Soil microbial community responses to contamination with silver, aluminium oxide and silicon dioxide nanoparticles.

Soil microorganisms are key contributors to nutrient cycling and are essential for the maintenance of healthy soils and sustainable agriculture. Although the antimicrobial effects of a broad range of nanoparticulate substances have been characterised in vitro, little is known about the impact of these compounds on microbial communities in environments such as soil. In this study, the effect of three widely used nanoparticulates (silver, silicon dioxide and aluminium oxide) on bacterial and fungal communities in an agricultural pastureland soil was examined in a microcosm-based experiment using a combination of enzyme analysis, molecular fingerprinting and amplicon sequencing. A relatively low concentration of silver nanoparticles (AgNPs) significantly reduced total soil dehydrogenase and urease activity, while Al2O3 and SiO2 nanoparticles had no effect. Amplicon sequencing revealed substantial shifts in bacterial community composition in soils amended with AgNPs, with significant decreases in the relative abundance of Acidobacteria and Verrucomicrobia and an increase in Proteobacteria. In particular, the relative abundance of the Proteobacterial genus Dyella significantly increased in AgNP amended soil. The effects of Al2O3 and SiO2 NPs on bacterial community composition were less pronounced. AgNPs significantly reduced bacterial and archaeal amoA gene abundance in soil, with the archaea more susceptible than bacteria. AgNPs also significantly impacted soil fungal community structure, while Al2O3 and SiO2 NPs had no effect. Several fungal ribotypes increased in soil amended with AgNPs, compared to control soil. This study highlights the need to consider the effects of individual nanoparticles on soil microbial communities when assessing their environmental impact.

Differential degradation of polycyclic aromatic hydrocarbon mixtures by indigenous microbial assemblages in soil.

UNLABELLED: Environmental contamination by polycyclic aromatic hydrocarbons (PAHs) typically occurs as mixtures of compounds. In this study, the response of indigenous soil bacterial and fungal communities to mixtures containing phenanthrene, fluoranthene and benzo(a)pyrene in various combinations was examined using molecular fingerprinting techniques and quantification of a key PAH degradative gene. Results were compared to a parallel study by Sawulski et al. (2014) which examined the effect of these PAHs on soil microbial communities when added as single contaminants. The rate of degradation of individual PAHs varied depending on whether the PAH was present as a single contaminant or in a mixture; phenanthrene was degraded most rapidly when present as a sole contaminant, fluoranthene was removed faster in the presence of the lower molecular weight phenanthrene and the rate of benzo(a)pyrene degradation was reduced in the presence of the 4-ring PAH, fluoranthene. Bacterial and fungal assemblages differed significantly between treatments regardless of which PAH was added to soil. Although less abundant than the Gram-negative PAH-RHDα gene, the gene associated with Gram-positive bacteria responded to a greater extent to the presence of PAHs, either as single compounds or as mixtures and this increase was significantly correlated with PAH degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Contaminated sites generally contain complex mixtures of pollutants. Development of effective bioremediation strategies for contaminated soils requires knowledge of the response of soil microbial communities to such mixtures. This study provides information on the degradation of different mixtures of three priority pollutants in soil with a history of polycyclic aromatic hydrocarbon contamination and examines the response of soil bacterial and fungal communities to the presence of these pollutants as sole contaminants or as part of a mixture. This is one of few studies to-date to compare the effects of single compounds and pollutant mixtures on more than one soil microbial community.

Microbial community structure during fluoranthene degradation in the presence of plants.

