Tool boxes for soil bioremediation assessment in heavy metal polluted areas

The thesis is divided into seven separate chapters, each of them has a specific purpose. The first chapter is a general introduction. It is based on the accurate bibliographic research and aims to clarify main issues concerning soil and heavy metals (e.g. the concept of soil quality and its indicators, heavy metals bioavailability, the plant-microorganisms interactions in soils, the possible sustainable methods for soil remediation). The general goal of the thesis and the specific aims of the experimental researches are described in chapter 2. The third chapter entitled “Set up a tool box of soil quality bioindicators” describes which “experimental tools” were chosen to assess the soil quality improvement during the bioremediation processes. In the following fourth chapter “Heavy metal bioavailability in soil and bioaccumulation into soil microbial biomass” the methods selected to measure the heavy metal bioavailability and accumulation into soil microbial biomass are described. The fifth and sixth chapters show as the previously selected soil bioindicators respond to two different bioremediation systems. The fifth chapter “Changes of soil quality in a phytoremediation system of a former dump” refers to a phytoremediation system applied to an ex-dump located in “Madonna dell’Acqua”, Municipality of San Giuliano Terme - Pisa (Italy). The discussions of the obtained results are important to evaluate the efficiency of this soil reclamation technology. The sixth chapter, entitled “Use of Plant Growth-Promoting Bacteria to enhance heavy metals phytoremediation in mining soils: a preliminary study under Umbrella project” refers to a pot experiment conducted to test the efficiency of the microbial-assisted phytoremediation based on the soil inoculation of different selected Plant-Growth-Promoting Bacteria. This preliminary test useful for the next pot experiments of the European Umbrella project, was conducted to convey a reflection about the use of PGPB applied to mining soils during a phytoremediation process. Finally, in the general discussion of the seventh chapter, the most important results are summarized and general conclusions are reported, focusing on the aspects of bioremediation to be developed and improved

Tool boxes for soil bioremediation assessment in heavy metal polluted areas

The thesis is divided into seven separate chapters, each of them has a specific purpose. The first chapter is a general introduction. It is based on the accurate bibliographic research and aims to clarify main issues concerning soil and heavy metals (e.g. the concept of soil quality and its indicators, heavy metals bioavailability, the plant-microorganisms interactions in soils, the possible sustainable methods for soil remediation). The general goal of the thesis and the specific aims of the experimental researches are described in chapter 2. The third chapter entitled “Set up a tool box of soil quality bioindicators” describes which “experimental tools” were chosen to assess the soil quality improvement during the bioremediation processes. In the following fourth chapter “Heavy metal bioavailability in soil and bioaccumulation into soil microbial biomass” the methods selected to measure the heavy metal bioavailability and accumulation into soil microbial biomass are described. The fifth and sixth chapters show as the previously selected soil bioindicators respond to two different bioremediation systems. The fifth chapter “Changes of soil quality in a phytoremediation system of a former dump” refers to a phytoremediation system applied to an ex-dump located in “Madonna dell’Acqua”, Municipality of San Giuliano Terme - Pisa (Italy). The discussions of the obtained results are important to evaluate the efficiency of this soil reclamation technology. The sixth chapter, entitled “Use of Plant Growth-Promoting Bacteria to enhance heavy metals phytoremediation in mining soils: a preliminary study under Umbrella project” refers to a pot experiment conducted to test the efficiency of the microbial-assisted phytoremediation based on the soil inoculation of different selected Plant-Growth-Promoting Bacteria. This preliminary test useful for the next pot experiments of the European Umbrella project, was conducted to convey a reflection about the use of PGPB applied to mining soils during a phytoremediation process. Finally, in the general discussion of the seventh chapter, the most important results are summarized and general conclusions are reported, focusing on the aspects of bioremediation to be developed and improved

Assessing the role of EO in biodiversity monitoring: options for integrating in-situ observations with EO within the context of the EBONE concept

