Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing

Deterioration of enhanced biological phosphorus removal (EBPR) has been linked to the proliferation of glycogen-accumulating organisms (GAOs), but few organisms possessing the GAO metabolic phenotype have been identified. An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional 16S rRNA gene cloning had failed. Therefore, rRNA-based stable isotope probing followed by full-cycle rRNA analysis was used to specifically identify the putative GAOs based on their characteristic metabolic phenotype. The study obtained sequences from a group of Alphaproteobacteria not previously shown to possess the GAO phenotype, but 90% identical by 16S rRNA gene analysis to a phylogenetic clade containing cloned sequences from putative GAOs and the isolate Defluvicoccus vanus. Fluorescence in situ hybridization (FISH) probes (DF988 and DF1020) were designed to target the new group and post-FISH chemical staining demonstrated anaerobic-aerobic cycling of polyhydroxyalkanoates, as per the GAO phenotype. The successful use of probes DF988 and DF1020 required the use of unlabelled helper probes which increased probe signal intensity up to 6.6-fold, thus highlighting the utility of helper probes in FISH. The new group constituted 33% of all Bacteria in the lab-scale bioreactor from which they were identified and were also abundant (51 and 55% of Bacteria) in two other similar bioreactors in which phosphorus removal had deteriorated. Unlike the previously identified Defluvicoccus-related organisms, the group identified in this study were also found in two full-scale treatment plants performing EBPR, suggesting that this group may be industrially relevant.

Reduction of the in vitro pro-inflammatory response by macrophages to poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

This study evaluates the pro-inflammatory response to the thermoplastic biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) through the analysis of cellular responses in vitro. The murine macrophage RAW264.7 cell line was cultured on solvent cast PHBV films, which was found to induce pro-inflammatory activity that required direct contact between the material and the macrophages. The identity of the pro-inflammatory stimulus was determined by culturing bone marrow-derived macrophages from bacterial lipopolysaccharide (LPS) hyporesponsive C3H/HeJ mice and CpG non-responsive TLR9-/- mice on PHBV. The lack of a pro-inflammatory response by the C3H/HeJ cells indicates that the pro-inflammatory agent present within PHBV is predominately LPS while the TLR9-/- macrophages confirmed that CpG-containing bacterial DNA is unlikely to contribute to the activity. A series of purification procedures was evaluated and one procedure was developed that utilized hydrogen peroxide treatment in solution. The optimized purification was found to substantially reduce the pro-inflammatory response to PHBV without adversely affecting either the molecular structure or molecular weight of the material thereby rendering it more amenable for use as a biomaterial in vivo. Crown Copyright (c) 2006 Published by Elsevier Ltd. All rights reserved.

Evaluation of immune effects of fowlpox vaccine strains and field isolates

The immune effects of fowlpox virus (FPV) field isolates and vaccine strains were evaluated in chickens infected at the age of 1 day and 6 weeks. The field isolates and the obsolete vaccine strain (FPV S) contained integrated reticuloendotheliosis virus (REV) provirus, while the current vaccine strain (FPVST) carries only REV LTR sequences. An indirect antibody ELISA was used to measure the FPV-specific antibody response. The non-specific humoral response was evaluated by injection of two T-cell-dependent antigens, sheep red blood cells (SRBC) and bovine serum albumin (BSA). There was no significant difference in the antibody response to FPV between chickens infected with FPV various isolates and strains at either age. In contrast, antibody responses to both SRBC and BSA were significantly lower in 1-day-old chickens inoculated with field isolates and FPV S at 2-3 weeks post-inoculation. Furthermore, cell-mediated immune (CMI) responses measured by in vitro lymphocyte proliferation assay and in vivo using a PHA-P skin test were significantly depressed in chickens inoculated with field isolates and FPV S at the same periods. In addition, thymus and bursal weights were lower in infected chickens. These immunosuppressive effects were not observed in chickens inoculated with the current vaccine strain, FPVST, at any time. The results of this study suggest that virulent field isolates and FPV S have immunosuppressive effects when inoculated into young chickens, which appeared in the first 3 weeks post infection. REV integrated in the FPV field isolates and FPV S may have played a central role in the development of immunosuppression. (c) 2006 Elsevier B.V. All rights reserved.

