Degradative solvent extraction of cyanobacteria: From reaction kinetics to potential organic matter evolution mechanism.

This study proposed a new continuous lumped reaction kinetics model to accurately reveal the control mechanism of cyanobacteria at each stage of degradative solvent extraction and discussed the potential evolution mechanism of organic matter. Results showed that degradation solvent extraction successfully separated nitrogen and phosphorus from cyanobacteria. The solute has high carbon and volatile contents, is almost ash-free, and forms a phosphorus-rich residue. The lowest fitting degree of the continuous lumped reaction model kinetics was 94.5%, suggesting that this model worked well. The depolymerization of the residue dominated between 200 and 350 °C, whereas solute decomposition dominated at 400 °C. Nitrogen-containing compounds, which originate from protein decarboxylation or deamination to generate amides, are the main components of the solute, and amino acids react with reducing sugars to generate nitrogen heterocyclic compounds, which are useful for preparing nitrogen-containing chemicals.

Biodegradation of 2-methylpentane in fluid fine tailings amended with a mixture of iso-alkanes under sulfate-reducing conditions.

Anaerobic microorganisms in Canada Natural Upgrading Limited (CNUL) fluid fine tailings (FFT) are sustained by residual solvent hydrocarbons. Although FFT are methanogenic in nature, sulfate-reducing microorganisms represent a significant portion of FFT bacterial community. In this study, we examined biodegradation of three iso-alkanes (2-methylbutane, 2-methylpentane, and 3-methylpentane), representing major iso-alkanes in paraffinic solvent, in CNUL FFT under sulfate-reducing conditions. During ∼1100 days of incubation, only 2-methylpentane was degraded partially, whereas 2-methylbutane and 3-methylpentane were not degraded. During active degradation of 2-methylpentane, the bacterial community was dominated by Anaerolineaceae followed by Syntrophaceae, Peptococcaceae, Desulfobacteraceae, and Desulfobulbaceae. The archaeal community was co-dominated by acetoclastic (Methanosaetaceae) and hydrogenotrophic (Methanobacteriaceae) methanogens. This study underlines the limited capability of the microbial community indigenous to CNUL FFT in degrading recalcitrant iso-alkanes under sulfate-reducing conditions.

Discovery of New Phenylacetone Monooxygenase Variants for the Development of Substituted Indigoids through Biocatalysis.

Indigoids are natural pigments obtained from plants by ancient cultures. Romans used them mainly as dyes, whereas Asian cultures applied these compounds as treatment agents for several diseases. In the modern era, the chemical industry has made it possible to identify and develop synthetic routes to obtain them from petroleum derivatives. However, these processes require high temperatures and pressures and large amounts of solvents, acids, and alkali agents. Thus, enzyme engineering and the development of bacteria as whole-cell biocatalysts emerges as a promising green alternative to avoid the use of these hazardous materials and consequently prevent toxic waste generation. In this research, we obtained two novel variants of phenylacetone monooxygenase (PAMO) by iterative saturation mutagenesis. Heterologous expression of these two enzymes, called PAMOHPCD and PAMOHPED, in E. coli was serendipitously found to produce indigoids. These interesting results encourage us to characterize the thermal stability and enzyme kinetics of these new variants and to evaluate indigo and indirubin production in a whole-cell system by HPLC. The highest yields were obtained with PAMOHPCD supplemented with L-tryptophan, producing ~3000 mg/L indigo and ~130.0 mg/L indirubin. Additionally, both enzymes could oxidize and produce several indigo derivatives from substituted indoles, with PAMOHPCD being able to produce the well-known Tyrian purple. Our results indicate that the PAMO variants described herein have potential application in the textile, pharmaceutics, and semiconductors industries, prompting the use of environmentally friendly strategies to obtain a diverse variety of indigoids.

Impact of winter savory extract (Satureja montana L.) on biochemical parameters in serum and oxidative status of liver with application of the principal component analysis in extraction solvent selection.

