Exploitation of endophytes for sustainable agricultural intensification

Intensive agriculture, which depends on unsustainable levels of agrochemical inputs, is environmentally harmful, and the expansion of these practices to meet future needs is not economically feasible. Other options should be considered to meet the global food security challenge. The plant microbiome has been linked to improved plant productivity and, in this microreview, we consider the endosphere – a subdivision of the plant microbiome. We suggest a new definition of microbial endophyte status, the need for synergy between fungal and bacterial endophyte research efforts, as well as potential strategies for endophyte application to agricultural systems.     

Tracing Sources of Streamwater Sulfate During Snowmelt Using S and O Isotope Ratios of Sulfate and <Superscript>35</Superscript>S Activity

The biogeochemical cycling of sulfur (S) was studied during the 2000 snowmelt at Sleepers River Research Watershed in northeastern Vermont, USA using a hydrochemical and multi-isotope approach. The snowpack and 10 streams of varying size and land use were sampled for analysis of anions, dissolved organic carbon (DOC), ³⁵S activity, and δ³⁴S and δ¹⁸O values of sulfate. At one of the streams, δ¹⁸O values of water also were measured. Apportionment of sulfur derived from atmospheric and mineral sources based on their distinct δ³⁴S values was possible for 7 of the 10 streams. Although mineral S generally dominated, atmospheric-derived S contributions exceeded 50% in several of the streams at peak snowmelt and averaged 41% overall. However, most of this atmospheric sulfur was not from the melting snowpack; the direct contribution of atmospheric sulfate to streamwater sulfate was constrained by ³⁵S mass balance to a maximum of 7%. Rather, the main source of atmospheric sulfur in streamwater was atmospheric sulfate deposited months to years earlier that had microbially cycled through the soil organic sulfur pool. This atmospheric/pedospheric sulfate (pedogenic sulfate formed from atmospheric sulfate) source is revealed by δ¹⁸O values of streamwater sulfate that remained constant and significantly lower than those of atmospheric sulfate throughout the melt period, as well as streamwater ³⁵S ages of hundreds of days. Our results indicate that the response of streamwater sulfate to changes in atmospheric deposition will be mediated by sulfate retention in the soil.     

Process development and intensification for enhanced production of Bacillus lipopeptides

The growing interest in Bacillus lipopeptides for high-value applications has driven process design, development and optimization for enhanced lipopeptide production. Traditional optimization approaches have been directed towards improving the overall titres by modification of media components and environmental parameters, almost exclusively in submerged cultures. Carbon and nitrogen sources, trace elements and oxygen availability have all been demonstrated to exhibit significant influences on lipopeptide yield, productivity and selectivity. This insight into process-linked kinetics, especially selectivity, has led to the introduction of novel process intensification and integration strategies which further promote process efficiency, and which include foam fractionation, inverse fluidization, rotating disc contacting and microfiltration with recycle. These strategies have not only transformed the production capabilities, but have also successfully integrated upstream production with downstream purification through cell retention and in situ product removal. This review analyses and critically discusses the impact of process conditions and process optimization strategies for improving lipopeptide production kinetics, specifically highlighting the emerging trend of process intensification and integration strategies and further, proposes a heuristic route to enhance lipopeptide production.     

Bioprocess intensification: a potential aqueous two-phase process for the primary recovery of B-phycoerythrin from Porphyridium cruentum

A process for the primary recovery of B-phycoerythrin from Porphyridium cruentum exploiting aqueous two-phase systems (ATPS) was developed in order to reduce the number of unit operations and benefit from an increased yield of the protein product. The evaluation of system parameters such as poly(ethylene glycol) (PEG) molecular mass, concentration of PEG as well as salt, system pH and volume ratio was carried out to determine under which conditions the B-phycoerythrin and contaminants concentrate to opposite phases. PEG 1450-phosphate ATPS proved to be suitable for the recovery of B-phycoerythrin because the target protein concentrated to the top phase whilst the protein contaminants and cell debris concentrated in the bottom phase. An extraction ATPS stage comprising volume ratio (Vr) equal to 1.0, PEG 1450 24.9% (w/w), phosphate 12.6% (w/w) and system pH of 8.0 allowed B-phycoerythrin recovery with a purity of 2.9 (estimated as the relation of the 545-280 nm absorbances). The use of ATPS resulted in a primary recovery process that produced a protein purity of 2.9 +/- 0.2 and an overall product yield of 77.0% (w/w). The results reported demonstrated the practical implementation of ATPS for the design of a primary recovery process as a first step for the commercial purification of B-phycoerythrin produced by P. cruentum.     

