Alan D. W. Dobson

Strategies to prevent mycotoxin contamination of food and animal feed : A review

Mycotoxins are fungal secondary metabolites that have been associated with severe toxic effects to vertebrates produced by many important phytopathogenic and food spoilage fungi including Aspergillus, Penicillium, Fusarium, and Alternaria species. The contamination of foods and animal feeds with mycotoxins is a worldwide problem. We reviewed various control strategies to prevent the growth of mycotoxigenic fungi as well as to inhibit mycotoxin biosynthesis including pre-harvest (resistance varieties, field management and the use of biological and chemical agents), harvest management, and post-harvest (improving of drying and storage conditions, the use of natural and chemical agents, and irradiation) applications. While much work in this area has been performed on the most economically important mycotoxins, aflatoxin B1 and ochratoxin A much less information is available on other mycotoxins such as trichothecenes, fumonisin B1, zearalenone, citrinin, and patulin. In addition, physical, chemical, and biological detoxification methods used to prevent exposure to the toxic and carcinogenic effect of mycotoxins are discussed. Finally, dietary strategies, which are one of the most recent approaches to counteract the mycotoxin problem with special emphasis on in vivo and in vitro efficacy of several of binding agents (activated carbons, hydrated sodium calcium aluminosilicate, bentonite, zeolites, and lactic acid bacteria) have also been reviewed.

Parameters affecting biological phosphate removal from wastewaters

This paper reviews some of the key wastewater composition parameters, which influence the biological removal of phosphate from wastewaters, such as COD content, volatile fatty acid (VFA) content, cation concentration, phosphorus load, pH and food to microorganism ratio. The discussion also focuses on operational parameters affecting successful nutrient removal in wastewater treatment plants, such as temperature, sludge quality, sludge settlement, dissolved oxygen (DO) concentration, anaerobic P-release and secondary P-release. The aim of this review is to compile an updated document for researchers and operators of biological nutrient removal (BNR) systems. In addition, the article will provide a good foundation for readers with no prior knowledge of the process.

Differential regulation of laccase gene expression in Pleurotus sajor-caju

Four laccase isozyme genes, Psc lac1, 2, 3 and 4 have been cloned from the edible mushroom, Pleurotus sajor-caju. The genes display a high degree of homology with other basidiomycete laccases (55-99%) at the amino acid level. Of the laccase genes isolated, Psc lac1 and 4 displayed the highest degree of similarity (85% at the amino acid level), while Psc lac3 showed the highest degree of divergence, exhibiting only 52-57% amino acid similarity to the other PL: sajor-caju laccase gene sequences. Laccase activity in PL: sajor-caju is affected by nutrient nitrogen and carbon, and by the addition of copper and manganese to the growth medium. In addition, 2,5-xylidine, ferulic acid, veratric acid and 1-hydroxybenzotriazole induced laccase activity in the fungus. Induction of individual laccase isozyme genes by carbon, nitrogen, copper, manganese and the two aromatic compounds, 2,5-xylidine and ferulic acid, occurred at the level of gene transcription. While Psc lac3 transcript levels appeared to be constitutively expressed, transcript levels for the other laccase isozyme genes, lac1, 2 and 4, were differentially regulated under the conditions tested.

Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which is present within these marine environments.

Isolation and analysis of bacteria with antimicrobial activities from the marine sponge haliclona simulans collected from irish waters

Samples of the marine sponge Haliclona simulans were collected from Irish coastal waters, and bacteria were isolated from these samples. Phylogenetic analyses of the cultured isolates showed that four different bacterial phyla were represented; Bacteriodetes, Actinobacteria, Proteobacteria, and Firmicutes. The sponge bacterial isolates were assayed for the production of antimicrobial substances, and biological activities against Gram-positive and Gram-negative bacteria and fungi were demonstrated, with 50% of isolates showing antimicrobial activity against at least one of the test strains. Further testing showed that the antimicrobial activities extended to the important pathogens Pseudomonas aeruginosa, Clostridium difficile, multi-drug-resistant Staphylococcus aureus, and pathogenic yeast strains. The Actinomycetes were numerically the most abundant producers of antimicrobial activities, although activities were also noted from Bacilli and Pseudovibrio isolates. Surveys for the presence of potential antibiotic encoding polyketide synthase and nonribosomal peptide synthetase genes also revealed that genes for the biosynthesis of these secondary metabolites were present in most bacterial phyla but were particularly prevalent among the Actinobacteria and Proteobacteria. This study demonstrates that the culturable fraction of bacteria from the sponge H. simulans is diverse and appears to possess much potential as a source for the discovery of new medically relevant biological active agents.