OBJECTIVE: Satureja montana L. is traditionally used as spice and for treatment various diseases. Many studies have shown antioxidative effect of Satureja species. Our thorough study in an animal model was performed through measurement of biochemical parameters in the serum, histology analysis and determination of oxidative status of the liver, coupled with investigation of extraction solvent selection using principal component analysis (PCA). MATERIALS AND METHODS: Winter savory dry extract (500 mg/kg) dispersion and saline solution were given to Wistar rats for 7 days after exposure to oxidative stress using toxic doses of paracetamol (600 mg/kg). Rats were sacrificed, after which a complete autopsy was performed, the blood obtained was used to determine biochemical parameters, and the liver was sliced for histological analysis and determination of oxidative stress enzymes. RESULTS: Indicators of hepatic and kidney functions, as well as the concentration of oxidative stress enzymes, were statistically significantly lower in animals treated with Satureja montana L. extract compared to the paracetamol group alone before the toxic dose of paracetamol. Liver enzymes were unaltered by pre-treatment with the extract, but the level of lipid peroxidase was decreased, and the level of catalase, glutathione reductase and superoxide dismutase increased proving in vivo antioxidant effect. In addition, the number of inflammatory cells is decreased coupled with activity of CYP2E1 enzymes proving hepatoprotective effect. CONCLUSIONS: Satureja montana L. extract in our research has shown hepatoprotective, anti-inflammatory and antioxidative effect. PCA analyses indicated that extraction mediums have a great impact on the antioxidative effect.

Pressurized Natural Deep Eutectic Solvent Extraction of Galanthamine and Related Alkaloids from Narcissus pseudonarcissus.

The isolation of a compound from a natural source involves many organic and mostly toxic solvents for extraction and purification. Natural deep eutectic solvents have been shown to be efficient options for the extraction of natural products. They have the advantage of being composed of abundantly available common primary metabolites, being nontoxic and environmentally safe solvents. The aim of this study was to develop a natural deep eutectic solvent-based extraction method for galanthamine, an important therapeutic agent for the treatment of Alzheimer's disease. This alkaloid can be produced by synthesis or by extraction from Narcissus bulbs. To develop an efficient extraction method, a number of different natural deep eutectic solvents was first tested for their solubilization capacity of galanthamine bromide salt. Promising results were obtained for ionic liquids, as well as some amphoteric and acidic natural deep eutectic solvents. In a two-cycle extraction process, the best solvents were tested for the extraction of galanthamine from bulbs. The ionic liquids produced poor yields, and the best results were obtained with some acid and sugar mixtures, among which malic acid-sucrose-water (1 : 1 : 5) proved to be the best, showing similar yields to that of the exhaustive Soxhlet extraction with methanol. Furthermore, the natural deep eutectic solvent was more selective for galanthamine.

A metabolic study to decipher amino acid catabolism-directed biofuel synthesis in Acetoanaerobium sticklandii DSM 519.

Acetoanaerobium sticklandii DSM 519 is a hyper-ammonia-producing anaerobe. It has the ability to produce organic solvents and acids from protein catabolism through Stickland reactions and specialized pathways. Nevertheless, its protein catabolism-directed biofuel production has not yet been understood. The present study aimed to decipher such growth-associated metabolic potential of this organism at different growth phases using metabolic profiling. A seed culture of this organism was grown separately in metabolic assay media supplemented with gelatin and or a mixture of amino acids. The extracellular metabolites produced by this organism were qualitatively analyzed by gas chromatography-mass spectrometry platform. The residual amino acids after protein degradation and amino acids assimilation were identified and quantitatively measured by high-performance liquid chromatography (HPLC). Organic solvents and acids produced by this organism were detected and the quantity of them determined with HPLC. Metabolic profiling data confirmed the presence of amino acid catabolic products including tyramine, cadaverine, methylamine, and putrescine in fermented broth. It also found products including short-chain fatty acids and organic solvents of the Stickland reactions. It reported that amino acids were more appropriate for its growth yield compared to gelatin. Results of quantitative analysis of amino acids indicated that many amino acids either from gelatin or amino acid mixture were catabolised at a log-growth phase. Glycine and proline were poorly consumed in all growth phases. This study revealed that apart from Stickland reactions, a specialized system was established in A. sticklandii for protein catabolism-directed biofuel production. Acetone-butanol-ethanol (ABE), acetic acid, and butyric acid were the most important biofuel components produced by this organism. The production of these components was achieved much more on gelatin than amino acids. Thus, A. sticklandii is suggested herein as a potential organism to produce butyric acid along with ABE from protein-based wastes (gelatin) in bio-energy sectors.

Enhanced and green extraction of bioactive compounds from Lippia citriodora by tailor-made natural deep eutectic solvents.

