Bio-processing of macroalgae Palmaria palmata: metabolite fractionation from pressed fresh material and ensiling considerations for long-term storage.

Red algae, belonging to the phylum Rhodophyta, contain an abundance of useful chemicals including bioactive molecules and present opportunities for the production of different products through biorefinery cascades. The rhodophyte Palmaria palmata, commonly termed dulse or dillisk, grows predominantly on the northern coasts of the Atlantic and Pacific Oceans and is a well-known snack food. Due to its abundance, availability and cultivation capacity, P. palmata was selected for study as a potential candidate for a biorefinery process. In addition to studying juice and solid fractions of freshly harvested P. palmata, we have investigated the novel possibility of preserving algal biomass by ensilaging protocols similar to those employed for terrestrial forage crops. In the metabolite partitioning within the solid and liquid fractions following screw-pressing, the majority of the metabolites screened for-water soluble carbohydrates, proteins and amino acids, lipids, pigments, phenolics and antioxidant activity-remained in the solid fraction, though at differing proportions depending on the metabolite, from 70.8% soluble amino acids to 98.2% chlorophyll a and 98.1% total carotenoids. For the ensiling study, screw-pressed P. palmata, with comparative wilted and chopped, and chopped only samples, were ensiled at scale with and without Safesil silage additive. All samples were successfully ensiled after 90 days, with screw-pressing giving lower or equal pH before and after ensiling compared with the other preparations. Of particular note was the effluent volumes generated during ensiling: 26-49% of the fresh weight, containing 16-34% of the silage dry matter. This may be of advantage depending on the final use of the biomass.

Biological processing in oscillatory baffled reactors: operation, advantages and potential.

The development of efficient and commercially viable bioprocesses is essential for reducing the need for fossil-derived products. Increasingly, pharmaceuticals, fuel, health products and precursor compounds for plastics are being synthesized using bioprocessing routes as opposed to more traditional chemical technologies. Production vessels or reactors are required for synthesis of crude product before downstream processing for extraction and purification. Reactors are operated either in discrete batches or, preferably, continuously in order to reduce waste, cost and energy. This review describes the oscillatory baffled reactor (OBR), which, generally, has a niche application in performing 'long' processes in plug flow conditions, and so should be suitable for various bioprocesses. We report findings to suggest that OBRs could increase reaction rates for specific bioprocesses owing to low shear, good global mixing and enhanced mass transfer compared with conventional reactors. By maintaining geometrical and dynamic conditions, the technology has been proved to be easily scaled up and operated continuously, allowing laboratory-scale results to be easily transferred to industrial-sized processes. This is the first comprehensive review of bioprocessing using OBRs. The barriers facing industrial adoption of the technology are discussed alongside some suggested strategies to overcome these barriers. OBR technology could prove to be a major aid in the development of commercially viable and sustainable bioprocesses, essential for moving towards a greener future.

Copper(II)-mediated thermolysis of alginates: a model kinetic study on the influence of metal ions in the thermochemical processing of macroalgae.

Thermochemical processing methods such as pyrolysis are of growing interest as a means of converting biomass into fuels and commodity chemicals in a sustainable manner. Macroalgae, or seaweed, represent a novel class of feedstock for pyrolysis that, owing to the nature of the environments in which they grow coupled with their biochemistry, naturally possess high metal contents. Although the impact of metals upon the pyrolysis of terrestrial biomass is well documented, their influence on the thermochemical conversion of marine-derived feeds is largely unknown. Furthermore, these effects are inherently difficult to study, owing to the heterogeneous character of natural seaweed samples. The work described in this paper uses copper(II) alginate, together with alginic acid and sodium alginate as model compounds for exploring the effects of metals upon macroalgae thermolysis. A thermogravimetric analysis-Fourier transform infrared spectroscopic study revealed that, unusually, Cu(2+) ions promote the onset of pyrolysis in the alginate polymer, with copper(II) alginate initiating rapid devolatilization at 143°C, 14°C lower than alginic acid and 61°C below the equivalent point for sodium alginate. Moreover, this effect was mirrored in a sample of wild Laminaria digitata that had been doped with Cu(2+) ions prior to pyrolysis, thus validating the use of alginates as model compounds with which to study the thermolysis of macroalgae. These observations indicate the varying impact of different metal species on thermochemical behaviour of seaweeds and offer an insight into the pyrolysis of brown macroalgae used in phytoremediation of metal-containing waste streams.

