New constructions of one- and two-stage pooling designs.

The study of gene functions requires a DNA library of high quality, such a library is obtained from a large mount of testing and screening. Pooling design is a very helpful tool for reducing the number of tests for DNA library screening. In this paper, we present new one- and two-stage pooling designs, together with new probabilistic pooling designs. The approach in this paper works for both error-free and error-tolerance scenarios.     

Rhodobacter sphaeroides RV cultivation and hydrogen production in a one- and two-stage chemostat

Rhodobacter sphaeroides RV cultivation and hydrogen production were studied in a one- and two-stage chemostat using lactic acid as substrate. Light saturation was observed when light intensities equal to or above 10 klx were applied. Under light saturation, the two-stage chemostat appeared to be very effective for hydrogen production, allowing complete nitrogen removal by bacterial growth in the first reactor. The hydrogen evolution rate in the second reactor was up to 75 ml H₂ (g dry weight)^–1 h^–1. Accumulation of storage material was observed in the second reactor of the two-stage chemostat under a large carbon excess and limiting light irradiance. The optimal hydraulic residence time was 15 h for both stages, leading to a total hydrogen production about 40% higher than in the one-stage chemostat. Under increasing influent ammonium and yeast extract concentrations, opposite trends of decreasing bacterial activity and increasing concentration resulted in a linear increase of the overall hydrogen production to 1.4–1.6lH₂ (l reactor)^–1 day^–1. Hydrogen production quickly fell when nitrogen was not completely metabolised. The hydrogen evolution rate was also found to depend on lactic acid concentration, and maximum bacterial activity was observed at 100 mM influent lactic acid.     

Microbial compositions and metabolic interactions in one- and two-phase partitioning airlift bioreactors treating a complex VOC mixture

Engineered microbial ecosystems in bioscrubbers for the treatment of volatile organic compounds (VOCs) have been complicated by complex VOC mixtures from various industrial emissions. Microbial associations with VOC removal performance of the bioscrubbers are still not definitive. Here, one- and two-phase partitioning airlift bioreactors were used for the treatment of a complex VOC mixture. Microbial characteristics in both bioreactors were uncovered by high-throughput metagenomics sequencing. Results showed that dominant species with specialized VOC biodegradability were mainly responsible for high removal efficiency of relative individual VOC. Competitive enzyme inhibitions among the VOC mixture were closely related to the deterioration of removal performance for individual VOC. Relative to the mass transfer resistance, the specialized biodegrading functions of microbial inoculations and enzymatic interactions among individual VOC biodegradation also must be carefully evaluated to optimize the treatment of complex VOC mixtures in bioreactors.     

Efficiency of one- and two-stage selection indexes for improvement of net economic merit in White Leghorns

Summary Mitochondrial (mt) and chloroplast (ct) DNAs from sugar beet lines carrying normal and introduced sources of male sterile cytoplasms have been characterized and compared on the basis of restriction enzyme analysis. Normal cytoplasm was shown to contain mt and ctDNAs which differed from those of the male sterile cytoplasms examined in the present investigation. On the other hand, four groups of male sterile cytoplasms could be differentiated by their own characteristic mtDNA digest patterns, while two were separated by ctDNA comparisons. In addition, a greater degree of variability of the mitochondrial genome is suggested. Our results also imply strict maternal inheritance of mt and ctDNAs. Thus, the organelle DNA assay provides a positive and alternative means of identifying various male sterile cytoplasms.     

Silent substitutions predictably alter translation elongation rates and protein folding efficiencies

Genetic code redundancy allows most amino acids to be encoded by multiple codons that are non-randomly distributed along coding sequences. An accepted theory explaining the biological significance of such non-uniform codon selection is that codons are translated at different speeds. Thus, varying codon placement along a message may confer variable rates of polypeptide emergence from the ribosome, which may influence the capacity to fold toward the native state. Previous studies report conflicting results regarding whether certain codons correlate with particular structural or folding properties of the encoded protein. This is partly due to different criteria traditionally utilized for predicting translation speeds of codons, including their usage frequencies and the concentration of tRNA species capable of decoding them, which do not always correlate. Here, we developed a metric to predict organism-specific relative translation rates of codons based on the availability of tRNA decoding mechanisms: Watson-Crick, non-Watson-Crick or both types of interactions. We determine translation rates of messages by pulse-chase analyses in living Escherichia coli cells and show that sequence engineering based on these concepts predictably modulates translation rates in a manner that is superior to codon usage frequency, which occur during the elongation phase, and significantly impacts folding of the encoded polypeptide. Finally, we demonstrate that sequence harmonization based on expression host tRNA pools, designed to mimic ribosome movement of the original organism, can significantly increase the folding of the encoded polypeptide. These results illuminate how genetic code degeneracy may function to specify properties beyond amino acid encoding, including folding.     

Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources

Inteins excise themselves out of precursor proteins by the protein splicing reaction and have emerged as valuable protein engineering tools in numerous and diverse biotechnological applications. Split inteins have recently attracted particular interest because of the opportunities associated with generating a protein from two separate polypeptides and with trans-cleavage applications made possible by split intein mutants. However, natural split inteins are rare and differ greatly in their usefulness with regard to the achievable rates and yields. Here we report the first functional characterization of new split inteins previously identified by bioinformatics from metagenomic sources. The N- and C-terminal fragments of the four inteins gp41-1, gp41-8, NrdJ-1, and IMPDH-1 were prepared as fusion constructs with model proteins. Upon incubation of complementary pairs, we observed trans-splicing reactions with unprecedented rates and yields for all four inteins. Furthermore, no side reactions were detectable, and the precursor constructs were consumed virtually quantitatively. The rate for the gp41-1 intein, the most active intein on all accounts, was k = 1.8 ± 0.5 × 10(-1) s(-1), which is ～10-fold faster than the rate reported for the Npu DnaE intein and gives rise to completed reactions within 20-30 s. No cross-reactivity in exogenous combinations was observed. Using C1A mutants, all inteins were efficient in the C-terminal cleavage reaction, albeit at lower rates. C-terminal cleavage could be performed under a wide range of reaction conditions and also in the absence of native extein residues flanking the intein. Thus, these inteins hold great potential for splicing and cleavage applications.     

Oxygen-transfer strategy and its regulation effects in serine alkaline protease production by Bacillus licheniformis

The effects of oxygen transfer on the production and product distribution in serine alkaline protease (SAP) fermentation by Bacillus licheniformis and oxygen-transfer strategy in relation to the physiology of the bacilli were investigated on a defined medium with citric acid as sole carbon source in 3.5-dm(3) batch bioreactor systems. By forming a 3 x 3 matrix with the parameters air-inlet rates of Q(O)/V(R) = 0.2, 0.5, 1.0 vvm, and agitation rates of N = 150, 500, 750 min(-1), the effects of oxygen transfer were investigated at nine different conditions. The concentrations of the product SAP and by-products, i.e., neutral protease, alpha-amylase, amino acids, and organic acids, and SAP activities were determined throughout the bioprocess. Among the constant air-flow and agitation-rate fermentations, Q(O)/V(R) = 0.5 vvm, N = 750 min(-1) oxygen-transfer conditions produced maximum SAP activity that was 500 U cm(-3), at t = 37 h. With the increase in Q(O)/V(R) and/or N, Damk?hler number that is the oxygen-transfer limitation decreases; and the process passes from oxygen-transfer limited conditions to biochemical-reaction limited conditions. Further increase in SAP activity, A = 680 U cm(-3) was achieved by applying an oxygen-transfer strategy based on the analysis of the data obtained with the constant oxygen-transfer condition experiments, with a step increase in air-inlet rate, from Q(O)/V(R) = 0.2 to Q(O)/V(R) = 0.5 vvm at N = 750 min(-1) constant agitation rate at t = 24 h. Organic acids and amino acids that were excreted to the fermentation medium varied depending on the oxygen-transfer conditions. With the increase in oxygen-transfer rate acetic acid concentration increased; contrarily, with the decrease in the oxygen-transfer rate the TCA-cycle organic acids alpha-ketoglutaric and succinic acids, and gluconic acid were excreted to the fermentation broth; nevertheless, the application of the oxygen-transfer strategy prevented the increase in acetic acid concentration between t = 35-38 h. Under all the oxygen-transfer conditions, the amino acid having the highest concentration and the amino acid that was not excreted to the fermentation broth were lysine and asparagine, respectively; both of which belong to the aspartic acid-group amino acids. Further, this result indicates the requirement of the genetic regulation directed to the aspartic acid-group enzymes for the progress in SAP production in B. licheniformis.     

