Biological Control of Aflatoxin Contamination in U.S. Crops and the Use of Bioplastic Formulations of Aspergillus flavus Biocontrol Strains To Optimize Application Strategies

Aflatoxin contamination has a major economic impact on crop production in the southern United States. Reduction of aflatoxin contamination in harvested crops has been achieved by applying nonaflatoxigenic biocontrol Aspergillus flavus strains that can out-compete wild aflatoxigenic A. flavus, reducing their numbers at the site of application. Currently, the standard method for applying biocontrol A. flavus strains to soil is using a nutrient-supplying carrier (e.g., pearled barley for Afla-Guard). Granules of Bioplastic (partially acetylated corn starch) have been investigated as an alternative nutritive carrier for biocontrol agents. Bioplastic granules have also been used to prepare a sprayable biocontrol formulation that gives effective reduction of aflatoxin contamination in harvested corn kernels with application of much smaller amounts to leaves later in the growing season. The ultimate goal of biocontrol research is to produce biocontrol systems that can be applied to crops only when long-range weather forecasting indicates they will be needed

Present status and perspective on the future use of aflatoxin biocontrol products

Aflatoxin contamination of important food and feed crops occurs frequently in warm tropical and subtropical regions. The contamination is caused mainly by Aspergillus flavus and A. parasiticus. Aflatoxin contamination negatively affects health and trade sectors and causes economic losses to agricultural industries. Many pre- and post-harvest technologies can limit aflatoxin contamination but may not always reduce aflatoxin concentrations below tolerance thresholds. However, the use of atoxigenic (non-toxin producing) isolates of A. flavus to competitively displace aflatoxin producers is a practical strategy that effectively limits aflatoxin contamination in crops from field to plate. Biocontrol products formulated with atoxigenic isolates as active ingredients have been registered for use in the US, several African nations, and one such product is in final stages of registration in Italy. Many other nations are seeking to develop biocontrol products to protect their crops. In this review article we present an overview of the biocontrol technology, explain the basis to select atoxigenic isolates as active ingredients, describe how formulations are developed and tested, and describe how a biocontrol product is used commercially. Future perspectives on formulations of aflatoxin biocontrol products, along with other important topics related to the aflatoxin biocontrol technology are also discussed.Fil: Moral, Juan. Universidad de Córdoba; EspañaFil: Garcia-Lopez, Maria Teresa. Universidad de Córdoba; EspañaFil: Camiletti, Boris Xavier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Agropecuarias. Departamento de Recursos Naturales. Cátedra de Microbiología Agrícola; ArgentinaFil: Jaime, Ramon. University of California at Davis; Estados UnidosFil: Michailides, Themis J.. University of California at Davis; Estados UnidosFil: Bandyopadhyay, Ranajit. International Institute of Tropical Agriculture; NigeriaFil: Ortega-Beltran, Alejandro. International Institute of Tropical Agriculture; Nigeri

Present status and perspective on the future use of aflatoxin biocontrol products

Open Access Journal; Published online: 01 Apr 2020Aflatoxin contamination of important food and feed crops occurs frequently in warm tropical and subtropical regions. The contamination is caused mainly by Aspergillus flavus and A. parasiticus. Aflatoxin contamination negatively affects health and trade sectors and causes economic losses to agricultural industries. Many pre- and post-harvest technologies can limit aflatoxin contamination but may not always reduce aflatoxin concentrations below tolerance thresholds. However, the use of atoxigenic (non-toxin producing) isolates of A. flavus to competitively displace aflatoxin producers is a practical strategy that effectively limits aflatoxin contamination in crops from field to plate. Biocontrol products formulated with atoxigenic isolates as active ingredients have been registered for use in the US, several African nations, and one such product is in final stages of registration in Italy. Many other nations are seeking to develop biocontrol products to protect their crops. In this review article we present an overview of the biocontrol technology, explain the basis to select atoxigenic isolates as active ingredients, describe how formulations are developed and tested, and describe how a biocontrol product is used commercially. Future perspectives on formulations of aflatoxin biocontrol products, along with other important topics related to the aflatoxin biocontrol technology are also discussed

