Plasmid uptake by bacteria: a comparison of methods and efficiencies

The ability to introduce individual molecules of plasmid DNA into cells by transformation has been of central importance to the recent rapid advancement of plasmid biology and to the development of DNA cloning methods. Molecular genetic manipulation of bacteria requires the development of plasmid-mediated transformation systems that include (1) chemical transformation, (2) electro-transformation, (3) biolistic transformation, and (4) sonic transformation, leading to the introduction of exogenous plasmid DNA into bacterial cells. In this review, the manipulation properties and transformation efficiencies of these techniques are described. In addition to these methods, a conceptually novel transformation technique, namely the hydrogel exposure method, was developed. The hydrogel exposure method, based on the Yoshida effect, provides a significant advance over chemical means for transforming many strains of Escherichia coli and a variety of other bacterial species. The new term “tribos transformation” has been proposed for this novel technique. We also determined that, compared to conventional methods, the hydrogel exposure method is a novel and convenient method by which to transform bacteria.     

Availability and utilization of waste fish feed by mussels Mytilus edulis in a commercial integrated multi-trophic aquaculture (IMTA) system: A multi-indicator assessment approach

Fish feed waste enhancement of the particulate food supply and performance of mussels Mytilus edulis suspended near salmon cages at an integrated multi-trophic aquaculture (IMTA) site was assessed using a multi-indicator approach. Dietary indicators included bulk measurements of seston quantity and nutritional quality, proximate analysis (PA), fatty acid (FA) and stable isotope (SI) composition. Mussel tissue indicators consisted of PA and FA composition. Mussel performance was assessed from physiological integrations (scope for growth, SFG), growth efficiency (K₂) and condition index (CI). All measurements were made over 2 days at a commercial IMTA farm and a monoculture mussel farm in the Bay of Fundy (Canada). Significant differences detected in seston quantity and quality were within the range of natural spatial variability. The SFG of IMTA mussels was lower (28.71 J h⁻¹) than monoculture mussels (38.71 J h⁻¹) and reflected site differences in natural food availability and composition that affected absorption rate. PA of mussel organs didn't reflect a significant fish feed contribution to the mussel diet. However, dietary enhancement and assimilation of fish feed waste was demonstrated by significantly higher levels of feed FA biomarkers 20:1ω9, 18:2ω6, 18:1ω9 and low ω3/ω6 ratio in seston, mussel tissues and feces at the IMTA site than at the mussel farm. SI (δ¹³C and δ¹⁵N) in seston and mussel feces significantly differed among sites and IMTA mussels had significantly higher CI (21%) than monoculture individuals (16%). It was concluded that bulk indicators of the diet, short-term physiological integrations, and PA of mussel tissues have a limited capacity to detect dietary enhancement at IMTA sites. FA and SI tracers of fish feed waste were shown to be more sensitive for detecting the low-levels of diet enhancement within the large range of natural seston variation.     

A preliminary study of the bioremediation potential of Codium fragile applied to seaweed integrated multi-trophic aquaculture (IMTA) during the summer

In integrated multi-trophic aquaculture (IMTA), seaweeds have the capacity to reduce the environmental impact of nitrogen-rich effluents in coastal ecosystems. To establish such bioremediation systems, selection of suitable seaweed species is important. The distribution and productivity of seaweeds vary seasonally based on water temperature and photoperiod. In Korea, candidate genera such as Pophyra, Laminaria, and Undaria grow from autumn to spring. In contrast, Codium grows well at relatively high water temperatures in summer. Thus, aquaculture systems potentially could capitalize on Codium’s capacity for rapid growth in the warm temperatures of late summer and early fall. In this study, we investigated ammonium uptake and removal efficiency by Codium fragile. In laboratory experiments, we grew C. fragile under various water temperatures (10, 15, 20, and 25°C), irradiances (dark, 10, and 100 μmol photons m⁻² s⁻¹), and initial ammonium concentrations (150 and 300 μM); in all cases, C. fragile exhausted the ammonium supply for 6 h. At 150 μM of , ammonium removal efficiency was greatest (99.5 ± 2.6%) when C. fragile was incubated at 20°C under 100 μmol photons m⁻² s⁻¹. At 300 μM of , removal efficiency was greatest (86.3 ± 2.1%) at 25°C under 100 μmol photons m⁻² s⁻¹. Ammonium removal efficiency was significantly greater at 20 and 25°C under irradiance of 100 μmol photons m⁻² s⁻¹ than under other conditions tested.     