Cell passage-associated transient high oxygenation causes a transient decrease in cellular glutathione and affects T cell responses to apoptotic and mitogenic stimuli

Routine cell line maintenance involves removal of waste products and replenishment of nutrients via replacement of cell culture media. Here, we report that routine maintenance of three discrete cell lines (HSB-CCRF-2 and Jurkat T cells, and phaeo-chromocytoma PC12 cells) decreases the principal cellular antioxidant, glutathione, by up to 42% in HSB-CCRF-2 cells between 60 and 120 min after media replenishment. However, cellular glutathione levels returned to baseline within 5 h after passage. The decrease in glutathione was associated with modulation of the response of Jurkat T cells to apoptotic and mitogenic signals. Methotrexate-induced apoptosis over 16 h, measured as accumulation of apoptotic nucleoids, was decreased from 22 to 17% if cells were exposed to cytotoxic agent 30 min after passage compared with cells exposed to MTX in the absence of passage. In contrast, interleukin-2 (IL-2) production over 24 h in response to the toxin phytohaemagglutinin (PHA), was increased by 34% if cells were challenged 2 h after passage compared with PHA treatment in the absence of passage. This research highlights the presence of a window of time after cell passage of non-adherent cells that may lead to over- or under-estimation of subsequent cell responses to toxins, which is dependent on cellular antioxidant capacity or redox state. © 2007 Elsevier B.V. All rights reserved.

The fabrication of biodegradable nanospheres from novel hydroxalkanoates for the delivery of actives of pharmaceutical and agricultural interest

This work describes the fabrication of nanospheres from a range of novel polyhydroxyalkanoates supplied by Monsanto, St Louis, Missouri, USA for the delivery of selected actives of both pharmaceutical and agricultural interest. Initial evaluation of established microsphere and nanosphere fabrication techniques resulted in the adoption and optimisation of a double sonication solvent evaporation method involving the synperonic surfactant F68. Nanospheres could be consistently generated with this method. Studies on the incorporation and release of the surrogate protein Bovine Serum Albumin V demonstrated that BSA could be loaded with between 10-40% w/w BSA without nanosphere destabilisation. BSA release from nanospheres into Hanks Balanced Salts Solution, pH 7.4, could be monitored for up to 28 days at 37°C. The incorporation and release of the Monsanto actives - the insecticide Admire® ({ 1-[(6-chloro-3-pyridinyl)methyIJ-N-nitro-2-imidazolidinimine}) and the plant growth hormone potassium salt Gibberellic acid (GA3K) from physico-chemically characterised polymer nanospheres was monitored for up to 37 days and 28 days respectively, at both 4°C and 23°C. Release data was subsequently fitted to established kinetic models to elaborate the possible mechanisms of release of actives from the nanospheres. The exposure of unloaded nanospheres to a range of physiological media and rural rainwater has been used to investigate the role polymer biodegradation by enzymatic and chemical means might play in the in vivo release of actives and agricultural applications. The potential environmental biodegradation of Monsanto polymers has been investigated using a composting study (International Standard ISO/FDIS 14855) in which the ultimate aerobic biodegradation of the polymers has been monitored by the analysis of evolved carbon dioxide. These studies demonstrated the potential of the polymers for use in the environment, for example as a pesticide delivery system.

