A Global Assessment of the Water Footprint of Farm Animal Products

The increase in the consumption of animal products is likely to put further pressure on the world’s freshwater resources. This paper provides a comprehensive account of the water footprint of animal products, considering different production systems and feed composition per animal type and country. Nearly one-third of the total water footprint of agriculture in the world is related to the production of animal products. The water footprint of any animal product is larger than the water footprint of crop products with equivalent nutritional value. The average water footprint per calorie for beef is 20 times larger than for cereals and starchy roots. The water footprint per gram of protein for milk, eggs and chicken meat is 1.5 times larger than for pulses. The unfavorable feed conversion efficiency for animal products is largely responsible for the relatively large water footprint of animal products compared to the crop products. Animal products from industrial systems generally consume and pollute more ground- and surface-water resources than animal products from grazing or mixed systems. The rising global meat consumption and the intensification of animal production systems will put further pressure on the global freshwater resources in the coming decades. The study shows that from a freshwater perspective, animal products from grazing systems have a smaller blue and grey water footprint than products from industrial systems, and that it is more water-efficient to obtain calories, protein and fat through crop products than animal products.     

Nonnatural substrates for polyketide synthases and their associated modifying enzymes

Polyketides are a large class of structurally diverse, biologically active natural products. Recent experiments add evidence that many of the enzymes involved in the biosynthesis of these natural products are intrinsically tolerant of nonnatural substrates. In addition, an increasing understanding of structure-function relationships in various enzyme-substrate systems is aiding efforts to begin engineering these proteins for even greater synthetic utility.     

Downstream processing of recombinant proteins from transgenic feedstock

The search for inexpensive production systems capable of producing large quantities of recombinant protein has resulted in the development of new technology platforms based on transgenic plants and animals. Over the past decade, these transgenic systems have been used to produce several products and potential therapeutic proteins. Improvements continue to be made, not only in how the proteins are expressed but also in how the end products are obtained. As improvements in expression are realized, cost-saving measures will increasingly focus on downstream processing.     

In vitro translation of infectious flacherie virus RNA in a wheat germ and a rabbit reticulocyte system

Infectious flacherie virus is an insect picornavirus isolated from the silkworm, Bombyx mori. Its RNA was found to act as an efficient mRNA in a wheat germ extract and a rabbit reticulocyte lysate translation system. In either system the sum of molecular weights of translation products far exceeded the coding capacity of the virus genome, which suggests the occurrence of proteolytic cleavage of large primary products to smaller polypeptides as reported for other picornaviruses and/or premature termination of translation. The highest molecular weight product of 200 000 (polyprotein-like product) could be translated in both systems. One of the antigenic products common to both systems had a molecular weight of 130 000, which corresponds to the sum of molecular weights of the four major viral proteins. Another product, which comigrated with viral protein 0, the largest viral structural protein in SDS-polyacrylamide gel electrophoresis, also showed antigenicity. Peptide mapping of these polypeptides showed that the two in vitro systems translated the same cistron in the viral RNA and that the smaller polypeptide was a part of the 130 000 Da product.     

Photobioreactors: production systems for phototrophic microorganisms

Microalgae have a large biotechnological potential for producing valuable substances for the feed, food, cosmetics and pharmacy industries as well as for biotechnological processes. The design of the technical and technological basis for photobioreactors is the most important issue for economic success in the field of phototrophic biotechnology. For future applications, open pond systems for large-scale production seem to have a lower innovative potential than closed systems. For high-value products in particular, closed systems of photobioreactors seem to be the more promising field for technical developments despite very different approaches in design.     