AIMS: To investigate bacterial and fungal community structure during degradation of varying concentrations (0-5000 mg kg(-1) ) of the polycyclic aromatic hydrocarbon (PAH) fluoranthene in the presence or absence of tomato plants. METHOD AND RESULTS: Fluoranthene amended or unamended growing media, with or without a plant, were incubated in pots in a glasshouse for 30 days. Fluoranthene concentration was quantified using GC-FID, while bacterial and fungal community structure was investigated using trflp and arisa, respectively. The abundance of two ring hydroxylating dioxygenase genes was measured using qPCR. More fluoranthene was degraded in the presence of tomato plants at 500 and 5000 mg fluoranthene kg(-1) (P < 0·001), and it had a toxic effect on plant growth. Bacterial and fungal community composition in the rhizosphere was significantly different from that in nonrhizospheric and unplanted samples. The influence of fluoranthene on bacterial communities overcame that of the plant root such that community composition in rhizosphere and planted nonrhizospheric samples was broadly similar when fluoranthene was present. PAH-RHDα-GP genes were more abundant than PAH-RHDα-GN genes in all treatments. PAH-RHDα-GN abundance was unaffected by either PAH concentration or growing medium type, while the abundance of PAH-RHDα-GP was greatest in the rhizosphere. CONCLUSIONS: Plants promoted microbial growth and diversity and led to increases in abundance of Gram-positive dioxygenase genes, correlated with increased degradation of fluoranthene. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to knowledge in the broad area of biodegradation and also provides useful information for the design of future bioremediation strategies.

Responses of anaerobic rumen fungal diversity (phylum Neocallimastigomycota) to changes in bovine diet.

AIMS: Anaerobic rumen fungi (Neocallimastigales) play important roles in the breakdown of complex, cellulose-rich material. Subsequent decomposition products are utilized by other microbes, including methanogens. The aim of this study was to determine the effects of dietary changes on anaerobic rumen fungi diversity. METHODS AND RESULTS: Altered diets through increasing concentrate/forage (50 : 50 vs 90 : 10) ratios and/or the addition of 6% soya oil were offered to steers and the Neocallimastigales community was assessed by PCR-based fingerprinting with specific primers within the barcode region. Both a decrease in fibre content and the addition of 6% soya oil affected Neocallimastigales diversity within solid and liquid rumen phases. The addition of 6% soya oil decreased species richness. Assemblages were strongly affected by the addition of 6% soya oil, whereas unexpectedly, the fibre decrease had less effect. Differences in volatile fatty acid contents (acetate, propionate and butyrate) were significantly associated with changes in Neocallimastigales assemblages between the treatments. CONCLUSIONS: Diet clearly influences Neocallimastigales assemblages. The data are interpreted in terms of interactions with other microbial groups involved in fermentation processes within the rumen. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge on the influence of diet on anaerobic fungi is necessary to understand changes in microbial processes occurring within the rumen as this may impact on other rumen processes such as methane production.

Effect of dietary prebiotic (mannan oligosaccharide) supplementation on the caecal bacterial community structure of turkeys.

The identification of specific bacterial species influenced by mannan oligosaccharide (MOS) supplementation may assist in the formulation of new and improved diets that promote intestinal health and improve bird performance, offering suitable alternatives to antimicrobials in feed for sustainable poultry production. This study has been conducted to evaluate the use of a MOS compound derived from the yeast cell wall of Saccharomyces cerevisiae on turkey performance, bacterial community structure and their phylogenetic associations. A 42-day turkey trial was carried out on birds fed control and MOS-supplemented diets. Bird performance data (weight gains, feed consumption and feed efficiency ratios) were collected, and caecal contents were extracted from randomly caught poults on days 28, 35 and 42 posthatch. Bird performance data showed no improvements as a result of dietary supplementation. Automated ribosomal intergenic spacer analysis (ARISA) revealed the bacterial community structure to be significantly altered on days 28 and 35 posthatch but not day 42 as a result of dietary supplementation. This technique was coupled with 16S rRNA gene sequence analysis to elucidate phylogenetic identities of bacteria. The dominant bacteria of the caecum on all days in both treatment groups were members of phylum Firmicutes, followed by the Bacteroidetes and Proteobacteria phyla, respectively. Statistical analysis of the 16S rRNA gene libraries showed that the composition of the MOS clone library differed significantly to the control on day 35 posthatch. It can be concluded that MOS alters the bacterial community structure in the turkey caecum.

Effect of dietary supplementation with a Saccharomyces cerevisiae mannan oligosaccharide on the bacterial community structure of broiler cecal contents.