Seed coat mediated resistance against Aspergillus flavus infection in peanut

Toxic metabolites known as aflatoxins are produced via certain species of the Aspergillus genus, specifically A. flavus, A. parasiticus, A. nomius, and A. tamarie. Although various pre- and post-harvest strategies have been employed, aflatoxin contamination remains a major problem within peanut crop, especially in subtropical environments. Aflatoxins are the most well-known and researched mycotoxins produced within the Aspergillus genus (namely Aspergillus flavus) and are classified as group 1 carcinogens. Their effects and etiology have been extensively researched and aflatoxins are commonly linked to growth defects and liver diseases in humans and livestock. Despite the known importance of seed coats in plant defense against pathogens, peanut seed coat mediated defenses against Aspergillus flavus resistance, have not received considerable attention. The peanut seed coat (testa) is primarily composed of a complex cell wall matrix consisting of cellulose, lignin, hemicellulose, phenolic compounds, and structural proteins. Due to cell wall desiccation during seed coat maturation, postharvest A. flavus infection occurs without the pathogen encountering any active genetic resistance from the live cell(s) and the testa acts as a physical and biochemical barrier only against infection. The structure of peanut seed coat cell walls and the presence of polyphenolic compounds have been reported to inhibit the growth of A. flavus and aflatoxin contamination; however, there is no comprehensive information available on peanut seed coat mediated resistance. We have recently reviewed various plant breeding, genomic, and molecular mechanisms, and management practices for reducing A. flavus infection and aflatoxin contamination. Further, we have also proved that seed coat acts as a physical and biochemical barrier against A. flavus infection. The current review focuses specifically on the peanut seed coat cell wall-mediated disease resistance, which will enable researchers to understand the mechanism and design efficient strategies for seed coat cell wall-mediated resistance against A. flavus infection and aflatoxin contamination

Estudio de la dinámica de inóculo de los agentes de biocontrol de aflatoxinas en frutos secos, resistencia varietal al patógeno y caracterización de la población de Aspergillus spp. sección Flavi en España

