Optimising parameters for pilot scale steam explosion and continuous pressurised disc refining of Miscanthus and sugarcane bagasse for xylose and xylo-oligosaccharide release.

The first comparative pre-treatment study of Miscanthus (Mxg) and sugarcane bagasse (SCB) using steam explosion (SE) and pressurised disc refining (PDR) pretreatment to optimise xylose and xylo-oligosaccharide release is described. The current investigation aimed to 1) Develop optimised batch-wise steam explosion parameters for Mxg and SCB, 2) Scale from static batch steam explosion to dynamic continuous pressurised disc refining, 3) Identify, understand, and circumvent scale-up production hurdles. Optimised SE parameters released 82% (Mxg) and 100% (SCB) of the available xylan. Scaling to PDR, Miscanthus yielded 85% xylan, highlighting how robust scouting assessments for boundary process parameters can result in successful technical transfer. In contrast, SCB technical transfer was not straightforward, with significant differences observed between the two processes, 100% (SE) and 58% (PDR). This report underlines the importance of feedstock-specific pretreatment strategies to underpin process development, scale-up, and optimisation of carbohydrate release from biomass.

HPLC-DAD-qTOF Compositional Analysis of the Phenolic Compounds Present in Crude Tomato Protein Extracts Derived from Food Processing.

The conversion of raw fruits and vegetables, including tomatoes into processed food products creates side streams of residues that can place a burden on the environment. However, these processed residues are still rich in bioactive compounds and in an effort to valorize these materials in tomato by-product streams, the main aim of this study is to extract proteins and identify the main phenolic compounds present in tomato pomace (TP), peel and skins (TPS) by HPLC-DAD-ESI-QTOF. Forty different phenolic compounds were identified in the different tomato extracts, encompassing different groups of phenolic compounds, including derivatives of simple phenolic acid derivatives, hydroxycinnamoylquinic acid, flavones, flavonones, flavonol, and dihydrochalcone. In the crude protein extract (TPE) derived from tomatoes, most of these compounds were still present, confirming that valuable phenolic compounds were not degraded during food processing of these co-product streams. Moreover, phenolic compounds present in the tomato protein crude extract could provide a valuable contribution to the required daily intake of phenolics that are usually supplied by consuming fresh vegetables and fruits.

Electrochemical quantification of d-glucose during the production of bioethanol from thermo-mechanically pre-treated wheat straw.

Mechanical pre-treatment (disc refining) of wheat straw, at both atmospheric and elevated pressure, is shown to be an efficient process to access fermentable monosaccharides, with the potential to integrate within the infrastructure of existing first-generation bioethanol plants. The mild, enzymatic degradation of this sustainable lignocellulosic biomass affords ca. 0.10-0.13 g/g (dry weight) of d-glucose quantifiable voltammetrically in real time, over a two hundred-fold range in experimental laboratory scales (25 mL to 5.0 L), with pressure disc refining of the wheat straw enabling almost twice the amount of d-glucose to be generated during the hydrolysis stage than experiments using atmospheric refining (0.06-0.09 g/g dry weight). Fermentation of the resulting hydrolysate affords 0.08-0.10 g/g (dry weight) of ethanol over similar scales, with ethanol productivity at ca. 37 mg/(L h). These results demonstrate that minimal cellulose decomposition occurs during pressure refining of wheat straw, in contrast to hemicellulose, and suggest that the development of green, mechanochemical processes for the scalable and cost-effective manufacture of second-generation bioethanol requires improved cellulose decomposition.

Developing an Olive Biorefinery in Slovenia: Analysis of Phenolic Compounds Found in Olive Mill Pomace and Wastewater.

