus sp. I-1 respectively. This observation has significant implications for the use of a stable isotope mass balance approach in monitoring degradation processes that rely on indigenous bacterial populations. The effects of the metabolic pathway utilised in degradation and inter-species variation on the magnitude of isotope fractionation are discussed. Possible explanations for the observed isotope fractionation include differences in the metabolic pathways utilised by the organisms and differences in specific growth rates and physiology. (C) 1999 Elsevier Science Ltd. All rights reserved.

Physicochemical and drug diffusion analysis of rifampicin containing polyethylene glycol-poly(epsilon-caprolactone) networks designed for medical device applications

This study reports the physicochemical and drug diffusion properties of rifampicin containing poly(epsilon-caprolactone) (PCL)/polyethylene glycol (PEG) networks, designed as bioactive biomaterials. Uniquely, the effects of the states of both rifampicin and PEG and the interplay between these components on these properties are described. PCL matrices containing rifampicin (1-5%, w/w) and PEG 200 (0-15%, w/w) were prepared by casting from an organic solvent (dichloromethane). The films were subsequently characterized in terms of their thermal/thermorheological, surface and tensile properties, biodegradation and drug diffusion/release properties. Incorporation of PEG and/or rifampicin significantly affected the tensile and surface properties of PCL, lowering the ultimate tensile strength, % elongation at break, Young modulus and storage and loss moduli. Both in the absence and presence of PEG, solubilisation of rifampicin within the crystalline domains of PCL was observed. PEG was present as a dispersed liquid phase. The release of rifampicin (3% loading) was unaffected by the presence of PEG. Similarly the release of rifampicin (5%) was unaffected by low concentrations of PEG (5-10%) however, at higher loadings, the release rate of rifampicin was enhanced by the presence of PEG. Rifampicin release (10% loading) was enhanced by the presence of PEG in a concentration dependent fashion. These observations were accredited to enhanced porosity of the matrix. In all cases, diffusion-controlled release of rifampicin occurred which was unaffected by polymer degradation. This study has uniquely illustrated the effect of hydrophilic pore formers on the physicochemical properties of PCL. Interestingly, enhanced diffusion controlled release was only observed from biomaterials containing high loadings of PEG and rifampicin (5, 10%), concentrations that were shown to affect the mechanical properties of the biomaterials. Care should therefore be shown when adopting this strategy to enhance release of bioactive agents from biomaterials. (C) 2011 Elsevier B.V. All rights reserved.

Degradation of carbon disulphide (CS2) in soils and groundwater from a CS2 -contaminated site

This study is the first investigation of biodegradation of carbon disulphide (CS₂) in soil that provides estimates of degradation rates and identifies intermediate degradation products and carbon isotope signatures of degradation. Microcosm studies were undertaken under anaerobic conditions using soil and groundwater recovered from CS₂-contaminated sites. Proposed degradation mechanisms were validated using equilibrium speciation modelling of concentrations and carbon isotope ratios. A first-order degradation rate constant of 1.25 × 10-2 h-1 was obtained for biological degradation with soil. Carbonyl sulphide (COS) and hydrogen sulphide (H₂S) were found to be intermediates of degradation, but did not accumulate in vials. A 13C/12C enrichment factor of -7.5 ± 0.8 ‰ was obtained for degradation within microcosms with both soil and groundwater whereas a 13C/12C enrichment factor of -23.0 ± 2.1 ‰ was obtained for degradation with site groundwater alone. It can be concluded that biological degradation of both CS2-contaminated soil and groundwater is likely to occur in the field suggesting that natural attenuation may be an appropriate remedial tool at some sites. The presence of biodegradation by-products including COS and H₂S indicates that biodegradation of CS₂ is occurring and stable carbon isotopes are a promising tool to quantify CS₂ degradation.

Survival and persistence of opportunistic Burkholderia species in host cells

Burkholderia are microorganisms that have a unique ability to adapt and survive in many different environments. They can also serve as biopesticides and be used for the biodegradation of organic compounds. Usually harmless while living in the soil, these bacteria are opportunistic pathogens of plants and immunocompromised patients, and occasionally infect healthy individuals. Some of the species in this genus can also be utilised as biological weapons. They all possess very large genomes and have two or more circular chromosomes. Their survival and persistence, not only in the environment but also in host cells, offers a remarkable example of bacterial adaptation.

The Influence of Aspect on the Biological Colonization of Stone in Northern Ireland.

The rate and type of biological colonization of stone is influenced by a wide array of environmental factors in addition to substrate characteristics. A series of experiments was designed to compare the rate and type of biological colonization of stone at varying locations over a 21-month time period. Exposure
trials were set up at nine different sites across Northern Ireland that covered a wide variety of environmental conditions. To determine aspect-related differences in colonization, blocks of Peakmoor sandstone and Portland limestone were placed on the north- and south-facing sides of purpose-designed exposure racks. Colorimetry and visual analysis were carried out on collected samples at increasing time intervals. Results showed significantly different rates of darkening and greening over time between north-facing and south-facing blocks, for both sandstone and limestone. This difference is likely to be representative of the fact that in Northern Ireland’s wet climate and northern-latitude position, the north face of a building will receive less direct sunlight. Therefore north-facing blocks, once wet, will remain damp for much longer than blocks on other façades. This slow-drying phenomenon is much more hospitable for biological colonization and continued growth than the hostile environment of rapid wetting and drying cycles experienced on the south face.