Natural deep eutectic solvents (NADESs) have been postulated as alternative green solvents for the isolation of valuable bioactive compounds from Lippia citriodora. Thus, 11 different NADESs, based on choline chloride (ChCl) as the hydrogen bond acceptor in combination with different hydrogen bond donors (organic acids, polyalcohols, sugars, and urea) were tested. According to the results obtained, ChCl-lactic acid exhibited the highest extraction yield for iridoids, 7.25 mg g-1, phenylpropanoids, 17.23 mg g-1, and flavonoids, 9.02 mg g-1 being significantly greater than phenylpropanoid and flavonoid yields, 15.63 and 5.43 mg g-1 respectively, extracted with methanol as conventional solvent. Subsequently, in order to optimise the most influential microwave assisted extraction (MAE) parameters, a Box-Behnken design paired with a response surface methodology were implemented. Temperature and water content showed a strong effect on the extraction of polyphenol sub-classes, while the effect of irradiation time was less noticeable on extraction yields. Temperature of 63.68 °C, a water content of 32.19% and a microwave irrdiation time of 17.08 min were the optimum conditions provided by the statistical program. The use of NADESs showed potential to facilitate the design and customisation of green tailor-made solvents which have greater extraction capacity than conventional organic solvents.

Mutants of Yarrowia lipolytica NCIM 3589 grown on waste cooking oil as a biofactory for biodiesel production.

BACKGROUND: Oleaginous yeasts are fast emerging as a possible feedstock for biodiesel production. Yarrowia lipolytica, a model oleaginous yeast is known to utilize a variety of hydrophobic substrates for lipid accumulation including waste cooking oil (WCO). Approaches to increase lipid content in this yeast include metabolic engineering which requires manipulation of multiple genes in the lipid biosynthesis pathway. A classical and cost-effective approach, namely, random chemical mutagenesis on the yeast can lead to increased production of biodiesel as is explored here. RESULTS: In this study, chemical mutagenesis using the alkylating agent, N- methyl-N'-nitro-N-nitrosoguanidine (MNNG) as well as an additional treatment with cerulenin, a fatty acid synthase inhibitor generated 800 mutants of Y. lipolytica NCIM 3589 (761 MNNG treated and 39 MNNG + cerulenin treated). A three-stage screening using Sudan Black B plate technique, Nile red fluorimetry and total lipid extraction using solvent was performed, which enabled selection of ten high lipid yielding mutants. Time course studies of all the ten mutants were further undertaken in terms of biomass, lipid yield and lipid content to select three stable mutants (YlB6, YlC7 and YlE1) capable of growing and accumulating lipid on WCO, with lipid contents of 55, 60 and 67% as compared to 45% for the wild type. The mutants demonstrated increased volumetric lipid productivities (0.062, 0.044 and 0.041 g L-1 h-1) as compared to the wild type (0.033 g L-1 h-1). The fatty acid profile of the three mutants consisted of a high content of C16 and C18 saturated and monounsaturated fatty acids and was found to be suitable for biodiesel production. The fuel properties, namely, density, kinematic viscosity, total acid number, iodine value of the three mutants were evaluated and found to lie within the limits specified by internationally accepted standards. Additionally, it was noted that the mutants demonstrated better cetane numbers and higher heating values than the wild type strain. CONCLUSION: The chemical mutagenesis strategy adopted in this study resulted in the successful isolation of three stable high SCO yielding mutants. The mutants, namely, YlB6, YlC7 and YlE1 exhibited a 1.22, 1.33 and 1.49-fold increase in lipid contents when grown on 100 g L-1 waste cooking oil than the parental yeast strain. The fatty acid methyl ester (FAME) profiles of all the three mutants was determined to be suitable for biodiesel suggesting their potential applicability while simultaneously addressing the management of waste cooking oil.

Transcriptional analysis of degenerate strain Clostridium beijerinckii DG-8052 reveals a pleiotropic response to CaCO3-associated recovery of solvent production.

