Ken Killham

The role of ecological theory in microbial ecology

Microbial ecology is currently undergoing a revolution, with repercussions spreading throughout microbiology, ecology and ecosystem science. The rapid accumulation of molecular data is uncovering vast diversity, abundant uncultivated microbial groups and novel microbial functions. This accumulation of data requires the application of theory to provide organization, structure, mechanistic insight and, ultimately, predictive power that is of practical value, but the application of theory in microbial ecology is currently very limited. Here we argue that the full potential of the ongoing revolution will not be realized if research is not directed and driven by theory, and that the generality of established ecological theory must be tested using microbial systems.

A stable bioluminescent construct of Escherichia coli O157:H7 for hazard assessments of long-term survival in the environment.

ABSTRACT A chromosomally lux-marked (Tn5 luxCDABE) strain of nontoxigenic Escherichia coli O157:H7 was constructed by transposon mutagenesis and shown to have retained the O157, H7, and intimin phenotypes. The survival characteristics of this strain in the experiments performed (soil at −5, −100, and −1,500 kPa matric potential and artificial groundwater) were indistinguishable from the wild-type strain. Evaluation of potential luminescence was found to be a rapid, cheap, and quantitative measure of viable E. coli O157:H7 Tn5 luxCDABE populations in environmental samples. In the survival studies, bioluminescence of the starved populations of E. coli O157:H7 Tn5 luxCDABE could be reactivated to the original levels of light emission, suggesting that these populations remain viable and potentially infective to humans. The attributes of the construct offer a cheap and low-risk substitute to the use of verocytotoxin-producing E. coli O157:H7 in long-term survival studies.

Influence of mineral soil on the palatability of organic matter for lumbricid earthworms: a simple food preference study

Abstract The food-preference behaviour of earthworms was examined in order to develop earthworm feeds which might act as a microbial carrier in earthworm-mediated dispersal of beneficial microorganisms in soil. A circular choice chamber containing 18 feeding stations was used to assess the food-type preference of four earthworm species ( Aporrectodea caliginosa, A. longa, Lumbricus rubellus, L. terrestris ). Representatives of each species were presented with four types of organic matter (cow dung, sheep dung, decomposed leaf litter, sewage sludge) either alone or mixed 1 to 4 (on a dry weight basis) with a sandy loam soil; soil alone was also tested in the same chamber. The nine types of potential food were each inoculated with Pseudomonas corrugata 2140R lux 8 at a rate of 10 7 cfu (colony forming units) g −1 wet wt of material. All earthworm species showed a strong preference for pure mineral soil over pure organic matter. Also, mineral-organic mixtures were clearly preferred to pure organic sources, with leaf litter + soil being the preferred mixture for all four earthworm species tested. In addition, cow dung + soil and sewage sludge + soil were acceptable to all species except A. caliginosa . Inoculation of the food stuffs with Pseudomonas corrugata 2140R lux 8 did not deter the earthworms from feeding and earthworm casts contained from 10 7 to 10 9 cfu g −1 wet wt. The choice chamber method offers a simple, rapid and inexpensive test for studying food preferences of earthworms for various applications.

Toxicity of mono-, di- and tri-chlorophenols to lux marked terrestrial bacteria, Burkholderia species Rasc c2 and Pseudomonas fluorescens.

Burkholderia species RASC and Pseudomonas fluorescens were marked with lux genes, encoding for bioluminescence and used to assess the toxicity of mono-, di- and tri-chlorophenols by determining the decline in bioluminescence following exposure to the compounds in aqueous solution. Toxicity was expressed as a 50% effective concentration value (EC50, equating to the concentration of compound which caused a 50% decline in bioluminescence. Comparing the toxicity values of the compounds showed that, in general, increasing the degree of chlorination, increased toxicity. By carrying out forward multiple linear regressions with log10 EC50 values and physio-chemical descriptors, it was shown that molecular parameters describing the hydrogen bonding nature of a chlorophenol provided a better fit than regressions between toxicity data and log10 Kow alone. Utilising these descriptor variables in equations, it was shown that the toxicity of chlorophenols to the lux marked bacteria could be predicted from the compounds physio-chemical characteristics. By correlating lux marked RASC c2 and P. fluorescens EC50 values with toxicity values using Pimephales promelas (fathead minnow), Tetrahymena pyriformis (ciliate) and marine bacterium Vibriofischeri, it was apparent that lux marked RASC c2 correlated well with the freshwater aquatic species (P. promelas and T. pyriformis). This implied that for predictions of toxicity of organic xenobiotic compounds to higher organisms, lux marked RASC c2 could be utilised as a rapid surrogate.