The valorization of olive pomace through the extraction of phenolic compounds at an industrial scale is influenced by several factors that can have a significant impact on the feasibility of this approach. These include the types and levels of phenolic compounds that are present, the impact that seasonal variation and cultivar type have on the phenolic compound content in both olive pomace and mill effluents and the technological approach used to process the olive crop. Chemical analysis of phenolic compounds was performed using an HPLC-diode-array detector (DAD)-qTOF system, resulting in the identification of 45 compounds in olive mill wastewater and pomace, where secoiridoids comprised 50-60% of the total phenolic content. This study examined three different factors that could impact the phenolic compound content of these processing streams, including cultivar types typically grown on local farms in Slovenia, the type of downstream processing used and seasonality effects. Olive crop varieties sourced from local farms showed high variability, and the highest phenolic content was associated with the local variety "Istrska Belica". During processing, the phenolic content was on average approximately 50% higher during two-phase decanting compared to three-phase decanting and the type of compound present significantly different. An investigation into the seasonal effects revealed that the phenolic content was 20% higher during the 2019 growing season compared to 2018. A larger sample size over additional growing seasons is required to fully understand the annual variation in phenolic compound content. The methods and results used in this study provide a basis for further analysis of phenolic compounds present in the European Union's olive crop processing residues and will inform techno-economic modelling for the development of olive biorefineries in Slovenia.

A case of systemic lupus erythematosus associated auto-splenectomy presenting as invasive pneumococcal sepsis.

A 21-year-old female with a history of systemic lupus erythematosus (SLE) presented to the emergency department with septic shock. She had been maintained on 5 mg prednisolone daily and hydroxychloroquine 400 mg once daily and been investigated three years prior for recurrent left upper quadrant chest pain. Her previous SLE complications included pericardial effusion and high-risk pregnancy. Intensive care support was required due to septic shock, and a diagnosis of primary invasive Streptococcus pneumoniae bacteraemia was made following positive blood cultures. Computer tomography imaging of the abdomen demonstrated asplenia, with a diagnosis of auto-splenectomy thought most likely. Retrospective analysis of blood films from the two years prior was consistent with hyposplenism, including Howell-Jolly Bodies. The patient recovered from her sepsis and is maintained on amoxicillin prophylaxis. She was vaccinated according to post splenectomy guidelines and registered to the spleen registry. We report a case of auto-splenectomy and subsequent invasive pneumococcal disease in a SLE patient.

Pressurised disc refining of wheat straw as a pre-treatment approach for agricultural residues: A preliminary assessment of energy consumption and fibre composition.

This preliminary study assesses a potential pre-treatment approach for agricultural residues, in order to improve enzyme access and cellulose digestibility that increased with increasing refining pressure. Wheat straw, an important European agri-residue, was chopped then refined at pilot-scale under different pressures (4-10 bar) and two refiner plate configurations. The most energy efficient runs used 0.94-0.96 kWh electricity; 8.9-11.0 MJ heat per kg dry matter fibre. A scaling factor specific to the machinery used in the trial suggested that wheat straw could be refined using approximately 160 kWh electricity and 980-1900 MJ heat per tonne DM yield at commercial-scale. Hemicellulose content in wheat straw at 31.8% decreased to the lowest level of 14.6% after refining at 10 bar. Pressurised disc refining did not appear to produce significant quantities of acetic acid, a key fermentation inhibitor, that could limit microbial fermentation.

Quantification of residual AEBSF-related impurities by reversed-phase liquid chromatography.

During research of a broadly neutralizing antibody (bNAb) for HIV-1 infection, site-specific clipping was observed during cell culture incubation. Protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), was supplemented to the cell culture feeding to mitigate clipping as one of the control strategies. It led to the need and development of a new assay to monitor the free AEBSF-related impurities during the purification process. In this work, a reversed-phase liquid chromatography (RPLC-UV) method was developed to measure the total concentration of AEBSF and its major degradant product, 4-(aminoethyl) benzenesulfonic acid (AEBS-OH). This quantitative approach involved hydrolysis pre-treatment to drive all AEBSF to AEBS-OH, a filtration step to remove large molecules, followed by RPLC-UV analysis. The method was qualified and shown to be capable of measuring AEBS-OH down to 0.5 µM with good accuracy and precision, which was then applied for process clearance studies. The results demonstrated that a Protein A purification step in conjunction with a mock ultrafiltration/diafiltration (UF/DF) step could remove AEBSF-related impurities below the detection level. Overall, this study is the first to provide a unique approach for monitoring the clearance of free AEBSF and its related degradant, AEBS-OH, in support of the bNAb research.

Selective precipitation and characterization of lignin-carbohydrate complexes (LCCs) from Eucalyptus.

