Düsseldorf, Universiẗat, Diss., 2002.

Variovorax paradoxus EPS is a gram-negative rod isolated from the sunflower rhizosphere at CSUSB. Preliminary research has shown that Variovorax paradoxus EPS has anti-staphylococcal activity in liquid and solid co-culture. Anti-staphylococcal activity of Wild type and V. paradoxus EPS 𝚫4519 on 0.5% YE agar with embedded S. aureus AH1710 supports the idea that a soluble molecule is responsible for this activity, as the agar acted as a physical barrier between V. paradoxus EPS and S. aureus colonies. Preliminary genetic analysis of V. paradoxus EPS identified three loci that suitable candidates for the synthesis of a potential anti-staphylococcal small molecule. Preliminary data failed to detect expression at two of the three identified loci and a strain with a mutation at the third locus continues to produce anti-staphylococcal activity. We hypothesize that the microbial agent is expressed at a different locus or loci that have not yet been identified. These gene products are responsible for the synthesis of the microbial agent and are controlled by exposure to Staphylococcus aureus. Optimal growth conditions were identified for V. paradoxus EPS and S. aureus to demonstrate the formation of a zone of inhibition on Tryptic Soy Agar. The use of a V. paradoxus EPS Δ 4519 transposon library at optimal growth conditions allowed us candidate mutants with altered antimicrobial activity phenotypes. We identified 28 insertion sites that resulted in altered antimicrobial activities, which will allow us to identify the genes involved in this biosynthetic pathway.

The weathering of shales is a globally important process affecting both natural and built environments. Shales form roughly 70 % of worldwide sedimentary rock deposits and therefore the weathering of these rocks has substantial effects on the geochemical cycling of elements such as carbon, iron and sulfur. Microbes have been shown to play a key role in weathering shales, primarily through the oxidation of the iron and sulfur of embedded pyrite and the resultant production of sulfuric acid. Despite significant interest in the microbial weathering of shales within industrial sectors such as biohydrometallurgy and civil engineering, comparatively few studies have investigated microbial shale weathering in natural environments. Furthermore, the role of microbes in natural shale weathering processes beyond iron oxidation has largely remained unexplored. In this thesis, the weathering capabilities of microbial communities from natural weathered shale was investigated. The North Yorkshire coastline was used as a study location, due to the abundance and diversity of natural cliffs and historic, disused industrial sites. Cliff erosion and recession on the North Yorkshire coastline is a major concern for local authorities and is the focus of current research. The aim of this work has been to evaluate microbial shale weathering processes within these environments, and hypothesise the possible contribution they may have to erosive processes. Phenotypic plate assays inoculated with weathered shale material were used to obtain rock weathering bacterial isolates that tested positive for a specific weathering phenotype, such as iron oxidation or siderophore production. Subsequent 16S rRNA sequencing enabled genera level identification, revealing 15 genera with rock weathering capabilities with several being associated with multiple weathering phenotypes including Aeromonas sp., Pseudomonas sp. and Streptomyces sp. Shale enrichment liquid cultures were incubated with shale rock chips to simulate natural biological weathering conditions, and the concentration of rock-leached elements in the fluid measured. No evidence of microbially-enhanced leaching was found consistently for any element, however the significant reduction in leachate iron concentration under biological conditions indicates that iron precipitation occurred via microbial iron oxidation. Enrichment cultures inoculated with weathered shale and containing organic matter (OM) rich rocks in water or M9 medium, both liquids lacking an organic carbon source, were grown over several months. The cultures yielded microbial isolates that could utilise rock bound OM sources and one bacterial isolate, Variovorax paradoxus, was taken forward for ecophysiological study. The shale rock that the organism was isolated from, along with other OM rich rocks (mudstones and coals), elicited complex responses from V. paradoxus including enhanced growth and motility. Finally, mineral microcosms in vitro and mesocosms in situ investigated microbial colonization and weathering of shale-comprising minerals (albite, calcite, muscovite, pyrite and quartz). Microcosms were established using iron oxidizing enrichment cultures, as based on the results of the simulated rock weathering experiments, while the in situ mesocosms were buried within weathered shale scree within a disused mine level. Levels of colonization significantly varied between minerals within the microcosms (pyrite > albite, muscovite > quartz > calcite). Although differences in mineral colonization were seen in the mesocosms, they did not match those in the microcosms and were not statistically significant. Pyrite incubated in the microcosms became significantly weathered, with extensive pit formation across the mineral surface that is consistent with microbial iron oxidation. In the mesocosms, pit formation was not identified on pyrite surfaces but dark etchings into the pyrite surface were found underneath fungi hyphal growth. The results of this thesis highlights that a range of microbial rock weathering mechanisms are abundant across weathered shale environments. Microbial iron oxidizing activity was a dominant biogeochemical process that altered rock-fluid geochemistry and weathered pyrite surfaces. However, the impact on rock or mineral weathering of other microbial mechanisms was not elucidated by this work. Given the known capabilities of these mechanisms, the conditions under which they are active may not have been met within the experimental setup used. Microbial iron oxidation in shale and shale-derived materials has previously been demonstrated to weaken rock structure through acid production and secondary mineral formation. From the results of this thesis, it is clear that microbial iron oxidation is an active process within some of the weathered shale environments studied, including cliff surfaces. Therefore, it can be hypothesised that microbial activity could play a role in structurally weakening shale rock within cliffs and accelerate their erosion. Future work should attempt to quantify the rate and extent of microbial iron oxidizing activity within shale cliff environments and investigate its contribution to erosive processes.

Staphylococcus aureus is one of the leading causes of fatal nosocomial infections. Often, S. aureus can grow as a biofilm which protects the population from the surrounding environment. Strains of S. aureus are resistant to virtually all known antibiotics on the market. Variovorax paradoxus is a soil microbe with many unusual metabolic activities. It has been previously observed that, V. paradoxus can inhibit the growth of S. aureus when in co-culture. In this work we report on inhibition of S. aureus biofilm formation by V. paradoxus due to a suspected inhibitory soluble factor.