Cost-effective production of Bacillus thuringiensis by solid-state fermentation

Production of Bacillus thuringiensis (Bt) was standardized on wheat bran based media in 250 ml Erlenmeyer flasks. Scale-up of Bt production on the best medium in plastic tubs with aeration at 8 h intervals starting 16 h after incubation yielded a significant increase in spore count and toxin content of the product. Maximum lysis of Bt cells was obtained by 60 h of incubation at 30 degrees C. This protocol was suitable for production of Bt strains and local isolates. The Bt produced proved highly effective at 0.1% concentration against larvae of castor semilooper, Achaea janata L, resulting in complete mortality by three days in laboratory bioassays. In field trials, the population of castor semilooper larvae on the castor bean crop was reduced significantly by three days after application. The cost for material production of 1 kg of Bt was approximately US dollars 0.70.     

Thermoanaerobacter ethanolicus in a comparison of the growth efficiencies of thermophilic and mesophilic anaerobes.

Maintenance coefficients and theoretical maximum growth yields, with respect to both substrate and ATP, were estimated for Thermoanaerobacter ethanolicus growing in a glucose-limited, continuous culture. A comparison of these values with those for other bacteria showed that, contrary to predictions by others, anaerobic thermophiles had neither low observed growth yields nor high maintenance energy coefficients.     

Recent trends of seaweed production in Chile

In the last fourteen years the production of seaweeds in Chile has ranged from 74 000 to 229 000 wet metric tons per year and has included about twenty species belonging to Phaeophyta and Rhodophyta. The only source of this production has been the exploitation of natural beds, except for Gracilaria, which is the only case of commercial cultivation and contributes significant quantities to total production. Initially most of the raw material was exported but currently important quantities of Gracilaria and several carrageenophytes are being processed by local industry. Changes in production of the main resources are analyzed with consideration of potential demand, level of knowledge about natural beds, and the situation of total Gracilaria farming, in order to attempt predictions for the supply. Current possibilities of applying new technologies to cultivate other economically important Chilean seaweeds are also analyzed and discussed.     

Prospects and challenges for industrial production of seaweed bioactives

Large‐scale seaweed cultivation has been instrumental in globalizing the seaweed industry since the 1950s. The domestication of seaweed cultivars (begun in the 1940s) ended the reliance on natural cycles of raw material availability for some species, with efforts driven by consumer demands that far exceeded the available supplies. Currently, seaweed cultivation is unrivaled in mariculture with 94% of annual seaweed biomass utilized globally being derived from cultivated sources. In the last decade, research has confirmed seaweeds as rich sources of potentially valuable, health‐promoting compounds. Most existing seaweed cultivars and current cultivation techniques have been developed for producing commoditized biomass, and may not necessarily be optimized for the production of valuable bioactive compounds. The future of the seaweed industry will include the development of high value markets for functional foods, cosmeceuticals, nutraceuticals, and pharmaceuticals. Entry into these markets will require a level of standardization, efficacy, and traceability that has not previously been demanded of seaweed products. Both internal concentrations and composition of bioactive compounds can fluctuate seasonally, geographically, bathymetrically, and according to genetic variability even within individual species, especially where life history stages can be important. History shows that successful expansion of seaweed products into new markets requires the cultivation of domesticated seaweed cultivars. Demands of an evolving new industry based upon efficacy and standardization will require the selection of improved cultivars, the domestication of new species, and a refinement of existing cultivation techniques to improve quality control and traceability of products.     

Intracellular concentration measurements in adherent cells: a comparison of import efficiencies of cell-permeable peptides

A protocol was developed for performing intracellular concentration measurements in flat adherent tissue culture cells by fluorescence correlation microscopy (FCM). Determination of the number of molecules in the confocal detection volume had to account for background fluorescence caused by molecules adsorbed to the surface of the measurement chamber. Such a background signal leads to a decrease in the amplitude of the autocorrelation function, and thereby to the calculation of an erroneously high number of molecules. Because of the spatial heterogeneity of the background intensity, a method was devised by which its contribution to the total fluorescence could be determined directly from each individual autocorrelation measurement. This method was applied to a comparison of the import efficiencies of different cell-permeable peptides at nanomolar concentrations. The Antennapedia homeodomain-derived peptide penetratin was imported about three times as efficient as the basic fibroblast growth factor-derived MTS peptide. Both peptides equilibrated between cytoplasm and nucleus. A relatively high mobility of these molecules inside the cells indicated that they may be rapidly degraded by cytosolic proteases. Based on these results, it will be possible to determine intracellular concentrations of inhibitors linked to import peptides directly by FCM at nanomolar concentrations and to optimise such constructs for proteolytic stability.     