MAIN CONCLUSION: Six types of lignin-carbohydrate complex (LCC) fractions were isolated from Eucalyptus. The acidic dioxane treatment applied significantly improved the yield of LCCs. The extraction conditions had a limited impact on the LCC structures and linkages. Characterization of the lignin-carbohydrate complex (LCC) structures and linkages promises to offer insight on plant cell wall chemistry. In this case, Eucalyptus LCCs were extracted by aqueous dioxane, and then precipitated sequentially by 70% ethanol, 100% ethanol, and acidic water (pH = 2). The composition and structure of the six LCC fractions obtained by selective precipitation were investigated by sugar analysis, molecular weight determination, and 2D HSQC NMR. It was found that the acidic (0.05-M HCl) dioxane treatment significantly improved the yield of LCCs (66.4% based on Klason lignin), which was higher than the neutral aqueous dioxane extraction, and the extraction condition showed limited impact on the LCC structures and linkages. In the fractionation process, the low-molecular-weight LCCs containing a high content of carbohydrates (60.3-63.2%) were first precipitated by 70% ethanol from the extractable solution. The phenyl glycoside (PhGlc) bonds (13.0-17.0 per 100Ar) and highly acetylated xylans were observed in the fractions recovered by the precipitation with 100% ethanol. On the other hand, such xylan-rich LCCs exhibited the highest frequency of ß-O-4 linkages. The benzyl ether (BE) bonds were only detected in the fractions obtained by acidic water precipitation.

Fructan synthesis, accumulation and polymer traits. II. Fructan pools in populations of perennial ryegrass (Lolium perenne L.) with variation for water-soluble carbohydrate and candidate genes were not correlated with biosynthetic activity and demonstrated constraints to polymer chain extension.

Differences have been shown between ryegrass and fescue within the Festulolium subline introgression family for fructan synthesis, metabolism, and polymer-size traits. It is well-established that there is considerable variation for water-soluble carbohydrate and fructan content within perennial ryegrass. However there is much still to be discovered about the fructan polymer pool in this species, especially in regard to its composition and regulation. It is postulated that similar considerable variation for polymer traits may exist, providing useful polymers for biorefining applications. Seasonal effects on fructan content together with fructan synthesis and polymer-size traits have been examined in diverse perennial ryegrass material comprising contrasting plants from a perennial ryegrass F2 mapping family and from populations produced by three rounds of phenotypic selection. Relationships with copy number variation in candidate genes have been investigated. There was little evidence of any variation in fructan metabolism across this diverse germplasm under these conditions that resulted in substantial differences in the complement of fructan polymers present in leaf tissue at high water-soluble carbohydrate concentrations. The importance of fructan synthesis during fructan accumulation was unclear as fructan content and polymer characteristics in intact plants during the growing season did not reflect the capacity for de novo synthesis. However, the retention of fructan in environmental conditions favoring high sink/low source demand may be an important component of the high sugar trait and the roles of breakdown and turnover are discussed.

Fructan synthesis, accumulation, and polymer traits. I. Festulolium chromosome substitution lines.

The fructans found as storage carbohydrates in temperate forage grasses have a physiological role in regrowth and stress tolerance. They are also important for the nutritional value of fresh and preserved livestock feeds, and are potentially useful as feedstocks for biorefining. Seasonal variation in fructan content and the capacity for de novo fructan synthesis have been examined in a Festulolium monosomic substitution line family to investigate variation in the polymers produced by grasses in the ryegrass-fescue complex. There were significant differences between ryegrass and fescue. Fescue had low polymeric fructan content and a high oligomer/polymer ratio; synthesis of polymers longer than degree of polymerization 6 (DP6) from oligomers was slow. However, extension of polymer length from DP10/DP20 upward appeared to occur relatively freely, and, unlike ryegrass, fescue had a relatively even spread of polymer chain lengths above DP20. This included the presence of some very large polymers. Additionally fescue retained high concentrations of fructan, both polymeric and oligomeric, during conditions of low source/high sink demand. There were indications that major genes involved in the control of some of these traits might be located on fescue chromosome 3 opening the possibility to develop grasses optimized for specific applications.

Increased delignification by white rot fungi after pressure refining Miscanthus.