Macrophages and lymphocyte responiveness to mitogen

Purified B-cells fail to proliferate in response to the strong thymus-independent (TI) antigen Lipopolysaccharide (LPS) in the absence of macrophages (Corbel and Melchers, 1983). The fact that macrophages, or factors derived from them are required is supported by the inability of marginal zone B-cells in infants to respond to highly virulent strains of bacteria such as Neisseria meningitidis and Streptococcus pneumoniae (Timens, 1989). This may be due to the lack of CD21 expression on B-cells in infants which could associate with its co-receptor (C3d) on adjacent macrophages. It is not clear whether cell surface contacts and/or soluble products are involved in lymphocyte-macrophage interactions in response to certain antigens. This thesis describes the importance of the macrophage in lymphocyte responses to T-dependent (TD) and TI antigens. The major findings of this thesis were as follows: (1). Macrophages were essential for a full proliferative response to a range of T - and B-cell mitogens and TI-1 and TI-2 antigens, including Concanavalin A, LPS, Pokeweed mitogen (PWM), Dextran sulphate, Phytohaemagglutinin-P (PHA-P) and Poly[I][C]. (2). A ratio of 1 macrophage to 1000 lymphocytes was sufficient for the mitogens to exert their effects. (3). The optimal conditions were established for the activation of an oxidative burst in cells of the monocyte/macrophage lineage as measured by luminometry. The order of ability was OpZ >PMA/lonomycin >f-MLP >Con A >DS >PHA >Poly[I][C] >LPS >PWM. Responses were only substantial and protracted with OpZ and PMA. Peritoneal macrophages were the most responsive cells, whereas splenic and alveolar macrophages were significantly less active and no response could be elicited with Kupffer cells, thus demonstrating heterogeneity between macrophages. (4). Activated macrophages that were then fixed with paraformaldehyde were unable to restore mitogenic responsiveness, even with a ratio of 1 macrophage to 5 lymphocytes. (5). Although highly purified T- and B-cells could respond to mitogen provided live macrophages were present, maximum activation was only observed when all 3 cell types were present. (6). Supernatants from purified macrophage cultures treated with a range of activators were able to partially restore lymphocyte responses to mitogen in macrophage-depleted splenocyte cultures, and purified T - and B-cell cultures. In fact supernatants from macrophages treated with LPS for only 30 minutes could restore responsiveness. Supernatants from OpZ treated macrophages were without effect. (7). Macrophage supernatants could not induce proliferation in the absence of mitogen. They therefore provide a co-mitogenic signal required by lymphocytes in order to respond to mitogen. (8). Macrophage product profiles revealed that LPS and Con A-treated macrophage supernatants showed elevated levels of IL-1β, TNF -α L TB4 and TXB2. These products were therefore good candidates as the co-mitogenic factor. The possible inhibitory factors secreted by OpZ-treated macrophages were PGE2, IL-10 and NO. (9). The removal of cytokines, eicosanoids and TNF-α from LPS-treated macrophage supernatants using Cycloheximide, Dexamethasone and an MMPI respectively, resulted in the inability of these supernatants to restore macrophage-depleted lymphocyte responses to mitogen. (10). rIL-1β and rTNF-α are co-mitogenic factors, as macrophage-depleted lymphocytes incubated with rIL-1β and rTNF-α can respond to mitogen.

Ceramide and reactive oxygen species (ROS) as signal transduction molecules in inflammation

