The search for a cheap and readily available feedstock for biodiesel production has driven researchers to investigate the potential use of activated sludge-derived lipid. Due to the low lipid yield of activated sludge, the utility of subcritical water (SCW) is being considered as a means of increasing the extractable lipids. In this work, operating parameters (temperature, time and biomass loading) identified in our previous work to influence lipid yield from activated sludge under SCW treatment were optimized, using face-centered central composite design (FCCCD). The results show that the maximum lipid yield obtained from the liquid and solid products, post SCW treatment, at the optimum conditions of temperature 80 degrees C, extraction time 20 min and biomass loading 1%, was 41.0 (wt./wt.)%. This gives approximately 454 (wt./wt.)% increase from 7.4 (wt./wt.)% obtained by solvent extraction without SCW treatment. The results suggest that activated sludge is a potential feedstock for biodiesel production.

Lignin is the second most abundant naturally occurring biopolymer from lignocellulosic biomass. While there are several lignin applications, attempts to add value to lignin are hampered by its inherent complex and heterogenous chemical structure. This work assesses the organosolv lignin aggregates behaviour of soluble lignin extract derived from Miscanthus × giganteus using different ethanol concentrations (50%, 40%, 30%, 20%, 10% and 1%). The effect of two different lignin concentrations using similar ethanol concentration on the efficacy of esterification was studied. Overall, particle size of lignin analysis showed that the particle size of lignin aggregates decreased with lower ethanol concentrations. 50% ethanol concentration of soluble lignin extract showed the highest particle size of lignin (3001.8 nm), while 331.7 nm of lignin particle size was recorded at 1% ethanol concentration. Such findings of particle size correlated well with the morphology of the lignin macromolecules. The lignin aggregates appeared to be disaggregated from population of large aggregates to sub-population of small aggregates when the ethanol concentration was reduced. Light microscopy images analysis by ImageJ shows that the average diameter and circularity of the corresponding lignin macromolecules differs according to different ethanol concentrations. The dispersion of lignin aggregates at low ethanol concentration resulted in high availability of hydroxyl group in the soluble lignin extract. The efficacy of the lignin modification via esterification was evidenced directly via FTIR using the similar ethanol concentration of soluble lignin extract at different lignin concentrations. This study provided the understanding of detail analysis on particle size determination, microscopic properties and structural insights of lignin aggregates at wider ethanol concentrations. The esterified lignin derived at 5 mg/mL is suggested to expand greater lignin functionality in the preparation of lignin bio-based materials.

Lignin is an industrial by-product produced from the pulping and paper industry, where the process generates lignin in the form of lignosulphonates. While there are many applications for lignin, there are all low value and attempts to add value to lignin are hindered by its complex physicochemical nature and the presence of sulphur. Adopting the biorefining concept, the study evaluates the impact of direct (DE) and sequential extraction (SE) of Miscanthus x giganteus using sub-critical water with associated modifiers; ethanol and carbon dioxide on the physical and chemical properties of the extracted lignin. Isolated lignins were characterised by a Fourier Transform Infrared Spectroscopy (FTIR). Although higher delignification was achieved by DE and SE about 81.5% and 58.0%, respectively, the lignin recovered from the SE process showed remarkably higher purity with 91.5%. Lignin recovery did not differ considerably for either processing method. FTIR revealed a qualitative reduction in the intensity of bonds corresponding to hydroxyl groups for the lignin derived from DE rather than SE processing routes. These indicated that the lignin derived from SE had potential for subsequent preparation in lignin value-added bio-based materials.