Almond and pistachio nuts can be occasionally colonized with the fungal species Aspergillus flavus and A. parasiticus and, concomitantly, contaminated with aflatoxins, potent carcinogenic mycotoxins for humans. As reviewed in chapter I, the most effective pre-harvest management strategy for limiting aflatoxin contamination is the massive release of native atoxigenic strains of A. flavus for the competitive displacement (or competitive exclusion) of wild toxigenic isolates from the agroecosystem. Many farmers from the U.S.A., Africa, and Italy and diverse crop industries benefit from using this technology. Thereafter, in temperate and tropical regions, where the biocontrol strategy is still not implemented, the search for native atoxigenic A. flavus strains is a necessity. In California, for example, the US Environmental Protection Agency granted registration of the atoxigenic strain AF36 of A. flavus for use in pistachio and almond in 2012 and 2017, respectively. This strain is applied using sorghum grains as the AF36 spores carrier with the commercial name AF36 Prevail®. The use of AF36 Prevail® is a clear competitive advantage for U.S. farmers since commercial biocontrol agents to reduce aflatoxin contamination are not available in other nut-growing regions such as Australia, Spain, or Turkey. Although AF36 Prevail® was primarily developed for applying to row crops (e.g. maize, cotton), it has also been effective in limiting aflatoxin contamination in nut trees in California. Even so, AF36 Prevail® often fails in nut orchards because of differential characteristics between row and tree crops. Then, we evaluated (chapter II) the sporulation of the biocontrol strain AF36 in pistachio orchards and advised farmers to spread the AF36 Prevail® product in the moist soil area but avoid the site where the irrigation drops fall. Our study about the dynamics of A. flavus’ spores suggested that AF36 Prevail® could be applied every two rows obtaining an overlapping effect on the non-treated row whether the distance between tree rows is ≤ 10 m. Furthermore, we detected that tree debris in the canopy act as an inoculum source for Aspergillus species included in section Nigri, ochratoxins producers, and biocontrol strategies may act parallelly to protect against both mycotoxins. It is essential to monitor how the atoxigenic AF36 strain survives and competes with aflatoxin-producing species populations in the target agroecosystem to understand how it can displace wild isolates of Aspergillus spp. Traditionally, biocontrol strains of A. flavus have been monitored through vegetative compatibility assays (VCA), but these are tedious and time-consuming. Thus, we tackled this concern by developing and validating a mismatch-qPCR assay to quantify the proportion of AF36 vs. toxigenic genotypes of A. flavus and A. parasiticus from diverse soil and plant samples. Our mismatch-qPCR efficiently quantifies AF36 proportions in the Aspergillus population. To overcome the disadvantages (loss of grains, poor sporulation, etcetera) of applying the strain AF36 using sorghum grains as carriers, we studied (chapter II) the pistachio male inflorescences as an inoculum source of atoxigenic strains. Male inflorescences are an abundant and free substrate, regularly distributed in the orchard. In our trials, the density of AF36 spores on the pistachio canopy of the inflorescence-treated trees was similar (P > 0.05) to this of Prevail®-treated trees. Furthermore, our results indicated that in pistachio orchards, where biocontrol practices are not conducted, eliminating this critical source of toxigenic Aspergillus inoculum is recommended. In chapter III, we characterized the resistance of various almond cultivars against A. flavus and A. parasiticus colonization and aflatoxin contamination. Remarkably, we found high variability in response to aflatoxin contamination of almond cultivars caused by both Aspergillus species. In addition, the shells were an insurmountable barrier to the pathogen, regardless of their type of shell (hard, semihard, or paper shell). However, natural-opening shells often occur in paper shell almond cultivars in the field. Our results also pointed out the importance of peach for introgressing resistance to the pathogen in almond breeding programs. Finally, we presented the possibility of combining both cultivar resistance and biocontrol, which offers a particularly promising aflatoxin control strategy. In a final chapter IV, we surveyed two leading Spanish almond- and pistachio-producing regions, Andalusia and Castilla La Mancha. In these surveys, we isolated 78 strains of Aspergillus section Flavi. Remarkably, four A. flavus were identified as atoxigenic (i.e., no-aflatoxin and no-cyclopiazonic acid producers) and, to our knowledge, this is the first report of atoxigenic strains of A. flavus native to Spain. Besides, six A. tamarii strains resulted, for the first time, described as slightly aflatoxigenic. With the work advocated in this Ph. D Thesis, we have contributed definitely to the optimized use of this biological control strain in Californian tree nut crops. In addition, we are closer to offering a safe product option to be used infield shortly by the Spanish almond and pistachio producers.Las almendras y los pistachos son colonizados ocasionalmente por las especies fúngicas Aspergillus flavus y A. parasiticus y, por consiguiente, pueden contaminarse con aflatoxinas, potentes micotoxinas cancerígenas para los humanos. En el capítulo I de la presente Tesis Doctoral, revisamos una de las estrategias de control más efectiva para limitar la contaminación por aflatoxinas en campo: la liberación masiva de cepas atoxigénicas (no productoras de micotoxinas) nativas de A. flavus para el desplazamiento competitivo (o exclusión competitiva) de los aislados toxigénicos del agroecosistema. Muchos agricultores de EE. UU., África e Italia tienen acceso