This study investigated the effects of dietary supplementation with a prebiotic mannan oligosaccharide (MOS) on broiler performance, bacterial community structure, and phylogenetic populations of cecal contents. Bird performance data were collected, and cecal samples were extracted from randomly caught poults from each treatment group every 7 days from hatching to the age of 42 days. Weight gain, feed consumption, and feed efficiency ratios did not differ significantly between groups. Automated ribosomal intergenic spacer analysis (ARISA) of the bacterial communities in birds receiving MOS-supplemented diets indicated that dietary supplementation with MOS at either of 2 levels significantly altered the bacterial community structure from that of the control group on all sample days. The phylogenetic identities of bacteria contained within the cecum were determined by constructing a 16S rRNA gene clone library. A total of 594 partial 16S rRNA gene sequences from the cecal contents were analyzed and compared for the three dietary treatments. The dominant bacteria of the cecum belonged to three phyla, Firmicutes, Bacteroidetes, and Proteobacteria; of these, Firmicutes were the most dominant in all treatment groups. Statistical analysis of the bacterial 16S rRNA gene clone libraries showed that the compositions of the clone libraries from broilers receiving MOS-supplemented diets were, in most cases, significantly different from that of the control group. It can be concluded that in this trial MOS supplementation significantly altered the cecal bacterial community structure.

The effect of dietary concentrate and soya oil inclusion on microbial diversity in the rumen of cattle.

AIMS: Methane emissions from ruminants are a significant contributor to global greenhouse gas production. The aim of this study was to examine the effect of diet on microbial communities in the rumen of steers. METHODS AND RESULTS: The effects of dietary alteration (50 : 50 vs 90 : 10 concentrate-forage ratio, and inclusion of soya oil) on methanogenic and bacterial communities in the rumen of steers were examined using molecular fingerprinting techniques (T-RFLP and automated ribosomal intergenic spacer analysis) and real-time PCR. Bacterial diversity was greatly affected by diet, whereas methanogen diversity was not. However, methanogen abundance was significantly reduced (P = 0.009) in high concentrate-forage diets and in the presence of soya oil (6%). In a parallel study, reduced methane emissions were observed with these diets. CONCLUSIONS: The greater effect of dietary alteration on bacterial community in the rumen compared with the methanogen community may reflect the impact of substrate availability on the rumen bacterial community. This resulted in altered rumen volatile fatty acid profiles and had a downstream effect on methanogen abundance, but not diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding how rumen microbial communities contribute to methane production and how these microbes are influenced by diet is essential for the rational design of methane mitigation strategies from livestock.

Assessing the impact of various ensilage factors on the fermentation of grass silage using conventional culture and bacterial community analysis techniques.

AIMS: Grass silage is an important ruminant feedstuff on farms during winter. The ensilage of grass involves a natural lactic acid bacterial fermentation under anaerobic conditions, and numerous factors can influence the outcome of preservation. The aim of this study was to investigate the effect of dry matter concentration, ensiling system, compaction and air infiltration on silage bacterial community composition. METHODS AND RESULTS: The impact of these factors was examined using conventional methods of microbial analysis and culture-independent Terminal Restriction Fragment Length Polymorphism (T-RFLP). Silage fermentation was restricted in herbage with a high dry matter concentration, and this was reflected in a shift in the bacterial population present. In contrast, ensiling system had little effect on bacterial community composition. Air infiltration, in the absence of compaction, altered silage bacterial community composition and silage pH. CONCLUSIONS: Dry matter concentration and the absence of compaction were the main factors affecting silage microbial community composition, and this was reflected in both the conventional culture-based and T-RFLP data. SIGNIFICANCE AND IMPACT OF THE STUDY: T-RFLP proved a useful tool to study the factors affecting ensilage. Apart from monitoring the presence or absence of members of the population, shifts in the relative presence of members could be monitored.

Comparison of DNA- and RNA-based bacterial community structures in soil exposed to 2,4-dichlorophenol.

AIMS: To examine the effect of the pollutant 2,4-dichlorophenol on DNA- and RNA-based bacterial communities in soil. METHODS AND RESULTS: Soil was exposed to 100 mg kg(-1) of 2,4-dichlorophenol (2,4-DCP), and degradation was monitored over 35 days. DNA and RNA were coextracted, and terminal restriction fragment length polymorphism (T-RFLP) was used to report changes in bacterial communities in response to the presence of the chlorophenol. The phylogenetic composition of the soil during degradation was determined by creating a clone library of amplified 16S rRNA sequences from both DNA and reverse-transcribed RNA from exposed soil. Resulting clones were sequenced, and putative identities were assigned. CONCLUSIONS: A significant difference between active (RNA-based) and total (DNA-based) bacterial community structure was observed for both T-RFLP and phylogenetic analyses in response to 2,4-DCP, with more pronounced changes seen in RNA-based communities. Phylogenetic analysis indicated the dominance of Proteobacteria in both profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the response of soil bacterial communities to the addition of the xenobiotic compound 2,4-DCP, and highlights the importance of including RNA-based 16S rRNA analysis to complement any molecular study in a perturbed soil.

