Sand Microcosms Research Articles

The Effect of Phylogenetically Different Bacteria on the Fitness of Pseudomonas fluorescens in Sand Microcosms

In most environments many microorganisms live in close vicinity and can interact in various ways. Recent studies suggest that bacteria are able to sense and respond to the presence of neighbouring bacteria in the environment and alter their response accordingly. This ability might be an important strategy in complex habitats such as soils, with great implications for shaping the microbial community structure. Here, we used a sand microcosm approach to investigate how Pseudomonas fluorescens Pf0-1 responds to the presence of monocultures or mixtures of two phylogenetically different bacteria, a Gram-negative (Pedobacter sp. V48) and a Gram-positive (Bacillus sp. V102) under two nutrient conditions. Results revealed that under both nutrient poor and nutrient rich conditions confrontation with the Gram-positive Bacillus sp. V102 strain led to significant lower cell numbers of Pseudomonas fluorescens Pf0-1, whereas confrontation with the Gram-negative Pedobacter sp. V48 strain did not affect the growth of Pseudomonas fluorescens Pf0-1. However, when Pseudomonas fluorescens Pf0-1 was confronted with the mixture of both strains, no significant effect on the growth of Pseudomonas fluorescens Pf0-1 was observed. Quantitative real-time PCR data showed up-regulation of genes involved in the production of a broad-spectrum antibiotic in Pseudomonas fluorescens Pf0-1 when confronted with Pedobacter sp. V48, but not in the presence of Bacillus sp. V102. The results provide evidence that the performance of bacteria in soil depends strongly on the identity of neighbouring bacteria and that inter-specific interactions are an important factor in determining microbial community structure.

The metabolic pathways and environmental controls of hydrocarbon biodegradation in marine ecosystems.

The metabolic pathways and environmental controls of hydrocarbon biodegradation in marine ecosystems.

Patterns and persistence of antibiotic resistance in faecal indicator bacteria from freshwater recreational beaches

This study was conducted to determine antibiotic susceptibility patterns among the faecal indicator bacteria (FIB), Escherichia coli and enterococci, and to determine the potential for freshwater beaches to serve as reservoirs of resistance genes where transfer of resistant phenotypes takes place or de novo resistance may evolve. One hundred and forty-seven E. coli and 150 enterococci collected from sand and water at recreational beaches along Lake Huron, Michigan, USA were screened against commonly used antibiotics. Resistance was apparent in both E. coli (19% resistant) and enterococci (65% resistant). Antibiotic-resistant E. coli were capable of growing in beach sand microcosms and were able to transfer a plasmid-encoded kanamycin-resistance gene in sand microcosms. Furthermore, resistant phenotypes were stable in the sand environment even in the absence of the corresponding antibiotic. Antibiotic-resistant FIB were prevalent and persistent in the beach habitat. Active populations of FIB at beaches express antibiotic resistance phenotypes and have the ability to transfer antibiotic resistance. These human-associated bacteria may be intermediaries in the movement of resistance between environmental and clinical reservoirs.

Decay of genetic markers for fecal bacterial indicators and pathogens in sand from Lake Superior

Beach sands impact water quality and pathogen loads, however, the comparative decay of the fecal indicator bacteria (FIB) Enterococcus spp. and Escherichia coli, and pathogens in freshwater sand have not been examined. In this study, freshwater sand microcosms were inoculated with sewage and pure cultures of bacterial pathogens to compare relative decay rates. The abundance of culturable Enterococcus spp. and E. coli, genetic markers for Enterococcus spp. (Entero1), total Bacteroides (AllBac), and human-specific Bacteroides (HF183), and genetic markers for the pathogens Campylobacter jejuni, methicillin-resistant Staphylococcus aureus (MRSA), Salmonella enterica subsp. enterica serovar Typhimurium, and Shigella flexneri were monitored over the course of two weeks using conventional culture methods and quantitative PCR (qPCR). The effect of moisture on the persistence of culturable FIB and all genetic markers was also determined. In addition, propidium monoazide (PMA) treatment was used to examine differences in the persistence of total genetic markers and those from live cells. Decay rates were statistically compared using Tukey's test. Moisture had a significant (p ≤ 0.05) effect on the decay rates of culturable indicator bacteria, total AllBac markers, and genetic markers for FIB, Salmonella, and MRSA from live cells. At 14% sand moisture, the decay rate of total markers was slower than that of live cells for all qPCR assays, but at 28% moisture, there was no difference in the decay rates of total and live markers for any assay. AllBac and MRSA markers increased in sand at 28% moisture, probably indicating cellular growth. Overall, culturable FIB and HF183 had decay rates that were most comparable to the bacterial pathogen markers examined in this study, whereas Entero1 and AllBac rarely exhibited decay rates similar to the bacterial pathogens in this study. The choice of FIB for assessment of fecal contamination in freshwater sand should take into account the pathogen of concern and sand moisture conditions.