The European Biodiversity Observation Network (EBONE) is a European contribution on terrestrial monitoring to GEO BON, the Group on Earth Observations Biodiversity Observation Network. EBONE’s aims are to develop a system of biodiversity observation at regional, national and European levels by assessing existing approaches in terms of their validity and applicability starting in Europe, then expanding to regions in Africa. The objective of EBONE is to deliver: 1. A sound scientific basis for the production of statistical estimates of stock and change of key indicators; 2. The development of a system for estimating past changes and forecasting and testing policy options and management strategies for threatened ecosystems and species; 3. A proposal for a cost-effective biodiversity monitoring system. There is a consensus that Earth Observation (EO) has a role to play in monitoring biodiversity. With its capacity to observe detailed spatial patterns and variability across large areas at regular intervals, our instinct suggests that EO could deliver the type of spatial and temporal coverage that is beyond reach with in-situ efforts. Furthermore, when considering the emerging networks of in-situ observations, the prospect of enhancing the quality of the information whilst reducing cost through integration is compelling. This report gives a realistic assessment of the role of EO in biodiversity monitoring and the options for integrating in-situ observations with EO within the context of the EBONE concept (cfr. EBONE-ID1.4). The assessment is mainly based on a set of targeted pilot studies. Building on this assessment, the report then presents a series of recommendations on the best options for using EO in an effective, consistent and sustainable biodiversity monitoring scheme. The issues that we faced were many: 1. Integration can be interpreted in different ways. One possible interpretation is: the combined use of independent data sets to deliver a different but improved data set; another is: the use of one data set to complement another dataset. 2. The targeted improvement will vary with stakeholder group: some will seek for more efficiency, others for more reliable estimates (accuracy and/or precision); others for more detail in space and/or time or more of everything. 3. Integration requires a link between the datasets (EO and in-situ). The strength of the link between reflected electromagnetic radiation and the habitats and their biodiversity observed in-situ is function of many variables, for example: the spatial scale of the observations; timing of the observations; the adopted nomenclature for classification; the complexity of the landscape in terms of composition, spatial structure and the physical environment; the habitat and land cover types under consideration. 4. The type of the EO data available varies (function of e.g. budget, size and location of region, cloudiness, national and/or international investment in airborne campaigns or space technology) which determines its capability to deliver the required output. EO and in-situ could be combined in different ways, depending on the type of integration we wanted to achieve and the targeted improvement. We aimed for an improvement in accuracy (i.e. the reduction in error of our indicator estimate calculated for an environmental zone). Furthermore, EO would also provide the spatial patterns for correlated in-situ data. EBONE in its initial development, focused on three main indicators covering: (i) the extent and change of habitats of European interest in the context of a general habitat assessment; (ii) abundance and distribution of selected species (birds, butterflies and plants); and (iii) fragmentation of natural and semi-natural areas. For habitat extent, we decided that it did not matter how in-situ was integrated with EO as long as we could demonstrate that acceptable accuracies could be achieved and the precision could consistently be improved. The nomenclature used to map habitats in-situ was the General Habitat Classification. We considered the following options where the EO and in-situ play different roles: using in-situ samples to re-calibrate a habitat map independently derived from EO; improving the accuracy of in-situ sampled habitat statistics, by post-stratification with correlated EO data; and using in-situ samples to train the classification of EO data into habitat types where the EO data delivers full coverage or a larger number of samples. For some of the above cases we also considered the impact that the sampling strategy employed to deliver the samples would have on the accuracy and precision achieved. Restricted access to European wide species data prevented work on the indicator ‘abundance and distribution of species’. With respect to the indicator ‘fragmentation’, we investigated ways of delivering EO derived measures of habitat patterns that are meaningful to sampled in-situ observations

Micro analytical observation of elemental distribution in arbuscular mycorrhizal (AM) roots from mining sites in South Africa and identification of their AM fungi