Feeding rates,assimilation efficiencies and growth of two amphipod species on biodeposited material from zebra mussels

1. Accumulation of organic material by the zebra mussel Dreissena polymorpha is assumed to be the source of a biodeposition‐based food web. However, only little is known about the importance of the biodeposited material as a food source and its contribution to increased abundances of macroinvertebrates in the presence of D. polymorpha. 2. Feeding, assimilation and growth of the amphipods Gammarus roeselii and Dikerogammarus villosus on food sources directly and indirectly associated with D. polymorpha (biodeposited material and chironomids) and on conditioned alder leaves were measured. The stoichiometry of carbon, nitrogen and phosphorus of the diets was measured as an important determining factor of food quality. 3. Chironomids had the highest nitrogen and phosphorus contents, alder leaves were depleted in nitrogen and phosphorus, and the stoichiometry of biodeposited material was intermediate. 4. Both amphipod species had highest feeding rates and assimilation efficiencies on chironomids. Gammarus roeselii fed more on biodeposited material than on alder leaves, but assimilation efficiencies were similar; D. villosus also had similar feeding rates and assimilation efficiencies on the two diets. 5. Both amphipod species had highest growth rates on chironomids and lowest growth rates on alder leaves. Both grew at intermediate rates on biodeposited material of D. polymorpha. The growth rates of the amphipod species were related to food stoichiometry. Overall, the invasive D. villosus grew faster than the indigenous G. roeselii. 6. Food resources directly and indirectly associated with D. polymorpha are potential diets for amphipods, providing further evidence for a D. polymorpha biodeposition‐based food web.     

Chemical composition and ethanol production potential of Thai seaweed species

This study evaluated the potential use of several Thai seaweed species for ethanol production. The high biomass of the green algae Ulva intestinalis and Rhizoclonium riparium and the red algae Gracilaria salicornia and Gracilaria tenuistipitata in an earthen pond culture led us to select these species for our study. The seaweed species were analyzed for chemical composition, resulting in ash contents of 37.62?±?0.15 % and fiber of 11.93?±?0.16 %, with the highest values in R. riparium. Low lipid values were found in all species, with the highest value (p?<?0.05) in G. salicornia (1.69?±?0.07 %) and the lowest in R. riparium (0.28?±?0.01 %) and G. tenuistipitata (0.26?±?0.01 %). The highest carbohydrate contents were found in G. tenuistipitata (54.89 %), and the lowest were in R. riparium (29.53 %). G. tenuistipitata (8.58?±?0.36 %) and U. intestinalis (8.24?±?0.28 %) had higher sulfate contents compared with G. salicornia (4.69?±?0.04 %) and R. riparium (1.97?±?0.20 %). The monosugar algal tissue components were analyzed by HPLC; rhamnose, xylose, fucose, arabinose, mannose, glucose, and galactose were used as reference sugars. Total sugar was found to be highest in G. tenuistipitata (98.21 %). Arabinose, glucose, and galactose were the main sugar components in all species. Glucose obtained from G. tenuistipitata (6.55 %) and R. riparium (6.52 %) was higher than in G. salicornia (0.27 %) and U. intestinalis (2.78 %). G. tenuistipitata fermentation gave a higher yield of ethanol (4.17?×?10^?3 g ethanol g^?1 sugars; 139.12 μg ethanol g^?1 glucose) than R. riparium (0.086?×?10^?3 g ethanol g^?1 sugars; 33.84 μg ethanol g^?1 glucose), U. intestinalis (0.074?×?10^?3 g ethanol g^?1 sugars; 9.98 μg ethanol g^?1 glucose), and G. salicornia (0.031?×?10^?3 g ethanol g^?1 sugars; 1.43 μg ethanol g^?1 glucose).     

Cost-effective and concurrent production of industrially valuable xylano-pectinolytic enzymes by a bacterial isolate Bacillus pumilus AJK

Simultaneous production of xylanase and pectinase by Bacillus pumilus AJK under submerged fermentation was investigated in this study. Under optimized conditions, it produced 315?±?16 IU/mL acidic xylanase, 290?±?20 IU/mL alkaline xylanase, and 88?±?9 IU/mL pectinase. The production of xylano-pectinolytic enzymes was the highest after inoculating media (containing 2% each of wheat bran and Citrus limetta peel, 0.5% peptone, 10?mM MgSO₄, pH 7.0) with 2% of 21-hr-old culture and incubated at 37°C for 60?hr at 200?rpm. Xylanase retained 100% activity from pH 6.0 to10.0 after 3?hr of incubation, while pectinase showed 100% stability from pH 6.0 to 9.0 even after 6?hr of incubation. Cost-effective and concurrent production of xylanase and pectinase by a bacterial isolate in the same production media suggests its potential for various biotechnological applications. This is the first report of simultaneous production of industrially important extracellular xylano-pectinolytic enzymes by B. pumilus.     

