Enzymatic digestion--a safe and rapid technique for individual separation of Macrobrachium rosenbergii embryos for cryopreservation studies

A new, safe, and rapid technique for the individual separation of the embryos of giant freshwater prawn Macrobrachium rosenbergii de Man is described. Two protease enzymes, e.g., trypsin and collagenase were used. Embryos in the advanced stage of development (gray embryos with eyespot and heart beat) were selected for the study. Treatment with collagenase and trypsin at respective concentrations of 0.05 and 0.25% for 30 min resulted in 100% separation of 35-40 mg of embryonic mass (approximately 180 embryos). A chelating agent, EDTA (ethylenediaminetetraacetic acid disodium salt: dihydrate) at 400 mg l(-1) enhanced the activity of trypsin. Trypsin and collagenase, when used together, were found to act synergistically. The separated embryos revealed no morphological injury when observed under the microscope. Further, in vitro hatching of the separated embryos was successful indicating that the present technique is safe and effective in achieving individual separation of prawn embryos.     

Identification of disulfide-linked peptides by isotope profiles produced by peptic digestion of proteins in 50% (18)O water

Determination of the disulfide-bond arrangement of a protein by characterization of disulfide-linked peptides in proteolytic digests may be complicated by resistance of the protein to specific proteases, disulfide interchange, and/or production of extremely complex mixtures by less specific proteolysis. In this study, mass spectrometry has been used to show that incorporation of (18)O into peptides during peptic digestion of disulfide-linked proteins in 50% (18)O water resulted in isotope patterns and increases in average masses that facilitated identification and characterization of disulfide-linked peptides even in complex mixtures, without the need for reference digests in 100% (16)O water. This is exemplified by analysis of peptic digests of model proteins lysozyme and ribonuclease A (RNaseA) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry (MS). Distinct isotope profiles were evident when two peptide chains were linked by disulfide bonds, provided one of the chains did not contain the C terminus of the protein. This latter class of peptide, and single-chain peptides containing an intrachain disulfide bond, could be identified and characterized by mass shifts produced by reduction. Reduction also served to confirm other assignments. Isotope profiling of peptic digests showed that disulfide-linked peptides were often enriched in the high molecular weight fractions produced by size exclusion chromatography (SEC) of the digests. Applicability of these procedures to analysis of a more complex disulfide-bond arrangement was shown with the hemagglutinin/neuraminidase of Newcastle disease virus.     

Purification and characterization of a novel 7-kDa non-specific lipid transfer protein-2 from rice (Oryza sativa)

A novel 7-kDa non-specific lipid transfer protein-2 (nsLTP2) has been isolated from rice (Oryza sativa) seeds. In contrast to nsLTP1s, few nsLTP2s have been purified and characterized. Complete amino acid sequence of rice nsLTP2 was determined by N-terminal Edman degradation of the intact protein as well as the peptide fragments resulted from trypsin digestions. Rice nsLTP2 consists of 69 amino acid residues with eight conserved cysteines forming four disulfide bonds. The secondary structure of rice nsLTP2 is predominantly alpha-helical as determined by circular dichroism spectroscopy. Cysteine pairings of nsLTP2 have one miss match at Cys(35)-X-Cys(37) motif compared to nsLTP1. Primary structure analysis of various plant nsLTP2s revealed an interesting conservation of sequence features among nsLTP2 family.     

Simulation on long-term operation of an anaerobic bioreactor for Korean food wastes

A mathematical model was formulated to simulate the long-term performance of an anaerobic bioreactor designed to digest Korean food wastes. The system variables of various decomposition steps were built into the model, which predicts the temporal characters of solid waste, and volatile fatty acid (VFA) in the reactor, and gas production in response to various input loadings and temperatures. The predicted values of VFA and gas production were found to be in good agreement with experimental observations in batch and repeated-input systems. Finally, long-term reactor performance was simulated with respect to the seasonal temperature changes from 5°C in winter to 25°C in summer at different food waste input loadings. The simulation results provided us with information concerning the success or failure of a process during long-term operation.     

Determination of selenium in serum by FI-HG-AAS and calculation of dietary intake

A method was developed for the determination of selenium concentration in serum by flow injection-hydride generation-atomic absorption spectrometry (FI-HG-AAS) following microwave digestion of serum samples and reduction of selenate to selenite. The detection limit of the method was 0.3 μg Se/L and the characteristic concentration, corresponding to the 0.0044 absorbance signal, was 0.12 μg Se/L. The results from the analysis of two Seronorm standard reference materials showed good agreement with the certified values. The method was then used to analyze selenium in sera of Austrian and Slovenian people for the calculation of dietary intakes. The selenium concentrations in sera of mothers at delivery, their neonates, and the male and female adults were 71 ± 14, 42 ± 6, 75 ± 21, and 65 ± 16 μg/L for the Austrians and 62 ± 15, 34 ± 7, 70 ± 12, and 66 ± 15 μg/L for the Slovenians. The dietary intakes of selenium of the mothers and the male and the female adults were calculated as 52, 37, and 46 μg/d for the Austrians and 45, 38, and 32 μg/d for the Slovenians.     

