S Powell

Microbial community variation in pristine and polluted nearshore Antarctic sediments

Abstract Two molecular methods were used to investigate the microbial population of Antarctic marine sediments to determine the effects of petroleum and heavy metal pollution. Sediment samples were collected in a nested design from impacted and non-impacted locations. A detailed description of the diversity of the microbial population in two samples was obtained using 16S ribosomal DNA clone libraries constructed from an impacted and a non-impacted location. The clone libraries were very similar with the exception of two sequence clusters containing clones from only the impacted location. All samples were analysed by denaturing gradient gel electrophoresis. The band patterns generated were transformed into a presence/absence matrix and a multivariate approach was used to test for differences in the locations. Statistically significant differences were observed both between and within locations. Impacted locations showed a greater variability within themselves than the control locations. Correlations between the community patterns and environmental variables suggested that pollution was one of a number of factors affecting the microbial community composition.

Using Real-Time PCR to Assess Changes in the Hydrocarbon-Degrading Microbial Community in Antarctic Soil During Bioremediation

A real-time polymerase chain reaction (PCR) method to quantify the proportion of microorganisms containing alkane monooxygenase was developed and used to follow changes in the microbial community in hydrocarbon-contaminated Antarctic soil during a bioremediation field trial. Assays for the alkB and rpoB genes were validated and found to be both sensitive and reproducible (less than 2% intrarun variation and 25–38% interrun variation). Results from the real-time PCR analysis were compared to analysis of the microbial population by a culture-based technique [most probable number (MPN) counts]. Both types of analysis indicated that fertilizer addition to hydrocarbon-contaminated soil stimulated the indigenous bacterial population within 1 year. The proportion of alkB containing microorganisms was positively correlated to the concentration of n-alkanes in the soil. After the concentration of n-alkanes in the soil decreased, the proportion of alkane-degrading microorganisms decreased, but the proportion of total hydrocarbon-degrading microorganisms increased, indicating another shift in the microbial community structure and ongoing biodegradation.

Light-stimulated growth of proteorhodopsin-bearing sea-ice psychrophile Psychroflexus torquis is salinity dependent

Proteorhodopsins (PRs) are commonly found in marine prokaryotes and allow microbes to use light as an energy source. In recent studies, it was reported that PR stimulates growth and survival under nutrient-limited conditions. In this study, we tested the effect of nutrient and salinity stress on the extremely psychrophilic sea-ice bacterial species Psychroflexus torquis, which possesses PR. We demonstrated for the first time that light-stimulated growth occurs under conditions of salinity stress rather than nutrient limitation and that elevated salinity is related to increased growth yields, PR levels and associated proton-pumping activity. PR abundance in P. torquis also is post-transcriptionally regulated by both light and salinity and thus could represent an adaptation to its sea-ice habitat. Our findings extend the existing paradigm that light provides an energy source for marine prokaryotes under stress conditions other than nutrient limitation.

Microbial communities on Australian modified atmosphere packaged Atlantic salmon.

The role of specific spoilage organisms (SSO) in products such as Atlantic salmon has been well documented. However, little is known about what other micro-organisms are present and these organisms may indirectly influence spoilage by their interactions with the SS0. We used a combination of culture-based and DNA-based methods to explore the microbial communities found on Atlantic salmon fillets packed in a modified atmosphere of carbon dioxide and nitrogen. After 15 days the communities were dominated by Shewanella spp. or Carnobacterium spp. and a variety of other genera were present in smaller numbers. Variability in the microbial community composition in packages processed on the same day was also observed. This was mostly due to differences in the presence of minor members of the community including species from genera such as Iodobacter, Serratia, Morganella and Yersinia. The combination of culture-based and culture-independent methods provided greater insight into the development of microbial communities on Atlantic salmon than would have been possible using only one method. This work highlights the potential importance of lactic acid bacteria (LAB) in fresh Atlantic salmon stored under modified atmosphere conditions.

