David J. Saul

Culturable Bacteria in Subglacial Sediments and Ice from Two Southern Hemisphere Glaciers

Viable prokaryotes have been detected in basal sediments beneath the few Northern Hemisphere glaciers that have been sampled for microbial communities. However, parallel studies have not previously been conducted in the Southern Hemisphere, and subglacial environments in general are a new and underexplored niche for microbes. Unfrozen subglacial sediments and overlying glacier ice samples collected aseptically from the Fox Glacier and Franz Josef Glacier in the Southern Alps of New Zealand now have been shown to harbor viable microbial populations. Total direct counts of 2–7 × 106 cells g−1 dry weight sediment were observed, whereas culturable aerobic heterotrophs ranged from 6–9 × 105 colony-forming units g−1 dry weight. Viable counts in the glacier ice typically were 3–4 orders of magnitude smaller than in sediment. Nitrate-reducing and ferric iron–reducing bacteria were detected in sediment samples from both glaciers, but were few or below detection limits in the ice samples. Nitrogen-fixing bacteria were detected only in the Fox Glacier sediment. Restriction fragment analysis of 16S rDNA amplified from 37 pure cultures of aerobic heterotrophs capable of growth at 4°C yielded 23 distinct groups, of which 11 were identified as β-Proteobacteria. 16S rDNA sequences from representatives of these 11 groups were analyzed phylogenetically and shown to cluster with bacteria such as Polaromonas vacuolata and Rhodoferax antarcticus, or with clones obtained from permanently cold environments. Chemical analysis of sediment and ice samples revealed a dilute environment for microbial life. Nevertheless, both the sediment samples and one ice sample demonstrated substantial aerobic mineralization of 14C-acetate at 8°C, indicating that sufficient nutrients and viable psychrotolerant microbes were present to support metabolism. Unfrozen subglacial sediments may represent a significant global reservoir of biological activity with the potential to influence glacier meltwater chemistry.

Bioremediation of hydrocarbon-contaminated polar soils

Bioremediation is increasingly viewed as an appropriate remediation technology for hydrocarbon-contaminated polar soils. As for all soils, the successful application of bioremediation depends on appropriate biodegradative microbes and environmental conditions in situ. Laboratory studies have confirmed that hydrocarbon-degrading bacteria typically assigned to the genera Rhodococcus, Sphingomonas or Pseudomonas are present in contaminated polar soils. However, as indicated by the persistence of spilled hydrocarbons, environmental conditions in situ are suboptimal for biodegradation in polar soils. Therefore, it is likely that ex situ bioremediation will be the method of choice for ameliorating and controlling the factors limiting microbial activity, i.e. low and fluctuating soil temperatures, low levels of nutrients, and possible alkalinity and low moisture. Care must be taken when adding nutrients to the coarse-textured, low-moisture soils prevalent in continental Antarctica and the high Arctic because excess levels can inhibit hydrocarbon biodegradation by decreasing soil water potentials. Bioremediation experiments conducted on site in the Arctic indicate that land farming and biopiles may be useful approaches for bioremediation of polar soils.

Aromatic hydrocarbon-degrading bacteria from soil near Scott Base, Antarctica.

Abstract Hydrocarbons persist in Antarctic soils when fuel oils such as JP8 jet fuel are spilled. For clean-up of hydrocarbon-contaminated soils in Antarctica, bioremediation has been proposed using hydrocarbon-degrading microbes indigenous to Antarctic soils. A number of alkane-degrading bacteria have been isolated previously from Antarctic soils. In this paper we describe the direct isolation of aromatic hydrocarbon-degrading bacteria from oil-contaminated Antarctic soil. Isolates that grew on JP8 jet fuel were characterised for their ability to degrade aromatic and aliphatic hydrocarbons and for growth at a range of temperatures. All isolates were gram-negative, oxidase-positive, rod-shaped bacteria. Representative strains were identified using 16S rDNA sequence analysis as either Sphingomonas spp. or Pseudomonas spp. Aromatic-degrading bacteria from Antarctic soils were psychrotolerant and appear similar to those found worldwide.

Cold-tolerant alkane-degrading Rhodococcus species from Antarctica

Abstract Bioremediation is a possible mechanism for clean-up of hydrocarbon-contaminated soils in the Antarctic. Microbes indigenous to the Antarctic are required that degrade the hydrocarbon contaminants found in the soil, and that are able to survive and maintain activity under in situ conditions. Alkane- degrading bacteria previously isolated from oil-contaminated soil from around Scott Base, Antarctica, grew on a number of n-alkanes from hexane (C6) through to eicosane (C20) and the branched alkane pristane. Mineralization of 14C-dodecane was demonstrated with four strains. Representative isolates were identified as Rhodococcus species using 16S rDNA sequence analysis. Rhodococcus spp. strains 5/14 and 7/1 grew at −2°C but numbers of viable cells declined when incubated at 37°C. Both strains appear to have the major cold-shock gene cspA. Partial nucleotide sequence analyses of the PCR-amplified cspA open reading frame from Rhodococcus spp. strains 5/14 and 7/1 were approximately 60% identical to cspA from Escherichia coli.