Reactive oxygen species (ROS) and the sphingolipid ceramide are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. Furthermore, the enhanced production of many of these agents, that utilise ROS and ceramide as signalling intermediates, is associated with the aetiologies of several vascular diseases (e.g. atherosclerosis) or disorders of inflammatory origin (e.g. rheumatoid arthritis; RA). Excessive monocyte recruitment and uncontrolled T cell activation are both strongly implicated in the chronic inflammatory responses that are associated with these pathologies. Therefore the aims of this thesis are (1) to further elucidate the cellular responses to modulations in intracellular ceramide/ROS levels in monocytes and T cells, in order to help resolve the mechanisms of progression of these diseases and (2) to examine both existing agents (methotrexate) and novel targets for possible therapeutic manipulation. Utilising synthetic, short chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide or, stimulation of CD95 to induce ceramide formation, it is described here that ceramide targets and manipulates two discrete sites responsible for ROS generation, preceding the cellular responses of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells. In both cell types, transient elevations in mitochondrial ROS generation were observed. However, the prominent redox altering effects appear to be the ceramide-mediated reduction in cytosolic peroxide, the magnitude of which dictates in part the cellular response in U937 monocytes, Jurkat T-cells and primary human peripheral blood resting or PHA-activated T-cells in vitro. The application of synthetic ceramides to U937 monocytes for short (2 hours) or long (16 hours) treatment periods reduced the membrane expression of proteins associated with cell-cell interaction. Furthermore, ceramide treated U937 monocytes demonstrated reduced adhesion to 5 or 24 hour LPS activated human umbilical vein endothelial cells (HUVEC) but not resting HUVEC. Consequently it is hypothesised that the targeted treatment of monocytes from patients with cardiovascular diseases with short chain synthetic ceramide may reduce disease progression. Herein, the anti-inflammatory and immunosuppressant drug, methotrexate, is described to require ROS production for the induction of cytostasis or cytotoxicity in U937 monocytes and Jurkat T-cells respectively. Further, ROS are critical for methotrexate to abrogate monocyte interaction with activated HUVEC in vitro. The histological feature of RA of enhanced infiltration, survivability and hyporesponsiveness of T-cells within the diseased synovium has been suggested to arise from aberrant signalling. No difference in the concentrations of endogenous T-cell ceramide, the related lipid diacylglycerol (DAG) and cytosolic peroxide ex vivo was observed. TCR activation following PHA exposure in vitro for 72 hours did not induced maintained perturbations in DAG or ceramide in T-cells from RA patients or healthy individuals. However, T-cells from RA patients failed to upregulate cytosolic peroxide in response to PHA, unlike those from normals, despite expressing identical levels of the activation marker CD25. This inability to upregulate cytosolic peroxide may contribute to the T-cell pathology associated with RA by affecting the signalling capacity of redox sensitive biomolecules. These data highlight the importance of two distinctive cellular pools of ROS in mediating complex biological events associated with inflammatory disease and suggest that modulation of cellular ceramides represents a novel therapeutic strategy to minimise monocyte recruitment.

Immunological studies in multiple low dose streptozotocin induced diabetes in mice

1. Multiple low doses of streptozotocin (MSZ) treatment successfully induced diabetes in male TO, MFI and HO lean mice. In contrast however, BALB/c mice failed to develop persistent hyperglycaemia. Single streptozotocin (SSZ) treatment also produced diabetes in TO mice. SSZ treatment however, produced severe weight loss and atrophy of the lymphoid organs. MSZ treatment on the other hand, was not cytotoxic towards lymphoid organs and, whilst there was no loss of body weight, growth rates were reduced in MSZ treated mice. 2. Following sheep red blood cell (SRBC) immunisation of MSZ-treated mice, haemagglutination titres, and numbers of antigen reactive cells and plaque forming cells were all significantly lower than control values. 3. In vitro proliferation of spleen cells in response to phytohaemagglutinin (PHA) and conconavalin A (ConA) was found to be significantly depressed in MSZ treated mice. However, T-lymphocyte responses were intact when the mice were not overtly hyperglycaemic. In contrast, however, T cell independent responses to lipopolysaccharide (LPS) were generally intact throughout the study period. 4. Cell mediated immunity, as assessed by measurements of delayed (Type IV) hypersensitivity, was also depressed in MSZ treated mice. This suppression could be reversed by insulin therapy. 5. Both natural killer cell activity and antibody dependent cell mediated cytotoxicity were found to be significantly increased in MSZ treated mice. 6. Histological examination of the pancreas showed the presence of insulitis, in MSZ treated mice, and cytotoxic effector cells against obese mice islet cells (as assessed by 51Cr release) and HIT-T15 cells (as assessed by insulin secretion) were found to be significantly increased. Furthermore, these effector cells were also found to show increased proliferation in the presence of homogenates prepared from HIT-T15 cells. Examination of the Sera from MSZ treated mice showed that islet cell surface antibodies were present.