Opportunities for marine bioprocess intensification using novel bioreactor design: frequency of barotolerance in microorganisms obtained from surface waters

In the context of marine biochemical systems, opportunities exist for the development of novel reactors, with optimization and conversion of current technologies having the potential to yield more efficient units. A limiting factor in the widespread commercial acceptance of a large range of marine metabolites is the efficient production of, for example, sufficient quantities of antibiotics and nutraceuticals to allow for structural analysis and clinical testing. Conventional methods utilised for physical and chemical process intensification require careful analysis of their potential application to shear-sensitive bioprocess systems. Stress induction, for example, provides one route to marine bioprocess intensification due to the expression of metabolites not otherwise possible. Use of high pressure as a stressing agent and/or intensification tool is discussed, and its potential, demonstrated by showing the existence of barotolerant (at 120 MPa) marine microorganisms obtained from shallow surface waters (<1.5 m deep), is shown. Microorganisms associated with the surface of, for example, seaweed show a greater likelihood of being barotolerant.     

Microbial structural diversity estimated by dilution-extinction of phenotypic traits and T-RFLP analysis along a land-use intensification gradient

The present work tested whether the relationship between functional traits and inoculum density reflected structural diversity in bacterial communities from a land-use intensification gradient applying a mathematical model. Terminal restriction fragment length polymorphism (T-RFLP) analysis was also performed to provide an independent assessment of species richness. Successive 10-fold dilutions of a soil suspension were inoculated onto Biolog GN(R) microplates. Soil bacterial density was determined by total cell and plate counts. The relationship between phenotypic traits and inoculum density fit the model, allowing the estimation of maximal phenotypic potential (Rmax) and inoculum density (KI) at which Rmax will be half-reduced. Though Rmax decreased with time elapsed since clearing of native vegetation, KI remained high in two of the disturbed sites. The genetic pool of bacterial community did not experience a significant reduction, but the active fraction responding in the Biolog assay was adversely affected, suggesting a reduction in the functional potential.     

Process intensification in fed-batch production bioreactors using non-perfusion seed cultures

ABSTRACT

Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 10⁶ cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2–10 × 10⁶ cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22–34 × 10⁶ cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3–6 × 10⁶ cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.     

Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis

A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.     

A simple technique for the preparation and storage of sucrose gradients

A method for preparing multiple sucrose gradients by quickly freezing layers of sucrose has been developed. These gradients may be stored in the freezer indefinitely, and thawed from 8 to 24 h at 4 degrees C before use. The middle region of the resulting sucrose gradients was linear. Thawing time and centrifugation had little effect on the shape of the gradient. The method is applicable for both small- and large-volume centrifuge tubes. Gradients prepared in the same batch were nearly identical.     

Ecological degradation and agricultural intensification in the Andean highlands

Andean cultural ecologists have made two claims in recent years: ecological decomposition is absent due to effective indigenous management of communal resources, and agricultural intensification is inversely related to altitude. Drawing on material from the Jukumani Indians of Northern Potosi, Bolivia, these assertions are challenged. First, there is little evidence to prove or disprove ecological degradation. Second, the location of agricultural intensification, as the Jukumani data suggests, is influenced by altitude as well as by the presence of market.Fieldwork in Bolivia was carried out between January 1979 and May 1981. This essay was originally presented at a conference entitled, How Communities Resolve Common Property Problems, sponsored by the Harvard Institute for International Development and the Center for Population Studies of Harvard University in the spring semester of 1983.     