Degenerate Clostridium beijerinckii strain (DG-8052) can be partially recovered by supplementing CaCO3 to fermentation media. Genome resequencing of DG-8052 showed no general regulator mutated. This study focused on transcriptional analysis of DG-8052 and its response to CaCO3 treatment via microarray. The expressions of 5168 genes capturing 98.6% of C. beijerinckii NCIMB 8052 genome were examed. The results revealed that with addition of CaCO3 565 and 916 genes were significantly up-regulated, and 704 and 1044 genes significantly down-regulated at acidogenic and solventogenic phase of DG-8052, respectively. These genes are primarily responsible for glycolysis to solvent/acid production (poR, pfo), solventogensis (buk, ctf, aldh, adh, bcd) and sporulation (spo0A, sigE, sigma-70, bofA), cell motility and division (ftsA, ftsK, ftsY, ftsH, ftsE, mreB, mreC, mreD, rodA), and molecular chaperones (grpE, dnaK, dnaJ, hsp20, hsp90), etc. The functions of some altered genes in DG-8052, totalling 5.7% at acidogenisis and 8.0% at sovlentogenisis, remain unknown. The response of the degenerate strain to CaCO3 was suggested significantly pleiotropic. This study reveals the multitude of regulatory function that CaCO3 has in clostridia and provides detailed insights into degeneration mechanisms at gene regulation level. It also enables us to develop effective strategies to prevent strain degeneration in future.

Membrane fluidity does not explain how solvents act on the middle-ear reflex.

Some volatile aromatic solvents have similar or opposite effects to anesthetics in the central nervous system. Like for anesthetics, the mechanisms of action involved are currently the subject of debate. This paper presents an in vivo study to determine whether direct binding or effects on membrane fluidity best explain how solvents counterbalance anesthesia's depression of the middle-ear reflex (MER). Rats were anesthetized with a mixture of ketamine and xylazine while also exposed to solvent vapors (toluene, ethylbenzene, or one of the three xylene isomers) and the amplitude of their MER was monitored. The depth of anesthesia was standardized based on the magnitude of the contraction of the muscles involved in the MER, determined by measuring cubic distortion product oto-acoustic emissions (DPOAEs) while triggering the bilateral reflex with contralateral acoustic stimulation. The effects of the aromatic solvents were quantified based on variations in the amplitude of the DPOAEs. The amplitude of the alteration to the MER measured in anesthetized rats did not correlate with solvent lipophilocity (as indicated by logKow values). Results obtained with the three xylene isomers indicated that the positions of two methyl groups around the benzene ring played a determinant role in solvent/neuronal cell interaction. Additionally, Solid-state Nuclear Magnetic Resonance (NMR) spectra for brain microsomes confirmed that brain lipid fluidity was unaffected by solvent exposure, even after three days (6h/day) at an extremely high concentration (3000ppm). Therefore, aromatic solvents appear to act directly on the neuroreceptors involved in the acoustic reflex circuit, rather than on membrane fluidity. The affinity of this interaction is determined by stereospecific parameters rather than lipophilocity.

Comparison of solvent-wetted and kneaded l-sulpiride-loaded solid dispersions: Powder characterization and in vivo evaluation.

The purpose of this study was to compare the powder properties, solubility, dissolution and oral absorption of solvent-wetted (SWSD) and kneaded (KNSD) l-sulpiride-loaded solid dispersions. The SWSD and KNSD were prepared with silicon dioxide, sodium laurylsulfate and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) using a spray dryer and high shear mixer, respectively. Their powder properties, solubility, dissolution and oral absorption were assessed compared to l-sulpiride powder. The drug in SWSD was in the amorphous state; however, in KNSD, it existed in the crystalline state. The SWSD with a drug/sodium laurylsulphate/TPGS/silicon dioxide ratio of 5/1/2/12 gave the higher drug solubility and dissolution compared to the KNSD with the same composition. The oral absorption of drug in the SWSD was 1.4 fold higher than the KNSD and 3.0 fold higher than the l-sulpiride powder (p<0.05) owing to better solubility and reduced crystallinity. Furthermore, the SWSD at the half dose was bioequivalent of commercial l-sulpiride-loaded product in rats. Thus, the SWSD with more improved oral absorption would be recommended as an alternative for the l-sulpiride-loaded oral administration.

Glutamate and histidine improve both solvent yields and the acid tolerance response of Clostridium beijerinckii NCP 260.