Bacterial community dynamics during the ensilage of wilted grass.

AIMS: Grass silage is the product formed by a natural lactic acid bacterial fermentation when grass is stored under anaerobic conditions, and represents an important ruminant feedstuff on farms during winter. Of the two commonly employed methods of ensiling forage, baled silage composition frequently differs from that of comparable precision-chop silage reflecting a different ensiling environment. The aim of this study was to investigate the dynamics of the silage fermentation in wilted grass and between ensiling systems. METHODS AND RESULTS: Fermentation dynamics were examined using traditional methods of silage analyses, including microbial enumeration and analysis of fermentation products, and culture-independent terminal restriction fragment length polymorphism (T-RFLP). A successful fermentation was achieved in both systems, with the fermentation (increase in lactic acid bacteria and lactic acid concentration, decrease in pH) proceeding rapidly once the herbage was ensiled. CONCLUSIONS: Under controlled conditions, little difference in silage quality and microbial composition were observed between ensiling systems and this was further reflected in the T-RFLP community analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: T-RFLP proved a potentially useful tool to study the ensilage process and could provide valid support to traditional methods, or a viable alternative to these methods, for investigating the dynamics of the bacterial community over the course of the fermentation.

SALT TOLERANCE IN THE HALOPHYTE SUAEDA MARITIMA (L.) DUM.: THE EFFECT OF SALINITY ON THE CONCENTRATION OF SODIUM IN THE XYLEM.

Rates of ion transport and transpiration were measured during the day and night in whole seedlings of Suaeda maritima growing over a range of salinities, in order to calculate concentrations of sodium and potassium in the xylem during these periods. Mean sodium concentration in the xylem was maximal at 56 mol m-3 Na with an external salinity of 200 mol m-3 NaCl. The sodium concentration in the xylem was greater in the dark than in the light at all external salinities investigated. Comparison of the external sodium with that in the xylem indicated that sodium was more strongly excluded from the transpiration stream as salinity increased. The mean concentration of potassium in the xylem declined as external NaCl concentration increased, although selectivity for potassium increased at higher salinities. Results are discussed in relation to osmotic adjustment in S. maritima.

THE PHYSIOLOGY OF BASIDIOMYCETE LINEAR ORGANS: I. PHOSPHATE UPTAKE BY CORDS AND MYCELIUM IN THE LABORATORY AND THE FIELD.

Phosphate uptake as a function of external medium concentration has been determined for mycelium grown in the laboratory, segments of cords collected from the field and cords in the field for a range of wood-decay basidiomycetes. Hofstee plots in all cases can be interpreted as indicating the presence of two uptake systems. Uptake of phosphate by mycelium is reduced by increasing the concentration of phosphate in the growth medium from 10 µM to 10 mM. The major portion absorbed by cords in the field remains within the segment exposed to radioactive solution, suggesting conversion of the phosphate to an immobile form unavailable for translocation.

ribosort: a program for automated data preparation and exploratory analysis of microbial community fingerprints.

ribosort is a computer package for convenient editing of automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphism (TRFLP) data. It is designed to eliminate the labourious task of manually classifying community fingerprints in microbial ecology studies. This program automatically assigns detected fragments and their respective relative abundances to appropriate ribotypes. It permits simultaneous sorting of multiple profiles and facilitates direct workflow from TRFLP and ARISA output through to community analyses. ribosort also provides several options to merge repeat profiles of a sample into a single composite profile. By creating a 'ribotypes by samples' matrix ready for statistical analyses, use of the package saves time and simplifies the preparation of DNA fingerprint data sets for statistical analysis. In addition, ribosort performs exploratory analysis on the data by creating multidimensional scaling plots that compare the similarity of sample profiles using the statistical software r.