comercial a este tipo de agentes de biocontrol. En cambio, en las regiones templadas y tropicales donde aún no se implementa esta estrategia de biocontrol es necesaria la búsqueda de cepas atoxigénicas de A. flavus. En California, la Agencia de Protección Ambiental (EPA) de EE. UU. otorgó el registro de la cepa atoxigénica AF36 de A. flavus para uso en pistachero y almendro en 2012 y 2017, respectivamente. La cepa AF36 se aplica en campo utilizando granos de sorgo recubiertos de esporas con el nombre comercial AF36 Prevail®. El uso de AF36 Prevail® supone una ventaja competitiva para los agricultores estadounidenses ya que, en otras regiones productoras de frutos secos, como Australia, España o Turquía, no hay agentes de control biológico comerciales disponibles. El producto AF36 Prevail® se desarrolló para su uso en cultivos extensivos (ej. maíz y algodón), aunque también se ha mostrado eficaz disminuyendo la contaminación por aflatoxinas en frutos secos. Aun así, el control biológico de aflatoxinas en frutos secos mediante el uso de AF36 Prevail® fracasa con frecuencia debido a características agronomicas propias de este tipo de cultivos arbóreos. En el capítulo II, por lo tanto, evaluamos la esporulación y dispersión del producto AF36 Prevail® en campos de pistachero y recomendamos a los agricultores aplicar el producto en el área de suelo irrigada por los microaspersores aunque, evitando la zona donde impactan las gotas del agua de riego ya que afecta negativamente a su esporulación. Según nuestro estudio sobre la dispersión de las esporas de A. flavus, las esporas de AF36 fácilmente alcanzan la copa de los pistacheros próximos al punto de aplicación aunque disminuye marcadamente con la distancia a la fuente de inóculo y la altura, ajustándose a distintas ecuaciones de difusión. Nuestros datos apuntan a que AF36 Prevail® podría aplicarse en filas alternas de pistacheros obteniéndose un efecto de protección (densidad de esporas por árbol) similar en el conjunto de los árboles si la distancia entre filas es ≤ 10 m. Además, detectamos que los restos de tejido que quedan en la copa de los pistacheros actúan como fuente de inóculo para las especies de Aspergillus de la sección Nigri, productores de ocratoxinas, por lo tanto, la estrategia de biocontrol puede actuar de forma paralela contra ambas micotoxinas. Para comprender cómo la cepa atoxigénica AF36 sobrevive, compite y desplaza a las cepas silvestres de Aspergillus spp. productoras de aflatoxinas, es esencial la monitorización en los campos donde ha sido liberada. Tradicionalmente, las cepas de A. flavus se han monitoreado mediante ensayos de compatibilidad vegetativa (VCA), pero son tediosos y requieren varias semanas para su ejecución. Por lo tanto, desarrollamos y validamos (capítulo II) un protocolo de qPCR basado en un Mismatch para cuantificar la proporción de AF36 frente a los genotipos toxigénicos de A. flavus y la población general de A. parasiticus. Nuestra qPCR-Mismatch cuantifica de manera eficiente las proporciones de AF36 respecto a la población de Aspergillus en el suelo y la planta. Debido a los problemas derivados de aplicar la cepa AF36 en granos de sorgo (pérdida de granos, mala esporulación, etcétera), estudiamos (capítulo II) la posibilidad de utilizar las inflorescencias masculinas de pistachero como fuente de inóculo para las cepas atoxigénicas. Las inflorescencias masculinas constituyen un sustrato abundante, gratuito, y que se distribuye regularmente en la plantación. En nuestros ensayos, la densidad de esporas de AF36 en la copa de los pistacheros con inflorescencias del suelo inoculadas con AF36 fue similar (P > 0.05) a la de los árboles tratados con AF36 Prevail®. Estos resultados apuntan indirectamente a que, en los campos de pistacheros donde no se llevan a cabo prácticas de biocontrol, es recomendable eliminar las inflorescencias masculinas en el suelo al constituir una importante fuente de inóculo. En el capítulo III, caracterizamos la resistencia de cultivares y selecciones avanzadas de almendro a la colonización por A. flavus y A. parasiticus y, la subsiguiente, contaminación por aflatoxinas. Sorprendentemente, encontramos una alta variabilidad en la resistencia/susceptibilidad de los genotipos de almendro a la colonización por ambas especies. Además, la cáscara (endocarpo) intacta resultó ser una barrera infranqueable para el patógeno, independientemente del tipo (dura, semidura o de papel). Sin embargo, las aperturas de la cáscara, que naturalmente pueden aparecer en cultivares de almendro de cáscara de papel, constituyen un punto de entrada para las esporas del patógeno. En este capítulo, además, destacamos la importancia del melocotonero para la introgresión de genes de resistencia al patógeno en los programas de mejora del almendro. Finalmente, presentamos la posibilidad de combinar tanto la resistencia del cultivar como el biocontrol, lo que ofrece una estrategia de control de aflatoxina particularmente prometedora. En un capítulo final (IV), caracterizamos la población de Aspergillus spp. en dos de las principales regiones españolas productoras de almendras y pistachos, Andalucía y Castilla La Mancha. Durante las prospecciones realizadas, aislamos 78 cepas de Aspergillus sección Flavi y seis cepas de A. tamarii que sorprendentemente fueron caracterizadas como ligeramente aflatoxigénicas. Cabe destacar, que identificamos cuatro cepas de A. flavus como atoxigénicas (es decir, no productoras de aflatoxinas ni ácido ciclopiazónico) lo que constituye la primera descripción de cepas atoxigénicas de A. flavus españolas. En la presente Tesis Doctoral, hemos contribuido notablemente a la optimización del control biológico de aflatoxinas en los cultivos de frutos secos de California. Además, estamos más cerca de ofrecer agentes de control biológicos para reducir la contaminación por aflatoxinas que puedan emplear los productores españoles