This work assesses the behavior of organosolv lignin aggregates derived from Miscanthus x giganteus using different ethanol concentrations (10%, 25%, 50%, and 75% by volume). The percentage of lignin recovery was found to decrease from 75.8% to 71.4% and 25.1%, as the ethanol concentration was increased from 10% to 25% and 50%, respectively. Increasing the ethanol concentration further to 75% led to zero recovery. The purity of the precipitated lignin was consistently found to be ≥90%. Lignin derived from the dried supernatant obtained at 50% ethanol concentration resulted in high lignin purity (51.6%) in comparison with the other ethanol concentrations used. Fourier transform infrared spectroscopy analysis showed that the precipitated lignin and dried supernatant at 50% ethanol concentration possessed the highest peak intensity apportioned to wavenumber of lignin as compared to that of at 25% and 10% ethanol concentrations, and the results linked with the percentage of lignin purity. The results of particle size analysis for precipitated lignin demonstrated particle sizes of 306, 392, and 2050 nm for 10%, 25%, and 50% ethanol concentrations, respectively, and the remaining supernatant with average particle sizes of 1598, 1197, and 875 nm, respectively. These results were verified with the morphology of lignin macromolecules in scanning electron microscopy images. Results of the particle size distribution of lignin revealed that the overall size of lignin aggregates decreased with decreasing ethanol concentration. In summary, these findings suggest that ethanol concentration affected the behavior of lignin aggregates in water–ethanol solution.

Response surface methodology (RSM) was applied to provide predictive models for optimisation of extraction of selected polyphenolic compounds from cider apple pomace under aqueous acetone.The design of experiment (DoE) was conducted to evaluate the influence of acetone concentration % (v/v), solid-to solvent ratio % (w/v), temperature (˚C) and extraction time (min) and their interaction on phenolic contents, using the Central Composite Rotatable Design (CCRD).The experimental data were analysed to fit statistical models for recovery of phenolic compounds.The selected models were significant (P < 0.05) and insignificant lack of fits (P > 0.05), except for Chlorogenic acid and Quercetin 3-glucoside which had significant lack of fits (P < 0.05).All models had satisfactory level of adequacies with coefficients of regression R 2 > 0.9000 and adjusted 2 Adj R reasonable agrees with predicted 2 Pri R .Coefficient of variation < 5% for each determination at the 95% confidence interval.These models could be relied upon to achieve optimal concentrations of polyphenolic compounds for applications in nutraceutical, pharmaceutical and cosmetic industries.

Activated sludge is currently being investigated as a potential feedstock to produce biodiesel. Reports have shown that its lipid yield is usually low, compared to the commercially available feedstocks, such as soybean, sunflower, and rapeseed oils. The current work is geared toward investigating the potential of enhancing the total lipid yield (neutral and phospholipid) by the application of subcritical water. The results show that subcritical water treatment increased the total lipid yield by 145% at the optimum conditions 280 degrees C, 40 min, 15 mL of water and biomass loading of 5%. The lipid classes identified were waxes, acylglycerol, sterol, free fatty acid, and phospholipids. The results of the fatty acid methyl esters analysis revealed palmitic acid as the predominant fatty acid. Practical applications The practical applications of the results are informed from the improved lipid yield obtained, suggesting that subcritical water treatment is essential if activated sludge were to be a viable feedstock for biodiesel, renewable diesel, and oleochemicals productions. This will serve as a sustainable activated sludge disposal strategy.

Polyphenolic compounds with relatively high antioxidant activity obtained from subcritical water extraction of apple pomace were assessed for encapsulation by spray drying technique, making use of polymeric substances co-extracted with the polyphenolic compounds. Comparative assessments were carried out of the directly encapsulated subcritical water extract (SWE) products with particles formed when encapsulated with the addition of hydroxyl propyl-β-Cyclodextrin (SWE + HPβ-CD). The powders were characterized for their physico-chemical properties such as, moisture content, density, particle size, hygroscopicity to assess their suitability within cosmetic formulations. The SWE and SWE + HPβ-CD encapsulated products resulted in different physical properties. Although the particle size was less than 4 μm for both products, the direct encapsulation (SWE) was highly hygroscopic and this property was significantly reduced with addition of HPβ-Cyclodextrin (SWE + HPβ-CD). Scanning electron microscopy (SEM) and Fourier Transform Infrared (FT-IR) spectroscopic were em-ployed to analyse the micronised powders to support evidence of encapsulation. Both techniques revealed the interaction between compounds in extract and the carrier HPβ-Cyclodextrin suggesting successful encapsulation. The effect of storage conditions on retention of antioxidant activity of the subcritical water extract was evaluated within 35 days for extracts with and without the carrier HPβ-Cyclodextrin. Hydroxyl propyl-β-Cyclodextrin offered protection against degradation of antioxidant compounds thereby potentially extending the shelf-life and making the encapsulated powder suitable for incorporation in cosmetic and pharmaceutical applications.