Feasible boundaries of aqueous two-phase systems with NH(3) and CO(2) as recyclable volatile salts

Aqueous two-phase systems (ATPSs) have great potential for use in the downstream processing of fermentation products. A major drawback of these systems, limiting application in industrial practice up till now, is the consumption of large amounts of auxiliary materials such as polymers and salts. Making use of alternative auxiliaries can diminish this relatively large discharge. A possible approach is to make use of volatile salts induced by combinations of ammonia and carbon dioxide that can be recycled to the extraction system. As part of an ongoing research effort on ATPSs with volatile salts, this work aims at getting more information on the system boundaries or operating conditions of these systems in terms of phase behavior. The results show that the NH(3)/CO(2) ratio is an important parameter that has a large influence on the system boundaries. Both for systems with PEG 2000 and PEG 4000, this ratio has to be larger than about 1.75 to make a liquid-liquid phase separation possible. The most optimal ratio seems to be 2.0 for reasons of solution composition and absence of solid salt.     

Growing Phototrophic Cells without Light

Many phototrophic microorganisms contain large quantities of high-value products such as n-3 polyunsaturated fatty acids and carotenoids but phototrophic growth is often slow due to light limitation. Some phototrophic microorganisms can also grow on cheap organic substrate heterotrophically. Heterotrophic cultivation can be well controlled and provides the possibility to achieve fast growth and high yield of valuable products on a large scale. Several strategies have been investigated for cultivation of phototrophic microorganisms without light. These include trophic conversion of obligate photoautotrophic microorganisms by genetic engineering, development of efficient cultivation systems and optimization of culture conditions. This paper reviews recent advances in heterotrophic cultivation of phototrophic cells with an emphasis on microalgae.     

Principles of the design and operational considerations of large scale high performance liquid chromatography (HPLC) systems for proteins and peptides purification.

This review introduces concepts of design of large scale HPLC systems for purification of proteins and peptides. It is addressed to users of large scale HPLC systems to aid in system selection and help in customizing the design. Major techniques used for large scale HPLC purification of proteins and peptides are briefly reviewed. Engineering aspects of system design are discussed in detail. The review of selected relevant literature is provided as well as author's experience with the existing designs and his own systems. Commercial publications have been used in preparation of this review but only the most significant are listed as examples. The design process for any new system should be related to the performance of existing systems, if possible of a large scale. Laboratory data are also very useful in aiding the design process since they provide a lead, as to which chromatography techniques may succeed in providing required separation levels. The expertise needed for system design and operation comes from many areas: protein and peptide chemistry, chromatographic theory, mass transfer and hydrodynamics, machine design and material science. All these factors have to be blended together during the system design process. The controls must ensure flexibility in adapting to changing system configuration, depending on the operational requirements for various products. Extensive interfacing with the operator during the process run is essential. This work is focused mostly on system design and operation for reversed-phase chromatography and hydrophobic interaction chromatography, but it also covers aspects associated with other chromatographic techniques. The reviewed design principles would also apply to design other than HPLC large scale chromatography systems for biotechnology and pharmaceutical operations.     

By-product inhibition effects on ethanolic fermentation by Saccharomyces cerevisiae

Inhibition by secondary fermentation products may limit the ultimate productivity of new glucose to ethanol fermentation processes. New processes are under development whereby ethanol is selectively removed from the fermenting broth to eliminate ethanol inhibition effects. These processes can concentrate minor secondary products to the point where they become toxic to the yeast. Vacuum fermentation selectively concentrates nonvolatile products in the fermentation broth. Membrane fermentation systems may concentrate large molecules which are sterically blocked from membrane transport. Extractive fermentation systems, employing nonpolar solvents, may concentrate small organic acids. By-product production rates and inhibition levels in continuous fermentation with Saccharomyces cerevisiae have been determined for acetaldehyde, glycerol, formic, lactic, and acetic acids, 1-propanol, 2-methyl-1-butanol, and 2,3-butanediol to assess the potential effects of these by-products on new fermentation processes. Mechanisms are proposed for the various inhibition effects observed.     

Oxygen solubilities in fermentation fluids

Summary Fermentation media consist of a large number of chemicals whose composition undergoes alteration during the course of fermentation. As a result of this, conventional methods and correlations for oxygen solubility measurement and prediction do not apply in these systems. Using a physical method, oxygen solubilities were measured in simulated chemical systems and in fermentation broths. Sugars, salts, and fermentation products were identified as major factors influencing oxygen solubility. Salt effect was correlated with electrical conductivity of the medium, which was easy to measure during fermentation. For mixtures and for fermentation medium, individual influences were found to be log-additive in accordance with Danckwerts (1970).     