Biological control of aflatoxins in Africa: current status and potential challenges in the face of climate change

Article purchased; in PressAflatoxin contamination of crops is frequent in warm regions across the globe, including large areas in sub-Saharan Africa. Crop contamination with these dangerous toxins transcends health, food security, and trade sectors. It cuts across the value chain, affecting farmers, traders, markets, and finally consumers. Diverse fungi within Aspergillus section Flavi contaminate crops with aflatoxins. Within these Aspergillus communities, several genotypes are not capable of producing aflatoxins (atoxigenic). Carefully selected atoxigenic genotypes in biological control (biocontrol) formulations efficiently reduce aflatoxin contamination of crops when applied prior to flowering in the field. This safe and environmentally friendly, effective technology was pioneered in the US, where well over a million acres of susceptible crops are treated annually. The technology has been improved for use in sub-Saharan Africa, where efforts are under way to develop biocontrol products, under the trade name Aflasafe, for 11 African nations. The number of participating nations is expected to increase. In parallel, state of the art technology has been developed for large-scale inexpensive manufacture of Aflasafe products under the conditions present in many African nations. Results to date indicate that all Aflasafe products, registered and under experimental use, reduce aflatoxin concentrations in treated crops by >80% in comparison to untreated crops in both field and storage conditions. Benefits of aflatoxin biocontrol technologies are discussed along with potential challenges, including climate change, likely to be faced during the scaling-up of Aflasafe products. Lastly, we respond to several apprehensions expressed in the literature about the use of atoxigenic genotypes in biocontrol formulations. These responses relate to the following apprehensions: sorghum as carrier, distribution costs, aflatoxin-conscious markets, efficacy during drought, post-harvest benefits, risk of allergies and/or aspergillosis, influence of Aflasafe on other mycotoxins and on soil microenvironment, dynamics of Aspergillus genotypes, and recombination between atoxigenic and toxigenic genotypes in natural conditions

MICRORGANISMOS E SEUS PRODUTOS DE FERMENTAÇÃO INTERFEREM NA QUALIDADE DE SEMENTES E PLÂNTULAS DE MILHO?

A microbiota do solo e suas funções ecológicas são responsáveis por relações diretas e indiretas com a planta. Objetivou-se avaliar a sanidade e germinação em sementes de milho, bem como a emergência e desenvolvimento de plântulas submetidas a microrganismos capturados de dois ambientes. Sementes de milho foram inoculadas ou irrigadas com microrganismos e seus produtos de fermentação, provenientes de duas áreas (mata ou cultivo de cana-de-açúcar) sob diferentes concentrações e avaliadas quanto a porcentagem de germinação, índice de velocidade de germinação, sanidade em teste em BOD, além da emergência, altura de plântula, massa de matéria seca da parte aérea e raiz e volume de raiz em ensaios em bandejas com solo. Não houve comprometimento na germinação, independentemente do tratamento utilizado. Houve a mitigação de Aspergillus spp. e Penicillium spp., porém, a potencialização de Fusarium spp em condições de laboratório sob aplicação de 50 e 100% de microrganismos capturados da área de mata e cana. De forma geral, houve efeito negativo da aplicação do tratamento sobre os parâmetros fisiológicos, provocados provavelmente por desequilíbrio na ecologia microbiana associado a sementes e plântulas.Palavras-chave: Fusarium spp.; microrganismos eficientes; ecossistemas. DO MICROORGANISMS AND THEIR FERMENTATION PRODUCTS INTERFER ON SEED AND SEEDLINGS CORN QUALITY? ABSTRACT: Soil microbiota and its ecological functions are responsible for direct and indirect relations with the plant. The aim of this study was to evaluate the corn seeds health and germination and seedling emergence and development submitted to microorganisms captured from two environments. Corn seeds were inoculated or irrigated with microorganisms and their fermentation products, coming from two areas (forest or sugarcane cultivation) under different concentrations and evaluated for the germination percentage, germination speed index, health in test in BOD, seedling emergence, seedling height, shoot and root dry matter mass, and root volume in soil tray tests. There was no effect on germination, regardless of the treatment used. There was mitigation of Aspergillus spp. and Penicillium spp., but the increase of Fusarium spp occurrence in laboratory conditions under concentration of 50 and 100% of microorganisms captured from the forest and sugarcane areas. In general, there was a negative effect of all treatments on physiological parameters, probably caused by an imbalance in the microbial ecology associated with seeds and seedlings.Keywords: Fusarium spp.; efficient microorganisms; ecosystems