Industrial wet cider apple pomace was subjected to subcritical water mediated hydrolysis in a batch reactor with varying experimental conditions such as solid-to- solvent ratio (1-8 % w/v), temperature (100- 200 oC) and a residency time (10-30 min) to understand the effects of the experimental conditions on overall recovery of polyphenolic compounds. Chlorogenic acid and some flavonoids were identified and quantified by high performance liquid chromatography (HPLC-DAD). Higher yields of chlorogenic acid and the flavonoids were obtained between 100- 150 oC for residence time of 20 minutes. Solid-to solvent ratio and temperature played a significant role in the recovery of the polyphenolic compounds (p< 0.05). The results demonstrated that, residence time at a fixed extraction condition was less significant (p> 0.05). However, it was significant for yield of degradation compounds such as protocatechuic aldehyde, 5- HMF and furfural. Subcritical water selectively influenced the chemical structure of the polyphenolic compounds.

Renewable diesel, a potential substitute for petroleum diesel, is usually produced from biological sources by deoxygenating fatty acids in the presence of organic solvents with the addition of hydrogen. This could pose a health challenge and increase the cost of production. The current work was conducted in subcritical water without the addition of hydrogen using palmitic and oleic acids as fatty acids. The impact of temperature and reaction time was assessed using 5% palladium on activated carbon (Pd/C) catalyst. The recovered organic products were analysed for hydrocarbons using gas chromatography. The results show that the main hydrocarbon products from palmitic and oleic acids were pentadecane and heptadecane, respectively. The maximum value of the molar yield of pentadecane was 35.4% at the optimum conditions of 290 degrees C and 1.5 h, and that from heptadecane was 16.6% at 330 degrees C and 4 h. This shows that 5% Pd/C is active for catalysing the hydrothermal decarboxylation of the fatty acids to hydrocarbons. With these results, the cost of production can probably be reduced and the capacity to use hydrocarbons from a renewable resource to displace conventional diesel can be increased.

Polyphenolic compounds extraction from industrial apple pomace was optimised by applying design of experiments (DoE) and surface response methodology using the Central Composite Rotatable Design (CCRD). The degree solubilisation and the yield of total phenolic content from the apple pomace using organic solvents was shown to be influenced by process parameters including solvent type, solvent concentration, temperature, apple pomace to solvent ratio, and extraction time (residency time). Optimal conditions of extracting phenolic compounds were as follows: acetone concentration, 65 % (v/v); solid to solvent ratio 1 %; extraction time 30 minutes and temperature 60oC. Optimum condition for solubilisation was as follows: acetone concentration 78 % (v/v); solid to solvent ratio 4.7 %; extraction time 54 minutes and temperature 21oC. Under these conditions, the total phenolic content and solubilisation were 21.70 ± 0.2 mg GAE/g dw and 19.20 ± 0.1g/100g of the dried apple pomace respectively and largely agreed with those predicted by the Stat-Ease software. Independent variables for optimisation of total phenolic content and solubilisation were completely different. The reverse phase HPLC analysis of the extract revealed the major polyphenolic compounds were chlorogenic acid, procyanidin B2, caffeic acid, epicatechin, ferulic acid, quercetin-3-galactoside, quercetin-3-glucoside and phloridzin.

