Toxicity of basil and orange essential oils and their components against two coleopteran stored products insect pests

Two commercialized essential oils and their constituent compounds were investigated for fumigant and contact activities against two grain storage insects, adults of the maize weevil (Sitophilus zeamais) and the red flour beetle (Tribolium castaneum). The two commercialized basil and orange oils showed strong fumigant and contact activities against S. zeamais and T. castaneum. The constituents of the basil oil were linalool (21.83%), estragole (74.29%), and α-humulene (2.17%), and those of the orange oil were α-pinene (0.54%), sabinene (0.38%), β-myrcene (1.98%), limonene (96.5%), and linalool (0.6%). As a toxic fumigant, the basil oil was more effective (24-h LC₅₀ = 0.014 and 0.020 mg cm^− 3) than the orange oil (24-h LC₅₀ = 0.106 and 0.130 mg cm^− 3) against S. zeamais and T. castaneum adults, respectively. Among the constituents of the two essential oils, the toxicity of estragole was the highest (0.004 and 0.013), followed by linalool (0.016 and 0.023), limonene (0.122 and 0.171), α-pinene (0.264 and 0.273), and β-myrcene (0.274 and 0.275) based on 24-h LC₅₀ values (mg cm^− 3). Similar results were obtained in a contact toxicity test. The contact activity of basil oil was more toxic than orange oil, and estragole and linalool showed pronounced contact toxicity against S. zeamais and T. castaneum adults. Alpha-humulene had no activity as a fumigant at the tested doses, but it did have an effect as a contact poison, having 24-h LD₅₀ values of 0.040 and 0.045 mg adult^− 1 to S. zeamais and T. castaneum, respectively. Although basil oil, orange oil, and their components displayed both contact and fumigant toxicities, their effects were mainly exerted by fumigant action via the vapor phase. Thus, basil oil, orange oil, and their components could be potential candidates as new fumigants for the control of S. zeamais and T. castaneum adults.     

Bio-resolution of a chiral epoxide using whole cells of Bacillus megaterium ECU1001 in a biphasic system

Optically active epoxides can be prepared by kinetic resolution of racemic mixtures using stereospecific epoxide hydrolases. To increase the bio-resolution efficiency of a sparingly water-soluble epoxide (glycidyl phenyl ether, GPE), we investigated the use of organic/aqueous two-phase system. Various conditions were systematically examined and optimized in shake flasks. Isooctane was found to be the most suitable solvent as the organic phase. The phase volume ratio (ϕ_o/w) and biocatalyst concentration were shown to be sensitive parameters affecting both the reaction rate and the enzyme enantiospecificity in the biphase system. An isooctane/aqueous system was developed to overcome the low solubility and instability of GPE in the aqueous phase, resulting in a significant improvement of enatiomeric ratio (E-value) from 39.5 to 94.0 and an average productivity of 18.8 mg GPE/(h g) biocatalyst to 48.9 mg GPE/(h g) biocatalyst, respectively. Resolution of a 90.1 g/l solution of racemic glycidyl phenyl ether in isooctane phase was successfully carried out in a mechanically stirred reactor (120 ml), affording (S)-glycidyl phenyl ether in high (100%) enantiomeric excess with a yield of 44.5%.     

Performance of Rhizopus,Rhizomucor, and Mucor in ethanol production from glucose,xylose, and wood hydrolyzates