Free-living heterotrophic nitrogen-fixing bacteria isolated from fuel-contaminated antarctic soils.

ABSTRACT Five bacterial isolates enriched from fuel-contaminated Antarctic soils fixed nitrogen in the dark heterotrophically and nonsymbiotically. Two isolates utilized jet fuel vapors and volatile hydrocarbons for growth but not in N-deficient medium. Bacteria such as these may contribute to in situ biodegradation of hydrocarbons in Antarctic soils.

Sequencing and expression of additional xylanase genes from the hyperthermophile Thermotoga maritima FjSS3B.1.

ABSTRACT Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking–PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87°C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity toxynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.

Crenarchaeota affiliated with group 1.1b are prevalent in coastal mineral soils of the Ross Sea region of Antarctica

The objective of this study was to examine the presence and diversity of Archaea within mineral and ornithogenic soils from 12 locations across the Ross Sea region. Archaea were not abundant but DNA sufficient for producing 16S rRNA gene clone libraries was extracted from 18 of 51 soil samples, from four locations. A total of 1452 clones were analysed by restriction fragment length polymorphism and assigned to 43 operational taxonomic units from which representatives were sequenced. Archaea were primarily restricted to coastal mineral soils which showed a predominance of Crenarchaeota belonging to group 1.1b (> 99% of clones). These clones were assigned to six clusters (A through F), based on shared identity to sequences in the GenBank database. Ordination indicated that soil chemistry and water content determined archaeal community structure. This is the first comprehensive study of the archaeal community in Antarctic soils and as such provides a reference point for further investigation of microbial function in this environment.

Cold tolerance of Pseudomonas sp. 30-3 isolated from oil-contaminated soil, Antarctica

Abstract. Pseudomonas sp. 30-3 was enriched from oil-contaminated soil from Wright Valley, Antarctica using JP8 jet fuel as sole carbon source. This isolate exhibited tolerance to temperatures ranging from 0°C to 35°C when cultured in laboratory medium. In a freeze-thaw study, an 89% survival was observed when Pseudomonas sp. 30-3 was exposed to 4°C prior to freezing. PCR amplification of a 248-bp DNA fragment in Pseudomonas sp. 30-3 using capB-gene specific primers showed a 98% amino acid sequence homology with CapB of Pseudomonas fragi and 62% homology with CspA of Escherichia coli. Radiolabeling of total cellular proteins exhibited elevated expression of an 8-kDa protein at 4°C, which suggests that the CapB in Pseudomonas sp. 30-3 may play a pivotal role in survival and tolerance at cold and subzero temperatures. Tolerance to cold temperatures and the ability to degrade hydrocarbons by Pseudomonas sp. 30-3 provide support for the application of bioremediation for petroleum hydrocarbons in Antarctic soils.

Heterogeneity of SSU and LSU rDNA sequences of Alexandrium species

With the growth of aquaculture, toxic phytoplankton have become a significant problem. At present, the identification of specific algal species within blooms requires considerable expertise, and the methods are both costly and time consuming. It is clear that alternative diagnostic tests must be developed that allow rapid and simple assessments to be made on site. We report on the identification of unique sequences within the SSU and LSU rDNA genes of Alexandrium species that provide data for the design of species- and genus-specific molecular probes. In addition we examine some aspects of the phylogeny of Alexandrium isolates from New Zealand waters.

Eurythermalism and the temperature dependence of enzyme activity

The “Equilibrium Model” has provided new tools for describing and investigating enzyme thermal adaptation. It has been shown that the effect of temperature on enzyme activity is not only governed by ∆G‡cat and ∆G‡inact but also by two new intrinsic parameters, ∆Heq and Teq, which describe the enthalpy and midpoint, respectively, of a reversible equilibrium between active and inactive (but not denatured) forms of enzyme. Twenty‐one enzymes from organisms with a wide range of growth temperatures were characterized using the Equilibrium Model. Statistical analysis indicates that Teq is a better predictor of growth temperature than enzyme stability (∆G‡inact). As expected from the Equilibrium Model, ∆Heq correlates with catalytic temperature tolerance of enzymes and thus can be declared the first intrinsic and quantitative measure of enzyme eurythermalism. Other findings shed light on the evolution of psychrophilic and thermophilic enzymes. The findings suggest that the description of the Equilibrium Model of the effect of temperature on enzyme activity applies to all enzymes regardless of their temperature origins and that its associated parameters, ∆Heq and Teq, are intrinsic and necessary parameters for characterizing the thermal properties of enzymes and their temperature adaptation and evolution.—Lee C. K., Daniel, R. M., Shepherd, C., Saul, D., Cary, S. C., Danson, M. J., Eisenthal, R., Peterson M. E. Eurythermal‐ism and the temperature dependence of enzyme activity FASEB J. 21, 1934–1941 (2007)