T4 Bacteriophage and E. coli Interaction in the Murine Intestine: A Prototypical Model for Studying Host-Bacteriophage Dynamics In Vivo.

Bacteriophages (phages) are viruses that infect bacteria with species- and strain-level specificity and are the most abundant biological entities across all known ecosystems. Within bacterial communities, such as those found in the gut microbiota, phages are implicated in regulating microbiota population dynamics and driving bacterial evolution. There has been renewed interest in phage research in the last decade, in part due to the host-specific killing capabilities of lytic phages, which offer a promising tool to counter the increasing threat of antimicrobial resistant bacteria. Furthermore, recent studies demonstrating that phages adhere to intestinal mucus suggest they may have a protective role in preventing bacterial invasion into the underlying epithelium. Importantly, like bacterial microbiomes, disrupted phageomes have been associated with worsened outcomes in diseases such as inflammatory bowel disease. Previous studies have demonstrated that phages can modulate the microbiome of animals and humans through fecal filtrate transplants, benefiting the host's health. With this recent wave of research comes the necessity to establish and standardize protocols for studying phages in the context of the gut microbiome. This protocol provides a set of procedures to study isolated T4 phages and their bacterial host, Escherichia coli, in the context of the murine gastrointestinal tract. The methods described here outline how to start from a phage lysate, administer it to mice and assess effects on bacterial host and phage levels. This protocol can be modified and applied to other phage-bacterial pairs and provides a starting point for studying host-phage dynamics in vivo.

Single-strain behavior predicts responses to environmental pH and osmolality in the gut microbiota.

Changes to gut environmental factors such as pH and osmolality due to disease or drugs correlate with major shifts in microbiome composition; however, we currently cannot predict which species can tolerate such changes or how the community will be affected. Here, we assessed the growth of 92 representative human gut bacterial strains spanning 28 families across multiple pH values and osmolalities in vitro. The ability to grow in extreme pH or osmolality conditions correlated with the availability of known stress response genes in many cases, but not all, indicating that novel pathways may participate in protecting against acid or osmotic stresses. Machine learning analysis uncovered genes or subsystems that are predictive of differential tolerance in either acid or osmotic stress. For osmotic stress, we corroborated the increased abundance of these genes in vivo during osmotic perturbation. The growth of specific taxa in limiting conditions in isolation in vitro correlated with survival in complex communities in vitro and in an in vivo mouse model of diet-induced intestinal acidification. Our data show that in vitro stress tolerance results are generalizable and that physical parameters may supersede interspecies interactions in determining the relative abundance of community members. This study provides insight into the ability of the microbiota to respond to common perturbations that may be encountered in the gut and provides a list of genes that correlate with increased ability to survive in these conditions. IMPORTANCE To achieve greater predictability in microbiota studies, it is crucial to consider physical environmental factors such as pH and particle concentration, as they play a pivotal role in influencing bacterial function and survival. For example, pH is significantly altered in various diseases, including cancers, inflammatory bowel disease, as well in the case of over-the-counter drug use. Additionally, conditions like malabsorption can affect particle concentration. In our study, we investigate how changes in environmental pH and osmolality can serve as predictive indicators of bacterial growth and abundance. Our research provides a comprehensive resource for anticipating shifts in microbial composition and gene abundance during complex perturbations. Moreover, our findings underscore the significance of the physical environment as a major driver of bacterial composition. Finally, this work emphasizes the necessity of incorporating physical measurements into animal and clinical studies to better understand the factors influencing shifts in microbiota abundance.

Release of cell wall phenolic esters during hydrothermal pretreatment of rice husk and rice straw.