The need for sustainable wastewater treatment and search for cheap feedstock for renewable energy production have driven investigators to evaluate the potential of producing biofuels from waste materials such as sewage sludge. The current work is aimed at assessing the potential for producing hydrocarbons from the lipid fraction of activated sludge via a catalytic hydrothermal decarboxylation reaction under varying residence time. Impacts of platinum on carbon (Pt/C) and palladium on carbon (Pd/C) catalysts were evaluated at 290 degrees C and residence time of 0.5, 1, 3 and 4 h. The analyses of the lipids and hydrocarbons were done using high-temperature gas chromatography. The results show that at the optimum conditions of 1 h, 290 degrees C, 20 mg of catalyst, 100 mg of lipid extract and 15 mL of water, the maximum yield of hydrocarbon was 23.2% of pentadecane obtained using Pt/C. This shows that the dominant hydrocarbon is pentadecane and that Pt/C exhibited greater catalytic effect than did Pd/C. The hydrocarbons might be used directly as renewable diesel and as such will contribute to the mitigation of climate change since emission of greenhouse gases will be reduced, and the use of activated sludge as the feedstock is expected to help minimize the cost of production and to ensure sustainable wastewater treatment.

Apple pomace, a by-product of apple juice and cider production is considered a potential source of pectin, citric acid and bioethanol and polyphenolic compounds. Polyphenolic compounds are known to possess antioxidant properties; hence, their isolation is a rout to add value to the apple pomace waste. The current research investigated the efficacy of subcritical water in the recovery of the polyphenolic antioxidant compounds from the apple pomace using a batch reactor system at 100-bar over a temperature range of 100-200°C. Experimental factors such as; nature of apple pomace, solid-to-solvent ratio, temperature, and residence time were studied and their impact on the yields of polyphenolic content and antioxidant activity were examined. Response surface methodology was used to design the experiment to study the potential interaction of operational parameter effects on overall recovery of polyphenolic compounds. Designed conditions demonstrated selectivity towards significant recovery of the polyphenolic compounds and antioxidant activity by Oxygen Radical Absorbance Capacity (ORAC). Optimized total Phenolic content and antioxidant activity of the wet apple pomace were achieved at 200°C for 1% solid-to-solvent ratio at a residence time of 30 minutes. Optimum Total phenolic content and ORAC activity of subcritical water extract were 49.86 mg GAE /g dw and 99285 μmol TE/g dw of apple pomace respectively. High Performance Liquid Chromatography (HPLC-DAD) in a reverse mode was applied to identify polyphenolic contents of the pomace extracts. Representative Hydrocinnamic acid and flavonoids were, Chlorogenic acid, Phloridzin, Procyanidin B2, Epicatechin and Quercetin glycosides. Water soluble antioxidant compound Protocatechuic aldehyde was identified for the first time only in the subcritical water extract and to date have not been identified in solvent extracts of cider apple pomace.

Previously we have shown that subcritical water may be used as an alternative to enzymatic digestion in the proteolysis of proteins for bottom-up proteomics. Subcritical water hydrolysis of proteins was shown to result in protein sequence coverages greater than or equal to that obtained following digestion with trypsin; however, the percentage of peptide spectral matches for the samples treated with trypsin were consistently greater than for those treated with subcritical water. This observation suggests that in addition to cleavage of the peptide bond, subcritical water treatment results in other hydrolysis products, possibly due to modifications of amino acid side chains. Here, a model peptide comprising all common amino acid residues (VQSIKCADFLHYMENPTWGR) and two further model peptides (VCFQYMDRGDR and VQSIKADFLHYENPTWGR) were treated with subcritical water with the aim of probing any induced amino acid side-chain modifications. The hydrolysis products were analyzed by direct infusion electrospray tandem mass spectrometry, either collision-induced dissociation or electron transfer dissociation, and liquid chromatography collision-induced dissociation tandem mass spectrometry. The results show preferential oxidation of cysteine to sulfinic and sulfonic acid, and oxidation of methionine. In the absence of cysteine and methionine, oxidation of tryptophan was observed. In addition, water loss from aspartic acid and C-terminal amidation were observed in harsher subcritical water conditions.