Detailed comparison of the protein-ligand docking efficiencies of GOLD, a commercial package and ArgusLab, a licensable freeware

Molecular docking and virtual screening based on molecular docking have become an integral part of many modern structure-based drug discovery efforts. Hence, it becomes a useful endeavor to evaluate existing docking programs, which can assist in the choice of the most suitable docking algorithm for any particular study. The objective of the current study was to evaluate the ability of ArgusLab 4.0, a relatively new molecular modeling package in which molecular docking is implemented, to reproduce crystallographic binding orientations and to compare its accuracy with that of a well established commercial package, GOLD. The study also aimed to evaluate the effect of the nature of the binding site and ligand properties on docking accuracy. The three dimensional structures of a carefully chosen set of 75 pharmaceutically relevant protein-ligand complexes were used for the comparative study. The study revealed that the commercial package outperforms the freely available docking engine in almost all the parameters tested. However, the study also revealed that although lagging behind in accuracy, results from ArgusLab are biologically meaningful. This taken together with the fact that ArgusLab has an easy to use graphical user interface, means that it can be employed as an effective teaching tool to demonstrate molecular docking to beginners in this area.     

Bioreactor seaweed cell culture for production of bioactive oxylipins

Liquid cell suspension cultures derived from marine plants have the potential to biosynthesize novel biomedicinal compounds in a controlled environment. Of particular interest are the eicosanoids and related oxylipins emanating from the 15-lipoxygenase manifold of the arachidonic acid cascade, which is active in the brown algaLaminaria saccharina. Filamentous cell clumps ofL. saccharina isolated from female gametophytes were cultured in an illuminated bubble-column bioreactor in GP2 artificial seawater nutrient medium at 13 °C and air flow rate of 0.35 L air min^–1 L^–1 culture (vvm). Growth kinetics and biomass productivity data were obtained as a function of incident light intensity (2.4 to 98mol photon m^–2 s^–1) and initial cell density (27 to 149 mg DCW L^–1). Maximum cell densities exceeded 1200 mg DCW L^–1 after a 20 day cultivation time at optimal conditions of 98mol photon m^–2 s^–1 and 118 mg DCW L^–1 initial cell density. Qualitative analysis of chloroform/methanol extracts of the cell culture biomass by GC-MS confirmed the presence of the hydroxy fatty acids 13-HODTA and 13-HOTE, the likely products of 15-lipoxygenase catalyzed oxidation of linoleic or linolenic acids.     

A comparison of the efficiencies of the shotgun and the bow in neotropical forest hunting

Whenever introduced into Amazonia and its neighboring regions, the shotgun has quickly replaced the bow and arrow and other aboriginal weapons of the hunt. The quick and widespread adoption of the shotgun is plainly a matter of its superiority over most aboriginal weapons. This paper compares the hunting efficiencies of the shotgun and the bow by means of a controlled field experiment among the Ye'kwana and Yanomamö Indians of the Upper Orinoco River of southern Venezuela. It also examines the impact of the shotgun on local animal populations and the economic changes brought about by the need to cash-crop in order to purchase Western hunting technology.Funds for the research and writing of this paper were made possible by an NIMH predoctoral fellowship to Napoleon A. Chagnon, Grant No. NIMH 5 R01 MH 26008-SSR.     

Biogas production from the brown seaweed Saccharina latissima: thermal pretreatment and codigestion with wheat straw

Saccharina latissima is a carbohydrate-rich fast-growing seaweed (SW) which may be utilized as a marine energy crop. In this study, S. latissima was anaerobically digested for biogas production, and the effects of thermal pretreatment and codigestion with wheat straw (WS) were investigated. Batch experiments showed that pretreatment of SW increased the methane yield from 223 to 268 mL g^-1 VS. Codigestion of SW and steam-exploded WS had a clear positive effect on biogas production, giving yields that were considerably higher than what would be expected on the basis of the yields obtained with either of the individual feedstocks. Thus, anaerobic digestion of blends of SW and lignocellulosic substrates provides a promising strategy for biogas production. Although thermal pretreatment increased the yield of biogas from SW such a harsh pretreatment is probably more relevant for the more recalcitrant lignocellulosic substrates.     

Ultrasonic singing by the blue-throated hummingbird: a comparison between production and perception

Blue-throated hummingbirds produce elaborate songs extending into the ultrasonic frequency range, up to 30 kHz. Ultrasonic song elements include harmonics and extensions of audible notes, non-harmonic components of audible syllables, and sounds produced at frequencies above 20 kHz without corresponding hearing range sound. To determine whether ultrasonic song elements function in intraspecific communication, we tested the hearing range of male and female blue-throated hummingbirds. We measured auditory thresholds for tone pips ranging from 1 kHz to 50 kHz using auditory brainstem responses. Neither male nor female blue-throated hummingbirds appear to be able to hear above 7 kHz. No auditory brainstem responses could be detected between 8 and 50 kHz at 90 dB. This high-frequency cutoff is well within the range reported for other species of birds. These results suggest that high-frequency song elements are not used in intraspecific communication. We propose that the restricted hummingbird hearing range may exemplify a phylogenetic constraint.