BACKGROUND: Rice husk and rice straw represent promising sources of biomass for production of renewable fuels and chemicals. For efficient utilisation, lignocellulosic components must first be pretreated to enable efficient enzymatic saccharification and subsequent fermentation. Existing pretreatments create breakdown products such as sugar-derived furans, and lignin-derived phenolics that inhibit enzymes and fermenting organisms. Alkali pretreatments have also been shown to release significant levels of simple, free phenolics such as ferulic acid that are normally esterified to cell wall polysaccharides in the intact plant. These phenolics have recently been found to have considerable inhibitory properties. The aim of this research has been to establish the extent to which such free phenolic acids are also released during hydrothermal pretreatment of rice straw (RS) and rice husk (RH). RESULTS: RS and RH were subjected to hydrothermal pretreatments over a wide range of severities (1.57-5.45). FTIR analysis showed that the pretreatments hydrolysed and solubilised hemicellulosic moieties, leading to an enrichment of lignin and crystalline cellulose in the insoluble residue. The residues also lost the capacity for UV autofluorescence at pH 7 or pH 10, indicating the breakdown or release of cell wall phenolics. Saponification of raw RS and RH enabled identification and quantification of substantial levels of simple phenolics including ferulic acid (tFA), coumaric acid (pCA) and several diferulic acids (DiFAs) including 8-O-4'-DiFA, 8,5'-DiFA and 5,5'-DiFA. RH had higher levels of pCA and lower levels of tFA and DiFAs compared with RS. Assessment of the pretreatment liquors revealed that pretreatment-liberated phenolics present were not free but remained as phenolic esters (at mM concentrations) that could be readily freed by saponification. Many were lost, presumably through degradation, at the higher severities. CONCLUSION: Differences in lignin, tFA, DiFAs and pCA between RS and RH reflect differences in cell wall physiology, and probably contribute to the higher recalcitrance of RH compared with RS. Hydrothermal pretreatments, unlike alkali pretreatments, release cinnamic acid components as esters. The potential for pretreatment-liberated phenolic esters to be inhibitory to fermenting microorganisms is not known. However, the present study shows that they are found at concentrations that could be significantly inhibitory if released as free forms by enzyme activity.

Optimising conditions for bioethanol production from rice husk and rice straw: effects of pre-treatment on liquor composition and fermentation inhibitors.

BACKGROUND: Rice straw and husk are globally significant sources of cellulose-rich biomass and there is great interest in converting them to bioethanol. However, rice husk is reportedly much more recalcitrant than rice straw and produces larger quantities of fermentation inhibitors. The aim of this study was to explore the underlying differences between rice straw and rice husk with reference to the composition of the pre-treatment liquors and their impacts on saccharification and fermentation. This has been carried out by developing quantitative NMR screening methods. RESULTS: Air-dried rice husk and rice straw from the same cultivar were used as substrates. Carbohydrate compositions were similar, whereas lignin contents differed significantly (husk: 35.3% w/w of raw material; straw 22.1% w/w of raw material). Substrates were hydrothermally pre-treated with high-pressure microwave processing across a wide range of severities. 25 compounds were identified from the liquors of both pre-treated rice husk and rice straw. However, the quantities of compounds differed between the two substrates. Fermentation inhibitors such as 5-HMF and 2-FA were highest in husk liquors, and formic acid was higher in straw liquors. At a pre-treatment severity of 3.65, twice as much ethanol was produced from rice straw (14.22% dry weight of substrate) compared with the yield from rice husk (7.55% dry weight of substrate). Above severities of 5, fermentation was inhibited in both straw and husk. In addition to inhibitors, high levels of cellulase-inhibiting xylo-oligomers and xylose were found and at much higher concentrations in rice husk liquor. At low severities, organic acids and related intracellular metabolites were released into the liquor. CONCLUSIONS: Rice husk recalcitrance to saccharification is probably due to the much higher levels of lignin and, from other studies, likely high levels of silica. Therefore, if highly polluting chemical pre-treatments and multi-step biorefining processes are to be avoided, rice husk may need to be improved through selective breeding strategies, although more careful control of pre-treatment may be sufficient to reduce the levels of fermentation inhibitors, e.g. through steam explosion-induced volatilisation. For rice straw, pre-treating at severities of between 3.65 and 4.25 would give a glucose yield of between 37.5 and 40% (w/DW, dry weight of the substrate) close to the theoretical yield of 44.1% w/DW, and an insignificant yield of total inhibitors.