Three-phase extractive fermentation

Fermentation with simultaneous extraction of a product inside the fermentor is an attractive alternative where substrates and/or products otherwise inhibit processes. Although a large number of systems are being tested, the selection of solvent and the technology and design of the process have nevertheless to be improved.     

Assessment of yield gaps on global grazed‐only permanent pasture using climate binning

To meet rising demands for agricultural products, existing agricultural lands must either produce more or expand in area. Yield gaps (YGs)—the difference between current and potential yield of agricultural systems—indicate the ability to increase output while holding land area constant. Here, we assess YGs in global grazed‐only permanent pasture lands using a climate binning approach. We create a snapshot of circa 2000 empirical yields for meat and milk production from cattle, sheep, and goats by sorting pastures into climate bins defined by total annual precipitation and growing degree‐days. We then estimate YGs from intra‐bin yield comparisons. We evaluate YG patterns across three FAO definitions of grazed livestock agroecosystems (arid, humid, and temperate), and groups of animal production systems that vary in animal types and animal products. For all subcategories of grazed‐only permanent pasture assessed, we find potential to increase productivity several‐fold over current levels. However, because productivity of grazed pasture systems is generally low, even large relative increases in yield translated to small absolute gains in global protein production. In our dataset, milk‐focused production systems were found to be seven times as productive as meat‐focused production systems regardless of animal type, while cattle were four times as productive as sheep and goats regardless of animal output type. Sustainable intensification of pasture is most promising for local development, where large relative increases in production can substantially increase incomes or “spare” large amounts of land for other uses. Our results motivate the need for further studies to target agroecological and economic limitations on productivity to improve YG estimates and identify sustainable pathways toward intensification.     

Commercial production of microalgae: ponds, tanks, tubes and fermenters

The commercial culture of microalgae is now over 30 years old with the main microalgal species grown being Chlorella and Spirulina for health food, Dunaliella salina for β-carotene, Haematococcus pluvialis for astaxanthin and several species for aquaculture. The culture systems currently used to grow these algae are generally fairly unsophisticated. For example, Dunaliella salina is cultured in large (up to approx. 250 ha) shallow open-air ponds with no artificial mixing. Similarly, Chlorella and Spirulina also are grown outdoors in either paddle-wheel mixed ponds or circular ponds with a rotating mixing arm of up to about 1 ha in area per pond. The production of microalgae for aquaculture is generally on a much smaller scale, and in many cases is carried out indoors in 20–40 l carboys or in large plastic bags of up to approximately 1000 l in volume. More recently, a helical tubular photobioreactor system, the BIOCOIL™, has been developed which allows these algae to be grown reliably outdoors at high cell densities in semi-continuous culture. Other closed photobioreactors such as flat panels are also being developed. The main problem facing the commercialisation of new microalgae and microalgal products is the need for closed culture systems and the fact that these are very capital intensive. The high cost of microalgal culture systems relates to the need for light and the relatively slow growth rate of the algae. Although this problem has been avoided in some instances by growing the algae heterotrophically, not all algae or algal products can be produced this way.     

A Review of the Advancements in Probiotic Delivery: Conventional vs. Non-conventional Formulations for Intestinal Flora Supplementation

Probiotic delivery systems are widely used nutraceutical products for the supplementation of natural intestinal flora. These delivery systems vary greatly in effectiveness to exert health benefits for a patient. Probiotic delivery systems can be categorized into conventional, pharmaceutical formulations, and non-conventional, mainly commercial food-based, products. The degree of health benefits provided by these probiotic formulations varies in their ability to deliver viable, functional bacteria in large enough numbers (effectiveness), to provide protection against the harsh effects of the gastric environment and intestinal bile (in vivo protection), and to survive formulation processes (viability). This review discusses the effectiveness of these probiotic delivery systems to deliver viable functional bacteria focusing on the ability to protect the encapsulated probiotics during formulation process as well as against harsh physiological conditions through formulation enhancements using coatings and polymer enhancements. A brief overview on the health benefits of probiotics, current formulation, patient and legal issues facing probiotic delivery, and possible recommendations for the enhanced delivery of probiotic bacteria are also provided. Newer advanced in vitro analyses that can accurately determine the effectiveness of a probiotic formulation are also discussed with an ideal probiotic delivery system hypothesized through a combination of the two probiotic delivery systems described.     