Synthesis and application of novel probes for the detection of cysteine sulphenic acids

Cysteine is a thiol containing amino acid that readily undergoes oxidation by reactive oxygen species (ROS) to form sulphenic (R-SOH) sulphinic (RSO2H) and sulphonic (RSO3H) acids. Thiol modifications of cysteine have been implicated as modulators of cellular processes and represent significant biological modifications that occur during oxidative stress and cell signalling. However, the different oxidation states are difficult to monitor in a physiological setting due to the limited availability of experimental tools. Therefore it is of interest to synthesise and use a chemical probe that selectively recognises the reversible oxidation state of cysteine sulphenic acid to understand more about oxidative signalling. The aim of this thesis was to investigate a synthetic approach for novel fluorescent probe synthesis, for the specific detection of cysteine sulphenic acids by fluorescence spectroscopy and confocal microscopy. N-[2-(Anthracen-2-ylamino)-2-oxoethyl]-3,5-dioxocyclohexanecarboxamide was synthesised in a multistep synthesis and characterised by nuclear magnetic resonance spectroscopy. The optimisation of conditions needed for sulphenic acid formation in a purified protein using human serum albumin (HSA) and the commercially available biotin tagged probe 3-(2,4-dioxocyclohexyl)propyl-5-((3aR,6S,6aS)-hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-6-yl)pentanoate (DCP-Bio1) were identified. This approach was extended to detect sulphenic acids in Jurkat T cells and CD4+ T cells pre- and post-stimulus. Buthionine sulfoximine (BSO) was used to manipulate the endogenous antioxidant glutathione (GSH) in human CD4+ T cells. Then the surface protein thiol levels and sulphenic acid formation was examined. T cells were also activated by the lectin phytohaemagglutinin-L (PHA-L) and formation of sulphenic acid was investigated using SDS-PAGE, western blotting and confocal microscopy. Resting Jurkat cells have two prominent protein bands that have sulphenic acid modifications whereas resting CD4+ T cells have an additional band present. When cells were treated with BSO the number of bands increased whereas activation reduced the number of proteins that were modified. The identities of the protein bands containing sulphenic acids were explored by mass spectrometry. Cysteine oxidation was observed in redox, metabolic and cytoskeletal proteins. In summary, a novel fluorescent probe for detection of cysteine sulphenic acids has been synthesised alongside a model system that introduces cysteine sulphenic acid in primary T cells. This probe has potential application in the subcellular localisation of cysteine oxidation during T cell signalling.

Cytokine analysis in Atlantic bottlenose dolphins: Molecular characterization of IL-4, IL-6, and IL-10

The health status of wild and captive Atlantic Bottlenose dolphins ( Tersiops truncatis) is difficult to ascertain. Mass strandings of these animals have been attributed to pollutants, as well as bacterial infections. Using human Enzyme Linked Immuno-Assays (ELISA) for immunological cytokines, I measured soluble cytokine levels with respect to their health status. In a retrospective analysis of dolphin sera, there was a trend of higher cytokine levels in “sick” animals. I cultured dolphin lymphocytes in the presence of a mitogen (PHA), a super antigen (Staph-A), Lipopolysaccharide (LPS), and a calcium flux inducer (PMA). Levels of messenger RNA, from these cultured cells, were assayed with Polymerase Chain Reaction (PCR) using primers for the human cytokines IL-2, IL-4, IL-6, IL-10, Tumor Necrosis Factor, and Interferon gamma. Only IL-4, IL-6, and IL-10 messages were obtained, inferring similar nucleotide homology to the human primer sequences. The PCR products were sequenced. Sixteen IL-4 sequences, twelve IL-6 sequences and seven IL-10 sequences were obtained and analyzed. Each cytokine exhibited the same nucleotide sequence in all dolphins examined. There was no difference in the cytokine profile in response to the various stimuli. The derived amino acid composition for each of the dolphin cytokines was used for molecular modeling, which showed that dolphin IL-4, IL-6, and IL-10 were structurally similar to the corresponding proteins of Perissodactyla. ^