Variation across a wheat genetic diversity panel for saccharification of hydrothermally pretreated straw.

BACKGROUND: Wheat straw forms an important, reliable source of lignocellulosic biomass for use in second-generation ethanol production. However, there is limited understanding of the variation in quality of straw from current breeding cultivars, and studies on such variation have generally employed suboptimal pretreatments. There is also a degree of confusion regarding phenotypic characteristics relevant to optimising the enzymatic saccharification of cellulose after suitable pretreatments for biorefining compared with those which determine good ruminant digestibility. The aim of this study has been to (a) evaluate and compare the levels of glucose enzymatically released from straw obtained from 89 cultivars of winter wheat after optimised hydrothermal pretreatments and (b) identify the underlying phenotypic characteristics relevant to enhanced glucose production with special reference to the ratios of constituent tissue types. RESULTS: Optimised pretreatment involved hydrothermal extraction at 210 °C for 10 min. Using excess cellulases, quantitative saccharification was achieved within 24 h. The amount of glucose released ranged from 192 to 275 mg/g. The extent of glucose release was correlated with (a) the level of internode tissue (R = 0.498; p = 6.84 × 10-7), (b) stem height (R = 0.491; p = 1.03 × 10-6), and (c) chemical characteristics particular to stem tissues including higher levels of cellulose (R = 0.552; p = 2.06 × 10-8) and higher levels of lignin R = 0.494; p = 8.67 × 10-7. CONCLUSIONS: In order to achieve maximum yields of cellulosic glucose for second-generation ethanol production, a predisposition for wheat to produce cellulose-enriched internode stem tissue, particularly of longer length, would be beneficial. This contrasts with the ideotype for ruminant nutrition, in which an increased proportion of leaf tissue is preferable.

Yeast diversity in relation to the production of fuels and chemicals.

In addition to ethanol, yeasts have the potential to produce many other industrially-relevant chemicals from numerous different carbon sources. However there remains a paucity of information about overall capability across the yeast family tree. Here, 11 diverse species of yeasts with genetic backgrounds representative of different branches of the family tree were investigated. They were compared for their abilities to grow on a range of sugar carbon sources, to produce potential platform chemicals from such substrates and to ferment hydrothermally pretreated rice straw under simultaneous saccharification and fermentation conditions. The yeasts differed considerably in their metabolic capabilities and production of ethanol. A number could produce significant amounts of ethyl acetate, arabinitol, glycerol and acetate in addition to ethanol, including from hitherto unreported carbon sources. They also demonstrated widely differing efficiencies in the fermentation of sugars derived from pre-treated rice straw biomass and differential sensitivities to fermentation inhibitors. A new catabolic property of Rhodotorula mucilaginosa (NCYC 65) was discovered in which sugar substrate is cleaved but the products are not metabolised. We propose that engineering this and some of the other properties discovered in this study and transferring such properties to conventional industrial yeast strains could greatly expand their biotechnological utility.

Comparison of saccharification and fermentation of steam exploded rice straw and rice husk.