AIMS: This study aims to examine the effect of amino acid supplementation on solvent production by Clostridium beijerinckii during the acetone-butanol fermentation and to determine whether amino acids are involved in the acid tolerance response (ATR), which results in increased solvents. METHODS AND RESULTS: Fermentation studies with Cl. beijerinckii NCP 260 in limited-nitrogen media supplemented with glutamate, glutamine, lysine, proline, histidine or asparagine revealed that only glutamate, glutamine or histidine increased butanol titres comparable to control media. Acid survival tests at pH 5 showed that glutamate and histidine were effective in protecting Cl. beijerinckii cells against acid shock, and may be involved in the ATR. Using quantitative PCR, the transcription of the glutamine synthetase, nitrogen regulator and glutamate synthase operon (glnA-nitR-gltAB) was monitored during acid shock conditions, and expression of both the nitR and gltA genes was shown to be increased twofold. CONCLUSIONS: Glutamate and histidine specifically enhance the ATR in Cl. beijerinckii NCP 260, and the genes encoding glutamate synthase and the NitR regulator are both upregulated, predicted to lead to increased endogenous glutamate pools during acidogenesis. This may enhance the ATR and allow more viable cells to enter solventogenesis, thereby increasing butanol titres. Glutamine, glutamate and histidine may also afford protection from butanol stress directly. SIGNIFICANCE AND IMPACT OF THE STUDY: Using substrates naturally rich in glutamine, glutamate and histidine in industrial fermentations is a promising means to increase acid survival and solvent yields in solventogenic Clostridium.

Unorthodox methods for enhancing solvent production in solventogenic Clostridium species.

While production of biofuels from renewable resources is currently receiving increased attention globally, concerns on availability and sustainability of cheap substrates for their production are growing as well. Lignocellulose-derived sugars (LDS) remain underutilized and merit consideration as a key feedstock. Among other obstacles such as low yield and low solvent titer, mitigation of stresses stemming from lignocellulose-derived microbial inhibitory compounds (LDMICs) that severely impair cell growth and solvent production is a major area of research interest. In addition to attempts at developing LDMIC-tolerant strains via metabolic engineering to enhance utilization of LDS, unconventional approaches that elicit different metabolic perturbations in microorganisms to relieve solvent- and LDMIC-mediated stresses have been explored to increase solvent production from LDS. In this review, the impacts of metabolic perturbations including medium supplementation with glycerol; furfural and 5-hydroxymethyl furfural; allopurinol, an inhibitor of xanthine dehydrogenase; calcium (Ca(2+)) and zinc (Zn(2+)) ions); and artificial electron carriers, methyl viologen and neutral red, on butanol production are discussed. Although these approaches have brought about considerable increases in butanol production, both from LDS and defined glucose-based media, the modes of action for most of these perturbations have yet to be fully characterized. Better understanding of these mechanisms would likely inform development of LDMIC-tolerant, butanol-overproducing strains, as well as possible combinatorial application of these approaches for enhanced butanol production. Hence, delineating the underlying mechanisms of these perturbations deserves further attention.

Transcriptional analysis of micronutrient zinc-associated response for enhanced carbohydrate utilization and earlier solventogenesis in Clostridium acetobutylicum.

The micronutrient zinc plays vital roles in ABE fermentation by Clostridium acetobutylicum. In order to elucidate the zinc-associated response for enhanced glucose utilization and earlier solventogenesis, transcriptional analysis was performed on cells grown in glucose medium at the exponential growth phase of 16 h without/with supplementary zinc. Correspondingly, the gene glcG (CAC0570) encoding a glucose-specific PTS was significantly upregulated accompanied with the other two genes CAC1353 and CAC1354 for glucose transport in the presence of zinc. Additionally, genes involved in the metabolisms of six other carbohydrates (maltose, cellobiose, fructose, mannose, xylose and arabinose) were differentially expressed, indicating that the regulatory effect of micronutrient zinc is carbohydrate-specific with respects to the improved/inhibited carbohydrate utilization. More importantly, multiple genes responsible for glycolysis (glcK and pykA), acidogenesis (thlA, crt, etfA, etfB and bcd) and solventogenesis (ctfB and bdhA) of C. acetobutylicum prominently responded to the supplementary zinc at differential expression levels. Comparative analysis of intracellular metabolites revealed that the branch node intermediates such as acetyl-CoA, acetoacetyl-CoA, butyl-CoA, and reducing power NADH remained relatively lower whereas more ATP was generated due to enhanced glycolysis pathway and earlier initiation of solventogenesis, suggesting that the micronutrient zinc-associated response for the selected intracellular metabolisms is significantly pleiotropic.

Antifungal potential of Avicennia schaueriana Stapf & Leech. (Acanthaceae) against Cladosporium and Colletotrichum species.