Avaliação do potencial da fibra e casca de coco maduro, casca de coco verde e cacto pré-tratados visando à produção de etanol

The present work investigated the potential of different residual lignocellulosic materials generated in rural and urban areas (coconut fibre mature, green coconut shell and mature coconut shell), and vegetable cultivated in inhospitable environments (cactus) aimed at the production of ethanol, being all materials abundant in the Northeast region of Brazil. These materials were submitted to pretreatments with alkaline hydrogen peroxide followed by sodium hydroxide (AHP-SHP), autohydrolysis (AP), hydrothermal catalyzed with sodium hydroxide (HCSHP) and alkali ethanol organosolv (AEOP). These materials pretreated were submitted to enzymatic hydrolysis and strategies of simultaneous saccharification and fermentation (SSF) and saccharification and fermentation semi-simultaneous (SSSF) by Saccharomyces cerevisiae, Zymomonas mobilis and Pichia stipitis. It was also evaluated the presence of inhibitory compounds (hydroxymethylfurfural, furfural, acetic acid, formic acid and levulinic acid) and seawater during the fermentative process. Materials pretreated with AHP-SHP have resulted in delignification of the materials in a range between 54 and 71%, containing between 51.80 and 54.91% of cellulose, between 17.65 and 28.36% of hemicellulose, between 7.99 and 10.12% of lignin. Enzymatic hydrolysis resulted in the conversions in glucose between 68 and 76%. Conversion yields in ethanol using SSF and SSSF for coconut fibre mature pretreated ranged from 0.40 and 0.43 g/g, 0.43 and 0.45 g/g, respectively. Materials pretreated by AP showed yields of solids between 42.92 and 92.74%, containing between 30.65 and 51.61% of cellulose, 21.34 and 41.28% of lignin. Enzymatic hydrolysis resulted in glucose conversions between 84.10 and 92.52%. Proceeds from conversion into ethanol using green coconut shell pretreated, in strategy SSF and SSSF, were between 0.43 and 0.45 g/g. Coconut fibre mature pretreated by HCSHP presented solids yields between 21.64 and 60.52%, with increased in cellulose between 28.40 and 131.20%, reduction of hemicellulose between 43.22 and 69.04% and reduction in lignin between 8.27 and 89.13%. Enzymatic hydrolysis resulted in the conversion in glucose of 90.72%. Ethanol yields using the SSF and SSSF were 0.43 and 0.46 g/g, respectively. Materials pretreated by AEOP showed solid reductions between 10.75 and 43.18%, cellulose increase up to 121.67%, hemicellulose reduction up to 77.09% and lignin reduced up to 78.22%. Enzymatic hydrolysis resulted in the conversion of glucose between 77.54 and 84.27%. Yields conversion into ethanol using the SSF and SSSF with cactus pretreated ranged from 0.41 and 0.44 g/g, 0.43 and 0.46 g/g, respectively. Fermentations carried out in bioreactors resulted in yields and ethanol production form 0.42 and 0.46 g/g and 7.62 and 12.42 g/L, respectively. The inhibitory compounds showed negative synergistic effects in fermentations performed by P. stipitis, Z. mobilis and S. cerevisiae. Formic acid and acetic acid showed most significant effects among the inhibitory compounds, followed by hydroxymethylfurfural, furfural and levulinic acid. Fermentations carried out in culture medium diluted with seawater showed promising results, especially for S. cerevisiae (0.50 g/g) and Z. mobilis (0.49 g/g). The different results obtained in this study indicate that lignocellulosic materials, pretreatments, fermentative processes strategies and the microorganisms studied deserve attention because they are promising and capable of being used in the context of biorefinery, aiming the ethanol production.