BACKGROUND: Rice cultivation produces two waste streams, straw and husk, which could be exploited more effectively. Chemical pretreatment studies using rice residues have largely focussed on straw exploitation alone, and often at low substrate concentrations. Moreover, it is currently not known how rice husk, the more recalcitrant residue, responds to steam explosion without the addition of chemicals. RESULTS: The aim of this study has been to systematically compare the effects of steam explosion severity on the enzymatic saccharification and simultaneous saccharification and fermentation of rice straw and husk produced from a variety widely grown in Vietnam (Oryza sativa, cv. KhangDan18). Rice straw and husk were steam exploded (180-230 °C for 10 min) into hot water and washed to remove fermentation inhibitors. In both cases, pretreatment at 210 °C and above removed most of the noncellulosic sugars. Prolonged saccharification at high cellulase doses showed that rice straw could be saccharified most effectively after steam explosion at 210 °C for 10 min. In contrast, rice husk required more severe pretreatment conditions (220 °C for 10 min), and achieved a much lower yield (75 %), even at optimal conditions. Rice husk also required a higher cellulase dose for optimal saccharification (10 instead of 6 FPU/g DM). Hemicellulase addition failed to improve saccharification. Small pilot scale saccharification at 20 % (w/v) substrate loading in a 10 L high torque bioreactor resulted in similarly high glucose yields for straw (reaching 9 % w/v), but much less for husk. Simultaneous saccharification and fermentation under optimal pretreatment and saccharification conditions showed similar trends, but the ethanol yield from the rice husk was less than 40 % of the theoretical yield. CONCLUSIONS: Despite having similar carbohydrate compositions, pretreated rice husk is much less amenable to saccharification than pretreated rice straw. This is likely to attenuate its use as a biorefinery feedstock unless improvements can be made either in the feedstock through breeding and/or modern biotechnology, or in the pretreatment through the employment of improved or alternative technologies. Physiological differences in the overall chemistry or structure may provide clues to the nature of lignocellulosic recalcitrance.

Effect of steam explosion on waste copier paper alone and in a mixed lignocellulosic substrate on saccharification and fermentation.

This study evaluated steam (SE) explosion on the saccharification and simultaneous saccharification and fermentation (SSF) of waste copier paper. SE resulted in a colouration, a reduction in fibre thickness and increased water absorption. Changes in chemical composition were evident at severities greater than 4.24 resulting in a loss of xylose and the production of breakdown products known to inhibit fermentation (particularly formic acid and acetic acid). SE did not improve final yields of glucose or ethanol, and at severities 4.53 and 4.83 reduced yields probably due to the effect of breakdown products and fermentation inhibitors. However, at moderate severities of 3.6 and 3.9 there was an increase in initial rates of hydrolysis which may provide a basis for reducing processing times. Co-steam explosion of waste copier paper and wheat straw attenuated the production of breakdown products, and may also provide a basis for improving SSF of lignocellulose.

Characterization of cell wall components of wheat bran following hydrothermal pretreatment and fractionation.

BACKGROUND: Pretreatments are a prerequisite for enzymatic hydrolysis of biomass and production of ethanol. They are considered to open up the plant cell wall structure by altering, moving or solubilizing lignin and hydrolyzing a proportion of hemicellulosic moieties. However, there is little information concerning pretreatment-induced changes on wheat bran cell wall polymers and indeed on changes in cell wall phenolic esters in bran or other lignocellulosic biomass. Here, we evaluate polymeric changes (chemical and physical) as a result of selected hydrothermal pretreatment conditions on destarched wheat bran using controlled polymer extraction methods. Quantification of cell wall components together with soluble oligosaccharides, the insoluble residues and ease of extractability and fractionation of biomass residues were conducted. RESULTS: Pretreatment solubilized selected arabinoxylans and associated cross-linking ferulic and diferulic acids with a concomitant increase in lignin and cellulosic glucose. The remaining insoluble arabinoxylans were more readily extractable in alkali and showed considerable depolymerization. The degree of arabinose substitution was less in xylans released by higher concentrations of alkali. The recalcitrant biomass which remained after pretreatment and alkali extraction contained mostly cellulosic glucose and Klason lignin. Pretreatment generated small but insignificant amounts of yeast-inhibiting compounds such as furfural and hydroxymethyl furfural. As such, simultaneous saccharification and fermentation of the hydrothermally pretreated bran resulted in increased ethanol yields compared to that of the control (97.5% compared to 63% theoretical). CONCLUSION: Hydrothermal pretreatment of destarched wheat bran resulted in degradation and depolymerization of the hemicellulosic arabinoxylans together with some breakdown of cellulosic glucose. This was accompanied by a significant reduction in the cross-linking phenolic acids such as ferulic and diferulic acids. The results suggest that hydrothermal pretreatment enhances enzymatic digestibility of the cellulose not only by depolymerization and solubilization of the hemicelluloses but by breakdown of interpolymeric phenolic cross-links between the remaining insoluble polymers. This allows easier access of hydrolytic enzymes by opening or loosening of the cell wall thus resulting in enhanced saccharification of cellulose and subsequent fermentation to ethanol. The reduction in cinnamic acids by selected breeding or biotechnological approaches could provide a useful basis for improved saccharification and fractionation of wheat bran polysaccharides.

