Simultaneous saccharification and fermentation of steam exploded duckweed: Improvement of the ethanol yield by increasing yeast titre.

This study investigated the conversion of Lemna minor biomass to bioethanol. The biomass was pre-treated by steam explosion (SE, 210°C, 10 min) and then subjected to simultaneous saccharification and fermentation (SSF) using Cellic® CTec 2 (20 U or 0.87 FPU g(-1) substrate) cellulase plus ß-glucosidase (2 U g(-1) substrate) and a yeast inoculum of 10% (v/v or 8.0×10(7) cells mL(-1)). At a substrate concentration of 1% (w/v) an ethanol yield of 80% (w/w, theoretical) was achieved. However at a substrate concentration of 20% (w/v), the ethanol yield was lowered to 18.8% (w/w, theoretical). Yields were considerably improved by increasing the yeast titre in the inoculum or preconditioning the yeast on steam exploded liquor. These approaches enhanced the ethanol yield up to 70% (w/w, theoretical) at a substrate concentration of 20% (w/v) by metabolising fermentation inhibitors.

Chemical characterisation and analysis of the cell wall polysaccharides of duckweed (Lemna minor).

Duckweed is potentially an ideal biofuel feedstock due to its high proportion of cellulose and starch and low lignin content. However, there is little detailed information on the composition and structure of duckweed cell walls relevant to optimising the conversion of duckweed biomass to ethanol and other biorefinery products. This study reports that, for the variety and batch evaluated, carbohydrates constitute 51.2% (w/w) of dry matter while starch accounts for 19.9%. This study, for the first time, analyses duckweed cell wall composition through a detailed sequential extraction. The cell wall is rich in cellulose and also contains 20.3% pectin comprising galacturonan, xylogalacturonan, rhamnogalacturonan; 3.5% hemicellulose comprising xyloglucan and xylan, and 0.03% phenolics. In addition, essential fatty acids (0.6%, α-linolenic and linoleic/linoelaidic acid) and p-coumaric acid (0.015%) respectively are the most abundant fatty acids and phenolics in whole duckweed.

Investigation of viability of plant tissue in the environmental scanning electron microscopy.

The advantages of environmental scanning electron microscopy (ESEM) make it a suitable technique for studying plant tissue in its native state. There have been few studies on the effects of ESEM environment and beam damage on the viability of plant tissue. A simple plant tissue, Allium cepa (onion) upper epidermal tissue was taken as the model for study. The change of moisture content of samples was studied at different relative humidities. Working with the electron beam on, viability tests were conducted for samples after exposure in the ESEM under different operating conditions to investigate the effect of electron beam dose on the viability of samples. The results suggested that without the electron beam, the ESEM chamber itself can prevent the loss of initial moisture if its relative humidity is maintained above 90%. With the electron beam on, the viability of Allium cepa (onion) cells depends both on the beam accelerating voltage and the electron dose/unit area hitting the sample. The dose can be controlled by several of the ESEM instrumental parameters. The detailed process of beam damage on cuticle-down and cuticle-up samples was investigated and compared. The results indicate that cuticular adhesion to the cell wall is relatively weak, but highly resistant to electron beam damage. Systematic study on the effect of ESEM operation parameters has been done. Results qualitatively support the intuitive expectations, but demonstrate quantitatively that Allium cepa epidermal cells are able to be kept in a hydrated and viable state under relevant operation condition inside ESEM, providing a basis for further in situ experiments on plant tissues.

Characterization of asparagus lignin by HPLC.

Lignin is the cell wall component most frequently associated with hardening. Its characterization and quantification are very important to understand the biochemical modifications related to the changes in texture of vegetables such as asparagus (Asparagus officinalis), in which this organoleptic attribute is a very important quality factor. In this study, asparagus lignin from the basal sections of fresh and stored spears was analyzed using 2 methods, the traditional (Klason lignin) and the recently developed derivatization, followed by reductive cleavage (DFRC) method. The latter is a simple and reproducible technique for lignin characterization based on a degradation procedure that produces analyzable monomers and dimers by cleaving alpha- and beta-aryl ethers in lignins. The primary monomers derived from DFRC degradation of lignins are essentially p-coumaryl peracetate, coniferyl peracetate, and sinapyl peracetate. To evaluate the efficiency of the DFRC method, our investigations have been carried on distinct sample types, including wood (data not shown), straw, and asparagus samples. The results have confirmed that lignin composition is affected by plant nature. It has been found that whereas wood samples mostly contain coniferyl units, plant foods, such as straw and asparagus, contain both coniferyl and guaiacyl units.