Effect of scale-up on average shear rates for aerated non-Newtonian liquids in external loop airlift reactors

Average shear rates have been estimated experimentally in a 700-dm3 external loop airlift reactor. Aqueous pseudoplastic carboxymethylcellulose and xanthan gum solutions were used to simulate non-Newtonian behavior of biological media. Average shear rates of non-Newtonian solutions were found by analogy with Newtonian glycerol solutions using downcomer liquid velocity as the measurable parameter. Due to the complexity of local shear rate measurement, an average shear rate was assumed to exist and is proportional to superficial gas velocity. The data from this work and those in the literature were used in producing a new correlation for estimating average shear rates as a function of superficial gas velocity, geometry, and dispersion height. Wall shear rates were found to be significant. The ratio of wall shear rates to bulk shear rates were varied from 5% to 40%. Furthermore, it has been found that shear rates generated in airlift loop reactors are lower than those generated in bubble columns. Copyright 1999 John Wiley & Sons, Inc.     

Consumption rates and utilization efficiencies of four species of polyphagous Lepidoptera feeding on sycamore leaves

Summary Four species of Geometridae showed similar consumption rates and approximate digestibilities (100 x ingestion-egestion/ingestion) of sycamore leaves (Acer pseudoplatanus L.). A significant decline in E.C.I. (100 x growth/ingestion) and E.C.D. (100x growth/ingestion-egestion) with increasing larval weight is reported. The earlier descent from the sycamore canopy ofErannis defoliaria Cl. andOperophtera brumata L. larvae, to pupate in the soil, in comparison withAlsophila aescularia D. and S. andAgriopis aurantiaria Hb. larvae is discussed in relation to small differences in the utilization efficiencies of final instar larvae. Later descending species suffered greater predation by wood-ants in the field site studied.     

Warming up the system: higher predator feeding rates but lower energetic efficiencies

Predictions on the consequences of the rapidly increasing atmospheric CO₂ levels and associated climate warming for population dynamics, ecological community structure and ecosystem functioning depend on mechanistic energetic models of temperature effects on populations and their interactions. However, such mechanistic approaches combining warming effects on metabolic (energy loss of organisms) and feeding rates (energy gain by organisms) remain a key, yet elusive, goal. Aiming to fill this void, we studied the metabolic rates and functional responses of three differently sized, predatory ground beetles on one mobile and one more resident prey species across a temperature gradient (5, 10, 15, 20, 25 and 30 °C). Synthesizing metabolic and functional‐response theory, we develop novel mechanistic predictions how predator–prey interaction strengths (i.e., functional responses) should respond to warming. Corroborating prior theory, warming caused strong increases in metabolism and decreases in handling time. Consistent with our novel model, we found increases in predator attack rates on a mobile prey, whereas attack rates on a mostly resident prey remained constant across the temperature gradient. Together, these results provide critically important information that environmental warming generally increases the direct short‐term per capita interaction strengths between predators and their prey as described by functional‐response models. Nevertheless, the several fold stronger increase in metabolism with warming caused decreases in energetic efficiencies (ratio of per capita feeding rate to metabolic rate) for all predator–prey interactions. This implies that warming of natural ecosystems may dampen predator–prey oscillations thus stabilizing their dynamics. The severe long‐term implications; however, include predator starvation due to energetic inefficiency despite abundant resources.     

Modeling and dynamic simulation of a two-stage pre-denitrification MBBR system under increasing organic loading rates

Bioprocess and Biosystems Engineering - Biofilm-based wastewater treatment systems have become attractive due to their numerous advantages when compared to other suspended growth processes....     

Predation risk and habitat complexity modify intermediate predator feeding rates and energetic efficiencies in a tri‐trophic system

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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.     

Liquid circulation in external-loop airlift bioreactors

A simple model for prediction of liquid velocity in external-loop airlift bioreactors has been developed. Theoretical correlations for friction factor of gas-non-Newtonian two-phase flows and for liquid velocity in the riser were derived using the concept of an eddy diffusivity. The predictions of the proposed model were compared with the available experimental data for the friction factor and the liquid velocity in the riser of external-loop airlift contactors. Satisfactory agreement was obtained.     

Gas hold-up in external-loop airlift bioreactors

A new model of gas hold-up is proposed for external-loop airlift bioreactors. It is based on the similarity between the liquid circulation due to the local variation of gas hold-up in airlift bioreactors and the natural convection due to temperature difference. The model is developed to include the case of non-Newtonian fermentation media which are involved in many industrially bioprocesses. The capability of the model is examined using a wide range of experimental results including the present data. Reasonable agreement is obtained between the proposed model and the experimental data both for Newtonian and non-Newtonian media.     