Biorefining of waste paper biomass: increasing the concentration of glucose by optimising enzymatic hydrolysis.

Waste copier paper is a potential substrate for the production of glucose relevant for manufacture of platform chemicals and intermediates, being composed of 51 % glucan. The yield and concentration of glucose arising from the enzymatic saccharification of solid ink-free copier paper as cellulosic substrate was studied using a range of commercial cellulase preparations. The results show that in all cellulase preparations examined, maximum hydrolysis was only achieved with the addition of beta-glucosidase, despite its presence in the enzyme mixtures. With the use of Accellerase® (cellulase), high substrate loading decreased conversion yield. However, this was overcome if the enzyme was added between 12.5 and 20 FPU g substrate(-1). Furthermore, this reaction condition facilitated continual stirring and enabled sequential additions (up to 50 % w/v) of paper to be made to the hydrolysis reaction, degrading nearly all (99 %) of the cellulose fibres and increasing the final concentration of glucose whilst simultaneously making high substrate concentrations achievable. Under optimal conditions (50 °C, pH 5.0, 72 h), digestions facilitate the production of glucose to much improved concentrations of up to 1.33 mol l(-1).

Combined effects of dietary yeast supplementation and methoprene treatment on sexual maturation of Queensland fruit fly.

Yeast hydrolysate supplements promote maturation of many tephritid flies targeted for control using the sterile insect technique (SIT), including Queensland fruit fly (Bactrocera tryoni; 'Q-fly'). Recently, application of the juvenile hormone analogue methoprene has been demonstrated to further promote maturation in some species. We here investigate the separate and combined effects of yeast hydrolysate and methoprene treatment on sexual maturation of sterile male and female Q-flies. Two methods of applying methoprene solution were used; topical application to adults and dipping of pupae. Consistent with previous studies, access to yeast hydrolysate greatly increased maturation of both male and female Q-flies. Maturation was further promoted by methoprene treatment, with similar effects evident for males and females and for both application methods. For flies provided access to yeast hydrolysate supplements, methoprene treatment advanced maturation by approximately 2days. No effects of diet or methoprene treatment were found on timing of copulation or copula duration. Countering the positive effects on sexual maturation, dipping of pupae in methoprene/acetone solution did diminish emergence rates and flight ability indices, and increased rates of wing deformity. Promising results of the present study encourage further investigation of treatment methods that maximise maturation while minimising detrimental effects on other aspects of fly quality.

Automated locomotor activity monitoring as a quality control assay for mass-reared tephritid flies.

BACKGROUND: The Sterile Insect Technique (SIT) requires vast numbers of consistently high quality insects to be produced over long periods. Quality control (QC) procedures are critical to effective SIT, both providing quality assurance and warning of operational deficiencies. We here present a potential new QC assay for mass rearing of Queensland fruit flies (Bactrocera tryoni Froggatt) for SIT; locomotor activity monitoring. We investigated whether automated locomotor activity monitors (LAMs) that simply detect how often a fly passes an infrared sensor in a glass tube might provide similar insights but with much greater economy. RESULTS: Activity levels were generally lower for females than for males, and declined over five days in the monitor for both sexes. Female activity levels were not affected by irradiation, but males irradiated at 60 or 70 Gy had reduced activity levels compared with unirradiated controls. We also found some evidence that mild heat shock of pupae results in adults with reduced activity. CONCLUSION: LAM offers a convenient, effective and economical assay to probe such changes.