In searching for ethanol producing microorganisms also capable of fermenting pentoses, nine zygomycetes strains including three strains of Rhizopus oryzae, Mucor corticolous, M. hiemalis, M. indicus, Rhizomucor pusillus, R. miehei, and zygomycete IT were examined. Each strain was cultivated on glucose, xylose or dilute-acid hydrolyzate (DAH) as carbon sources, and the production of ethanol, lactic acid, glycerol, xylitol, and succinic acid were investigated. Great similarities but also conspicuous differences were seen between the species, to some extent linked to the genera. All strains were capable of growing on glucose or xylose as single carbon source. With the exception of the two Rhizomucor strains, all produced ethanol. All the strains produced glycerol as by-product, while Rhizopus and Rhizomucor but not Mucor produced lactic acid in significant amounts. All Mucor and Rhizopus strains and one strain of Rhizomucor produced xylitol in the xylose medium, but no xylitol was detected after growth on DAH. All Mucor and two R. oryzae strains were capable of growing on DAH. Two Mucor species, M. hiemalis and M. indicus showed greater ethanol production than the other strains. The ethanol yields by M. hiemalis on glucose, xylose, and DAH were 0.39, 0.18, and 0.44 g/g, respectively, whereas the corresponding results for M. indicus were 0.39, 0.22, and 0.44 g/g. The strains also rapidly consumed hydroxymethyl furfural present in DAH.     

Construction of recombinant Escherichia coli for enhanced bioconversion of colchicine into 3-demethylated colchicine at 70 l bioreactor level

A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5 l bioreactor with 3 l working volume. In 5 l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70 l bioreactor and resulted into ∼80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l⁻¹ h⁻¹. Scale-up factors were measured as volumetric oxygen transfer coefficient (k_La) i.e., 56 h⁻¹ and impeller tip velocity (V_tip) i.e., 7.065 m s⁻¹, respectively. The kinetic parameters K_m, k_cat, and k_cat/K_m of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 ± 30 μM, 8533 ± 25 min⁻¹, and 31.49 μM min⁻¹, respectively, when IPTG induced recombinant E. coli culture was used.     

Cultivation of Chlorella vulgaris in tubular photobioreactors: A lipid source for biodiesel production

Chlorella vulgaris was cultivated in two different 2.0 L-helicoidal and horizontal photobioreactors at 5 klux using the bicarbonate contained in the medium and ambient air as the main CO₂ sources. The influence of bicarbonate concentration on biomass growth as well as lipid content and profile was first investigated in shake flasks, where the stationary phase was achieved in about one half the time required by the control. The best NaHCO₃ concentration (0.2 g L⁻¹) was then used in both photobioreactors. While the fed-batch run performed in the helicoidal photobioreactor provided the best result in terms of biomass productivity, which was (84.8 mg L⁻¹ d⁻¹) about 2.5-fold that of the batch run, the horizontal configuration ensured the highest lipid productivity (10.3 mg L⁻¹ d⁻¹) because of a higher lipid content of biomass (22.8%). These preliminary results suggest that the photobioreactor configuration is a key factor either for the growth or the composition of this microalga. The lipid quality of C. vulgaris biomass grown in both photobioreactors is expected to meet the standards for biodiesel, especially in the case of the helicoidal configuration, provided that further efforts will be made to optimize the conditions for its production as a biodiesel source.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Purification and characterization of a novel protease-resistant α-galactosidase from Rhizopus sp. F78 ACCC 30795

A novel extracellular α-galactosidase, named Aga-F78, from Rhizopus sp. F78 ACCC 30795 was induced, purified and characterized in this study. This soybean-inducible α-galactosidase was purified to homogeneity by ammonium sulfate precipitation and fast protein liquid chromatography (FPLC), with a yield of 14.6% and a final specific activity of 74.6 U mg⁻¹. Aga-F78 has an estimated relative molecular mass of 78 kDa from SDS-PAGE while native mass of 210 kDa and 480 kDa from non-denaturing gradient PAGE. This α-galactosidase had no N- or O-glycosylated. Amino acid sequences of three internal fragments were determined, and fragment 1, NQLVLDLTR, shared high homology with bacterial and fungal GH-36 α-galactosidases. The optimum pH and temperature on activity of Aga-F78 were 4.8 and 50 °C, respectively. The properties of pH and temperature stability, effect of ions and chemicals were also studied. Furthermore, the resistant to neutral and alkaline proteases and substrate specificity of natural substrates (melibiose, raffinose, stachyose and guar gum) were also studied to enlarged the application of Aga-F78 in more fields. Kinetic studies revealed a K_m and V_max of 2.9 mmol l⁻¹ and 246.1 μmol (mg min)⁻¹, respectively, using pNPG as substrate. To our knowledge, this is the first report of purification and characterization of α-galactosidase from Rhizopus with some special properties, which may aid its utilization in the food and feed industries.     