PCR-based detection and quantification of mycotoxigenic fungi*

Mycotoxins are secondary metabolites produced by many phytopathogenic and food spoilage fungi including Penicillium, Fusarium and Aspergillus. The toxicity and carcinogenicity of many of these mycotoxins, and their potential to contaminate foods and animal feedstuffs is a cause of serious concern globally, both from a food safety and food trade standpoint. Thus the rapid identification of mycotoxigenic fungi would be desirable, such that early intervention steps could be applied to help limit the amounts of contaminated materials, particularly cereals and cereal-based products, gaining access to the human food chain. With this in mind a number of PCR-based methodologies have been developed for the identification of mycotoxin biosynthetic genes in different fungal genera, together with assays developed using other genes or random amplification of polymorphic DNA (RAPD) methodologies for the identification of specific toxigenic fungi. In addition, reverse transcription (RT)-PCR, competitive PCR and Real Time quantitative PCR methodologies have also been developed for this purpose. The development of each of these techniques, their usefulness, limitations and adaptability will be discussed together with descriptions of specific examples where these techniques have been utilised in different experimental settings.

Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host

The Psc lac4 gene from Pleurotus sajor-caju has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. The native Ple. sajor-caju laccase signal sequence was effective in directing the secretion of lac4 expressed in Pic. pastoris. The control of media pH and temperature was found to be important in obtaining sufficient quantities of the protein to allow purification and subsequent biochemical characterization. The recombinant Psc Lac4 was purified to electrophoretic homogeneity and was shown to be immunologically related to Pleurotus eryngii Lac1. The purified laccase was estimated to have a molecular mass of around 59 kDa, to have a carbohydrate content of approximately 7% and a calculated pI of 4.38. The enzyme oxidized the substrates 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol, syringaldazine and guaiacol, exhibiting optimal pHs of 3.3, 6, 6.5 and 7 respectively. With ABTS as substrate the enzyme displayed optimal activity at 35 degrees C and pH 3.5. The enzyme was strongly inhibited by sodium azide and thioglycolic acid but not by EDTA.

Marine Metagenomics: New Tools for the Study and Exploitation of Marine Microbial Metabolism

The marine environment is extremely diverse, with huge variations in pressure and temperature. Nevertheless, life, especially microbial life, thrives throughout the marine biosphere and microbes have adapted to all the divergent environments present. Large scale DNA sequence based approaches have recently been used to investigate the marine environment and these studies have revealed that the oceans harbor unprecedented microbial diversity. Novel gene families with representatives only within such metagenomic datasets represent a large proportion of the ocean metagenome. The presence of so many new gene families from these uncultured and highly diverse microbial populations represents a challenge for the understanding of and exploitation of the biology and biochemistry of the ocean environment. The application of new metagenomic and single cell genomics tools offers new ways to explore the complete metabolic diversity of the marine biome.

Metagenomic approaches to exploit the biotechnological potential of the microbial consortia of marine sponges

Natural products isolated from sponges are an important source of new biologically active compounds. However, the development of these compounds into drugs has been held back by the difficulties in achieving a sustainable supply of these often-complex molecules for pre-clinical and clinical development. Increasing evidence implicates microbial symbionts as the source of many of these biologically active compounds, but the vast majority of the sponge microbial community remain uncultured. Metagenomics offers a biotechnological solution to this supply problem. Metagenomes of sponge microbial communities have been shown to contain genes and gene clusters typical for the biosynthesis of biologically active natural products. Heterologous expression approaches have also led to the isolation of secondary metabolism gene clusters from uncultured microbial symbionts of marine invertebrates and from soil metagenomic libraries. Combining a metagenomic approach with heterologous expression holds much promise for the sustainable exploitation of the chemical diversity present in the sponge microbial community.

Functional metagenomic strategies for the discovery of novel enzymes and biosurfactants with biotechnological applications from marine ecosystems.

Marine ecosystems are home to bacteria which are exposed to a wide variety of environmental conditions, such as extremes in temperature, salinity, nutrient availability and pressure. Survival under these conditions must have necessitated the adaptation and the development of unique cellular biochemistry and metabolism by these microbes. Thus, enzymes isolated from these microbes have the potential to possess quite unique physiological and biochemical properties. This review outlines a number of function‐based metagenomic approaches which are available to screen metagenomic libraries constructed from marine ecosystems to facilitate the exploitation of some of these potentially novel biocatalysts. Functional screens to isolate novel cellulases, lipases and esterases, proteases, laccases, oxidoreductases and biosurfactants are described, together with approaches which can be employed to help overcome some of the typical problems encountered with functional metagenomic‐based screens.