Salmonella must be viable in order to attach to the surface of prepared vegetable tissues.

AIMS: The aims of the current study were to explore the site of bacterial attachment to vegetable tissues and to investigate the hypothesis that Salmonella must be living in order to attach to this site(s). METHODS AND RESULTS: Scanning electron micrographs of intact potato cells showed that Salm. serotype Typhimurium attached to cell-wall junctions; suggesting a high-level of site selectivity. Inactivation of Salm. Typhimurium using heat, ethanol, formalin or Kanamycin resulted in cells that could be no longer attached to these sites. Attachment of a Gfp(+) strain of Salm. Typhimurium to cell-wall material (CWM) was examined via flow cytometric analysis. Only live Salm. Typhimurium attached to the CWM. CONCLUSIONS: Salmonella serotype Typhimurium must be metabolically active to ensure attachment to vegetable tissues. Attachment preferentially occurs at the plant cell-wall junction and the cell-wall components found here, including pectate, may provide a receptor site for bacterial attachment. SIGNIFICANCE AND IMPACT OF THE STUDY: Further studies into individual plant cell-wall components may yield the specific bacterial receptor site in vegetable tissues. This information could in turn lead to the development of more targeted and effective decontamination protocols that block this site of attachment.

Production of feruloyl esterases and xylanases by Talaromyces stipitatus and Humicola grisea var. thermoidea on industrial food processing by-products.

Feruloyl esterase (FAE) and xylanase activities were detected in culture supernatants from Humicola grisea var. thermoidea and Talaromyces stipitatus grown on brewers' spent grain (BSG) and wheat bran (WB), two agro-industrial by-products. Maximum activities were detected from cultures of H. grisea grown at 150 rpm, with 16.9 U/ml and 9.1 U/ml of xylanase activity on BSG and WB, respectively. Maximum FAE activity was 0.47 U/ml and 0.33 U/ml on BSG and WB, respectively. Analysis of residual cell wall material after microbial growth shows the preferential solubilisation of arabinoxylan and cellulose, two main polysaccharides present in BSG and WB. The production of low-cost cell-wall-deconstructing enzymes on agro-industrial by-products could lead to the production of low-cost enzymes for use in the valorisation of food processing wastes.

In vitro evaluation of the prebiotic activity of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel.

The prebiotic effect of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel was studied using pure and mixed cultures of human faecal bacteria. This was compared to the prebiotic effect of fructo-oligosaccharides (FOS). Individual species of bifidobacteria and lactobacilli responded positively to the addition of the bergamot extract, which contained oligosaccharides in the range of three to seven. Fermentation studies were also carried out in controlled pH batch mixed human faecal cultures and changes in gut bacterial groups were monitored over 24 h by fluorescent in situ hybridisation, a culture-independent microbial assessment. Addition of the bergamot oligosaccharides (BOS) resulted in a high increase in the number of bifidobacteria and lactobacilli, whereas the clostridial population decreased. A prebiotic index (PI) was calculated for both FOS and BOS after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h incubation, with BOS showing a greater value (6.90) than FOS (6.12).

Synergy between xylanases from glycoside hydrolase family 10 and family 11 and a feruloyl esterase in the release of phenolic acids from cereal arabinoxylan.

The bioconversion of waste residues (by-products) from cereal processing industries requires the cooperation of enzymes able to degrade xylanolytic and cellulosic material. The type A feruloyl esterase from Aspergillus niger, AnFaeA, works synergistically with (1-->4)-beta-D-xylopyranosidases (xylanases) to release monomeric and dimeric ferulic acid (FA) from cereal cell wall-derived material. The esterase was more effective with a family 11 xylanase from Trichoderma viride in releasing FA and with a family 10 xylanase from Thermoascus aurantiacus in releasing the 5,5' form of diferulic acid from arabinoxylan (AX) derived from brewers' spent grain. The converse was found for the release of the phenolic acids from wheat bran-derived AXs. This may be indicative of compositional differences in AXs in cereals.