Physical, chemical and biological processes and fates of petroleum-based plastic and bioplastic pollutants in aquatic environments

By the mid to late 20th Century the use of petroleum-based plastics had become widespread. Much of this plastic has been and continues to be littered, leading to plastic pollution becoming ubiquitous in marine environments. Plastic pollutants can cause physical harm to marine organisms, via entanglement or ingestion. Persistent organic pollutants (POPs) such as some brominated flame retardants (BFRs) are known to accumulate onto plastic pollutant surfaces in marine environments, and may provide a novel pathway for exposure to these chemicals to organisms. Additionally, plastic surfaces containing microbial biofilms have been suggested as a vector for the transport of harmful algae and pathogens beyond their natural ranges. In recent decades, bioplastics (plastics derived from biological based materials) have been developed and utilised as an alternative to petroleum-based plastics. However, the environmental fates of bioplastic pollutants, the processes of BFR accumulation and biofilm development onto bioplastics remains undetermined. This thesis sought to advance current knowledge of the fate of pollutant petroleum-based plastics and bioplastics within aquatic ecosystems. This was addressed via an experimental approach in which polypropylene (PP) as a model petroleum-based plastic, polylactic acid (PLA) as a model bioplastic and glass slides as non-plastic control substrate were deployed in an exposure experiment at five sites along a freshwater-marine continuum of the Yarra River into Port Phillip Bay, Melbourne, Australia. The three specific objectives were to; compare variation in the structural properties of PP and PLA, via analysis of surface hydrophobicity, tensile strength, crystallinity and chemical structure (Chapter 3); determine the potential for BFRs to accumulate onto PP and PLA with comparison to glass substrates (Chapter 4); and compare spatial-, temporal- and substrate-specific (PP, PLA and glass) variation in the structure and composition of microbial (prokaryotic and eukaryotic) biofilm communities forming on polymer and glass surfaces and with comparison to those in the surrounding water (Chapter 5). Research in Chapter 3 revealed that between Day 1 and Month 12 of the exposure experiment there were no significant changes in either the water contact angle (WCA) (a proxy used to assess surface hydrophobicity), or Young's Modulus (a measure related to tensile strength) for either PP and PLA substrates. There was no overall trend of an increase of the Max load at break (Max Load) (a measure related to tensile strength) of the PLA substrates between the other sampling dates. However, there was a significant increase (P < 0.05) of the Max Load for the PLA substrates but not the PP substrates. Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) (crystallinity) analysis of the plastics were undertaken to compare polymers between Day 1 and Month 6. There was an increase in the crystallinity of the PLA substrates but not for the PP substrates. Neither the PP nor the PLA substrates exhibited any change in FTIR spectra between Day 1 and Month 6 and indicated no change in the chemical structure of either plastic type. Research in Chapter 4 investigated accumulation of two groups of BFRs onto the PP, PLA and glass substrates. The targeted BFRs were polybrominated diphenyl ethers (PBDEs) and novel brominated flame retardants (NBFRs). The selected BFR analytes were extracted from the PP and glass substrates using a selective pressurised liquid extraction (S-PLE) method. A novel dual vortex and sonication method was successfully developed and implemented for the extraction of BFR analytes from the PLA substrates. Analysis of the selected BFRs was undertaken using an Agilent 7000C gas chromatograph coupled to a triple quadrupole mass spectrometer (GC-MS/MS). Differences in BFR concentrations between the three substrate types was not able to be assessed due to sample loss from sample frames over the course of the experiment. At least one PBDE congener and one NBFR compound were detected in all samples, although the mean ∑PBDE and ∑NBFR concentrations on the substrates were low, 12.3 ng g-1 ± 7.4 ng g-1 and 23 ng g-1 ± 23 ng g-1, respectively. Research in Chapter 5 explored structural and compositional changes in the prokaryotic and eukaryotic microbial biofilm communities as well as water communities via high-throughput DNA amplicon sequencing of the 16S and 18S rRNA genes, respectively. The structure of the microbial biofilm communities on substrates were distinct from those in the surrounding water environment and differed principally with sample site, and then with sampling date. There was no significant (P > 0.05) difference in the composition of microbial biofilm communities between any of the three substrate types. The prokaryotic biofilms communities were dominated by Proteobacteria (alpha-, beta-, and gamma- classes) and Bacteroidetes, and the eukaryotic biofilm communities were dominated by diatoms and ciliates. Both the prokaryotic and eukaryotic water microbial community types had higher mean numbers of observed operational taxonomic units (OTUs) and Shannon diversity when compared to the coupon-biofilm communities. The relative abundance of three key functional guilds of potential plastic degraders, pathogenic bacteria and harmful algae were assessed. None of these three functional guilds had relative abundances greater than 1 % of the overall community; although three fish pathogens Pseudomonas anguilliseptica, Acinetobacter johnsonii and A. lwoffii, were frequently identified, being detected in over two thirds of the biofilm communities. This research has shown that PLA is as physically- and chemically- stable as PP over a 12- month period in aquatic environments. It was hypothesised that substantial degradation of the plastics did not occur because the plastics were quickly biofouled within days from deployment, and that biofouling in water would have reduced the rate of photo-oxidative degradation, one of the main degradation processes to occur to plastics in natural environments. PLA, PP and glass substrates were all found to have the capability to accumulate PBDEs and NBFRs. The plastic biofilm communities were shown to be diverse and distinct from those in the surrounding water communities, and the plastic biofilm (both PP and PLA) communities were highly similar to those forming on glass, demonstrating that plastic biofilm communities consists of predominantly generalist surface colonisers. Three fish pathogens (P. anguilliseptica, A. johnsonii and A. lwoffii) were frequently identified within the substrate biofilm communities, indicating that aquatic plastic debris may be a long- term novel exposure pathway for pathogen exposure in fish due to the high number of plastic fragments in aquatic environments, and the ability of plastics to passively travel vast distances. The lack of plastic degrading organisms identified on the plastics raises doubts that PLA, will be biodegraded to any significant extent in aquatic environments. Therefore, bioplastics should be held in the same regard as petroleum-based plastics by government, policy makers and industry leaders as they work towards solutions that reduce the impacts of both petroleum-based plastics and bioplastics within aquatic ecosystems