Impact of Matric Potential and Pore Size Distribution on Growth Dynamics of Filamentous and Non-Filamentous Soil Bacteria

The filamentous growth form is an important strategy for soil microbes to bridge air-filled pores in unsaturated soils. In particular, fungi perform better than bacteria in soils during drought, a property that has been ascribed to the hyphal growth form of fungi. However, it is unknown if, and to what extent, filamentous bacteria may also display similar advantages over non-filamentous bacteria in soils with low hydraulic connectivity. In addition to allowing for microbial interactions and competition across connected micro-sites, water films also facilitate the motility of non-filamentous bacteria. To examine these issues, we constructed and characterized a series of quartz sand microcosms differing in matric potential and pore size distribution and, consequently, in connection of micro-habitats via water films. Our sand microcosms were used to examine the individual and competitive responses of a filamentous bacterium (Streptomyces atratus) and a motile rod-shaped bacterium (Bacillus weihenstephanensis) to differences in pore sizes and matric potential. The Bacillus strain had an initial advantage in all sand microcosms, which could be attributed to its faster growth rate. At later stages of the incubation, Streptomyces became dominant in microcosms with low connectivity (coarse pores and dry conditions). These data, combined with information on bacterial motility (expansion potential) across a range of pore-size and moisture conditions, suggest that, like their much larger fungal counterparts, filamentous bacteria also use this growth form to facilitate growth and expansion under conditions of low hydraulic conductivity. The sand microcosm system developed and used in this study allowed for precise manipulation of hydraulic properties and pore size distribution, thereby providing a useful approach for future examinations of how these properties influence the composition, diversity and function of soil-borne microbial communities.

Volatiles produced by the mycophagous soil bacteriumCollimonas

It is increasingly recognized that volatile organic compounds play an import role during interactions between soil microorganisms. Here, we examined the possible involvement of volatiles in the interaction of Collimonas bacteria with soil fungi. The genus Collimonas is known for its ability to grow at the expense of living fungi (mycophagy), and antifungal volatiles may contribute to the attack of fungi by these bacteria. We analyzed the composition of volatiles produced by Collimonas on agar under different nutrient conditions and studied the effect on fungal growth. The volatiles had a negative effect on the growth of a broad spectrum of fungal species. Collimonas bacteria did also produce volatiles in sand microcosms supplied with artificial root exudates. The production of volatiles in sand microcosms was enhanced by the presence of fungi. The overall picture that we get from our study is that antifungal volatiles produced by Collimonas could play an important role in realizing its mycophagous lifestyle. The current work is also interesting for understanding the ecological relevance of volatile production by soil bacteria in general as we found strong influences of root exudates composition and incubation conditions on the spectrum of volatiles produced.

Biodegradation of diesel/biodiesel blends in saturated sand microcosms

The aim of the study was to evaluate the biodegradation extent of both aromatic and aliphatic hydrocarbon fractions in saturated sandy microcosm spiked with diesel/biodiesel blends (D, B10, B20, B30, B40, B50, B60, B70, B80, B90 and B100, where D is commercial petroleum diesel fuel and B is commercial biodiesel blend) augmented with a bacterial consortium of petroleum degraders. The biodegradation kinetics for blends were evaluated based on measuring the amount of emitted CO2 after 578days. Subsequently, the residual aromatic and aliphatic fractions were separated and determined by employing GC-FID and GC×GC–TOF-MS. Additionally, the influence of biodiesel-amendment on the community dynamics was assessed based on the results of real-time PCR analyzes. Our results suggest that the biodegradation extents of both aliphatic and aromatic hydrocarbon were uninfluenced by the addition of biodiesel, regardless of the concentration used. This observation leads to the conclusion that blending with biodiesel does not impact the long-term biodegradation of specific diesel oil fractions.