A mathematical model for dispersal of bacterial inoculants colonizing the wheat rhizosphere

Abstract A mathematical model has been constructed to describe bacterial growth and movement in the rhizosphere. In the model, bacteria are introduced into the soil on inoculated seeds and growth occurs, after seed germination, on material produced as root exudates. Movement of substrates away from the rhizosphere into the bulk soil is by diffusion and microbial movement is mediated by carriage on the root surface. The relationship between specific growth rate and substrate concentration is described by Monod kinetics and death occurs at a constant specific rate. An important component of the model is treatment of the effects of matric potential on the distribution and activity of bacteria in different microhabitats. Simulation of the model quantifies the distribution of both bacteria and substrate with depth and time in the rhizosphere and demonstrates significant differences between substrate concentrations at high and low matric potentials. Sensitivity analysis of model predictions indicates the parameters which govern microbial growth to be more important determinants of microbial movement than plant-associated parameters. Predictions of the model compared well with experimental data on microbial movement in the rhizosphere of wheat plants grown in microcosms, and inoculated with luminescence-marked Pseudomonas fluorescens, and provide the basis for quantitative risk assessment following environmental release of genetically-engineered microorganisms.

Use of lux-based biosensors for rapid diagnosis of pollutants in arable soils

A field trial, in Central Scotland, demonstrated that crop yields were reduced with increased application of paper mill sludge to land. A suite of ecotoxicity assays, including, luminescence response of lux-marked bacteria, respirometry and enzyme activity was used to assess toxicity of the paper mill sludge to the soil microbial biomass. The results from the use of the lux based biosensors correlated well with more traditional microbial indicators of soil pollution (respiration and enzyme activity). Concentrations of metals and organic contaminants in samples were confirmed using GFAAS and GC-MS, respectively. The main pollutant components of paper mill sludge were found to be Cd, Cu and PCP (pentachlorophenol). The range of environmental bioassays used, with chemical verification, was shown to offer a rapid and comprehensive battery test for rapid assessment of the ecotoxicity associated with papermill sludge application to land.

Dynamic response of microbial biomass, respiration rate and ATP to glucose additions

Abstract The dynamic responses of microbial biomass, respiration rate and ATP concentration to glucose solution additions to soil have been measured and compared. The relative response of the three determinants was found to be quite different and ATP per unit of biomass was found to change substantially over the short term. At no single measurement time did the three determinants give similar responses to glucose addition. This confirms that caution is needed in the interpretation of results when only one, or even a pair, of the variables studied is used to indicate soil microbial response to substrate addition or stress factors. All three variables together provide better insight into the dynamics of microbial response to substrate C flow in soil.

Response of a Rhizobium-based luminescence biosensor to Zn and Cu in soil solutions from sewage sludge treated soils

Abstract A luminescence based biosensor (Rhizotox-C) was used as an indicator of heavy metal pollution of soils. The response of the biosensor to increasing concentrations of total soil Zn, soil solution Zn, soil solution free Zn 2+ , total soil Cu and total soil solution Cu from soils of a long-term sewage sludge field experiment was investigated. The bioluminescence response of the Rhizotox-C biosensor declined as total soil Zn, soil solution Zn and free soil solution Zn 2+ concentrations increased. The EC 25 values for the biosensor for total soil Zn, soluble soil solution Zn and free soil solution Zn 2+ were 164±43 mg kg −1 soil, 4±0.7 and 2±0.3 mg l −1 , respectively. The EC 50 values were 403±57 mg kg −1 soil, 16±3 and 6±1.0 mg l −1 , respectively. The largest soil solution concentration of Cu was about 620 μg l −1 , but this had no significant effect on luminescence. This corresponded to a total Cu concentration in bulk soil of about 349 mg kg −1 .

Observations on oligotrophic growth of fungi on silica gel

A range of fungi obtained from culture collections or isolated from soils were able to grow on silica gel media to which no carbon or nitrogen sources were added. Presumably fungi scavenge nutrients from the atmosphere under these conditions although alternative sources are also commented on. The growth of numerous fungi isolated from a range of soils, on carbon-free silica gel media suggests that low levels of available carbon in soil should not prevent limited growth of the fungi.

Effect of Nematodes on Rhizosphere Colonization by Seed-Applied Bacteria

ABSTRACT There is much interest in the use of seed-applied bacteria for biocontrol and biofertilization, and several commercial products are available. However, many attempts to use this strategy fail because the seed-applied bacteria do not colonize the rhizosphere. Mechanisms of rhizosphere colonization may involve active bacterial movement or passive transport by percolating water or plant roots. Transport by other soil biota is likely to occur, but this area has not been well studied. We hypothesized that interactions with soil nematodes may enhance colonization. To test this hypothesis, a series of microcosm experiments was carried out using two contrasting soils maintained under well-defined physical conditions where transport by mass water flow could not occur. Seed-applied Pseudomonas fluorescens SBW25 was capable of rhizosphere colonization at matric potentials of −10 and −40 kPa in soil without nematodes, but colonization levels were substantially increased by the presence of nematodes. Our results suggest that nematodes can have an important role in rhizosphere colonization by bacteria in soil.