Recombinant Escherichia coli cultivation in a pressurized airlift bioreactor: assessment of the influence of temperature on oxygen transfer and uptake rates

Bioprocess and Biosystems Engineering - Temperature influences the rates of oxygen transfer (OTR) and uptake (q O2) in aerobic bioprocesses. Hence, joint analysis of q O2 and OTR at variable...     

Stock-specific changes in growth rates, food conversion efficiencies, and energy allocation in response to temperature change in juvenile Atlantic cod

Under common environments, populations of laboratory reared (Grand Banks, GB and Gulf of Maine, GOM) and wild caught (Fortune Bay, FB and Bonavista Bay, BB; Newfoundland) juvenile cod Gadus morhua responded similarly to temperature change in specific growth rates, food conversion efficiencies, condition factors, liver water content, and muscle water content. However, GOM cod had higher condition factors, and showed differences from GB cod in phenotypic plasticity of hepatosomatic index to temperature. These differences were not present in a different population comparison between FB and BB cod. All populations had higher growth rates and food conversion efficiencies at warmer temperatures, and exhibited compensatory growth when temperature was increased. The results suggest relatively larger genetic differences between GB and GOM cod than between FB and BB cod, and indicate that the faster growth of southern populations in the wild is not due to a higher genetic capacity for growth rate.     

Effects of Equex, one- or two-step dilution, and two freezing and thawing rates on post-thaw survival of dog spermatozoa

The objectives of the present study were to evaluate the effects of adding Equex to a TRIS-extender, diluting the semen in 1 or 2 steps, freezing according to 2 methods, thawing at 2 rates, and the interactions between these treatments, on the post-thaw survival of dog spermatozoa at 38 degrees C. Ten ejaculates were obtained from 8 dogs. Each ejaculate was centrifuged, and the seminal plasma was discarded. Each sperm pellet was diluted with 2 mL of a TRIS-glucose-egg yolk extender containing 3% glycerol (Extender 1 [Ext-1]). Ejaculates were then pooled (9 x 10(9) spermatozoa), and Ext-1 was added to obtain 200 x 10(6) spermatozoa/mL. The semen pool was carefully mixed and divided into aliquots, and processed according to a 2 x 2 x 2 x 2 factorial design to evaluate the effects of 1) adding the same volume of a second TRIS-glucose-egg yolk extender with 7% glycerol that contained (Ext-2-E) or didn't contain (Ext-2) 1% of Equex STM Paste (final concentration of spermatozoa 100 x 10(6) spermatozoa/mL, glycerol 5%, Equex 0% [Ext-2] or 0.5% [Ext-2-E]); 2) diluting the semen in 1 step (adding Ext-2 or Ext-2-E before equilibration) or in 2 steps (adding Ext-2 or Ext-2-E after equilibration, just before the freezing operation); 3) freezing the straws horizontally in a styrofoam box 4 cm above liquid nitrogen (LN2) or by lowering them vertically into a LN2 tank in 3 steps; and 4) thawing at 70 degrees C for 8 sec or at 37 degrees C for 15 sec. A total of 16 treatment combinations were evaluated. Sperm motility was evaluated after thawing and at 1-h intervals during 7 h of incubation at 38 degrees C by subjective examination and by using a CASA-system. Plasma membrane integrity and acrosomal status were evaluated simultaneously at 1, 3 and 6 h post-thaw using a triple fluorescent staining procedure and flow cytometry. The best post-thaw survival and thermoresistance of spermatozoa was obtained when Equex was present in the extender (P<0.0001); the semen dilution was performed in 2 steps instead of 1 (P<0.0001); the freezing was carried out using the box instead of the tank (P<0.05); and the straws were thawed at 70 degrees C for 8 sec instead of at 37 degrees C for 15 sec (P<0.0001).     

Distribution and seasonality ofLegionella pneumophila in colling towers

The numbers of presumptiveLegionella pneumophila cells in waters and sediments of nine different cooling towers located on the same site in the northeastern United States were determined at approximately monthly intervals for 18 months. All systems received makeup water from the same source and received the same chemical treatments. PresumptiveL. pneumophila were found in both water and sediment samples from all systems on all sampling dates. An important result of this study was the finding that tower sediments represent large reservoirs ofL. pneumophila. The only correlation between levels of presumptiveL. pneumophila and any of the physical, chemical, or operating characteristics evaluated was with winter shutdown and drainage followed by a nonoperational period. These systems showed a definite seasonal response with the highest levels of presumptiveL. pneumophila found in the summer and fall. Systems operated year round showed relatively constant numbers ofL. pneumophila in both water and sediments.