Functional morphology of digestion in the stomachless,piscivorous needlefishes Tylosurus gavialoides and Strongylura leiura ferox (Teleostei: Beloniformes)

Belonidae are unusual in that they are carnivorous but lack a stomach and have a straight, short gut. To develop a functional morphological model for this unusual system the gut contents and alimentary tract morphology of Tylosurus gavialoides and Strongylura leiura ferox were investigated. The posterior orientation of the majority of the pharyngeal teeth supports the swallowing of whole large prey, but not their mastication. Mucogenic cells are abundant in the mucosa lining, particularly the esophagus, and their secretions are likely to protect the gut lining from damage while lubricating passage of the prey. Esophagus, anterior intestine, posterior intestine, and rectum all have highly reticulate mucosae. The anterior three gut sections are distensible to accommodate the passage of prey. However, following ingestion large prey are passed to the highly distensible posterior intestine where they rest head first against the ileorectal valve. Alimentary pH ranges from neutral to weakly acidic. Fish prey is digested head first with the head being largely digested while the remainder of the body is still intact. The nondistensibility of the rectum and the small aperture provided by the ileorectal valve suggest the products of intestinal digestion are either small particulates or fluids that pass into rectum where they are absorbed. J. Morphol. 2009. © 2009 Wiley‐Liss, Inc.     

Development of a pilot scale anaerobic digester for biogas production from cow manure and whey mix

This paper presents results from anaerobic digestion of cow manure and whey mix. A pilot scale anaerobic digester, 128 l in volume, has been developed, to operate under batch and fed-batch conditions. The versatile and unique characteristics of the instrument allowed testing the methane production directly in the farm. The digester performance was evaluated with two calibration tests, the main for a period of 56 days. The study test was divided into three phases, one for each type of feeding operation (batch, fed-batch, batch). The initial phase of digestion resulted in 57 l-CH₄/kg-VS, the second phase had a yield of 86.6 l-CH₄/kg-VS and the third one had a production of 67 l-CH₄/kg-VS. The total methane yield was equal to 211.4 l-CH₄/kg-VS. Using the obtained pilot plant results to a real scale diary production cycle, it was possible to evaluate an electricity production equal to 8.86 kwh per 1 t/d. The conducted tests did show that there is a good potential to the use of a cow manure and whey biomass mix for biogas production.     

Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry

Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells.     

Cattle feed or bioenergy? Consequential life cycle assessment of biogas feedstock options on dairy farms

On‐farm anaerobic digestion (AD) of wastes and crops can potentially avoid greenhouse gas (GHG) emissions, but incurs extensive environmental effects via carbon and nitrogen cycles and substitution of multiple processes within and outside farm system boundaries. Farm models were combined with consequential life cycle assessment (CLCA) to assess plausible biogas and miscanthus heating pellet scenarios on dairy farms. On the large dairy farm, the introduction of slurry‐only AD led to reductions in global warming potential (GWP) and resource depletion burdens of 14% and 67%, respectively, but eutrophication and acidification burden increases of 9% and 10%, respectively, assuming open tank digestate storage. Marginal GWP burdens per Mg dry matter (DM) feedstock codigested with slurry ranged from –637 kg CO₂e for food waste to +509 kg CO₂e for maize. Codigestion of grass and maize led to increased imports of concentrate feed to the farm, negating the GWP benefits of grid electricity substitution. Attributing grass‐to‐arable land use change (LUC) to marginal wheat feed production led to net GWP burdens exceeding 900 kg CO₂e Mg^?1 maize DM codigested. Converting the medium‐sized dairy farm to a beef‐plus‐AD farm led to a minor reduction in GWP when grass‐to‐arable LUC was excluded, but a 38% GWP increase when such LUC was attributed to marginal maize and wheat feed required for intensive compensatory milk production. If marginal animal feed is derived from soybeans cultivated on recently converted cropland in South America, the net GWP burden increases to 4099 kg CO₂e Mg^?1 maize DM codigested – equivalent to 55 Mg CO₂e yr^?1 per hectare used for AD‐maize cultivation. We conclude that AD of slurry and food waste on dairy farms is an effective GHG mitigation option, but that the quantity of codigested crops should be strictly limited to avoid potentially large international carbon leakage via animal feed displacement.     

Mass balance and life cycle assessment of biodiesel from microalgae incorporated with nutrient recycling options and technology uncertainties

This article presents mass balances and a detailed life cycle assessment (LCA) for energy and greenhouse gases (GHGs) of a simulated microalgae biodiesel production system. Key parameters of the system include biomass productivity of 16 and 25 g m^?2 day^?1 and lipid content of algae of 40% and 25% for low and normal nitrogen conditions respectively. Based on an oil extraction efficiency from wet biomass of 73.6% and methane yields from anaerobically digested lipid‐extracted biomass of 0.31 to 0.34 l per gram of volatile solids, the mass balance shows that recycling growth media and recovering nutrients from residual biomass through anaerobic digestion can reduce the total demand for nitrogen by 66% and phosphorus by 90%. Freshwater requirements can be reduced by 89% by recirculating growth media, and carbon requirements reduced by 40% by recycling CO₂ from biogas combustion, for normal nitrogen conditions. A variety of technology options for each step of the production process and allocation methods for coproducts used outside the production system are evaluated using LCA. Extensive sensitivity and scenario analysis is also performed to provide better understanding of uncertainty associated with results. The best performing scenario consists of normal nitrogen cultivation conditions, bioflocculation and dissolved air flotation for harvesting, centrifugation for dewatering, wet extraction with hexane, transesterification for biodiesel production, and anaerobic digestion of biomass residual, which generates biogas used in a combined heat and power unit for energy recovery. This combination of technologies and operating conditions results in life cycle energy requirements and GHG emissions of 1.02 MJ and 71 g CO₂‐equivalent per MJ of biodiesel, with cultivation and oil extraction dominating energy use and emissions. Thus, even under optimistic conditions, the near‐term performance of this biofuel pathway does not achieve the significant reductions in life cycle GHG emissions hoped for from second‐generation biofuel feedstocks.