Two-stage biological treatment of olive mill wastewater with whey as co-substrate

Olive mill wastewater (OMW) is a highly polluting wastewater, caused by a high organic load and phenol content. These characteristics suggest that it may be suitable for aerobic treatment and anaerobic bacterial digestion. Aerobic treatment coupled with anaerobic bacterial digestion may be economically feasible as the methane produced is a valuable energy source while simultaneously purifying the OMW. In an attempt to improve the overall performance of the process, the addition of a co-substrate such as whey to the aerobic treatment pre-treatment of OMW by the yeast Candida tropicalis was studied.The two-stage system operated satisfactorily up to an organic loading rate (OLR) of 3.0 kg COD L⁻¹ day⁻¹ with a biogas production rate of 1.25 L_biogas L_reactor⁻¹ day⁻¹ and a total COD reduction in excess of 93% (62% COD reduction in aerobic pretreatment and 83% COD reduction in anaerobic digestion). Fifty-four percent of the phenol was biodegraded during the aerobic treatment stage, and biogas with between 68% and 75% methane was produced during anaerobic digestion.     

Genetic Diversity and Potential Function of Microbial Symbionts Associated with Newly Discovered Species of Osedax Polychaete Worms

We investigated the genetic diversity of symbiotic bacteria associated with two newly discovered species of Osedax from Monterey Canyon, CA, at 1,017-m (Osedax Monterey Bay sp. 3 “rosy” [Osedax sp. MB3]) and 381-m (Osedax Monterey Bay sp. 4 “yellow collar”) depths. Quantitative PCR and clone libraries of 16S rRNA gene sequences identified differences in the compositions and abundances of bacterial phylotypes associated with the newly discovered host species and permitted comparisons between adult Osedax frankpressi and juveniles that had recently colonized whalebones implanted at 2,891 m. The newly discovered Osedax species hosted Oceanospirillales symbionts that are related to Gammaproteobacteria associated with the previously described O. frankpressi and Osedax rubiplumus (S. K. Goffredi, V. J. Orphan, G. W. Rouse, L. Jahnke, T. Embaye, K. Turk, R. Lee, and R. C. Vrijenhoek, Environ. Microbiol. 7:1369-1378, 2005). In addition, Osedax sp. MB3 hosts a diverse and abundant population of additional bacteria dominated by Epsilonproteobacteria. Ultrastructural analysis of symbiont-bearing root tissues verified the enhanced microbial diversity of Osedax sp. MB3. Root tissues from the newly described host species and O. frankpressi all exhibited collagenolytic enzyme activity, which covaried positively with the abundance of symbiont DNA and negatively with mean adult size of the host species. Members of this unusual genus of bone-eating worms may form variable associations with symbiotic bacteria that allow for the observed differences in colonization and success in whale fall environments throughout the world's oceans.     

Plant–herbivore interactions in Alaskan arctic tundra change with soil nutrient availability

Herbivores in nutrient‐limited systems such as arctic tundra have been suggested to play a minor role in controlling plant growth simply because they are relatively few in number. However, theory predicts that as net primary productivity (NPP) increases because of greater inputs of nutrients or energy, herbivores may have greater effects on plant growth. This prediction has not been tested in the context of climate warming in arctic tundra, which may increase soil nutrient availability and thus NPP. We examined a long‐term experiment that excluded small and large mammalian herbivores and increased soil nutrients in two arctic Alaskan tundra communities: dry heath (DH) and moist acidic tussock (MAT). In the ninth year of manipulations, we measured weekly growth of three plant species of three growth forms: tussock‐forming graminoid, rhizomatous graminoid, and dwarf deciduous shrub, in each community. All species grew better when fertilized. In DH, this increase in growth was exaggerated when plants were protected from herbivores, confirming that herbivory had a negative effect on plant growth under increased nutrient conditions, but was unimportant under ambient soil conditions. However, in MAT, the importance of herbivory differed among species with fertilization. The tussock‐forming sedge at MAT, Eriophorum vaginatum, grew better and flowered more when fenced under both ambient and amended nutrients compared to plants exposed to herbivores. This species decreases in abundance in long‐term fertilized plots when mammals are present, and our results suggest that herbivory may be accounting for at least some of that loss, in addition to shifts in competitive relationships. Although we only focused on individual plants here rather than the entire community, our results suggest that under the increased soil nutrient conditions expected with continued climate warming in the Arctic, herbivores may become more important in affecting several abundant tundra plant populations, and should not be ignored.     