Arabinoxylan and mono- and dimeric ferulic acid release from brewer's grain and wheat bran by feruloyl esterases and glycosyl hydrolases from Humicola insolens.

An enzyme preparation from the thermophilic fungus Humicola insolens, Ultraflo L, was able to solubilise more than half of the biomass of brewer's grain and wheat bran, two agro-industrial co-products. While almost all of the ferulic acid was released in the free form, the majority of diferulates were released still attached to soluble feruloylated oligosaccharides, except for the 8,5' benzofuran form, which remained mostly in the residue. H. insolens also produced an esterase capable of releasing over 50% of p-coumaric acid present in wheat bran, but only 9% from the brewer's grain. The polysaccharide content in the residues after enzyme treatment comprised mostly cellulose and arabinoxylan, which suggests that part of the arabinoxylan in these residues is inaccessible to the xylanases of H. insolens. Differences in the solubilised arabinose-to-xylose ratio coupled to high free ferulate release suggest that the structure of feruloylated arabinoxylan in barley and wheat may differ.

Fracture of plant tissues and walls as visualized by environmental scanning electron microscopy.

The environmental scanning electron microscope (ESEM) provides a highly relevant and controllable environment in which to study hydrated systems without the artefacts of other highly prepared specimens. The instrument facilitates control of turgor through hydration using different chamber vapour pressures. Deformation of a simple plant tissue-upper epidermal layers in Allium cepa (onion)-was observed at the scale of the two principal failure mechanisms: cell breakage; and cell separation induced by treatment with a chelating agent. Cell rupture and release of contents occurred at cellular junctions ahead of an imposed growing notch, indicating that disruption of cells occurred remotely from the creation of a new surface. Cells that separated usually maintained their turgor and the separation process took place through progressive failure of middle lamellar material seen as strands between separating cells. These mechanisms were compared with the rupture of excised Chara corallina walls that occurred by formation and breakage of strands between separating wall layers. This study provides in situ visual characterization of wall rupture and cell separation at the microscopic level in hydrated plant material.

Plant Cell Walls and Food Quality.

Plant cell walls constitute the key structural components of plants and many plant-based foods. They are well known for contributing to a range of "quality" characteristics, from organoleptic texture to the properties of dietary fiber. Much of the research on cell walls has focused on the physiological aspects of plant growth and development with the belief that this route holds the key to controlling quality characteristics. In addition, consideration of quality has often been determined by what is easily measurable. This review assesses critically the role of plant cell walls in relation to the ultimate determinant of quality - the consumer, but within a whole food-chain context. We conclude that effective exploitation of cell-wall research in relation to optimizing quality requires an integrated approach taking into account the multi-functional roles of plant cell walls, and the diversity of consumer-related quality dimensions.

Microbial colonization of naturally black olives during fermentation and associated biochemical activities in the cover brine.

AIMS: To establish the site of microbial growth on naturally black fermented table olives, and to monitor the population dynamics of yeasts and selected micro-organisms together with the changes in organic acid profile and pH in the cover brine during fermentation. METHODS AND RESULTS: During fermentation, the numbers of Enterobacteriaceae and Pseudomonas spp. in the brine decreased whilst lactic acid bacteria and yeast populations increased. Scanning electron microscopy showed that a yeast-rich biofilm developed on the epicuticular wax of the olive skin during fermentation. Yeasts also predominated in the stomatal openings, but bacteria were more numerous in intercellular spaces in the sub-stomatal flesh. Citric, malic and tartaric acids were the major organic acids accumulating in the brine during fermentation. CONCLUSIONS: Micro-organisms associated with the skin, stomata and flesh in fermenting black olives may experience different local conditions to those prevailing in the cover brine. SIGNIFICANCE AND IMPACT OF THE STUDY: These are the first observations of the micro-organisms associated with the fruit of naturally fermented black olives and of the accumulation of specific organic acids during fermentation.