Chemical composition and free radical-scavenging,anticancer and anti-inflammatory activities of the essential oil from Ocimum kilimandscharicum

ObjectiveThe essential oil from the leaves of Ocimum kilimandscharicum (EOOK), collected in Dourados-MS, was investigated for anticancer, anti-inflammatory and antioxidant activity and chemical composition.Materials and methodsThe essential oil was extracted by hydrodistillation, and the chemical composition was performed by gas chromatography–mass spectrometry. The essential oil was evaluated for free radical-scavenging activity using the DPPH assay and was tested in an anticancer assay against ten human cancer cell lines. The response parameter (GI₅₀) was calculated for the cell lines tested. The anti-inflammatory activity was evaluated using carrageenan-induced pleurisy in mice.ResultsThe chemical composition showed 45 components with a predominance of monoterpenes, such as camphor (51.81%), 1,8 cineole (20.13%) and limonene (11.23%). The EOOK exhibited potent free radical-scavenging activity by the DPPH assay with a GI₅₀ of 8.31 μg/ml. The major constituents, pure camphor (IC₅₀ = 12.56 μg/ml) and mixture of the limonene: 1, 8 cineole (IC₅₀ = 23.25 μg/ml) displayed a potent activity. The oral administration of EOOK (at 30 and 100 mg kg⁻¹), as well as the pure camphor or a mixture of 1,8 cineole with limonene, significantly inhibited the carrageenan (Cg) induced pleurisy, reducing the migration of total leukocytes in mice by 82 ± 4% (30 mg kg⁻¹ of EOOK), 95 ± 4% (100 mg kg⁻¹ of EOOK), 83 ± 9% (camphor) and 80 ± 5% (mixture of 1,8 cineole:limonene 1:1). In vitro cytotoxicity screening against a human ovarian cancer cell line displayed high selectivity and potent anticancer activity with GI₅₀ = 31.90 mg ml⁻¹. This work describes the anti-inflammatory, anticancer and antioxidant effects of EOOK for the first time.ConclusionsThe essential oil exhibited marked anti-inflammatory, antioxidant and anticancer effects, an effect that can be attributed the presence of majorital compounds, and the response profiles from chemical composition differed from other oils collected in different locales.     

Investigation on the properties and kinetics of glucose-fed aerobic granular sludge

Aerobic granulation is a process in which suspended biomass aggregate and form discrete well-defined granules in aerobic systems. To investigate the properties and kinetics of aerobic granular sludge, aerobic granules were cultivated with glucose synthetic wastewater in a series of sequencing batch reactors (SBR). The spherical shaped granules were observed on 8th day with the mean diameter of 0.1 mm. With the organic loading rate (OLR) being increased to 4.0 g COD L⁻¹ d⁻¹, aerobic granules grew matured with spherical shape. The size of granules ranged from 1.2 to 1.8 mm, and the corresponding settling velocity of individual granule was 24.2–36.4 m h⁻¹. The oxygen utilization rate (OUR) of mature granules was 41.90 g O₂ kg MLSS⁻¹ h⁻¹, which was two times higher than that of activated sludge (18.32 g O₂ kg MLSS⁻¹ h⁻¹). The experimental data indicated that the substrate utilization and biomass growth kinetics generally followed Monod's kinetics model. The corresponding kinetic coefficients of k (maximum specific substrate utilization rate), K_s (half velocity coefficient), Y (growth yield coefficient) and K_d (decay coefficient) were determined as follows, k_c = 23.65 d⁻¹, K_c = 3367.05 mg L⁻¹, K_N = 0.038 d⁻¹, K_N = 29.65 mg L⁻¹, Y = 0.1927–0.2022 mg MMLS (mg COD)⁻¹ and K_d = 0.00845–0.0135 d⁻¹, respectively. Those properties of aerobic granules made aerobic granules system had a short setup period, high substrate utilization rate and low sludge production.     