Recycled paper mill sludge (RPMS) was hydrothermally treated by subcritical water (SBW) in two modes of operation, namely semi-continuous and batch, at temperatures in the range 150-250 degrees C, and 100 bar. The performance of both systems was analyzed in terms of degree of hydrolysis, product yield and formation of degradation products. The highest degree of RMPS hydrolysis was achieved using the batch reactor configuration, while in the semi-continuous system higher yields of water soluble compounds (WSC) and monosaccharides were achieved. By using the semi-continuous system at 100 bar, 250 degrees C and a water flow rate of 5 mL/min, 40% of the organic carbon content of RPMS was recovered in the form of soluble compounds. Additionally 20% of total carbohydrates and 75% of total lignin of RPMS were recovered as soluble material. Detailed mass balances performed for both operational modes revealed that under batch mode, the naturally occurring compounds found in RPMS underwent significant transformation, generating derivatives referred to as degradation products. An analysis of the severity factor was performed for both operational modes. It was concluded that significant values of WSC were only obtained for severity factors higher than four.

Currently biodiesel, is produced from plant oils or animal’s fats by a liquid-phase catalysed transesterification process at low temperature. Although biodiesel is renewable and to a large extent sustainable, inherent properties such as poor cold flow, low oxidation stability, low cetane value restrict application to blends with fossil fuels. An alternative to biodiesel is renewable diesel produced by catalytic hydrotreating of oils and fats and is considered a drop in fuel because its properties are similar to petroleum diesel. In addition to developing alternative productions routes there is continued interest in reducing the cost of the feed stock, waste cooking oils and fats are increasingly used as the feedstocks due to low cost. However use of oils and fat are highly adulterated resulting in high free fatty acid content which turn impacts on the efficiency of FAME production. Therefore in light of the need to develop alternative lipid feed stocks and related efficient catalysis the present study investigates the potential of producing renewable diesel from the lipids-extracted from activated sludge, a waste water treatment by-product, through catalytic hydrothermal decarboxylation. The microbial lipids were first extracted from the activated sludge using the Folch et al method before hydrothermal decarboxylation reactions were carried out using palladium (Pd/C) and platinum (Pt/C) on activated carbon as the catalysts in a batch reactor. The impact of three temperatures 290, 300, 330C and residence time between 30 min and 4hrs was assessed. At the end of the reaction, the products were recovered using organic solvents and characterized using gas chromatography (GC). The principle products of the reaction were pentadecane and heptadecane. The highest yields of pentadecane and heptadecane from lipid-extract were 23.23% and 15.21%, respectively. These yields were obtained at 290C and residence time 1h using Pt/C. To the best of our knowledge, the current work is the first investigation on the hydrothermal decarboxylation of lipid-extract from activated sludge. Activated sludge, lipid, hydrothermal decarboxylation, renewable diesel

Subcritical water is an emerging tool in the processing of bioorganic waste. Subcritical water is an environmentally benign solvent which has the potential to provide an alternative to traditional methods of protein hydrolysis without the inclusion of expensive acids or enzymes. To date, most studies on the subcritical water mediated hydrolysis of proteins have focused on the production of amino acids, rather than the intermediate peptides. Here, we investigate the specificity of subcritical water with respect to the production of peptides from three model proteins, hemoglobin, bovine serum albumin, and β-casein, and compare the results with enzymatic digestion of proteins by trypsin. In addition, the effect of subcritical water (SCW) treatment on two protein post-translational modifications, disulfide bonds and phosphorylation, was investigated. The results show that high protein sequence coverages (>80%) can be obtained following subcritical water hydrolysis. These are comparable to those obtained following treatment with tryspin. Under mild subcritical water conditions (160 °C), all proteins showed favored cleavage of the Asp-X bond. The results for β-casein revealed favored cleavage of the Glu-X bond at subcritical water temperatures of 160 and 207 °C. That was similarly observed for bovine serum albumin at a subcritical water temperature of 207 °C. Subcritical water treatment results in very limited cleavage of disulfide bonds. Reduction and alkylation of proteins either prior to or post subcritical water treatment improve reported protein sequence coverages. The results for phosphoprotein β-casein show that, under mild subcritical water conditions, phosphorylation may be retained on the peptide hydrolysis products.