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2016.South Africa, as one of the leaders in mining industry, due to the variety and quantity of minerals produced, has been and is still producing a number of mine tailings which are contaminated by heavy metals. Heavy metals are very harmful to plants and especially to human beings and animals due to their non-biodegradable nature. The problem of environmental metal pollution could be combated by the establishment of Arbuscular Mycorrhiza (AM) vegetation on the surface of mine tailings. Besides the toxicity of the substrate, such areas usually lack essential nutrients (mainly N, P, and K) and organic matter. AM fungi contribute to soil structure by forming micro- and macro- soil aggregates within the net of external hyphae. Their presence may reduce stress caused by lack of nutrients or organic matter and increase plant resistance to pathogens, drought and heavy metals. Therefore, mycorrhizal fungi may become the key factor in successful plant revegetation of heavy-metal-polluted areas by promoting the success of plant establishment and increasing soil fertility and quality. The aim of this project was to identify AM fungi from a number of heavy metal sites in South Africa using both morphological and molecular techniques, followed by the evaluation of heavy metal distribution and localisation in mycorrhizal roots. Soil samples were collected from three different provinces, namely: Gauteng, Mpumalanga and North West provinces. The sites were selected based on their historical and current heavy metal contamination. Indigenous AM fungal isolates (which are adapted to local soil conditions) can stimulate plant growth better than non-indigenous isolates. AM fungal spores were isolated from 100g of representative soil sample by the wet sieving and decanting method, followed by assessment of spore numbers and infective propagules. The spores of a subset of the pot samples were mounted on microscope slides in polyvinyl lactic acid glycerol and identified by morphological characteristics to the level of genus or species. Most of the spores counted were observed in a 45 μm sieve. These spores were tiny and had different sizes, colours and shapes. The majority of the observed spores were small, brown and oval in shape. For morphological identification, plant roots were stained and hyphae were found to be the most abundant in roots. For molecular identification, two sets of nested PCR primers, namely NS1 & NS4 coupled with AML1 & AML2, were employed in this study due to their ability to amplify all subgroups of arbuscular mycorrhizal fungi (AM fungal, Glomeromycota), while excluding sequences from other organisms. Through both morphological characteristics and molecular identification, the following fungal genera were identified for the first time in the studied sites in South Africa. The study identified a total of 14 AM fungal genera and 55 AM fungal species, which are: Glomus (15), Acaulospora (11), Scutellospora (6), Gigaspora (6), Rhizophagus (3), Funneliformis (3), Archaeospora (2), Claroideoglomus (2), Ambispora (2), Sclerocystis (1), Fuscutata (1), Entrophospora (1), Diversispora (1), Paraglomus (1). Both Glomus and Acualospora have been observed to be the highest occurring genera in the analysed soil samples, followed by Scutellospora and Gigaspora and others mentioned. PIXE technique was successful in localising elemental concentration in both plant roots and AM fungal structures, as well as in indicating the large vesicles in root tissue. AM fungal structures in the outer cortex or outer epidermal layer of the root cross-sections were observable, as shown by the more significantly enriched Si in the vesicles and arbuscules. Distinctive elemental maps can be used to localise sites of colonisation and verification of the symbiotic nature of the tissue. This indicates that a range of metals can be sequestered in AM fungal structures above levels in surrounding host root tissue, and demonstrates the potential of Micro-PIXE to determine metal accumulation and elemental distribution in mycorrhizal plant roots and inter-and intracellular AM fungal structures. This research highlights the potential of AM fungi for inoculation of plants as a prerequisite for successful restoration of heavy metal contaminated soils. It also illustrates the importance of AM fungal diversity in selected high heavy metal (HM) sites in RSA, particularly in the North West and the Gauteng gold mining slime dams. Therefore, phytoremediation of mine tailings by mycorrhizal plants seems to be one of the most promising lines of research on mine tailings contamination by heavy metals. The strategies which evolved during this project have great potential for phytoremediation of toxic mining sites, and thus can help mitigate the environmental problems, especially in the mining waste sites.LG201

Ecology of freshwater turtles and other wetland wildlife in a north-central West Virginia watershed