Viral Membrane Protein Topology Is Dictated by Multiple Determinants in Its Sequence

The targeting, insertion, and topology of membrane proteins have been extensively studied in both prokaryotes and eukaryotes. However, the mechanisms used by viral membrane proteins to generate the correct topology within cellular membranes are less well understood. Here, the effect of flanking charges and the hydrophobicity of the N-terminal hydrophobic segment on viral membrane protein topogenesis are examined systematically. Experimental data reveal that the classical topological determinants have only a minor effect on the overall topology of p9, a plant viral movement protein. Since only a few individual sequence alterations cause an inversion of p9 topology, its topological stability is robust. This result further indicates that the protein has multiple, and perhaps redundant, structural features that ensure that it always adopts the same topology. These critical topogenic sequences appear to be recognized and acted upon from the initial stages of protein biosynthesis, even before the ribosome ends protein translation.     

Demulsification of oil-rich emulsion from enzyme-assisted aqueous extraction of extruded soybean flakes

Extraction of soybean oil from flaked and extruded soybeans using enzyme-assisted aqueous extraction processing (EAEP) is a promising alternative to conventional hexane extraction. The efficiencies of four proteases releasing oil from extruded material were compared. Protex 51FP, Protex 6L and Protex 7L each extracted 90% of the total oil available while Protex 50FP gave similar extraction yield as the control (without enzyme treatment). During EAEP, however, a stable emulsion forms that must be broken in order to recover free soybean oil. The potential of various proteases and phospholipases to destabilize the emulsion was determined. Two enzymes, a phospholipase A2 (LysoMax) and a protease (Protex 51FP) were selected to determine the effect of enzyme concentration on demulsification. Although at a 2% concentration (w/w, enzyme/(cream+free oil)), each enzyme tested was effective in totally destabilizing the cream; the protease released significantly more free oil than did the phospholipase at concentrations less than 2%. At 0.2% concentration, 88 and 48% of free oil were obtained with the protease and phospholipase, respectively. Reducing the pH of the cream also destabilized the cream with maximum demulsification at the isoelectric point of soy proteins. These results provide destabilization strategies for the oil-rich emulsion formed during aqueous extraction processing of extruded flakes and significantly contribute to the development of this environmentally-friendly technology.     

High-cell density shake-flask expression and rapid purification of the large fragment of Thermus aquaticus DNA polymerase I using a new chemically and temperature inducible expression plasmid in Escherichia coli

We have developed a new expression vector, pcI^ts ind⁺, based upon the powerful rightward promoter of bacteriophage lambda, which is controlled by a temperature-sensitive and chemically-inducible version of the lambda repressor on the same plasmid. Locating the repressor gene on the plasmid makes this vector “portable” in that it can be used to transform any strain of Escherichia coli. Hence, control over strains, induction conditions, and harvest times can be used to optimize yields of heterologous proteins. To provide a proof of concept, we show that E. coli recA⁺ and recA⁻ host cells transformed with pcI^ts ind⁺ modKlenTaq1 (a modified version of the large fragment of Thermus aquaticus DNA polymerase I) could be grown to high cell densities in multiple shake-flasks. A mutant version of modKlenTaq1 (V649C) could be induced by simply raising the thermostat setting from 30 to 37 °C and (in the case of recA⁺ cells) adding nalidixic acid to achieve full induction (12–13% of the total cellular protein). Using a rapid, two-step purification process, it was possible to purify nearly 300 mg of modKlenTaq1 V649C from six 2.8-L baffle-bottomed shake-flasks each holding 1.5 L of culture for a final yield of approximately 33 mg per liter or 3 mg of purified enzyme per gram of cells wet weight.     