Pressure refining, a pulp making process to separate fibres of lignocellulosic materials, deposits lignin granules on the surface of the fibres that could enable increased access to lignin degrading enzymes. Three different white rot fungi were grown on pressure refined (at 6 bar and 8 bar) and milled Miscanthus. Growth after 28 days showed highest biomass losses on milled Miscanthus compared to pressure refined Miscanthus. Ceriporiopsis subvermispora caused a significantly higher proportion of lignin removal when grown on 6 bar pressure refined Miscanthus compared to growth on 8 bar pressure refined Miscanthus and milled Miscanthus. RM22b followed a similar trend but Phlebiopsis gigantea SPLog6 did not. Conversely, C. subvermispora growing on pressure refined Miscanthus revealed that the proportion of cellulose increased. These results show that two of the three white rot fungi used in this study showed higher delignification on pressure refined Miscanthus than milled Miscanthus.

Tag removal by site-specific cleavage of recombinant fusion proteins.

Where an affinity tag has served its purpose it may become desirable to remove it from the protein of interest. This chapter describes the removal of such fusion partners from the intended protein product by cleavage with site-specific endoproteases. Methods to achieve proteolytic cleavage of the fusion proteins are provided, along with techniques for optimising the yield of authentic product.

Immobilized metal ion affinity chromatography of native proteins.

Immobilized metal affinity chromatography (IMAC) is a common place technique in modem protein purification. IMAC is distinct from most other affinity chromatography technologies in that it can operate on a native, unmodified protein without the need for a specialized affinity "tag" to facilitate binding. This can be particularly important where a protein of interest is to be separated from a complex mixture such as serum or an environmental isolate. Relying on the interaction of specific surface amino acids of the target protein and chelated metal ions, IMAC can provide powerful discrimination between small differences in protein sequence and structure. Additionally, IMAC supports have been demonstrated to function effectively as cation exchangers, allowing for two modes of purification with a single column. This chapter provides methodologies to perform IMAC in its most fundamental form, that of the interaction between histidine and immobilized metal ions, those that enable purification of proteins that lack surface histidines and the operation of IMAC supports in cation exchange mode.

Immobilized metal ion affinity chromatography of histidine-tagged fusion proteins.

Immobilized metal ion affinity chromatography (IMAC) is a ubiquitous technique in modem recombinant production and purification. The wide range of expression vectors for the production of histidine-tagged recombinant proteins as well as the variety of stationary supports for their separation make IMAC an attractive and versatile choice for fast and reliable protein purification. It is not uncommon for IMAC purification to yield near homogenous target protein, with purities over 95%. The small size of the histidine tag means that in many cases it can remain associated with the target protein without interference with its intended function, obviating the need for any potentially complicating tag removal steps. This chapter provides protocols for the routine purification of such histidine-tagged fusion proteins. As with any purification regime, complications with IMAC can arise. Lacking the absolute specificity of a biological ligand/ligate system such as the avidin/biotin interaction or an antibody and its cognate antigen, IMAC can sometimes display non-ideal product purity. The protocols described in this chapter provide strategies for the improvement in the purity of IMAC-purified proteins. Similarly, non-specific binding may reduce product yields and purity in some circumstances. Methodologies for enhancing the yield of the target protein are therefore provided.

Site-specific cleavage of fusion proteins.

Where an affinity tag has served its purpose, it may become desirable to remove it from the protein of interest. This chapter describes the removal of such fusion partners from the intended protein product by cleavage with site-specific endoproteases. Methods to achieve proteolytic cleavage of the fusion proteins are provided, along with techniques for optimizing the yield of authentic product.

Thiophene-Functionalized TTF pi-Electron Donors as Potential Precursors to Conducting Polymers and Organic Metals: Synthesis, Properties, Structure, and Electropolymerization Studies.

The synthesis and physical properties of a series of novel thiophene-substituted TTF electron donors (6a-d) are described. The cyclic voltammograms of 6a-d exhibited two reversible one-electron redox waves, characteristic of TTF derivatives. Electropolymerization studies on compound 6a in nitrobenzene indicate that no polymer formed on the working electrode, but a blue-colored intermediate was observed diffusing away from the electrode. In addition, the single-crystal X-ray structure of compound 6d indicates that the central five-membered C(3)S(2) rings are buckled and the ethylenedithiolo fragment adopts a twisted chair conformation. The pendant thiophene moieties are highly flexible, and short intermolecular interactions of 3.49 Å (S.S) and 3.45 Å (S.C) exist in the unit cell, resulting in the formation of a chain structure along the c-axis.