Use of near infrared spectroscopy to predict microbial numbers on Atlantic salmon

The potential of a near infrared spectroscopy (NIR) method to detect as well as predict microbial spoilage on Atlantic salmon (Salmo salar) was investigated. Principal component analysis (PCA) of the NIR spectra showed clear separation between the fresh salmon fillets and those stored for nine days at 4°C indicating that NIR could detect spoilage. A partial least squares regression (PLS) prediction model for total aerobic plate counts after nine days was established using the NIR spectra collected when the fish was fresh to predict the number of bacteria that would be present nine days later. The calibration equation was good (R(2) = 0.95 and RMSE = 0.12 log cfu/g) although the error of the validation curve was larger (R(2) = 0.64 and RMSE = 0.32 log cfu/g). These results indicate that with further model development, it may be possible to use NIR to predict bacterial numbers, and hence shelf-life, in Atlantic salmon and other seafood.

Phytoremediation of hydrocarbon contaminants in subantarctic soils: An effective management option

Accidental fuel spills on world heritage subantarctic Macquarie Island have caused considerable contamination. Due to the islands high latitude position, its climate, and its fragile ecosystem, traditional methods of remediation are unsuitable for on-site clean up. We investigated the tolerance of a subantarctic native tussock grass, Poa foliosa (Hook. f.), to Special Antarctic Blend (SAB) diesel fuel and its potential to reduce SAB fuel contamination via phytoremediation. Toxicity of SAB fuel to P. foliosa was assessed in an 8 month laboratory growth trial under growth conditions which simulated the islands environment. Single seedlings were planted into 1 L pots of soil spiked with SAB fuel at concentrations of 1000, 5 000, 10,000, 2000 and 40,000 mg/kg (plus control). Plants were harvested at 0, 2, 4 and 8 months and a range of plant productivity endpoints were measured (biomass production, plant morphology and photosynthetic efficiency). Poa foliosa was highly tolerant across all SAB fuel concentrations tested with respect to biomass, although higher concentrations of 20,000 and 40,000 mg SAB/kg soil caused slight reductions in leaf length, width and area. To assess the phytoremediation potential of P. foliosa (to 10 000 mg/kg), soil from the planted pots was compared with that from paired unplanted pots at each SAB fuel concentration. The effect of the plant on SAB fuel concentrations and the associated microbial communities found within the soil (total heterotrophs and hydrocarbon degraders) were compared between planted and unplanted treatments at the 0, 2, 4 and 8 month harvest periods. The presence of plants resulted in significantly less SAB fuel in soils at 2 months and a return to background concentration by 8 months. Microbes did not appear to be the sole driving force behind the observed hydrocarbon loss. This study provides evidence that phytoremediation using P. foliosa is a valuable remediation option for use at Macquarie Island, and may be applicable to the management of fuel spills in other cold climate regions.

Extensive Gene Acquisition in the Extremely Psychrophilic Bacterial Species Psychroflexus torquis and the Link to Sea-Ice Ecosystem Specialism

Sea ice is a highly dynamic and productive environment that includes a diverse array of psychrophilic prokaryotic and eukaryotic taxa distinct from the underlying water column. Because sea ice has only been extensive on Earth since the mid-Eocene, it has been hypothesized that bacteria highly adapted to inhabit sea ice have traits that have been acquired through horizontal gene transfer (HGT). Here we compared the genomes of the psychrophilic bacterium Psychroflexus torquis ATCC 700755T, associated with both Antarctic and Arctic sea ice, and its closely related nonpsychrophilic sister species, P. gondwanensis ACAM 44T. Results show that HGT has occurred much more extensively in P. torquis in comparison to P. gondwanensis. Genetic features that can be linked to the psychrophilic and sea ice-specific lifestyle of P. torquis include genes for exopolysaccharide (EPS) and polyunsaturated fatty acid (PUFA) biosynthesis, numerous specific modes of nutrient acquisition, and proteins putatively associated with ice-binding, light-sensing (bacteriophytochromes), and programmed cell death (metacaspases). Proteomic analysis showed that several genes associated with these traits are highly translated, especially those involved with EPS and PUFA production. Because most of the genes relating to the ability of P. torquis to dwell in sea-ice ecosystems occur on genomic islands that are absent in closely related P. gondwanensis, its adaptation to the sea-ice environment appears driven mainly by HGT. The genomic islands are rich in pseudogenes, insertional elements, and addiction modules, suggesting that gene acquisition is being followed by a process of genome reduction potentially indicative of evolving ecosystem specialism.