Pectin distribution at the surface of potato parenchyma cells in relation to cell-cell adhesion.

The crispness of fruits and vegetables is dependent, predominantly, on the maintenance of cell adhesion. There is a growing body of evidence to suggest that cell adhesion in plants is controlled at the edge of cell faces rather than across the entire cell surface. The aim of the current study has been to exploit antibody-labeling techniques in conjunction with methods that induce cell separation to explore the distribution of highly esterified and weakly esterified pectic polysaccharides on the cell surface. Potato parenchyma tissue was subjected to cooking and chemical treatments, which induced softening through cell separation. Scanning electron microscopy (SEM) revealed characteristic patterns on the surface of these separated cells, which outlined the imprint of neighboring cells. Monoclonal antibodies, JIM5 and JIM7, were used to locate weakly esterified and highly esterified pectin by silver-enhanced immunogold SEM. The edge-of-face structures labeled strongly with JIM5 but not JIM7, indicating that they contained polygalacturonic acid of low ester content. In addition, adhesion of the middle lamella to the face of the primary wall was found to differ from adhesion at the edge of each cell face. This, in conjunction with the antibody-labeling observations, complements previous transmission electron microscopy studies and is consistent with the edge-of-face regions having a specialist role in cell adhesion.

Fracture mechanics of the cell wall of Chara corallina.

Previous mechanical studies using algae have concentrated on cell extension and growth using creep-type experiments, but there appears to be no published study of their failure properties. The mechanical strength of single large internode cell walls (up to 2 mm diameter and 100 mm in length) of the charophyte (giant alga) Chara corallina was determined by dissecting cells to give sheets of cell wall, which were then notched and fractured under tension. Tensile tests, using a range of notch sizes, were conducted on cell walls of varying age and maturity to establish their notch sensitivity and to investigate the propagation of cracks in plant cell walls. The thickness and stiffness of the walls increased with age whereas their strength was little affected. The strength of unnotched walls was estimated as 47+/-13 MPa, comparable to that of some grasses but an order of magnitude higher than that published for model bacterial cellulose composite walls. The strength was notch-sensitive and the critical stress intensity factor K1c was estimated to be 0.63+/-0.19 MNm(-3/2), comparable to published values for grasses.

In vitro protein digestibility and physicochemical properties of dry red bean (Phaseolus vulgaris) flour: effect of processing and incorporation of soybean and cowpea flour.

A study was carried out to determine the effect of germination and drying temperature on the in vitro protein digestibility and physicochemical properties of dry red bean flours. A 2 x 3 factorial experiment with two treatments (germination and nongermination) and three drying temperatures was used for this purpose. The effect of particle size on water absorption capacity of bean flour was investigated. In addition, the effect of incorporating soybean and cowpea into the red bean flour on functional properties was equally investigated. Results reveal that protein digestibility increased with germination and also with drying temperature. Drying at 60 degrees C produced flours of optimum functional characteristics, although the hydrophilic/lipophilic index was high and the solubility index reduced. Germination and particle size as well as drying temperature all affected the water uptake properties of bean flours. Incorporation of soybean and cowpea flour into germinated bean flour at levels of 10 and 30%, respectively, produced a composite with higher functional properties.

Tissue-specific developmental changes in cell-wall ferulate and dehydrodiferulates in sugar beet.

Sugar beet (Beta valgaris L.) seedlings were grown for 8-14 weeks, and then separated into leaf, petiole, inner and outer storage root and absorptive root fractions. Cell-wall ferulate and dehydrodiferulate esters were analysed by HPLC. In leaves, ferulate dimers were mostly 8-8 linked, while 8-O-4 and sometimes 8-5 linkages were most abundant in all other tissues. The total dimer content and percentage of dimerisation were much higher in the absorptive root than in other tissues. These results indicated varying patterns of ferulate and dehydrodiferulate ester content in different tissues, suggesting corresponding variations in the biosynthetic processes. When [14C]-cinnamate was applied to the leaves at 4 weeks, and [14C]-dimers measured in root cell walls at 8 and 14 weeks, a much higher proportion of 8-5 linkages was found in the [14C]-dimers than in total (non-radioactive) dimers in all parts of the root, especially at 14 weeks, indicating further complexity in the metabolism of cell-wall phenolics.