Successional colonization of perennial ryegrass by rumen bacteria.

UNLABELLED: This study investigated successional colonization of perennial ryegrass (PRG) by the rumen microbiota. PRG grown for 6 weeks in a greenhouse was incubated in sacco in the rumens of three Holstein × Freisian cows over a period of 24 h. PRG incubated within the rumen was subsequently harvested at various time intervals postincubation to assess colonization over time. DGGE-based dendograms revealed the presence of distinct primary (0-2 h) and secondary (4 h onwards) attached bacterial communities. Moving window analysis, band number and Shannon-Wiener diversity indices suggest that after 2 h a proportion of primary colonizing bacteria detach, to be replaced with a population of secondary colonizing bacteria between 2 and 4 h after entry of PRG into the rumen. Sequencing and classification of bands lost and gained between 2 and 4 h showed that the genus Prevotella spp. was potentially more prevalent following 4 h of incubation, and Prevotella spp. 16S rDNA-based QPCR supported this finding somewhat, as 2- to 4-h Prevotella QPCR data were greater but not significantly so. Low-temperature scanning electron microscopy showed that attached bacteria were predominantly enveloped in extracellular polymeric substances. In conclusion, colonization of fresh PRG is biphasic with primary colonization completed within 2 h and secondary colonization commencing after 4 h of attachment in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: We investigated, over a 24-h period in sacco, whether attachment of rumen microbiota to perennial ryegrass (PRG) showed successional changes in diversity. Knowledge of the bacterial species that attach to PRG over time may aid our understanding of the temporal function of the attached microbiota and ultimately permit the development of novel strategies for improving animal production to meet the future demands for meat and milk.

Detection and monitoring of anaerobic rumen fungi using an ARISA method.

AIM: To develop an automated ribosomal intergenic spacer region analysis (ARISA) method for the detection of anaerobic rumen fungi and also to demonstrate utility of the technique to monitor colonization and persistence of fungi, and diet-induced changes in community structure. METHODS AND RESULTS: The method could discriminate between three genera of anaerobic rumen fungal isolates, representing Orpinomyces, Piromyces and Neocallimastix species. Changes in anaerobic fungal composition were observed between animals fed a high-fibre diet compared with a grain-based diet. ARISA analysis of rumen samples from animals on grain showed a decrease in fungal diversity with a dominance of Orpinomyces and Piromyces spp. Clustering analysis of ARISA profile patterns grouped animals based on diet. A single strain of Orpinomyces was dosed into a cow and was detectable within the rumen fungal population for several weeks afterwards. CONCLUSIONS: The ARISA technique was capable of discriminating between pure cultures at the genus level. Diet composition has a significant influence on the diversity of anaerobic fungi in the rumen and the method can be used to monitor introduced strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the use of ARISA analysis, a better understanding of the effect of diets on rumen anaerobic fungi populations is provided.

Effects of high-sugar ryegrass silage and mixtures with red clover silage on ruminant digestion. 1. In vitro and in vivo studies of nitrogen utilization.