Avaliação da influência de surfactantes químico e biológico na hidrólise enzimática de casca de coco verde após pré-tratamento ácido/alcalino e com peróxido de hidrogênio alcalino

In Brazil many types of bioproducts and agroindustrial waste are generated currently, such as cacashew apple bagasse and coconut husk, for example. The final disposal of these wastes causes serious environmental issues. In this sense, waste lignocellulosic content, as the shell of the coconut is a renewable and abundant raw material in which its use has an increased interest mainly for the 2nd generation ethanol production. The hydrolysis of cellulose to reducing sugars such as glucose and xylose is catalysed by a group of enzymes called cellulases. However, the main bottleneck in the enzymatic hydrolysis of cellulose is the significant deactivation of the enzyme that shows irreversible adsorption mechanism leading to reduction of the cellulose adsorption onto cellulose. Studies have shown that the use of surfactants can modify the surface property of the cellulose therefore minimizing the irreversible binding. The main objective of the present study was to evaluate the influence of chemical and biological surfactants during the hydrolysis of coconut husk which was subjected to two pre-treatment in order to improve the accessibility of the enzymes to the cellulose, removing this way, part of the lignin and hemicellulose present in the structure of the material. The pre-treatments applied to coconut bagasse were: Acid/Alkaline using 0.6M H2SO4 followed by 1M NaOH, and the one with Alkaline Hydrogen Peroxide at a concentration of 7.35% (v/v) and pH 11.5. Both the material no treatment and pretreated were characterized using analysis of diffraction X-ray (XRD), Scanning Electron Microscopy (SEM) and methods established by NREL. The influence of both surfactants, chemical and biological, was used at concentrations below the critical micelle concentration (CMC), and the concentrations equal to the CMC. The application of pre-treatment with coconut residue was efficient for the conversion to glucose, as well as for the production of total reducing sugars, it was possible to observe that the pretreatment fragmented the structure as well as disordered the fibers. Regarding XRD analysis, a significant increase in crystallinity index was observed for pretreated bagasse acid/alkali (51.1%) compared to the no treatment (31.7%), while that for that treated with PHA, the crystallinity index was slightly lower, around 29%. In terms of total reducing sugars it was not possible to observe a significant difference between the hydrolysis carried out without the use of surfactant compared to the addition of Triton and rhamnolipid. However, by observing the conversions achieved during the hydrolysis, it was noted that the best conversion was using the rhamnolipíd for the husk pretreated with acid/alkali, reaching a value of 33%, whereas using Triton the higher conversion was 23.8%. The coconut husk is a residue which can present a high potential to the 2nd generation ethanol production, being the rhamonolipid a very efficient biosurfactant for use as an adjuvant in the enzymatic process in order to act on the material structure reducing its recalcitrance and therefore improving the conditions of access for enzymes to the substrate increasing thus the conversion of cellulose to glucose.

Avaliação da influência de surfactantes químico e biológico na hidrólise enzimática de casca de coco verde após pré-tratamento ácido/alcalino e com peróxido de hidrogênio alcalino