UNLABELLED: There is significant interest in research to develop plant extracts with fungicidal activities that are less harmful to the environment and human health than synthetic fungicides. This study aimed to evaluate the antifungal activity of the extracts of Avicennia schaueriana against Colletotrichum and Cladosporium species and to identify the compounds responsible for the activity. Leaves and stems of A. schaueriana were extracted with ethanol and partitioned with petroleum ether, chloroform and ethyl acetate. The antifungal activity of such extracts was tested by bioautography against Cladosporium sphaerospermum, Cladosporium cladosporioides and Colletotrichum lagenarium. Ethanolic extracts, petroleum ether and chloroform fractions of stems had the highest antifungal activity with several active bands (Rf = 0·72 and Rf = 0·55). In the agar dilution assay, ethanolic extract, petroleum ether and chloroform fractions of stems were the most efficacious, presenting 85, 62 and 63% growth inhibition of Colletotrichum gloeopsporioides and minimum inhibitory concentration values between 1 and 1·5 mg ml(-1) , respectively. Analysis carried out using gas chromatography coupled to a mass spectrometry of petroleum ether and chloroform fractions allowed the identification of fatty acids methyl esters, lupeol and naphthoquinones such as lapachol, α-lapachone, naphtho[2,3-b]furan-4,9-dione, 2-isopropyl- and avicenol-C. We may infer that the antifungal activity of A. schaueriana is due to the abundance of these compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that Avicennia schaueriana extracts have a high potential for the growth inhibition of Colletotrichum and Cladosporium ssp. and will provide a starting point for discovering new natural products with antifungal activity. Their development is of particular interest to organic production systems where synthetic fungicides cannot be used.

Effects of Tulbaghia violacea Harv. (Alliaceae) rhizome methanolic extract on kidney function and morphology in Dahl salt-sensitive rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tulbaghia violacea has been used traditionally for the treatment of several ailments, including hypertension. The herb has been shown to have antihypertensive properties which have been attributed to its angiotensin-converting enzymeinhibitory (ACEI) activity. It could, therefore, prove beneficial in ameliorating renal pathology associated with hypertension. To evaluate the effects of long-term administration of Tulbaghia violacea on renal function and morphology in the Dahl salt-sensitive (DSS) rat model. MATERIALS AND METHODS: Male DSS rats were treated intra-peritoneally (i.p.) as follows: methanolic extract of Tulbaghia violacea: (TVL) (50 mg/kg/b.w.), captopril: (CAP) (25 mg/kg/b.w.), or distilled water, control: (CON) (3 ml/kg/b.w.). Blood pressure (BP) was measured bi-weekly, whilst 24-h urine volumes and electrolyte concentrations were assessed weekly. Animals were sacrificed on day 49 by halothane overdose. Blood was removed for determination of plasma and serum electrolytes. Left kidney tissues were harvested for the determination of nuclear factor-kappaß (NF-kß) and transforming growth factor-ß (TGF-ß) gene expressions. RESULTS: TVL significantly reduced mean arterial pressure (MAP) and diastolic blood pressure (DBP). TVL showed reduced blood urea nitrogen, serum creatinine, total protein in urine as well as increased serum total protein. TVL decreased thiobarbituric acid reactive substances (TBARS) and increased glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity and nitric oxide significantly. NF-kß and TGF-ß) gene expressions were significantly reduced in TVL and CAP treated rats. Moreover, renal morphology improved significantly in TVL and CAP treated animals. CONCLUSION: TVL and CAP demonstrated marked improvement in renal function and morphology.

Ionic liquid and deep eutectic solvent-activated CelA2 variants generated by directed evolution.