Rapid and sensitive microbial analysis by capillary isotachophoresis with continuous electrokinetic injection under field amplified conditions

A highly sensitive capillary isotachophoresis method with LIF detection for microbial analysis was developed. This allowed the reliable analysis of Escherichia coli bacteria with a LOD of 14 cells in a sample volume of 100 μL, or 1.35 × 102 cell/mL, which is 47 times lower than reported by CE‐LIF and 148 times lower than CE‐UV with on‐line concentration. A leading electrolyte of 50 mM Tris‐HCl was used while the cells were diluted in 5 mM Tris HEPES as the terminator. To facilitate detection, cells were stained with the universal nucleic acid fluorophore SYTO 9. Continuous electrokinetic injection of the cells from the terminator under field amplified conditions concentrated cells into a single peak at the leader/terminator boundary allowing quantitation by measurement of peak height. The method was applied to water collected from two local streams, with only filtration through a 5‐μm syringe filter to remove large particulate matter followed by a ten times dilution in terminator, with total analysis time approximately 40 min. The detected cell numbers in the water samples by the isotachophoresis method were 3.70 × 105 cell/mL and 2.62 × 104 cell/mL, which were slightly higher than the 9.50 × 104 cell/mL and 1.96 × 104 cell/mL obtained by conventional microbiological plate counting.

Location and DGGE methodology can influence interpretation of field experimental studies on the response to hydrocarbons by Antarctic benthic microbial community

A field experiment investigating the effect of oil contamination on benthic microbial communities was conducted near Casey Station, East Antarctica. Defaunated sediment was treated with a mixture of Special Antarctic Blend diesel and lubricating oil and deployed in three different bays for eleven weeks. A molecular fingerprinting technique, denaturing gradient gel electrophoresis (DGGE), was used to investigate the microbial community structure. The variation between replicate samples within treatment groups indicates that the benthic microbial populations are very diverse and evenly distributed. Comparisons to determine the significance of both deployment location and hydrocarbon treatment showed that the greatest effect was from a combination of location and treatment. Detailed analysis suggests that subtle differences may be obscured by variability introduced by PCR and gel stages in DGGE, undermining this experimental approach. It is concluded that both location and hydrocarbon contamination influenced the development of the microbial communities but that the effect of hydrocarbon treatment varied with location. This has important implications for the design of future experiments on the effect of hydrocarbons on benthic communities, especially if it is intended to generalize the conclusions drawn from site specific studies.

Evaluation of a permeable reactive barrier to capture and degrade hydrocarbon contaminants

A permeable reactive barrier (PRB) was installed during 2005/2006 to intercept, capture and degrade a fuel spill at the Main Power House, Casey Station, Antarctica. Here, evaluation of the performance of the PRB is conducted via interpretation of total petroleum hydrocarbon (TPH) concentrations, degradation indices and most probable number (MPN) counts of total heterotroph and fuel degrading microbial populations. Results indicate that locations which contained the lowest TPH concentrations also exhibited the highest levels of degradation and numbers of fuel degrading microbes, based on the degradation indices and MPN methods selected. This provides insights to the most appropriate reactive materials for use in PRB’s in cold and nutrient-limited environments.