In Brazil many types of bioproducts and agroindustrial waste are generated currently, such as cacashew apple bagasse and coconut husk, for example. The final disposal of these wastes causes serious environmental issues. In this sense, waste lignocellulosic content, as the shell of the coconut is a renewable and abundant raw material in which its use has an increased interest mainly for the 2nd generation ethanol production. The hydrolysis of cellulose to reducing sugars such as glucose and xylose is catalysed by a group of enzymes called cellulases. However, the main bottleneck in the enzymatic hydrolysis of cellulose is the significant deactivation of the enzyme that shows irreversible adsorption mechanism leading to reduction of the cellulose adsorption onto cellulose. Studies have shown that the use of surfactants can modify the surface property of the cellulose therefore minimizing the irreversible binding. The main objective of the present study was to evaluate the influence of chemical and biological surfactants during the hydrolysis of coconut husk which was subjected to two pre-treatment in order to improve the accessibility of the enzymes to the cellulose, removing this way, part of the lignin and hemicellulose present in the structure of the material. The pre-treatments applied to coconut bagasse were: Acid/Alkaline using 0.6M H2SO4 followed by 1M NaOH, and the one with Alkaline Hydrogen Peroxide at a concentration of 7.35% (v/v) and pH 11.5. Both the material no treatment and pretreated were characterized using analysis of diffraction X-ray (XRD), Scanning Electron Microscopy (SEM) and methods established by NREL. The influence of both surfactants, chemical and biological, was used at concentrations below the critical micelle concentration (CMC), and the concentrations equal to the CMC. The application of pre-treatment with coconut residue was efficient for the conversion to glucose, as well as for the production of total reducing sugars, it was possible to observe that the pretreatment fragmented the structure as well as disordered the fibers. Regarding XRD analysis, a significant increase in crystallinity index was observed for pretreated bagasse acid/alkali (51.1%) compared to the no treatment (31.7%), while that for that treated with PHA, the crystallinity index was slightly lower, around 29%. In terms of total reducing sugars it was not possible to observe a significant difference between the hydrolysis carried out without the use of surfactant compared to the addition of Triton and rhamnolipid. However, by observing the conversions achieved during the hydrolysis, it was noted that the best conversion was using the rhamnolipíd for the husk pretreated with acid/alkali, reaching a value of 33%, whereas using Triton the higher conversion was 23.8%. The coconut husk is a residue which can present a high potential to the 2nd generation ethanol production, being the rhamonolipid a very efficient biosurfactant for use as an adjuvant in the enzymatic process in order to act on the material structure reducing its recalcitrance and therefore improving the conditions of access for enzymes to the substrate increasing thus the conversion of cellulose to glucose.

Distribution of diatoms, coccolithophores and planktic foraminifera in the Arabian Sea

The distribution of diatoms, coccolithophores and planktic foraminifers mirrored the hydrographic and trophic conditions of the surface ocean (0-100 m) across the upwelling area off the Oman coast to the central Arabian Sea during May/June 1997 and July/August 1995. The number of diatoms was increased in waters with local temperature minimum and enhanced nutrient concentration (nitrate, phosphate, silicate) caused by upwelling. Vegetative cells of Chaetoceros dominated the diatom assemblage in the coastal upwelling area. Towards the more nutrient depleted and stratified surface waters to the southeast, the number of diatoms decreased, coccolithophore and planktic foraminiferal numbers increased, and floral and faunal composition changed. In particular, the transition between the eutrophic upwelling region off Oman and the oligotrophic central Arabian Sea was marked by moderate nutrient concentration, and high coccolithophore and foraminifer numbers. Florisphaera profunda, previously often referred as a 'lower-photic-zone-species', was frequent in water depths as shallow as 20 m, and at high nutrient concentration up to 14 µmol NO3/l and 1.2 µmol PO4/. To the oligotrophic southeast of the divergence, cell densities of coccolithophores declined and Umbellosphaera irregularis prevailed throughout the water column down to 100 m depth. In general, total coccolithophore numbers were limited by nutrient threshold concentration, with low numbers (<10*10**3 cells/l) at high [NO3] and [PO4], and high numbers (>70*10**3 cells/l) at low [NO3] and [PO4]. The components of the complex microplankton succession, diatoms, coccoliths and planktic foraminifers (and possibly others), should ideally be used as a combined paleoceanographic proxy. Consequently, models on plankton ecology should be resolved at least for the seasonality, to account for the bias of paleoceanographic transfer calculations.