Chemoenzymatic cellulose degradation is one of the key steps for the production of biomass-based fuels under mild conditions. An effective cellulose degradation process requires diverse physico-chemical dissolution of the biomass prior to enzymatic degradation. In recent years, "green" solvents, such as ionic liquids and, more recently, deep eutectic liquids, have been proposed as suitable alternatives for biomass dissolution by homogenous catalysis. In this manuscript, a directed evolution campaign of an ionic liquid tolerant ß-1,4-endoglucanase (CelA2) was performed in order to increase its performance in the presence of choline chloride/glycerol (ChCl:Gly) or 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), as a first step to identify residues which govern ionic strength resistance and obtaining insights for employing cellulases on the long run in homogenous catalysis of lignocellulose degradation. After mutant library screening, variant M4 (His288Phe, Ser300Arg) was identified, showing a dramatically reduced activity in potassium phosphate buffer and an increased activity in the presence of ChCl:Gly or [BMIM]Cl. Further characterization showed that the CelA2 variant M4 is activated in the presence of these solvents, representing a first report of an engineered enzyme with an ionic strength activity switch. Structural analysis revealed that Arg300 could be a key residue for the ionic strength activation through a salt bridge with the neighboring Asp287. Experimental and computational results suggest that the salt bridge Asp287-Arg300 generates a nearly inactive CelA2 variant and activity is regained when ChCl:Gly or [BMIM]Cl are supplemented (~5-fold increase from 0.64 to 3.37 µM 4-MU/h with the addition ChCl:Gly and ~23-fold increase from 3.84 to 89.21 µM 4-pNP/h with the addition of [BMIM]Cl). Molecular dynamic simulations further suggest that the salt bridge between Asp287 and Arg300 in variant M4 (His288Phe, Ser300Arg) modulates the observed salt activation.

A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: characterization and application to enzymatic biodiesel production.

To develop a robust whole-cell biocatalyst that works well at moderately high temperature (40-50°C) with organic solvents, a thermostable lipase from Geobacillus thermocatenulatus (BTL2) was introduced into an Aspergillus oryzae whole-cell biocatalyst. The lipase-hydrolytic activity of the immobilized A. oryzae (r-BTL) was highest at 50°C and was maintained even after an incubation of 24-h at 60°C. In addition, r-BTL was highly tolerant to 30% (v/v) organic solvents (dimethyl carbonate, ethanol, methanol, 2-propanol or acetone). The attractive characteristics of r-BTL also worked efficiently on palm oil methanolysis, resulting in a nearly 100% conversion at elevated temperature from 40 to 50°C. Moreover, r-BTL catalyzed methanolysis at a high methanol concentration without a significant loss of lipase activity. In particular, when 2 molar equivalents of methanol were added 2 times, a methyl ester content of more than 90% was achieved; the yield was higher than those of conventional whole-cell biocatalyst and commercial Candida antarctica lipase (Novozym 435). On the basis of the results regarding the excellent lipase characteristics and efficient biodiesel production, the developed whole-cell biocatalyst would be a promising biocatalyst in a broad range of applications including biodiesel production.

Inhaled toluene can modulate the effects of anesthetics on the middle-ear acoustic reflex.

Toluene (Tol) is an organic solvent widely used in the industry. It is also abused as an inhaled solvent, and can have deleterious effects on hearing. Recently, it was demonstrated that Tol has both anticholinergic and antiglutamatergic effects, and that it also inhibits voltage-dependent Ca(2+) channels. This paper describes a study of the effects of inhaled Tol on rats anesthetized with isoflurane, pentobarbital, or a mixture of ketamine/xylazine. Hearing was tested using distortion product oto-acoustic emissions (DPOAEs) associated with a contralateral noise to evaluate contraction of the middle-ear muscles. This allowed us to assess the interactions between the effects of Tol and anesthesia on the central nervous system (CNS). Although both anesthetics and Tol are known to inhibit the middle-ear acoustic reflex, our data indicated that inhaled Tol counterbalances the effects of anesthetic in a dose-dependent manner. In other terms, Tol can increase the amplitude of the middle-ear reflex in anesthetized rats, whatever the nature of the anesthetic used. This indicates that inhaling Tol (a Ca(2+)-channel-blocking drug) modifies the potency of anesthesia, and thereby the amplitude of the middle-ear reflex.

Continuous lactose fermentation by Clostridium acetobutylicum--assessment of energetics and product yields of the acidogenesis.

An assessment of both the growth and the metabolism of acidogenic cells Clostridium acetobutylicum DSM 792 is reported in the paper. Tests were carried out in a CSTR under controlled pH conditions. Cultures were carried out using a semi-synthetic medium supplemented with lactose as carbon source. Acids and solvents, that represent products of the ABE process, have been purposely added in controlled amounts to the culture medium to investigate their effects on the product yields. The mass fractional yield of biomass and products were expressed as a function of the specific growth rate taking into account the Pirt model. The maximum ATP yield and the maintenance resulted 29.1 g(DM)/mol(ATP) and 0.012 mol(ATP)/g(DM)h, respectively. Quantitative features of the C. acetobutylicum growth model were in good agreement with experimental results. The model proposes as a tool to estimate the mass fractional yield even for fermentations carried out under conditions typical of the solventogenesis.