Soil Water Properties of Kerangas Forest Soil after Invasion by Acacia

Soil water is important for forest ecosystems as infiltration and percolation process use soil water for plant growth. The presence of invasive Acacia species may limit the availability of soil water because these species absorb more water than native species. Hence, the objective was to investigate the effect of Acacia invasion on the soil water properties of Kerangas forests. In each invaded and non-invaded Acacia plots, holes a lysimeter was installed into the holes and used to extract soil water by direct contact to the soil. The results shows the invasion of Acacia has affected the Kerangas forest by higher absorption of water and higher fixation of nitrate

Chapter 34 - Biocompatibility of nanocellulose: Emerging biomedical applications

Nanocellulose already proved to be a highly relevant material for biomedical applications, ensued by its outstanding mechanical properties and, more importantly, its biocompatibility. Nevertheless, despite their previous intensive research, a notable number of emerging applications are still being developed. Interestingly, this drive is not solely based on the nanocellulose features, but also heavily dependent on sustainability. The three core nanocelluloses encompass cellulose nanocrystals (CNCs), cellulose nanofibrils (CNFs), and bacterial nanocellulose (BNC). All these different types of nanocellulose display highly interesting biomedical properties per se, after modification and when used in composite formulations. Novel applications that use nanocellulose includewell-known areas, namely, wound dressings, implants, indwelling medical devices, scaffolds, and novel printed scaffolds. Their cytotoxicity and biocompatibility using recent methodologies are thoroughly analyzed to reinforce their near future applicability. By analyzing the pristine core nanocellulose, none display cytotoxicity. However, CNF has the highest potential to fail long-term biocompatibility since it tends to trigger inflammation. On the other hand, neverdried BNC displays a remarkable biocompatibility. Despite this, all nanocelluloses clearly represent a flag bearer of future superior biomaterials, being elite materials in the urgent replacement of our petrochemical dependence