Two experiments were carried out to determine the effects of feeding grass silages differing in their water-soluble carbohydrate content, with or without red clover silage, on the efficiency of nutrient use. High-sugar grass, control grass, and red clover were ensiled in laboratory silos for use in an in vitro experiment (Exp. 1). For an in vivo experiment (Exp. 2), the same forage types were baled and ensiled. All silages were well preserved; within experiments the grass silages had similar composition, except for greater (P < 0.05) water-soluble carbohydrate concentrations in the high-sugar than the control grass silage. In Exp. 1, high-sugar grass, control grass, and red clover silages were fed alone or as mixtures (30:70, 50:50, or 70:30 on a DM basis, respectively) of each grass with the red clover silage to a simulated rumen culture system. There were no significant differences in microbial N flow or efficiency of microbial protein synthesis between individual forages. However, the corresponding values for the 70:30 ratio of high-sugar grass:red clover silage were greater (P < 0.05) than for the red clover silage. The value for the efficiency of N use (g of microbial N/g of feed N) was greater (0.86; P < 0.05) for high-sugar grass silage than the control grass silage. In addition, the high-sugar grass:red clover silage mixtures all gave greater (P < 0.05) values for the efficiency of N use than red clover silage alone; this difference was not achieved with the control grass mixture. Experiment 2 was an incomplete Latin square design conducted with 6 Here-ford x Friesian steers (163 +/- 5.9 kg of BW) with rumen and duodenal cannulas fed the following 5 silage diets: high-sugar grass silage; control grass silage; high-sugar grass and red clover silage (50:50 DM basis); control grass and red clover silage (50:50 DM basis); and red clover silage. Rumen NH3-N concentration was lowest (P < 0.05) with the high-sugar grass silage. Microbial N flows to the duodenum and efficiency of microbial protein synthesis were greater (P < 0.05) for steers fed the high-sugar grass silage than for control grass and red clover silages, and mixing red clover with grass silages increased (P < 0.05) these values compared with red clover silage alone. In both experiments, the efficiency of incorporation of silage N into microbial N was more than 20% greater (P < 0.05) for high-sugar grass than for control grass silage. These data suggest that grass silage with high-sugar content provides a forage-based strategy for balancing N and energy supply and improving the efficiency of use of grass silage N in the rumen.

Evidence in support of a role for plant-mediated proteolysis in the rumens of grazing animals.

The present work aimed to differentiate between proteolytic activities of plants and micro-organisms during the incubation of grass in cattle rumens. Freshly cut ryegrass was placed in bags of varying permeability and incubated for 16 h in the rumens of dairy cows that had previously grazed a ryegrass sward, supplemented with 4 kg dairy concentrate daily. Woven polyester bags (50 microm pore size) permitted direct access of the micro-organisms and rumen fluid enzymes to the plant material. The polythene was impermeable even to small molecules such as NH(3). Dialysis tubing excluded micro-organisms and rumen enzymes/metabolites larger than 10 kDa. DM loss was 46.3 % in polyester, 36.2 % in polythene and 38.1 % in dialysis treatments. It is possible that the DM loss within polythene bags occurred due to a solubilisation of plant constituents (e.g. water-soluble carbohydrates) rather than microbial attachment/degradation processes. The final protein content of the herbage residues was not significantly different between treatments. Regardless of bag permeability, over 97 % of the initial protein content was lost during incubations in situ. Electrophoretic separation showed that Rubisco was extensively degraded in herbage residues whereas the membrane-associated, light-harvesting protein remained relatively undegraded. Protease activity was detected in herbage residues and bathing liquids after all incubation in situ treatments. Although rumen fluid contains proteases (possibly of plant and microbial origin), our results suggest that, owing to cell compartmentation, their activity against the proteins of intact plant cells is limited, supporting the view that plant proteases are involved in the degradation of proteins in freshly ingested herbage.

Vacuum packing: a model system for laboratory-scale silage fermentations.