Exposure of insect cells to ionising radiation in vivo induces persistent phosphorylation of a H2AX homologue (H2AvB).

The response of eukaryotic cells to ionising radiation (IR)-induced double-strand DNA breaks is highly conserved and involves a DNA repair mechanism characterised by the early phosphorylation of histone protein H2AX (producing the active form γH2AX). Although the expression of an induced γH2AX variant has been detected in Drosophila melanogaster, the expression and radiation response of a γH2AX homologue has not been reported in economically important fruit flies. We use Bactrocera tryoni (Diptera: Tephritidae, Queensland fruit fly or 'Q-fly') to investigate this response with a view to developing molecular assays to detect/quantify exposure of fruit flies to IR and consequent DNA damage. Deep sequencing confirmed the presence of a H2AX homologue that we have termed H2AvB (i.e. variant Bactrocera) and has an identical sequence to a histone reported from the human disease vector Glossina morsitans. A linear dose-response of γH2AvB (0-400 Gy IR) was observed in whole Q-fly pupal lysates 24h post-IR and was detected at doses as low as 20 Gy. γH2AvB signal peaked at ~20min after IR exposure and at 24h post-IR the signal remained elevated but declined significantly by 5 days. Persistent and dose-dependent γH2AvB signal could be detected and quantified either by western blot or by laser scanning cytometry up to 17 days post-IR exposure in histone extracts or isolated nuclei from adult Q-flies (irradiated as pupae). We conclude that IR exposure in Q-fly leads to persistent γH2AvB signals (over a period of days) that can easily be detected by western blot or quantitative immunofluorescence techniques. These approaches have potential as the basis for assays for detection and quantification of prior IR exposure in pest fruit flies.

Spontaneous mutation reveals influence of exopolysaccharide on Lactobacillus johnsonii surface characteristics.

As a competitive exclusion agent, Lactobacillus johnsonii FI9785 has been shown to prevent the colonization of selected pathogenic bacteria from the chicken gastrointestinal tract. During growth of the bacterium a rare but consistent emergence of an altered phenotype was noted, generating smooth colonies in contrast to the wild type rough form. A smooth colony variant was isolated and two-dimensional gel analysis of both strains revealed a protein spot with different migration properties in the two phenotypes. The spot in both gels was identified as a putative tyrosine kinase (EpsC), associated with a predicted exopolysaccharide gene cluster. Sequencing of the epsC gene from the smooth mutant revealed a single substitution (G to A) in the coding strand, resulting in the amino acid change D88N in the corresponding gene product. A native plasmid of L. johnsonii was engineered to produce a novel vector for constitutive expression and this was used to demonstrate that expression of the wild type epsC gene in the smooth mutant produced a reversion to the rough colony phenotype. Both the mutant and epsC complemented strains had increased levels of exopolysaccharides compared to the wild type strain, indicating that the rough phenotype is not solely associated with the quantity of exopolysaccharide. Another gene in the cluster, epsE, that encoded a putative undecaprenyl-phosphate galactosephosphotransferase, was deleted in order to investigate its role in exopolysaccharide biosynthesis. The ΔepsE strain exhibited a large increase in cell aggregation and a reduction in exopolysaccharide content, while plasmid complementation of epsE restored the wild type phenotype. Flow cytometry showed that the wild type and derivative strains exhibited clear differences in their adhesive ability to HT29 monolayers in tissue culture, demonstrating an impact of EPS on surface properties and bacteria-host interactions.

High concentrations of cellulosic ethanol achieved by fed batch semi simultaneous saccharification and fermentation of waste-paper.