Inoculation of PAH-degrading strains of Fusarium solani and Arthrobacter oxydans in rhizospheric sand and soil microcosms: microbial interactions and PAH dissipation

Very little is known about the influence of bacterial-fungal ecological interactions on polycyclic aromatic hydrocarbon (PAH) dissipation in soils. Fusarium solani MM1 and Arthrobacter oxydans MsHM11 can dissipate PAHs in vitro. We investigated their interactions and their effect on the dissipation of three PAHs-phenanthrene (PHE), pyrene (PYR) and dibenz(a,h)anthracene (DBA)-in planted microcosms, in sterile sand or non-sterile soil. In sterile sand microcosms planted with alfalfa, the two microbes survived and grew, without any significant effect of co-inoculation. Co-inoculation led to the dissipation of 46% of PHE after 21days. In soil microcosms, whether planted with alfalfa or not, both strains persisted throughout the 46days of the experiment, without any effect of co-inoculation or of alfalfa, as assessed by real-time PCR targeting taxon-level indicators, i.e. Actinobacteria 16S rDNA and the intergenic transcribed spacer specific to the genus Fusarium. The microbial community was analyzed by temporal temperature gradient electrophoresis and real-time PCR targeting bacterial and fungal rDNA and PAH-ring hydroxylating dioxygenase genes. These communities were modified by PAH pollution, which selected PAH-degrading bacteria, by the presence of alfalfa and, concerning the bacterial community, by inoculation. PHE and PYR concentrations significantly decreased (91 and 46%, respectively) whatever the treatment, but DBA concentration significantly decreased (30%) in planted and co-inoculated microcosms only.

Phenolic removal processes in biological sand filters, sand columns and microcosms

This study evaluated the removal processes involved in the removal of the phenolic component of winery wastewater in biological sand filters, sand columns and sand microcosms. It was found that at low influent phenolic concentrations, complete organic removal was accomplished, but at high concentrations, there was incomplete substrate removal and an accumulation of potentially toxic metabolites, including catechol. The sand provided a suitable substrate for the treatment of phenolic-laden waste, and both biotic (48%) and abiotic (52%) removal mechanisms effected the removal of model phenolics. Prior acclimation of microbial communities increased the biodegradation rate of phenolic acids significantly.

Root exudates affect phenanthrene biodegradation, bacterial community and functional gene expression in sand microcosms

Polycyclic aromatic hydrocarbon (PAH) degradation may be increased in plant rhizosphere, but the role of plants in PAH degradation is not clearly understood. We investigated the effect of ryegrass root exudates on phenanthrene (PHE) degradation, PHE degrading bacterial population and functional gene expression in sand microcosms inoculated with bacteria from an industrial PAH polluted soil. Dominant bacteria were identified using 16S rDNA based temporal thermal gradient electrophoresis (TTGE). Functional PAH-ring hydroxylating dioxygenase (PAH-RHD α) genes (DNA) and transcripts (RNA) were quantified through real-time PCR and RT-PCR. Without root exudates PHE degradation was rapid and complete within 5 days. Herbaspirillum sp. and Pseudomonas putida were the dominant strains. Percentage of PAH-RHD α relative to 16S rRNA, both for genes and transcripts, was the highest after only two days and then decreased. With phenanthrene and root exudates as carbon sources, only 50% of the PHE was degraded in 10 days. P. putida-related bacteria were also detected with other strains related to Pseudomonas sp. and Paenibacillus. PHE degradation was reduced, and the percentage of PAH-RHD α transcripts was lower with root exudates than with only PHE, but increased over the incubation, while PAH-RHD α gene copy number was higher.