C-hordein in barley and ω-gliadins in wheat are members of the prolamins protein families. Prolamins are the major component of cereal storage proteins and composed of non-essential amino acids (AA) such as proline and glutamine therefore have low nutritional value. Using double stranded RNAi silencing technology directed towards C-hordein we obtained transgenic barley lines with up to 94.7 % reduction in the levels of C-hordein protein relative to the parental line. The composition of the prolamin fraction of the barley parental line cv. Golden Promise was resolved using SDS-PAGE electrophoresis, the protein band were excised and the proteins identified by quadrupole-time-of-flight mass spectrometry. Subsequent SDS-PAGE separation and analysis of the prolamin fraction of the transgenic lines revealed a reduction in the amounts of C-hordeins and increases in the content of other hordein family members. Analysis of the AA composition of the transgenic lines showed that the level of essential amino acids increased with a concomitant reduction in proline and glutamine. Both the barley C-hordein and wheat ω-gliadin genes proved successful for RNAi-gene mediated suppression of barley C-hordein level. All transgenic lines that exhibited a reduction for C-hordein showed off-target effects: the lines exhibited increased level of B/γ-hordein while D-hordein level was reduced. Furthermore, the multicopy insertions correlated negatively with silencing.

Lignin was isolated with subcritical water:ethanol:CO2 (Sub-CW) from biorefinery biomasses, such as giant reed (AD) and miscanthus (MG), with recovery yields about 30% on Klason lignin. Their structural composition assessed by IR and NMR techniques, as well as Derivatization Followed by Reductive Cleavage (DFRC/GC-MS). The 2D HSQC-NMR spectra elucidated that the Sub-CW extracts contained different lignin dimers and co-extracted carbohydrates. The DFRC/GC-MS revealed that syringyl molecules were more abundant in AD, while guaiacyl monomers were predominant in MG. Lignin residues were derivatized with phospholane to quantitatively estimate the amount of OH groups by 31P NMR, showing a marked predominance of aliphatic units for both lignins, due to the presence of either hydroxyls in lignin side-chain or residual carbohydrates. Lignin residues derivatized with phospholane allowed to record 31P-DOSY NMR spectra. AD-lignin showed a smaller diffusivity constant than for MG-substrate, possibly because of the larger content of disaccharides in MG lignin. We showed that the molecular composition of lignin isolated by the Sub-CW technique may differ depending on the type of biomass used for the extraction, suggesting a different industrial application of lignin from various biomass.

BACKGROUND:Cereal storage proteins represent one of the most important sources of protein for food and feed and they are coded by multigene families. The expression of the storage protein genes exhibits a temporal fluctuation but also a response to environmental stimuli. Analysis of temporal gene expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging.RESULTS:We designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and γ-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families, subfamilies or individual members. The specificity of the primer sets was validated before successfully applying them to a cDNA population derived from developing grains of field grown Hordeum vulgare cv. Barke. The results quantify the number of moles of transcript contributed to a particular gene family and its subgroups. More over the results indicate the genotypic specific gene expression.CONCLUSIONS:Quantitative RT-PCR with SYBR Green labelling can be a useful technique to follow gene expression levels of large gene families with highly homologues members. We showed variation in the temporal expression of genes coding for barley storage proteins. The results imply that our rapid qRT-PCR system was sensitive enough to identify the presence of alleles and their expression profiles. It can be used to check the temporal fluctuations in hordein expressions or to find differences in their response to environmental stimuli. The method could be extended for cultivar recognition as some of the sequences from the database originated from cv. Golden Promise were not expressed in the studied barley cultivar Barke although showed primer specificity with their cloned DNA sequences.