The role of volumetric power input in the growth,morphology, and production of a recombinant glycoprotein by Streptomyces lividans in shake flasks

The impact of flask geometry on Streptomyces lividans growth and morphology, production and O-mannosylation of a recombinant O-glycoprotein (APA from Mycobacterium tuberculosis) was described and associated to the evolution of the volumetric power input (P/V) in three shake flask geometries. During the exponential growth, the highest P/V was found in baffled flasks (BF) with 0.51 kW/m³, followed by coiled flasks (CF) with 0.44 kW/m³ and normal Erlenmeyer flasks (NF) with 0.20 kW/m³ (flasks volume of 250 mL, filling with 50 mL and agitated at 150 rpm). During the stationary phase, P/V decreased 20% in BF and CF, but increased two times in NF, surely due to changes in mycelial morphology and its effects on rheology. Also, NF cultures were carried out at a filling volume and agitation of 15 mL, 150 rpm (15 mL-NF), and 25 mL, 168 rpm (25 mL-NF), in order to raise P/V closely to the values obtained in CF. However, different growth, morphology and recombinant protein productivity were obtained. These data indicate that P/V is not a definitive parameter that can determine bacteria growth and morphology, not even glycoprotein production. But it can be proposed that the oxygen transfer in the center of the pellets and hydromechanical stress might be the more relevant parameters than P/V.     

Chromate reduction by resting cells of Achromobacter sp. Ch-1 under aerobic conditions

Chromate reduction was carried out by resting cells of Achromobacter sp. Ch-1 with lactate as electron donor under aerobic conditions. The reduction activity of the samples supplemented with lactate was two times as those without lactate. The reduction rate was influenced by initial pH and lactate concentration. Under the optimal conditions, pH 9.0 and 4000 mg l⁻¹ lactate supplement, reduction rate was 5.45 mg l⁻¹ min⁻¹. The reduction rate decreased with increasing of Cr(VI) concentrations and increased with cell densities proportionally. The maximum reduction limit of Ch-1 cells was obtained at 2107 mg l⁻¹ of Cr(VI).     

Investigation on the formation and kinetics of glucose-fed aerobic granular sludge

Aerobic granular sludge was cultivated in a glass sequencing batch reactor (SBR) with glucose synthetic wastewater. The spherical shaped granules were observed on 4th day with the mean diameter of 0.1 mm. With the increase of chemical oxygen demand (COD) concentration of the influent, aerobic granules grew matured, the size of which ranged from 1.2 to 1.9 mm. The aerobic granular sludge could sustain high organic loading rate (about 4.0 g COD L⁻¹ d⁻¹), with good settling ability (settling velocity 36 m/h) and high biomass concentration (MLSS 6.7 ±0.2 g/L). Experimental data indicated that the substrate utilization and biomass growth kinetics followed Monod's kinetics model approximately. The corresponding kinetic coefficients of maximum specific substrate utilization rate (k), half velocity coefficient (K_s), growth yield coefficient (Y) and decay coefficient (K_d) were 13.2 d⁻¹, 275.8 mg/L, 0.183–0.250 mg MLSS/mg COD and 0.023–0.075 d⁻¹, respectively, which made aerobic granules have short setup period, high rate of substrate utilization and little surplus sludge.     