An In Vivo Kinetic Model with L-[35S]Methionine for the Determination of Local Cerebral Rates for Methionine Incorporation into Protein in the Rat

A method has been developed for the simultaneous in vivo measurement of local rates for methionine incorporation into cerebral protein in the rat. It is based on the use of L-[35S]methionine as a tracer for reflecting the bidirectional exchange of methionine between plasma and brain and its incorporation into cerebral protein, using a dynamic three-compartment model. An operational equation based on this model has been derived in terms of determinable variables. The method has been applied to the normal freely moving rat and to the rat under chloral hydrate anesthesia. In the freely moving rat, the values of methionine incorporation into cerebral protein in the gray matter vary widely from structure to structure (50-300 nmol/100 g/min), with the highest values in structures related to neurosecretory functions, e.g., supraoptic and paraventricular nuclei. The values for white matter are more uniform (24-28 nmol/100 g/min) at levels approximately six- to seven-fold lower than for gray matter. Chloral hydrate anesthesia depresses the rate of methionine incorporation in all the structures examined. Anesthesia did not reduce the heterogeneity normally present within gray matter.     

Perfusion-Perls and -Turnbull methods supplemented by DAB intensification for nonheme iron histochemistry: demonstration of the superior sensitivity of the methods in the liver,spleen, and stomach of the rat

Perfusion-Perls and -Turnbull methods supplemented by the intensification with 3,3-diaminobenzidine (+ DAB) enabled stronger and more extensive staining of nonheme iron than the Perls + and Turnbull + DAB methods carried out on tissue sections fixed with 10% formalin in 0.9% saline or PBS. The section- and perfusion-Perls + DAB methods are not specific for the demonstration of nonheme ferric iron but also stain nonheme ferrous iron. However, owing to its high sensitivity, the perfusion-Perls + DAB method would provide useful information about nonheme iron deposition regardless of oxidation states in normal and pathological conditions. The perfusion-Turnbull + DAB method is specifically demonstrable of nonheme ferrous iron and the results from this method showed significant stores of nonheme ferrous iron in the hepatocytes, Kupffer cells, splenic macrophages, and gastric parietal cells of the rat. Since nonheme ferrous iron is considered to be critically involved in free radical generation, the perfusion-Turnbull + DAB method would visualize such populations of cells that are at risk from free radical damage.     

A simple procedure for obtaining autoradiographs of G-banded chromosomes

Chromosomal preparations from cells flash-labeled with ³H-dT were Giemsa-banded following trypsin digestion, allowed to air-dry, and then were coated with a layer of 1% Formvar. The slides were subsequently coated with radiographic emulsion (NTB2) and processed for autoradiography. The resulting chromosomes had distinct G-banding and radiographic labeling patterns. Chemographic grain formation in the emulsion, normally caused by Giemsa stain, was prevented by the film of Formvar, allowing a very low background grain level.     

Improved method for processing autoradiographs of cells grown on multiwell plates

Summary A modification of an established procedure for autoradiographic processing of cultured cells is described. This method eliminates the need for pipetting each individual well and also for cutting and dismantling the multiwell plate for slide preparation In this procedure the entire plate can be processed as a single unit and the cells can be analyzed in situ, thus eliminating the time consuming pipetting and cutting procedures. Furthermore, the entire experiment can be filed without use of additional slides or storage boxes. Hence, this is a simpler, time conserving, and economical way to process large numbers of cultures for thymidine labeling indices. This study was supported in part through a grant from the J. Graham Brown Regional Cancer Center, Louisville, Kentucky 40292.     

Rapid and Sensitive Single-Step Radiochemical Assay for Catechol-O-Methyltransferase

Abstract: A simple, rapid and reliable radiometric assay for the determination of catechol- O -methyltransferase activity is described. The method is based on the conversion of catechol to [³H]guaiacol by catechol- O -methyltransferase in the presence of Mg²⁺, adenosine deaminase and S -adenosyl l -[methyl-³H]methionine. Incubation and direct extraction of [³H]guaiacol into organic scintillation fluid, as well as counting, are performed in the same standard scintillation vial. The assay is easy to perform and more sensitive than previous analogous procedures. The method has been applied to the assay of catechol- O -methyltransferase activity in discrete brain areas and also peripheral organs of rat and in human erythrocytes.     

Synthesis of 1-Hydroxyalkyl-3-Substituted Ureas and Thioureas,Substrates for Alcohol Dehydrogenase

A series of 1-(2-hydroxyethyl)- and 1-(3-hydroxyethyl)-3-substituted ureas and thioureas were synthesized. 1-(3-Hydroxyethyl)-3-acylthioureas were shown to be specific substrates for alcohol dehydrogenase in vitro.