Schistosoma mansoni: patter of release of secretory products.

Adult Schistosoma mansoni were maintained in vitro for 1 hr with radioactively labeled precursors of protein, glycoprotein, and polysaccharides. The worms were then washed extensively and the supernates analyzed. The precursors N-acetylglucosamine, N-acetylgalactosamine, glucosamine, galactosamine, glucose, leucine, and fucose were incorporated into the worms and both large and small molecular weight products accumulated in the supernatant. For all the precursors except fucose, there was an initial rapid and then slower phase of release for both the large and small molecular weight materials. The amount of label retained by the worms as well as the proportion excreted as large molecular weight material was characteristic for the precursor used. In contrast, the products of fucose were released within 4 to 6 hr and therefore only exhibited the early secretory phase. There was no retention of fucose by the worms. Hydrolysis of large molecular weight products revealed that the N-acetylglucosamine-derived material was incorporated as amino sugars and fucose was incorporated as fucose. Therefore, N-acetylglucosamine and fucose precursors can specifically label secretory glycoproteins of schistosomes in a manner similar to that in mammalian systems.     

Transfer of metabolites across the peroxisomal membrane

Peroxisomes perform a large variety of metabolic functions that require a constant flow of metabolites across the membranes of these organelles. Over the last few years it has become clear that the transport machinery of the peroxisomal membrane is a unique biological entity since it includes nonselective channels conducting small solutes side by side with transporters for 'bulky' solutes such as ATP. Electrophysiological experiments revealed several channel-forming activities in preparations of plant, mammalian, and yeast peroxisomes and in glycosomes of Trypanosoma brucei. The properties of the first discovered peroxisomal membrane channel - mammalian Pxmp2 protein - have also been characterized. The channels are apparently involved in the formation of peroxisomal shuttle systems and in the transmembrane transfer of various water-soluble metabolites including products of peroxisomal β-oxidation. These products are processed by a large set of peroxisomal enzymes including carnitine acyltransferases, enzymes involved in the synthesis of ketone bodies, thioesterases, and others. This review discusses recent data pertaining to solute permeability and metabolite transport systems in peroxisomal membranes and also addresses mechanisms responsible for the transfer of ATP and cofactors such as an ATP transporter and nudix hydrolases. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.     

Genetic approaches to the identification of interactions between membrane proteins in yeast

The recent sequencing of entire eukaryotic genomes has renewed the interest in identifying and characterizing all gene products that are expressed in a given organism. The characterization of unknown gene products is facilitated by the knowledge of its binding partners. Thus, a novel protein may be classified by identifying previously characterized proteins that interact with it. If such an approach is carried out on a large scale, it may allow the rapid characterization of the thousands of predicted open reading frames identified by recent sequencing projects. Currently, the yeast two-hybrid system is the most widely used genetic assay for the detection of protein-protein interactions. The yeast two-hybrid system has become popular because it requires little individual optimization and because, as compared to conventional biochemical methods, the identification and characterization of protein-protein interactions can be completed in a relatively short time span. In this review, we briefly discuss the yeast two-hybrid system and its application to large scale screening studies that aim at deciphering all protein-protein interactions taking place in a given cell type or organism. We then focus on a class of proteins that is unsuitable for conventional yeast two-hybrid systems, namely integral membrane proteins and membrane-associated proteins, and describe several novel genetic systems that combine the advantages of the yeast two-hybrid system with the potential to identify interaction partners of membrane-associated proteins in their natural setting.     