Biodegradation in a Partially Saturated Sand Matrix: Compounding Effects of Water Content, Bacterial Spatial Distribution, and Motility

Bacterial pesticide degraders are generally heterogeneously distributed in soils, leaving soil volumes devoid of degradation potential. This is expected to have an impact on degradation rates because the degradation of pollutant molecules in such zones will be contingent either on degraders colonizing these zones or on pollutant mass transfer to neighboring zones containing degraders. In a model system, we quantified the role exerted by water on mineralization rate in the context of a heterogeneously distributed degradation potential. Alginate beads colonized by Pseudomonas putida KT2440 were inserted at prescribed locations in sand microcosms so that the initial spatial distribution of the mineralization potential was controlled. The mineralization rate was strongly affected by the matric potential (decreasing rate with decreasing matric potential) and by the initial distribution of the degraders (more aggregated distributions being associated with lower rates). The mineralization was diffusion-limited, as confirmed with a mathematical model. In wet conditions, extensive cell dispersal was observed for the flagellated wild type and, albeit to a lesser extent, for a nonflagellated mutant, partially relieving the diffusion limitation. Dry conditions, however, sustained low mineralization rates through the combined effects of low pollutant diffusivity and limited degrader dispersal.

Movement of newly assimilated 13C carbon in the grass Lolium perenne and its incorporation into rhizosphere microbial DNA

One of the key processes that drives rhizosphere microbial activity is the exudation of soluble organic carbon (C) by plant roots. We describe an experiment designed to determine the impact of defoliation on the partitioning and movement of C in grass (Lolium perenne L.), soil and grass-sterile sand microcosms, using a (13)CO(2) pulse-labelling method. The pulse-derived (13)C in the shoots declined over time, but that of the roots remained stable throughout the experiment. There were peaks in the atom% (13)C of rhizosphere CO(2) in the first few hours after labelling probably due to root respiration, and again at around 100 h. The second peak was only seen in the soil microcosms and not in those with sterilised sand as the growth medium, indicating possible microbial activity. Incorporation of the (13)C label into the microbial biomass increased at 100 h when incorporation into replicating cells, as indicated by the amounts of the label in the microbial DNA, started to increase. These results indicate that the rhizosphere environment is conducive to bacterial growth and replication. The results also show that defoliation had no impact on the pattern of movement of (13)C from plant roots into the microbial population in the rhizosphere.

Corrosion and transport of depleted uranium in sand-rich environments

The firing of depleted uranium (DU) weapons during conflicts and military testing has resulted in the deposition of DU in a variety of sand-rich environments. In this study, DU-amended dune sand microcosm and column experiments were carried out to investigate the corrosion of DU and the transport of corrosion products. Under field-moist conditions, DU corroded to metaschoepite ((UO 2) 8O 2(OH) 12·(H 2O) 10) at a rate of 0.10 ± 0.012 g cm −2 y −1. This loosely bound corrosion product detached easily from the coupon and became distributed heterogeneously within the sand. The corrosion of DU caused significant changes in the geochemical environment, with NO 3 - and Fe(III) reduction observed. Column experiments showed that transport of metaschoepite was mainly dependent on its dissolution and the subsequent interaction of the resulting dissolved uranyl ( UO 2 2 + ) species with sand particles. The modelling results predict that the transport of U released from metaschoepite dissolution is retarded, due to a slowly desorbing surface species (first order desorption rate constant = 5.0 (±1.0) × 10 −8 s −1). The concentrations of U eluting from the metaschoepite column were orders of magnitude higher than the World Health Organisation’s recommended maximum admissible concentration for U in drinking water of 15 μg L −1. Therefore, a relatively high level of mobile U contamination would be expected in the immediate proximity of a corroding penetrator in a sand-rich environment.