Nitrogen transformation in the hybrid bioreactor landfill

The hybrid bioreactor landfill was promising in solid waste management. In the work, the nitrogen removal and nitrogen transformation in hybrid bioreactor landfill with sequencing of facultative anaerobic and aerobic conditions was explored. The result showed that the combination of facultative anaerobic and aerobic conditions in the hybrid bioreactor landfill was indeed effective in eliminating ammonia both from the leachate and the refuse thoroughly. About 72% of nitrogen was reduced from the landfilled fresh refuse through the operation of 357 days. At the end of the experiment, the concentrations of COD, ammonia, nitrate and TN in the leachate decreased to 399.2 mg l^?1, 20.6 mg N l^?1, 3.7 mg N l^?1 and 25.3 mg N l^?1, respectively.     

Anthraquinones production in Rubia tinctorum cell suspension cultures: Down scale of shear effects

The effect of turbulence on suspended cells is one of the most complex problems in the scale-up of cell cultures. In the present paper, a direct comparison of the effects of turbulence on suspension cultures of Rubia tinctorum in a standard bioreactor and in shake flask cultures was done. A procedure derived from the well known global method proposed by Nishikawa et al. (1977) [39] was applied. Standard flasks and four-baffled shake flasks were used. The effect of turbulence and light irradiation on cell viability, biomass, and anthraquinones (AQs) production was evaluated. The biomass concentration and AQs production obtained using baffled shake flasks agitated at 360 rpm were similar to that achieved in R. tinctorum suspension cultures growing in a stirred tank bioreactor operating at 450 rpm, previously published (Busto et al., 2008 [17]). The effect of light on AQs production was found to be very significant, and a difference of up to 48% was found in cells with and without illumination after 7 days of culture. It is concluded that this down-scaled and simple flask culture system is a suitable and valid small scale instrument for the study of intracellular mechanisms of turbulence-induced AQs production in R. tinctorum suspension cultures.     

Production of lactic acid from soybean stalk hydrolysate with Lactobacillus sake and Lactobacillus casei

In order to make full use of soybean stalk produced in large quantity annually in China, a process is proposed for production of lactic acid from soybean stalk hydrolysate with Lactobacillus sake and Lactobacillus casei. Experiments were conducted using the proposed process and experimental results indicate that the potential of 242 mg (g stalk)⁻¹ fermentable sugar is released from hydrolysate through enzymatic saccharication with a saccharication of 51%. The main sugar released from pretreated soybean stalk through enzymatic hydrolysis was a mixture of glucose, xylose and cellobiose at a ratio of 3.9:1.7:1. Fermentation of soybean stalk hydrolysate by L. sake and L. casei yielded the lactic acid conversion of 48% and 56%, respectively, however, lactic acid conversion increased to 71% by co-inoculation of both strains. L. sake and L. casei were able to degrade glucose, but unable to completely assimilate xylose and cellobiose. The proposed process can be used to produce lactic acid from soybean stalk hydrolysate.     

Hydrolysis of orange peel by a pectin lyase-overproducing hybrid obtained by protoplast fusion between mutant pectinolytic Aspergillus flavipes and Aspergillus niveus CH-Y-1043

Pectin lyases cleave the internal glycosidic bonds of pectin by β-elimination, producing non-saturated galacturonic oligomers. Genetic improvement of pectin lyase-overproducing strains is still necessary to improve industrial processes based on this enzyme. In the present study hybrids were obtained by protoplast fusion between mutant pectinolytic Aspergillus flavipes and Aspergillus niveus CH-Y-1043 strains. Prototrophic segregants showed different isoenzymatic profiles and produced increased levels of pectin lyase in cultures containing lemon peel as a sole carbon source. Hybrid HZ showed an increase of 450% and 1300% in pectin lyase production compared with that of A. niveus CH-Y-1043 and A. flavipes, respectively. Pectin lyase produced by the hybrid HZ was partially purified and used for the hydrolysis of orange peel. Pectin lyase was able to hydrolyze 56% of orange peel biomass. However, addition of 2 RFU and 20 U of endo- and exo-polygalacturonase, respectively, induced the hydrolysis of 92% of orange peel solids. In conclusion HZ is a pectin lyase-overproducing hybrid with potential applications in the pectin industry.     