Seeing tissue as a 'phase of matter': exploring statistical mechanics for the cell

The field of tissue engineering aims to produce living, biological constructs which possess the appropriate spatial ordering of cells and their extra cellular matrix products. The complexity of a single cell and its interactions in a large collective have made development of useful models to assist in tissue culture difficult, and consequentially most tissue culture endeavors are limited to trial and error approaches. Some cell types display a natural tendency to spontaneously self-assemble into large domains of parallel-oriented cells. In this work, we show that these cell culture systems can be studied in the context of continuous disorder-order phase transformations. We suggest that collective ordering of the cells is controlled by the amount of noise in the walk of the individual cells (directional persistence) because undifferentiated mesenchymal stem cells display a seven-times higher directional persistence than mature fibroblasts and have a 24-times larger final-oriented domain size, an observation that corresponds with collective ordering in self-propelled particle systems. The study of cell culture systems using analogies derived from statistical mechanics yields simple, practical models offering insight into how a long-range order can be obtained in tissue-engineered constructs, providing a new paradigm for managing operations with large collectives of living cells.     

Aqueous two-phase recovery of bio-nanoparticles: a miniaturization study for the recovery of bacteriophage T4

Biotechnology industry has recently been demanding nanoparticulate products (20-200 nm) such as viruses, plasmids, virus-like particles and drug delivery assemblies. These products are mainly used as gene delivery systems in gene therapy protocols. During the process development for the manufacture of these products, it is crucial to optimize the recovery and purification steps. Unfortunately, the high value of some bio-nanoparticles complicates the optimization studies. The solvent extraction method with aqueous two-phase systems (ATPS) has been used to successfully recover bioproducts on a large scale. In this study, the potential miniaturization of ATPS is presented. The partition behavior of pure bovine serum albumin (BSA) in PEG-800-phosphate and bacteriophage T4 in PEG 8000-phosphate and PEG 600-sulphate systems were studied at three different scales (10 g, 2 g and 300 microl). The results obtained showed that the volume ratio (V(R)) for BSA (V(R)=1.0) was comparable to the blank systems at the scales studied. Additionally, the partition coefficient (K) was also similar (K=0.05) with more than 82% of BSA concentrated in the bottom phase. Same system was challenged with bacteriophage T4 showing a V(R)=1.0 and K greater than 5 with the infective particles concentrated in the top phase. The bacteriophage T4 was concentrated in opposite phase in the PEG-600-sulfate system with a consistent V(R)=0.8 and K<0.2 for the scales analyzed. The partition behavior the bacteriophage T4 was comparable to that reported previously for adenoviral vectors in same system at 15 ml scale. The results obtained demonstrated that the miniaturization of ATPS is feasible and reproducible for the two models selected. This provides significant information about the miniaturization process of such ATPS for their potential generic applications in the recovery of different bio-nanoparticle products.     

Synthesis and assembly of large subunits into ribulose bisphosphate carboxylase/oxygenase in chloroplast extracts

We have developed a new system for the in vitro synthesis of large subunits and their assembly into ribulose bisphosphate carboxylase oxygenase (Rubisco) holoenzyme in extracts of higher plant chloroplasts. This differs from previously described Rubisco assembly systems because the translation of the large subunits occurs in chloroplast extracts as opposed to isolated intact chloroplasts, and the subsequent assembly of large subunits into holoenzyme is completely dependent upon added small subunits. Amino acid incorporation in this system displayed the characteristics previously reported for chloroplast-based translation systems. Incorporation was sensitive to chloramphenicol or RNase but resistant to cycloheximide, required magnesium, and was stimulated by nucleotides. The primary product of this system was the large subunit of Rubisco. However, several lower molecular weight polypeptides were formed. These were structurally related to the Rubisco large subunit. The initiation inhibitor aurintricarboxylic acid (ATA) decreased the amount of lower molecular weight products accumulated. The accumulation of completed large subunits was only marginally reduced in the presence of ATA. The incorporation of newly synthesized large subunits into Rubisco holoenzyme occurred under conditions previously identified as optimal for the assembly of in organello-synthesized large subunits and required the addition of purified small subunits.