Residue chemistry and microbial community structure during decomposition of eucalypt, wheat and vetch residues

Previous studies have shown that residue chemistry and microbial community structure change during decomposition, however little is known about the relationship between C-chemistry and microbial community structure. To address this knowledge gap, we studied C-chemistry and microbial community structure during the decomposition of eucalypt, wheat and vetch residues with and without additional inorganic N. Bags containing ground residues of eucalypt, wheat, and vetch were buried in sand microcosms after inoculation with a diverse microbial community. Respiration was measured over an incubation period of 150 days. At different times during incubation, total C and N of the residues were analysed and residue carbon chemistry was determined by 13C-NMR (nuclear magnetic resonance) spectroscopy. Microbial communities were assessed by phospholipid fatty acid (PLFA) analyses. Results indicated that during decomposition, residue C-chemistry and microbial community composition changed over time and differed between residue types. Changes in microbial community structure were associated with changes in residue C-chemistry, mainly the relative content of aryl-C and O-alkyl-C. Addition of N increased cumulative respiration, altered C-chemistry during decomposition, particularly in high C/N residues (wheat and eucalypt), and changed microbial succession leading to an earlier establishment of a stable microbial community structure. N addition to eucalypt and wheat reduced the decomposition of aryl-C compounds.

Pilot- and bench-scale testing of faecal indicator bacteria survival in marine beach sand near point sources

Factors affecting faecal indicator bacteria (FIB) and pathogen survival/persistence in sand remain largely unstudied. This work elucidates how biological and physical factors affect die-off in beach sand following sewage spills. Solar disinfection with mechanical mixing was pilot-tested as a disinfection procedure after a large sewage spill in Los Angeles. Effects of solar exposure, mechanical mixing, predation and/or competition, season, and moisture were tested at bench scale. First-order decay constants for Escherichia coli ranged between -0.23 and -1.02 per day, and for enterococci between -0.5 and -1.0 per day. Desiccation was a dominant factor for E. coli but not enterococci inactivation. Effects of season were investigated through a comparison of experimental results from winter, spring, and fall. Moisture was the dominant factor controlling E. coli inactivation kinetics. Initial microbial community and sand temperature were also important factors. Mechanical mixing, common in beach grooming, did not consistently reduce bacterial levels. Inactivation rates are mainly dependent on moisture and high sand temperature. Chlorination was an effective disinfection treatment in sand microcosms inoculated with raw influent.

Mycophagous growth of Collimonas bacteria in natural soils, impact on fungal biomass turnover and interactions with mycophagous Trichoderma fungi

Bacteria of the genus Collimonas are widely distributed in soils, although at low densities. In the laboratory, they were shown to be mycophagous, that is, they are able to grow at the expense of living hyphae. However, so far the importance of mycophagy for growth and survival of collimonads in natural soil habitats is unknown. Using a Collimonas-specific real-time PCR assay, we show here that the invasion of field soils by fungal hyphae (Absidia sp.) resulted in a short-term, significant increase (average fourfold) of indigenous collimonads. No such responses were observed for other soil bacteria studied (Pseudomonas, Burkholderia, PCR-denaturing gradient gel electrophoresis patterns of total bacteria and Burkholderia). Hence, it appears that the stimulation of growth of Collimonas bacteria by fungal hyphae is not common among other soil bacteria. In the same field soils, Trichoderma, a fungal genus known for mycophagous (mycoparasitic) growth, increased upon introduction of Absidia hyphae. Hence, mycophagous growth by Collimonas and Trichoderma can occur in the same soils. However, in controlled experiments (sand microcosms), collimonads appeared to have a negative effect on mycophagous growth of a Trichoderma strain. The effect of mycophagous growth of collimonads on fungal biomass dynamics was studied in sand microcosms using the same Absidia sp. as a test fungus. The growth of collimonads did not cause a significant reduction in the Absidia biomass. Overall, the study indicates that mycophagous nutrition may be important for collimonads in natural soils, but the impact on fungal biomass turnover is likely to be minor.

Impact of laboratory-imposed physical disturbance on the abundance of four isolates of marine gymnamoebae

Four species of gymnamoebae isolated from a sandy shore were subjected to physical disturbance within sand microcosms containing either 250 or 125 μm sand. Firstly, the effect of two different strengths of physical disturbance on amoebae populations within sand was investigated. Secondly, whether the size of sand grains occupied by amoebae populations changed their response to disturbance events was assessed. Highest abundances of amoebae were recorded in control flasks containing 125 μm sand. Increasing the strength of disturbance decreased the abundance of all four species. Abundances of the two smallest isolates (Platyamoeba sp. and a limax amoeba) were least affected by increasing disturbance. The greatest decrease in abundance was observed in populations of two larger (>15 μm) Mayorella sp. and Vannella sp. The greatest impact of physical disturbance was seen on populations within 250 μm sand. This study shows that physical disturbance may be an important community structuring force within natural marine gymnamoebae communities.