AIMS: To determine the utility of vacuum-packed polythene bags as a convenient, flexible and cost-effective alternative to fixed volume glass vessels for lab-scale silage studies. METHODS AND RESULTS: Using perennial ryegrass or red clover forage, similar fermentations (as assessed by pH measurement) occurred in glass tube and vacuum-packed silos over a 35-day period. As vacuum-packing devices allow modification of initial packing density, the effect of four different settings (initial packing densities of 0.397, 0.435, 0.492 and 0.534 g cm(-3)) on the silage fermentation over 16 days was examined. Significant differences in pH decline and lactate accumulation were observed at different vacuum settings. Gas accumulation was apparent within all bags and changes in bag volume with time was observed to vary according to initial packing density. CONCLUSIONS: Vacuum-packed silos do provide a realistic model system for lab-scale silage fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of vacuum-packed silos holds potential for lab-scale evaluations of silage fermentations, allowing higher throughput of samples, more consistent packing as well as the possibility of investigating the effects of different initial packing densities and use of different wrapping materials.

Proteolysis and cell death in clover leaves is induced by grazing.

Programmed plant cell death is a widespread phenomenon resulting in the formation of xylem vessels, dissected leaf forms, and aerenchyma. We demonstrate here that some characteristics of programmed cell death can also be observed during the cellular response to biotic and abiotic stress when plant tissue is ingested by grazing ruminants. Furthermore, the onset and progression of plant cell death processes may influence the proteolytic rate in the rumen. This is important because rapid proteolysis of plant proteins in ruminants is a major cause of the inefficient conversion of plant to animal protein resulting in the release of environmental N pollutants. Although rumen proteolysis is widely believed to be mediated by proteases from rumen microorganisms, proteolysis and cell death occurred concurrently in clover leaves incubated in vitro under rumenlike conditions (maintained anaerobically at 39 degrees C) but in the absence of a rumen microbial population. Under rumenlike conditions, both red and white clover cells showed progressive loss of DNA, but this was only associated with fragmentation in white clover. Cell death was indicated by increased ionic leakage and the appearance of terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labelled nuclei. Foliar protein decreased to 50% of the initial values after 3 h incubation in white clover and after 4 h in red clover, while no decrease was observed in ambient (25 degrees C, aerobic) incubations. In white clover, decreased foliar protein coincided with an increased number of protease isoforms.

Plasma metabolites indicate energy metabolism disruption during the preclinical phase of bovine spongiform encephalopathy infection.

During the preclinical phase of bovine spongiform encephalopathy (BSE), significantly increased concentrations of lactic acid were measured in the blood of infected dairy cows. Other plasma metabolites, including alanine, leucine, serine, and glutamic acid, also showed significantly altered concentrations in the preclinical BSE animals compared to a control group. This appears consistent with the exaggerated stress response observed in clinical BSE, and precedes the development of clinical signs and overt behavioural changes. A number of other plasma metabolites including other amino acids and components of the plasma fatty acid content showed no association with BSE status.

Alternative forages--back to the future.

The BSE crisis, restrictions over export of UK beef and bans on animal-derived concentrate feeds are driving agriculture in the UK 'back' towards more sustainable, low input systems for feeding cattle and sheep using 'natural' home-grown forages. Research is in progress to answer some of the questions surrounding the use of protein- and energy-rich alternative forages as feeds for ruminants.

Efficient improvement of silage additives by using genetic algorithms.

The enormous variety of substances which may be added to forage in order to manipulate and improve the ensilage process presents an empirical, combinatorial optimization problem of great complexity. To investigate the utility of genetic algorithms for designing effective silage additive combinations, a series of small-scale proof of principle silage experiments were performed with fresh ryegrass. Having established that significant biochemical changes occur over an ensilage period as short as 2 days, we performed a series of experiments in which we used 50 silage additive combinations (prepared by using eight bacterial and other additives, each of which was added at six different levels, including zero [i.e. , no additive]). The decrease in pH, the increase in lactate concentration, and the free amino acid concentration were measured after 2 days and used to calculate a "fitness" value that indicated the quality of the silage (compared to a control silage made without additives). This analysis also included a "cost" element to account for different total additive levels. In the initial experiment additive levels were selected randomly, but subsequently a genetic algorithm program was used to suggest new additive combinations based on the fitness values determined in the preceding experiments. The result was very efficient selection for silages in which large decreases in pH and high levels of lactate occurred along with low levels of free amino acids. During the series of five experiments, each of which comprised 50 treatments, there was a steady increase in the amount of lactate that accumulated; the best treatment combination was that used in the last experiment, which produced 4.6 times more lactate than the untreated silage. The additive combinations that were found to yield the highest fitness values in the final (fifth) experiment were assessed to determine a range of biochemical and microbiological quality parameters during full-term silage fermentation. We found that these combinations compared favorably both with uninoculated silage and with a commercial silage additive. The evolutionary computing methods described here are a convenient and efficient approach for designing silage additives.