The goal of wetland mitigation in the United States is to achieve a no-net-loss of wetlands; however, mitigated wetlands must be monitored to ensure wetland function is comparable to natural wetlands. In this study, relationships between land use practices and freshwater turtle abundance, wetland connectivity, and heavy metal bioaccumulation were investigated. In Chapter 1, a summary of pre-restoration species abundance and diversity is provided for anurans, birds, benthic macroinvertebrates, fishes, small mammals, plants, and turtles in riparian wetlands along Ruby Run, a tributary of Deckers Creek in north-central West Virginia, USA. These data provide a baseline for comparison after mitigation is completed to monitor effectiveness of achieving near-natural wetland function. In Chapter 2, relationships between relative abundance of two common turtle species found in West Virginia (snapping turtles [Chelydra serpentina] and painted turtles [Chrysemys picta]) and habitat characteristics are investigated across 39 wetlands within the Upper Deckers Creek watershed in north-central West Virginia, USA. The effects of wetland and landscape characteristics on snapping turtle movement among wetlands are also assessed. Out of the 42 adult snapping turtles across 22 wetland sites (0.04–7.45 ha) that were equipped with radio transmitters, movement was documented among wetlands for 27 individuals (66%). Wetland perimeter, substrate depth, vegetative cover, and distance from roads were positively associated with snapping turtle relative abundance. Wetland perimeter and agriculture within 500 m were negatively associated with snapping turtle movement. Wetland vegetative cover and canopy cover were positively associated with painted turtle relative abundance. Landscape-level characteristics such as nearby farm ponds, wetlands, agriculture, and roads should be considered when developing wetland conservation plans to maximize turtle abundance. Wetland connectivity is important because snapping turtles and painted turtles regularly use a diversity of wetland types with abundant vegetation and natural surroundings. In Chapter 3, the presence of cadmium, chromium, lead, mercury, selenium, and zinc concentrations is reported across sites within the Upper Deckers Creek watershed and relate landscape characteristics to concentrations found in wetland soil and turtle tissues. A total of 33 painted turtles and 24 snapping turtles across 22 sites were tested for metal concentrations through non-destructive tissue sampling (i.e. blood and nails). All metal concentrations were higher in nails than blood, but concentration differences between species varied across metals. Selenium levels in soil and turtle nails were positively correlated with proximity to mine land. Turtle nail lead concentrations were positively correlated with wetland soil Pb concentrations. Percent agriculture within 30 m of wetlands was negatively correlated with mercury in blood but positively correlated in nails, and all samples analyzed had mercury levels that exceeded U.S. Environmental Protection Agency consumption limits. Proximity to these land use practices should be considered when implementing and managing wetlands and associated buffer areas. Guidelines for turtle consumption limits should be investigated further to ensure human health and safety

Myxomycete communities occurring in fragmented forest patches in two municipalities of Laguna, Philippines

In spite of the increasing number of myxomycete studies in the tropical Southeast Asia over the last decades, many forest patches are still left unexplored for the region, in particular in the Philippine archipelago. Thus, an assessment of myxomycete diversity, occurrence, and composition was carried out in forest fragments from two municipalities of the province of Laguna, Philippines. From the 12 established 5 m × 5 m plots, a total of 240 moist chamber cultures were prepared for this study and yielded 42 myxomycete species belonging to 14 genera. Rarefaction curves and several heterogeneity indices revealed higher species diversity in Los Baños than in Calauan. Moreover, the taxonomic diversity index also showed that the taxonomic diversity of myxomycete in Los Baños is more intuitively diverse than in Calauan. In terms of community analysis between the two municipalities, it showed that myxomycete communities clustered between different microhabitats. High values of coefficient of community and percentage similarity indices possibly implicates that spore dispersal in forest fragments in closer proximities may cause high number of shared species (60%). With an addition of Cribraria lepida as a new record for the country, this study now updates the myxomycete profile for Laguna, Philippines

Genomic and Culturomic Analysis of Gut Microbiota Function and Salmonella Enterica Expansion