Evaluation of cysteine ethyl ester as efficient inducer for glutathione overproduction in Saccharomyces spp.

Economical yeast based glutathione (GSH) production is a process that is influenced by several factors like raw material and production costs, biomass production and efficient biotransformation of adequate precursors into the final product GSH. Nowadays the usage of cysteine for the microbial conversion into GSH is industrial state of practice. In the following study, the potential of different inducers to increase the GSH content was evaluated by means of design of experiments methodology. Investigations were executed in three natural Saccharomyces strains, S. cerevisiae, S. bayanus and S. boulardii, in a well suited 50 ml shake tube system. Results of shake tube experiments were confirmed in traditional baffled shake flasks and finally via batch cultivation in lab-scale bioreactors under controlled conditions. Comprehensive studies showed that the usage of cysteine ethyl ester (CEE) for the batch-wise biotransformation into GSH led up to a more than 2.2 times higher yield compared to cysteine as inducer. Additionally, the intracellular GSH content could be significantly increased for all strains in terms of 2.29 ± 0.29% for cysteine to 3.65 ± 0.23% for CEE, respectively, in bioreactors. Thus, the usage of CEE provides a highly attractive inducing strategy for the GSH overproduction.     

Comparative kinetic study between moving bed biofilm reactor-membrane bioreactor and membrane bioreactor systems and their influence on organic matter and nutrients removal

New technologies regarding wastewater treatment have been developed. Among these technologies, the moving bed biofilm reactor combined with membrane bioreactor (MBBR-MBR) is a recent solution alternative to conventional processes. This paper presents the results obtained from three wastewater treatment plants working in parallel. The first wastewater treatment plant consisted of a membrane bioreactor (MBR), the second one was a MBBR-MBR system containing carriers both in anoxic and aerobic zones, and the last one consisted of a MBBR-MBR system which contained carriers only in the aerobic zone. The reactors operated with a hydraulic retention time of 26.47 h. During the study, the difference between the experimental plants was not statistically significant concerning organic matter and nutrients removal. However, different tendencies regarding nutrients removal are shown by the three wastewater treatment plants. In this sense, the performances in terms of nitrogen and phosphorus removal of the MBBR-MBR system which contained carriers only in the aerobic zone (67.34 ± 11.22% and 50.65 ± 11.13%, respectively) were slightly better than those obtained from another experimental plants. As a whole, the pilot plant which consisted of a MBR showed better performance from the point of view of the kinetics of the heterotrophic and autotrophic biomass with values of μ_m,H = 0.00858 h⁻¹, μ_m,A = 0.07646 h⁻¹, K_M = 2.37 mg O₂ L⁻¹ and K_NH = 1.31 mg N L⁻¹.     

Kinetic behavior of Candida guilliermondii yeast during xylitol production from highly concentrated hydrolysate

Rice straw hemicellulosic hydrolysate containing a high xylose concentration was used as fermentation medium to evaluate the kinetic behavior of Candida guilliermondii yeast (FTI 20037) during the bioconversion of xylose into xylitol. Assays were conducted first with detoxified and non-detoxified (raw) hydrolysates and semi-synthetic medium in agitated flasks, and second with detoxified hydrolysate in a stirred-tank bioreactor at a given oxygen transfer rate. The results for the agitated flasks showed that in detoxified hydrolysate the xylose-to-xylitol bioconversion by the yeast was as effective as in synthetic medium and 47% higher than in raw hydrolysate. In the stirred-tank bioreactor, the kinetic behavior of the yeast in detoxified hydrolysate was slower, resulting in smaller values of fermentative parameters, probably due to unsuitability of the oxygen transfer rate employed (K_La=22 h⁻¹).