Identification and characterization of anaerobic gut fungi using molecular methodologies based on ribosomal ITS1 and 185 rRNA.

The gut fungi are an unusual group of zoosporic fungi occupying a unique ecological niche, the anaerobic environment of the rumen. They exhibit two basic forms, with nuclear migration throughout the hyphal mass for polycentric species and with concentration of nuclear material in a zoosporangium for monocentric species. Differentiation between isolates of these fungi is difficult using conventional techniques. In this study, DNA-based methodologies were used to examine the relationships within and between two genera of monocentric gut fungi gathered from various geographical locations and host animals. The ribosomal ITS1 sequence from 16 mono- and 4 polycentric isolates was PCR-amplified and sequenced; the sequences obtained were aligned with published sequences and phylogenetic analyses were performed. These analyses clearly differentiate between the two genera and reflect the previously published physiological conclusions that Neocallimastix spp. constitute a more closely related genus than the relatively divergent genus Piromyces. The analyses place two type species N. frontalis and N. hurleyensis together but, contrary to a recent suggestion in the literature, place them apart from the other agreed species N. patriciarum. In situ hybridization and slot-blotting were investigated as potential methods for detection of and differentiation between monocentric gut fungi. DNA slot-blot analysis using ribosomal sequences is able to differentiate between gut fungal genera and thus has considerable potential for use in ecological studies of these organisms.

Tansley Review No. 118: Post-ingestion metabolism of fresh forage.

It is generally assumed that breakdown of plant material in the rumen is a process mediated by gut microorganisms. This view arose because of the identification of a pre-gastric fermentation in the rumen, brought about by a large and diverse microbial population. The extensive use of dried and ground feed particles in forage evaluation might have helped to promote this assumption. However, although the assumption might be correct in animals feeding on conserved forage (hay and silage) where the cells of ingested forage are dead, it is possible that with grazed (living) forage, the role played by plant enzymes in the rumen has been overlooked. In a grazing situation, plant cells that remain intact on entering the rumen are not inert, but will respond to the perceived stresses of the rumen environment for as long as they are metabolically viable. Metabolic adjustments could include anaerobic and heat-shock responses that could promote premature senescence, leading to remobilization of cell components, especially proteins. Moreover, contact of plant cells with colonizing microorganisms in the rumen might promote a type of hypersensitive response, in much the same way as it does outside the rumen. After fresh plant material enters the rumen and prior to extensive plant cell-wall degradation, there is often a phase of rapid proteolysis providing N in excess of that required to maintain the rumen microbial population. The inefficient use of this ingested N results in generation of ammonia and urea in exhaled breath and urine, which promotes welfare and environmental pollution concerns. Therefore an important research goal in livestock agriculture is to find ways of decreasing this initial rate of proteolysis in the rumen. This will benefit the farmer financially (through decreased use of feed supplements), but will also benefit the environment, as N pollution can adversely affect pasture diversity and ecology. This review considers the possible responses of plant metabolism to the rumen environment, and how such considerations could alter current thinking in ruminant agriculture. Contents Summary 37 I. INTRODUCTION 37 II. DIGESTION OF PLANTS IN THE RUMEN: OLD AND NEW CONCEPTS 39 III. RUMEN-INDUCED PLANT METABOLISM: CELL DEGRADATION AND DEATH 41 IV. FUTURE PROSPECTS 50 Acknowledgements 51 References 51.