A simple screening procedure for selecting fungi with potential for use in the bioremediation of contaminated land

A simple initial screening procedure for selecting strains of white-rot fungi with potential for use in bioremediation of contaminated sites is described. Besides the ability to degrade low molecular weight PAHs, isolates were screened for their growth rate on straw-based agar media, their potential to tolerate high concentrations of phenanthrene and their ability to out-grow the cellulolytic fungus Trichoderma harzianum on straw agar plates. Results from simple in vitro tests were correlated with the ability of the different strains to degrade PAHs in sand microcosms. It was found that fungal growth rate on straw-based agar media in the presence of phenanthrene correlated well with the ability of the different fungi to degrade PAHs in sand microcosms. Whereas growth rate on straw-based agar plates per se was indicative of the ability of white-rot fungi to establish in the presence of a competing fungus, it was a poor indicator of the fungus’ ability to degrade PAHs.

Root exudation from Hordeum vulgare in response to localized nitrate supply

Root proliferation as a response to exploit zones of nutrient enrichment in soil has been demonstrated for a wide range of plant species. However, the effectiveness of this as a strategy to acquire nutrients is also dependent on interactions with the soil microbial community. Specifically, C-flow from roots modifies microbial activity and probably the balance between nutrient mineralization and immobilization processes in the rhizosphere. In this study, near-natural abundance 13C-labelling and gene-reporter methods were applied to determine the effects of uneven nitrate supply to roots of Hordeum vulgare on assimilate partitioning and root exudation. Plants were initially grown in uniform nitrate supply in split-root, sand microcosms after which one treatment continued to receive uniform supply, and the other received nitrate to one root compartment only. At the time of imposing the treatments, the CO2 supplied to the plants was switched to a cylinder source, providing a distinct delta13C-signature and allowing the fate of new assimilate within the plants to be determined. The labelling approach allowed quantification of the expected preferential allocation of new C-assimilate to roots in enriched nitrate, prior to any measurable effect on whole biomass or root architecture. Biosensor (lux-marked Pseudomonas fluorescens 10586 pUCD607) bioluminescence, quantified spatially by CCD imaging, demonstrated that root exudation was significantly increased for roots in enriched nitrate. This response of root exudation, being primarily associated with root apices and concurrent with enhanced assimilate supply, strongly suggests that C-flow from roots is an integral component of the proliferation response to nitrate.

Predator–prey interaction in soil food web: functional response, size-dependent foraging efficiency, and the influence of soil texture

In a series of laboratory experiments, we presented carnivorous Macrobiotus richtersi (Tardigrada, Macrobiotidae) with nematode prey to assess their importance as predator. We investigated consumption rate for (a) different prey densities (10–400 prey individuals), (b) different prey biomasses (22–80 ng), (c) different prey species (Pelodera teres, Rhabditidae, versus Acrobeloides nanus, Cephalobidae) and (d) different environments (2-D agar surface versus 3-D sand fractions of three different textures). M. richtersi consumed up to 4.6 μg nematode prey in 4 h, that is, 43% of the tardigrade’s body mass. Predation rate was positively correlated with prey density. The optimal prey in the present investigation was the biggest prey because it yielded the highest biomass uptake per time. In addition, the size of M. richtersi played an important role in consumption rate. Bacterivorous nematodes reacted differently to attack. Even in a water film on stiff agar where nematode agility was limited, a vigorous undulation reaction of P. teres led to a measurable reduction in consumption rate. A. nanus, in contrast, showed little response to attack. Microcosm experiments with sands of different particle size demonstrated that M. richtersi is able to chase and consume small bacterivorous nematodes in a 3-D soil matrix. However, consumption rate in sand microcosms was significantly reduced compared with pure agar. The sand matrix improved nematode agility and possibly provided small pores as refuge for the nematodes. The lowest consumption rate was observed in fine sand. Effects of predatory tardigrades on nematode numbers in the field are discussed.