Microbial community changes during the bioremediation of creosote-contaminated soil.

AIMS: To investigate the effects of aeration on the ex situ biodegradation of polycyclic aromatic hydrocarbons (PAHs) in creosote-contaminated soil and its effect on the microbial community present. METHODS AND RESULTS: Aerated and nonaerated microcosms of soil excavated from a former timber treatment yard were maintained and sampled for PAH concentration and microbial community changes by terminal restriction fragment length polymorphism (TRFLP) analysis. After an experimental period of just 13 days, degradation was observed with all the PAHs monitored. Abiotic controls showed no loss of PAH. Results unexpectedly showed greater loss of the higher molecular weight PAHs in the nonaerated control. This may have been due to the soil excavation causing initial decompaction and aeration and the resulting changes caused in the microbial community composition, indicated by TRFLP analysis showing several ribotypes greatly increasing in relative abundance. Similar changes in both microcosms were observed but with several possible key differences. The species of micro-organisms putatively identified included Bacilli, pseudomonad, aeromonad, Vibrio and Clostridia species. CONCLUSIONS: Excavation of the contaminated soil leads to decompaction, aeration and increased nutrient availability, which in turn allow microbial biodegradation of the PAHs and a change in the microbial community structure. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the changes occurring in the microbial community during biodegradation of all PAHs is essential for the development of improved site remediation protocols. TRFLP allows useful monitoring of the total microbial community.

Bacterial community dynamics across a floristic gradient in a temperate upland grassland ecosystem.

Alterations in soil bacterial communities across a transect between a semi natural upland grassland and an agriculturally improved enclosure were assessed using culture-based methods and a nucleic-acid-based method, terminal restriction fragment length polymorphism (TRFLP). While plant diversity decreased across the transect towards the improved area, numbers of 16S rDNA terminal restriction fragments increased, indicating an increase in numbers of bacterial ribotypes. Bacterial numbers, microbial activity, and potential functional diversity also followed a similar trend, increasing with decreasing plant diversity. Alterations in bacterial community structure were coincident with changes in soil physicochemical properties which also changed across the transect. Increases in soil pH, nitrate, phosphorus, potassium, and calcium occurred toward the improved grassland, while organic matter and ammonium declined. The inverse relationship between floristic diversity and bacterial ribotype numbers suggests that soil physicochemical factors may be as influential in determining bacterial diversity in soils of upland grassland communities as floristic diversity.

Salt tolerance in the halophyte Suaeda maritima L. Dum. : The maintenance of turgor pressure and water-potential gradients in plants growing at different salinities.

Osmotic potentials and individual epidermal cell turgor pressures were measured in the leaves of seedlings of Suaeda maritima growing over a range of salinities. Leaf osmotic potentials were lower (more negative) the higher the salt concentration of the solution and were lowest in the youngest leaves and stem apices, producing a gradient of osmotic potential towards the apex of the plant. Epidermal cell turgor pressures were of the order of 0.25 to 0.3 MPa in the youngest leaves measured, decreasing to under 0.05 MPa for the oldest leaves. This pattern of turgor pressure was largely unaffected by external salinity. Calculation of leaf water potential indicated that the gradient between young leaves and the external medium was not altered by salinity, but with older leaves, however, this gradient diminished from being the same as that for young leaves in the absence of NaCl, to under 30% of this value at 400 mM NaCl. These results are discussed in relation to the growth response of S. maritima.

Evidence for sltA1 as a salt-sensitive allele of the arginase gene (agaA) in the ascomycete Aspergillus nidulans.

Strains of Aspergillus nidulans carrying the sltA1 mutation, conferring sensitivity to KCl and NaCl, also showed an arginine-sensitive phenotype whereby concentrations of the L-amino acid at or above 10 mM were toxic to growth. Sexual progeny of a cross between a sltA1 mutant and a wild-type strain showed a co-segregation of salt and arginine sensitivity. Similarly, revertants to salt tolerance showed a loss of arginine sensitivity as did sltA1 strains that were transformed with a cosmid carrying the putative sltA1+ wild-type allele. In addition, arginine sensitivity could be relieved by L-ornithine. It is suggested that sltA1 is a salt-sensitive allele of the arginase gene (agaA).