Evidence of a role for plant proteases in the degradation of herbage proteins in the rumen of grazing cattle.

Protein breakdown in the rumen is generally regarded as a two-stage process in which proteases produced by rumen microorganisms cleave plant protein into peptides and amino acids. However, many of the fiber-degrading cellulolytic species in the rumen are not in fact proteolytic, and the proteolytic activity of the entire rumen microbial population is only moderate when compared to the gastric and pancreatic secretions in the abomasum. Moreover, plant cell walls remain largely intact after initial chewing (particularly in cattle), presenting a physical barrier that must be breached prior to their effective colonization. The present study considers the hypothesis that the plant enzymes are at least partly responsible for herbage protein degradation in grazing ruminants. Ryegrass, red clover, white clover, and bird's-foot trefoil were incubated in the presence and absence of rumen microorganisms. The production of volatile fatty acids indicated the level of microbial activity, whereas the relative disappearance of the large subunit of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco LSU) indicated proteolytic activity. In all incubations, the relative abundance of the Rubisco LSU decreased as the incubation progressed. When rumen microorganisms were absent, low molecular weight peptides (below 20 kDa) accumulated as the incubation progressed. This accumulation was not observed in the presence of rumen microorganisms. Therefore we suggest that the intrinsic plant proteases contribute to the initial stages of proteolysis of grazed herbage.

Dilution rate increases production of plant cell-wall degrading enzymes by anaerobic fungi in continuous-flow culture.

The gut anaerobic fungi,Neocallimastix hurleyensis and aOrpinomyces sp., were grown in 100 mL batch and continuous-flow cultures on wheat straw at a concentration of 80 g dry matter/L of culture liquid. In batch cultures,N. hurleyensis and Orpinomyces sp. degraded only ca. 9% and 5% of the wheat straw, respectively. In continuous-flow cultures, however, the two fungi degraded 52-56% of the apparent dry matter of wheat straw. Both fungi were able to produce greater quantities (up to x 30) of cell-wall degrading enzymes (CMCase, xylanase, beta-glucosidase and beta-xylosidase) in continuous-flow cultures than in the corresponding batch cultures. Increasing the dilution rate in continuous-flow culture resulted in the production of increased enzyme activity for all the measured cell-wall degrading enzymes, with proportional relationships between dilution rate and the cumulative activities of beta-glucosidase and beta-xylosidase. Dilution rates, however, had no consistent effect on the cumulative production of the fermentation end-products, acetate, formate, D- and L-lactate from both fungi. In addition to acetate and formate,N. hurleyens is produced D- and L-lactate in both batch and continuous-flow cultures, whereas only trace amounts of L-lactate were detected in the Orpinomyces sp. cultures.

Evidence that linker sequences and cellulose-binding domains enhance the activity of hemicellulases against complex substrates.

Xylanase A (XYLA) and arabinofuranosidase C (XYLC) from Pseudomonas fluorescens subsp. cellulosa are modular enzymes consisting of discrete cellulose-binding domains (CBDs) and catalytic domains joined by serine-rich linker sequences. To evaluate the role of the CBDs and interdomain regions, the capacity of full-length and truncated derivatives of the two enzymes, lacking either the linker sequences or CBDs, to hydrolyse a range of substrates, and bind to cellulose, was determined. Removal of the CBDs did not affect either the activity of XYLA or XYLC against soluble arabinoxylan. Similarly, deletion of the linker sequences did not alter the affinity of the enzymes for cellulose or their activity against soluble substrates, even when bound to cellulose via the CBDs. Truncated derivatives of XYLA lacking either the linker sequences or the CBD were less active against xylan contained in cellulose-hemicellulose complexes, compared with the full-length xylanase. Similarly, removal of the CBD from XYLC diminished the activity of the enzyme (XYLC''') against plant-cell-wall material containing highly substituted arabinoxylan. The role of CBDs and linker sequences in the catalytic activity of hemicellulases against the plant cell wall is discussed.