Physicochemical characteristics of onion (Allium cepa L.) tissues.

The structure and mechanical properties of onions are important factors affecting their textural quality. The onion bulb consists of several layers of pigmented, papery scales surrounding fleshy storage scales that comprise an upper epidermis, an intermediate parenchyma tissue, and a lower epidermis. The purpose of this study was to examine the chemical composition of cell walls from the papery scales and outer fleshy scales of onion (Allium cepa L. cv. Sturon) in relation to their mechanical properties. Cell-wall material (CWM) was prepared from the component tissues and analyzed for its carbohydrate and phenolic composition. The CWMs were rich in uronic acid and glucose, with smaller quantities of arabinose, galactose, and xylose. In the fleshy scales, the lower epidermis contained relatively more galactose-rich pectic polysaccharides, whereas the upper epidermis and the papery scales contained virtually no galactose. Analysis of mechanical properties showed that the order of strength of the tissues was papery scales > fleshy scales, which were in the order lower epidermis > upper epidermis > intermediate parenchyma. The upper epidermis of fleshy scales was stronger in the vertical than the horizontal direction, and both orientations showed negligible notch sensitivity. Cyclohexane-trans-1,2-diaminetetraacetate-induced vortex-induced cell separation of the intermediate layer of fleshy scales indicated that calcium cross-linking may play an important role in cell-cell adhesion. A small but significant amount of ferulic acid was found in the walls, predominantly in the thick cuticle of the lower epidermis of fleshy scales. Alkali-labile wall-bound flavonoids were also detected.

The mechanical properties and molecular dynamics of plant cell wall polysaccharides studied by Fourier-transform infrared spectroscopy.

Polarized one- and two-dimensional infrared spectra were obtained from the epidermis of onion (Allium cepa) under hydrated and mechanically stressed conditions. By Fourier-transform infrared microspectroscopy, the orientation of macromolecules in single cell walls was determined. Cellulose and pectin exhibited little orientation in native epidermal cell walls, but when a mechanical stress was placed on the tissue these molecules showed distinct reorientation as the cells were elongated. When the stress was removed the tissue recovered slightly, but a relatively large plastic deformation remained. The plastic deformation was confirmed in microscopic images by retention of some elongation of cells within the tissue and by residual molecular orientation in the infrared spectra of the cell wall. Two-dimensional infrared spectroscopy was used to determine the nature of the interaction between the polysaccharide networks during deformation. The results provide evidence that cellulose and xyloglucan associate while pectin creates an independent network that exhibits different reorientation rates in the wet onion cell walls. The pectin chains respond faster to oscillation than the more rigid cellulose.

Esterified phenolics of the cell walls of chufa (Cyperus esculentusL. ) tubers and their role in texture.

Chufas (Cyperus esculentus) are edible tubers that, like Chinese waterchestnut (CWC), are very crisp when raw and do not soften when cooked. The present study compares the mechanical properties of chufas with those of potato and CWC in relation to the carbohydrate and phenolic compositions of the cell walls. The cutting toughness of raw chufa was higher than that of raw CWC and potato; its value decreased on boiling, as also observed with CWC, but remained over twice that of raw potato. Chufa cell walls were rich in xylose, arabinose, glucose, uronic acid, and galactose, with minor quantities of mannose. The cell walls of the parenchyma exhibited a uniform pH-dependent autofluorescence indicating the presence of cinnamic acid derivatives. Analysis of these revealed that peeled tuber cell walls are rich in ferulic acid, whereas p-coumaric acid dominates the monomeric phenol fraction of the skin. Cell wall material from both skin and peeled tubers contains a significant amount of different diferulic acids ( approximately 20% of the wall ferulic acid), consisting mainly of the 8-O-4'-, 8-5'-, and 5-5'-dimers. These are potentially available to form thermally stable cross-links between polysaccharides within the wall and between cells. This may confer thermal stability of texture.