Gwilym A. Williams

Microscopic characterisation of filamentous microbes: towards fully automated morphological quantification through image analysis

Mycelial morphology is a critically important process property in industrial fermentations of filamentous microorganisms, as particular phenotypes are associated with maximum productivity. The morphological form that develops in a given process results from the combination of various environmental factors, together with the genotype of the organism itself. The design of systems capable of rapidly and accurately characterising morphology within a given process represents a significant challenge to biotechnologists, as the complex phenotypes that are manifested are often not easily quantified.

Relating Fractal Dimension to Branching Behaviour in Filamentous Microorganisms

The productivity of an industrial fermentation process involving a filamentous microbe is heavily dependent on the morphological form adopted by the organism. The development of systems capable of rapidly and accurately characterising morphology within a given process represents a significant challenge to biotechnologists, as the complex phenotypes that are manifested are often not easily quantified. Conventional parameters employed in these analyses are of limited value, as they reveal little about the specific branching behaviour of the organism, which is an important consideration given the demonstrated link between branching frequency and metabolite production. More recently, fractal geometry has been employed in the analysis of microbes, but a clear link between fractal dimension and branching behaviour has not been demonstrated. This study presents an alternative means of enumerating the fractal dimension of fungal mycelial structures, by generating a ‘fractal signal’ from an object boundary. In the analysis of a population of Aspergillus oryzae mycelia, both fractal dimension and hyphal growth unit were found to increase together over time. An extensive analysis of different populations of Penicillium chrysogenum and A. oryzae mycelia, cultivated under a variety of different conditions, revealed a strong correlation between fractal dimension and hyphal growth unit. The technique has the potential to be adapted and applied to any morphological form that may be encountered in a fermentation process, providing a universally applicable process parameter for more complete data acquisition.

Emerging Technologies for the Pretreatment of Lignocellulosic Biomass

Pretreatment of lignocellulosic biomass to overcome its intrinsic recalcitrant nature prior to the production of valuable chemicals has been studied for nearly 200 years. Research has targeted eco-friendly, economical and time-effective solutions, together with a simplified large-scale operational approach. Commonly used pretreatment methods, such as chemical, physico-chemical and biological techniques are still insufficient to meet optimal industrial production requirements in a sustainable way. Recently, advances in applied chemistry approaches conducted under extreme and non-classical conditions has led to possible commercial solutions in the marketplace (e.g. High hydrostatic pressure, High pressure homogenizer, Microwave, Ultrasound technologies). These new industrial technologies are promising candidates as sustainable green pretreatment solutions for lignocellulosic biomass utilization in a large scale biorefinery. This article reviews the application of selected emerging technologies such as ionizing and non-ionizing radiation, pulsed electrical field, ultrasound and high pressure as promising technologies in the valorization of lignocellulosic biomass.

Nitrocellulose as a general tool for fungal slide mounts.

The microscopic examination of fungi is conventionally carried out by wet mount staining using lactophenol cotton blue, in combination with different modes of sample preparation: the use of mounting needles, the “adhesive tape lift,” and various agar block techniques are well documented in the

Automated analysis of filamentous microbial morphology with AnaMorf

The morphological quantification of filamentous microbes represents an important analytical technique in the optimization of bioprocesses involving such organisms, given the demonstrated links between morphology and metabolite yield. However, in many studies, much of this quantification has required some degree of manual intervention, if it has been conducted at all, burdening biotechnologists with a time‐consuming process and potentially introducing bias into analyses. Here, software for the automated quantification of filamentous microbes is presented, implemented as a plug‐in for the widely used, freely available image analysis package, ImageJ. The software, together with all related source code, documentation and test data, is freely available to the community via an online repository.

Lignocellulosic Biorefineries in Europe: Current State and Prospects

Lignocellulosic biorefining processes plant-derived biomass into a range of bio-based products. Currently, more than 40 lignocellulosic biorefineries are operating across Europe. Here, we address the challenges and future opportunities of this nascent industry by elucidating key elements of the biorefining sector, including feedstock sourcing, processing methods, and the bioproducts market.

A Review on Bioconversion of Agro-Industrial Wastes to Industrially Important Enzymes

Agro-industrial waste is highly nutritious in nature and facilitates microbial growth. Most agricultural wastes are lignocellulosic in nature; a large fraction of it is composed of carbohydrates. Agricultural residues can thus be used for the production of various value-added products, such as industrially important enzymes. Agro-industrial wastes, such as sugar cane bagasse, corn cob and rice bran, have been widely investigated via different fermentation strategies for the production of enzymes. Solid-state fermentation holds much potential compared with submerged fermentation methods for the utilization of agro-based wastes for enzyme production. This is because the physical–chemical nature of many lignocellulosic substrates naturally lends itself to solid phase culture, and thereby represents a means to reap the acknowledged potential of this fermentation method. Recent studies have shown that pretreatment technologies can greatly enhance enzyme yields by several fold. This article gives an overview of how agricultural waste can be productively harnessed as a raw material for fermentation. Furthermore, a detailed analysis of studies conducted in the production of different commercially important enzymes using lignocellulosic food waste has been provided.

Spectrophotometric assay of endo-β-N-acetylglucosaminidase H activity

Abstract An endoglycosidase H (Endo H) assay based on the use of a glycoprotein enzyme as substrate, in conjunction with immobilised concanavalin A (Con A), was devised and investigated. In this system, ribonuclease B (RNAase B) functions as both glycosidase substrate and reporter enzyme. Separation of glycosylated from deglycosylated RNAase B is effected by mixing with colloidal Con A-agarose, followed by centrifugation. The activity of carbohydrate-denuded RNAase B in the supernate is used to calculate endoglycosidase activity. Results obtained with this assay correlated closely with analysis of digest products by denaturing electrophoresis and lectin reactivity on western blots. Effective removal of non-hydrolysed RNAase B from assay digests was dependent on both lectin-agarose bead concentration and time of exposure. The assay was linear over the range 10–100 fmol of Endo H protein; a Km value of 0.48 mmol/l and turnover number of 7200 mol RNAase B/mol enzyme/min was determined for this endoglycosidas...