Enteric bacteria that are resident in the hindgut of mammals are critical in immune development, digestion, and colonization resistance against pathogens. One of the major pathogens that gut commensals provide resistance against is Salmonella enterica, a major foodborne pathogen capable of infecting almost every warm-blooded animal. Given the interplay between pathogens and commensals in the gut lumen, the gut microbiota of pigs was studied by combining two disparate techniques: shotgun metagenomics and high throughput culturomics. Metagenomics readily identifies major taxa present in samples and can give an estimation to total genetic catalogue from an environment. However, many rare or low abundance taxa were retrieved in culture that were not reliably obtained from metagenomics. Major gene pathways recovered from culture isolates were absent from metagenomics. In addition to studying the gut microbiota, two genomics studies were conducted on S. enterica. The first study was to establish and investigate the genomic population structure of a bovine-adapted serovar, S. enterica Dublin. The serovar is a primary pathogen of cattle and can establish carrier states with the pathogen being shed intermittently in the feces and milk. It was observed that the core and ancillary genomes are strongly influence by region of isolation. Virulence and prophage content were homogenous among the geographic clades. However, US genomes yielded multiple antibiotic resistance genes mediated by an IncA/C2 plasmid. Antibiotic resistance was not common in isolates outside of the US. The second S. enterica genome investigation was to identify co-occurrence between metal and antibiotic resistance. Co-occurrence between the genotypes was identified, but is isolated to one clade of S. enterica I 4,[5],12:i:-. However, multiple serovars contain resistance to copper and silver, which may permit the expansion into novel niches as metal use continues to rise in medicine and agriculture

Evaluation of a New Method for Large-Scale and Gene-targeted Next Generation DNA Sequencing in Nonmodel Species

The efficient method called exon capture provides for sequencing genes genome-wide, targeting candidate genes, and sampling specific exons within genes. Although developed for model species with available whole genome sequences, the method can capture exons in nonmodel species using the genomic resources of a related model species. How close the relatives must be for effective exon capture is not known. The work herein demonstrates cross-taxa capture in ungulates, using the domestic cow genome as a reference. It also describes a computer program designed for collecting exon sequences for exon capture, allowing users to set per-gene and overall base pair (bp) limits, and to prefer internal or external exons. Cross-taxa exon capture was tested with subject-reference divergence times from 0 to ~60 million years. Sequencing success decreased with increasing subject-reference phylogenetic divergence. With the domestic cow genome as reference, American bison exons, at 1-2 million years (MY) of divergence, were captured as successfully as those of a domestic cow. The cow and bison captures each yielded sequence from ~80% of the 3.6 million bp targeted. Two bighorn sheep, 7 mule deer, and 4 pigs at about 20, 30, and 60 MY of divergence from the cow, respectively, yielded averages of ~70%, ~60%, and ~55% of the targeted bp. A gene family with many closely related, duplicated loci was expected to show reduced success compared to the whole collection. This prediction was supported, as 63 exons in the MHC gene family sequences yielded 62% fully sequenced in the cow, and 32%, 20%, and 4% for the bighorn, deer, and pigs, respectively. A comparison of two sequence alignment programs showed that Stampy, designed for high sample-reference divergence, was dramatically better than BWA, designed for low divergence, only in the pig capture, in which Stampy yielded ~30% more bp than did BWA. A universal ungulate exon capture array could be developed using the 8,999 exons that were fully sequenced in all species, including the pig at ~60 MY. As this method helps us understand the genetic basis of evolutionary processes, so it can contribute to an informed study and stewardship of our ecological endowment