A fundamental goal of second generation ethanol production is to increase the ethanol concentration to 10% (v/v) or more to optimise distillation costs. Semi simultaneous saccharification and fermentations (SSSF) were conducted at small pilot scale (5L) utilising fed-batch additions of solid shredded copier paper substrate. Early addition of Accellerase® 1500 at 16 FPU/g substrate and 30 U/g ß-glucosidase followed by substrate only batch addition allowed low final equivalent enzyme concentrations to be achieved (3.7 FPU/g substrate) whilst maintaining digestion. Batch addition resulted in a cumulative substrate concentration equivalent to 65% (w/v). This in turn resulted in the production of high concentrations of ethanol (11.6% v/v). The success of this strategy relied on the capacity of the bioreactor to perform high shear mixing as required. Further research into the timing and number of substrate additions could lead to further improvement in overall yields from the 65.5% attained.

Characterization of cell wall components of wheat straw following hydrothermal pretreatment and fractionation.

Thermophysical pretreatment enhances the enzymatic hydrolysis of lignocellulose. However, its impact on cell wall chemistry is still poorly understood. This paper reports the effects of hydrothermal pretreatment on the degradation and alkali-extractability of wheat straw cell wall polymers. Pretreatment resulted in loss and/or solubilization of arabinoxylans (by 53%), ferulic and diferulic acids which are important cross-linking agents accompanied by concomitant increases in cellulose (up to 43%) and lignin (29%). The remaining water-insoluble hemicelluloses were more readily extractable in alkali and were reduced in molecular weight indicating substantial thermochemical depolymerization. They were also associated with smaller but significant amounts of (cellulose-derived) glucose. The alkali-insoluble residues consisted predominantly of cellulosic glucose and lignin and contained p-coumaric acid. The depolymerization of hemicelluloses, reduction in cinnamic acids and partial degradation of cellulose is likely to contribute significantly to the accessibility of cellulases during subsequent enzymolysis.

A role for copula duration in fertility of Queensland fruit fly females mated by irradiated and unirradiated males.

Females of many tephritid fruit flies can mate more than once, and can store ejaculates from multiple males. As well as being an important element of reproductive biology, multiple mating by females is of particular relevance for sterile insect technique programs used to control major tephritid pests. Here we investigate the consequences of multiple mating on fertility of Queensland fruit fly (Bactrocera tryoni) females sequentially mated to irradiated ('sterile') and unirradiated ('normal') males. Females mated by two normal males showed persistent high fertility whereas females mated by two sterile males showed persistent low fertility. Despite lack of association between copula duration and sperm number, fertility of females mated to a normal and then a sterile male increased with duration of the first copulation and decreased with duration of the second. Fertility of females mated to a sterile male and then a normal male was not influenced by duration of the first copulation but increased with duration of the second. These findings reveal a need for increased attention to how factors other than sperm number influence post-copulatory sexual selection in tephritid flies, and in particular how copula duration is linked to sperm storage and usage.

Enzymatic and chemical treatment limits on the controlled solubilization of brewers' spent grain.

The enzymatic hydrolysis of brewers' spent grain (BSG) has been investigated through treatment with commercial carbohydrases and proteases. Resultant residues were then chemically fractionated and delignified. Enzymatic treatments released 25-30% of the BSG mass and yielded precursors suitable for subsequent conversion to potentially value-added products. Controlled chemical fractionation selectively solubilized arabinoxylan but with no differences apparent due to prior enzyme treatment. The loss of non-polysaccharide components during alkali treatment suggests the presence of a high proportion of alkali-soluble lignin. Further delignification of the alkali-insoluble residues and further chemical fractionation released the remaining hemicellulose, to yield a residue which was >90% cellulose. Further knowledge of the properties and interaction between BSG polymers will facilitate an improved enzyme-assisted total deconstruction of BSG and hence the exploitation of its biomass.