Extracellular Nm23H1 stimulates neurite outgrowth from dorsal root ganglia neurons in vitro independently of nerve growth factor supplementation or its nucleoside diphosphate kinase activity

The nucleoside diphosphate (NDP) kinase, Nm23H1, is a highly expressed during neuronal development, whilst induced over-expression in neuronal cells results in increased neurite outgrowth. Extracellular Nm23H1 affects the survival, proliferation and differentiation of non-neuronal cells. Therefore, this study has examined whether extracellular Nm23H1 regulates nerve growth. We have immobilised recombinant Nm23H1 proteins to defined locations of culture plates, which were then seeded with explants of embryonic chick dorsal root ganglia (DRG) or dissociated adult rat DRG neurons. The substratum-bound extracellular Nm23H1 was stimulatory for neurite outgrowth from chick DRG explants in a concentration-dependent manner. On high concentrations of Nm23H1, chick DRG neurite outgrowth was extensive and effectively limited to the location of the Nm23H1, i.e. neuronal growth cones turned away from adjacent collagen-coated substrata. Nm23H1-coated substrata also significantly enhanced rat DRG neuronal cell adhesion and neurite outgrowth in comparison to collagen-coated substrata. These effects were independent of NGF supplementation. Recombinant Nm23H1 (H118F), which does not possess NDP kinase activity, exhibited the same activity as the wild-type protein. Hence, a novel neuro-stimulatory activity for extracellular Nm23H1 has been identified in vitro, which may function in developing neuronal systems.     

The structure of the Lingo-1 ectodomain, a module implicated in central nervous system repair inhibition

Nogo receptor (NgR)-mediated control of axon growth relies on the central nervous system-specific type I transmembrane protein Lingo-1. Interactions between Lingo-1 and NgR, along with a complementary co-receptor, result in neurite and axonal collapse. In addition, the inhibitory role of Lingo-1 is particularly important in regulation of oligodendrocyte differentiation and myelination, suggesting that pharmacological modulation of Lingo-1 function could be a novel approach for nerve repair and remyelination therapies. Here we report on the crystal structure of the ligand-binding ectodomain of human Lingo-1 and show it has a bimodular, kinked structure composed of leucine-rich repeat (LRR) and immunoglobulin (Ig)-like modules. The structure, together with biophysical analysis of its solution properties, reveals that in the crystals and in solution Lingo-1 persistently associates with itself to form a stable tetramer and that it is its LRR-Ig-composite fold that drives such assembly. Specifically, in the crystal structure protomers of Lingo-1 associate in a ring-shaped tetramer, with each LRR domain filling an open cleft in an adjacent protomer. The tetramer buries a large surface area (9,200 A2) and may serve as an efficient scaffold to simultaneously bind and assemble the NgR complex components during activation on a membrane. Potential functional binding sites that can be identified on the ectodomain surface, including the site of self-recognition, suggest a model for protein assembly on the membrane.     

Geographic ranges, population structure, and ages of sexual and parthenogenetic snail lineages

Abstract A sexual reproduction is thought to doom organisms to extinction due to mutation accumulation and parasite exploitation. Theoretical models suggest that parthenogens may escape the negative effects of conspecific and biological enemiecs through escape in space. Through intensive sequencing of a mitochondrial DNA (mtDNA) and a nuclear intron locus in sexual and pathenogenetic freshwater snails (Campelom), I examine three questionss: (1) Are sexual mtDNA lineage more restricted geographically than parthenogenetic mtDNA lineages? (2) Are independent pathenogenetic lineages shorter lived than sexual lineages? (3) Do pathenogens have higher intraindividual nuclear sequence diversity and form well‐differentiated monophyletic groups as expected under the Meselson effect? Geographic ranges of parthenogenetic lineages are significantly larger than geographic ranges of sexual lineages. Based on coalescence times under different deographic assumptions, asexual lineages are short lived, but there is variation in clonal ages. Although alternative explanations exit, these results suggest that asexual lineages may persist in the short term through dispersal, and that various constraints may cause geographic restriction of sexual lineagess. Both allotriploid and diploid Campleloma parthenogens have significantly higher allelic divergence within individuals, but show limited nuclear sequence divergence from sexual ancestors. In contrast to previous allozyme evidence for nonhybrid origins of diploid Campeloma parthenogens, cryptic hybridization may account for elevated heterozygosity.