Establishment of Pollinator Habitat within a Livestock Pasture Ecosystem

Pollinators are important for fertilization, setting fruits, and seed development of more than 78% of the flowering plants that provide food for human beings and other species. Use of pollinators to maximize crop production is a proven agricultural practice; however, it has been less explored in livestock forage production systems. This study investigated pollinator abundance and diversity in pastures using different sampling methods and determined the impact of different pasture management practices on insect pollinators in a livestock pasture ecosystem. In Chapter 2, utility of four different colors of pan trap (blue, green, yellow, and purple) for sampling bees in a livestock pasture ecosystem of native forage species was examined. Blue color traps were the most attractive to bees followed by purple, yellow and green color traps. In Chapter 3, vanes with different colors (dark blue, bright blue, dark yellow, bright yellow, purple, and red) were designed and evaluated for their light reflectance properties and attractiveness to wild bees in livestock pasture. Bright blue traps captured the highest number of individuals and species of bees. This could be due to appropriate match between the visual spectrum of bees and light reflectance spectrum of vanes. Bees responded similarly to traps with other colors of vanes, except for red vane traps, which captured the lowest number of bees. In Chapter 4, impacts of grazing native forb and grasses on insect pollinators and other arthropod communities in a pasture system was determined. The abundance, diversity, and evenness of bee communities and other insects were greater in non-grazed plots as compared to grazed pasture plots. However, species richness, as measured by rates of species accumulation relative to sampling effort, was not different among treatments. In Chapter 5, a two-year study assessed how organic and non-organic pasture management practices affect bee abundance and diversity in pastures. Bee diversity, abundance, and species richness of bees (as measured by rarefaction curve) were similar between pastures under organic or non-organic management. Different factors such as low availability of floral resources in organic pastures, use of synthetic fertilizers (to promote the growth of plants) and herbicides for weed management in non-organic pastures might have affected abundance and richness in organic and non-organic pastures. In Chapter 6, impact of commercial native forb/legume/grass (FLG) or warm season grasses (WSG; equal mix of Andropogon gerardi, Tripsacum dactyloides, and Sorghastrum nutans) on bees and non-bee insect communities in livestock pasture was determined. Total number of bees collected was higher in FLG than in WSG (3380 in FLG vs 3158 in WSG). Similarly, 3692 non-bee insects were collected from FLG whereas WSG contained 2346 non-bee insects. Findings from these studies will be helpful in selecting appropriate sampling methods for monitoring bees and other insect communities and also be helpful in developing pollinator-friendly pastures that support diversity of native bee species and other beneficial insects in livestock pasture ecosystems

Variation in fungal endophyte communities of pedunculate oak (Quercus robus L.) : spatial, temporal and environmental aspects

Fungal endophytes are a taxonomically and ecologically heterogeneous group of ubiquitous intercellular fungi that live part of their life within the tissues of the plants without causing apparent harm to the host. They may contribute to the stress tolerance and resistance of the plants, and endophytes may also be involved in decomposition processes. Thus, these fungi are an interesting part of biodiversity e.g. in forest ecosystems. Little is still known about the temporal and spatial patterns in endophyte community structures in trees. Moreover, while it is known that endophytes spread to trees from the environment (horizontally) it is not clear at which scales their spreading occurs, and if e.g. neighbouring vegetation determines endophyte communities in trees. In addition, it is not known if the general vitality of the trees affects the frequency and diversity of endophyte infections in trees. Using culture-dependent method (isolations), endophyte communities were compared in young pedunculate oak (Quercus robur L.) trees that were surrounded by herbs and shrubs in different combinations and that showed different vitality status. The isolations were done from current and previous year’s twigs (xylem and bark) in early and late summer to study the temporal and within-tree spatial patterns. A total of 384 samples were plated with bark and xylem pieces. Altogether 172 of them produced a total number of 285 isolates, which could be grouped in 22 morphological groups (morphotypes). The colonisation and isolation rate values showed the succession of endophyte communities during the season and inside the twigs but there were no significant effects of tree vitality on these rates. However, in June the declining trees had higher morphotype evenness, indicating that declining trees were compromised in their ability to defend themselves against fungal infections, which may have reduced the competition between the different invading fungi. The Jaccard’s similarity index and Shannon’s diversity index showed that there was no clear pattern of infection from the closest surrounding vegetation, suggesting that the spreading of these fungi occurs at a larger scale