Characterization of one novel cry8 gene from Bacillus thuringiensis strain Q52-7

Bacillus thuringiensis (Bt) is the most widely used insecticidal microbe due to its specific toxicity and safe use with respect to animals and the environment. In this study, we isolated Bt strain Q52-7 from a soil sample collected in the Qian Shan District, Liao Ning Province, China. We observed that the Q52-7 strain produced spherical crystals. The Bt Q52-7 strain had high toxicity against Asian Cockchafer (Holotrichia parallela), exhibiting an LC₅₀ of 3.80 × 10⁹ cfu/g, but is not toxic for Anomala corpulenta Motschulsky and Holotrichia oblita. Using general cry8 primers, we amplified a 1.3 kb fragment with the polymerase chain reaction. Specific primers were designed for the amplified fragment to clone the full-length coding region. A novel gene, cry8Na1, had 69 % sequence similarity with cry8Ca1. cry8Na1 gene was successfully expressed in the HD-73^– acrystalliferous mutant of Bt subsp. Kurstaki HD-73. Bioassays demonstrated that the Cry8Na1 protein is highly toxic for the H. parallela, with a 50 % lethal concentration of 8.18 × 10¹⁰ colony forming units per gram.     

Overexpression of polyphosphate kinase gene (ppk) increases bioinsecticide production by Bacillus thuringiensis

Polyphosphate (polyP), synthesized by polyP kinase (PPK) using the terminal phosphate of ATP as substrate, performs important functions in every living cell. The present work reports on the relationship between polyP metabolism and bioinsecticide production in Bacillus thuringiensis subsp. israelensis (Bti). The ppk gene of Bti was cloned into vector pHT315 and the effect of its overexpression on endotoxin production was determined. Endotoxin production by the recombinant strain was found to be consistently higher than that by the wild type strain and the strain that carried the empty plasmid. The toxicity of the recombinant mutant strain (LC₅₀ 5.8 ± 0.6 ng ml^?1) against late 2nd instar Culex quinquefasciatus was about 7.7 times higher than that of Bti (LC₅₀ 44.9 ± 7 ng ml^?1). To our knowledge this is the first reported study which relates polyP metabolism with bioinsecticide biosynthesis.     

Characterization of a novel <Emphasis Type="Italic">cry8</Emphasis> gene specific to Melolonthidae pests: <Emphasis Type="Italic">Holotrichia oblita</Emphasis> and <Emphasis Type="Italic">Holotrichia parallela</Emphasis>

A new polymerase chain reaction–restriction fragment length polymorphism method for the identification of cry8-type genes from Bacillus thuringiensis has been established by designing a pair of new universal primers. By this method, a novel gene, cry8Ga1, encoding a polypeptide of 1,157 amino acids with a deduced molecular mass of 131.2 kDa was identified and cloned from B. thuringiensis HBF-18. Recombinant B. thuringiensis strain HD8G, harboring cry8Ga1, has insecticidal activity against larvae of Melolonthidae pests: Holotrichia oblita and Holotrichia parallela. This is the first report of a Cry toxin that has insecticidal activity to Melolonthidae pest H. oblita.     

Ochrosin,a new biosurfactant produced by halophilic Ochrobactrum sp. strain BS-206 (MTCC 5720): Purification,characterization and its biological evaluation

A new bioactive molecule characterized as 4-dimethylaminobenzaldehyde (designated as Ochrosin) was isolated from a halophilic Ochrobactrum sp. strain BS-206 (MTCC 5720). Ochrosin exhibited good antimicrobial activity against both Gram-positive and Gram-negative bacteria and different Candida strains with minimum inhibitory concentration (MIC) values ranging from 4.68 to 150 μg mL⁻¹. It also exhibited anti-adhesive activity against various bacterial pathogens. Ochrosin caused an increase in the reactive oxygen species (ROS) levels to 32.4% and 28% and increased the NAD⁺/NADH ratio by 2.61 and 2.26 in S. aureus and E. coli, respectively. Ochrosin appears to interfere with ergosterol biosynthetic pathway in different Candida strains as it reduced the ergosterol content. Ochrosin showed insecticidal activity against three major stored-product grain pests like Tribolium castaneum, Callosobruchus chinensis and Sitophilus oryzae with LC₅₀ values of 7.3, 8.9 and 7.6 μg cc⁻¹, respectively. The anti-feedant index (ED₅₀) values observed were 1.07 and 1.35 μg cm⁻² of leaf area against Achaea janata and Spodoptera litura, respectively. This is the first report on Ochrosin, a multifunctional biosurfactant, produced by Ochrobactrum sp. BS-206 exhibiting promising antimicrobial, anti-adhesive, anti-feedant and insecticidal activities.     

Functional consortium for hydrogen production from cellobiose: Concentration-to-extinction approach

A functional bacterial consortium that can effectively hydrolyze cellobiose and produce bio-hydrogen was isolated by a concentration-to-extinction approach. The sludge from a cattle feedlot manure composting plant was incubated with 2.5–20 g l^?1 cellobiose at 35 °C and pH 6.0. The microbial diversity of serially concentrated suspensions significantly decreased following increasing cellobiose concentration, finally leaving only two viable strains, Clostridium butyricum strain W4 and Enterococcus saccharolyticus strain. This consortium has a maximum specific hydrogen production rate of 2.19 mol H₂ mol hexose^?1 at 5 g l^?1 cellobiose. The metabolic pathways shifted from ethanol-type to acetate-butyrate type as cellobiose concentration increased from 2.5 to >7 g l^?1. The concentration-to-extinction approach is effective for isolating functional consortium from natural microflora. In this case the functional strains of interest are more tolerant to the increased loadings of substrates than the non-functional strains.     

Effect of Zinc nanoparticles on oxidative stress-related genes and antioxidant enzymes activity in the brain of Oreochromis niloticus and Tilapia zillii

This study was carried out to determine the median lethal concentrations (LC₅₀) of Zinc nanoparticles (ZnNPs) on Oreochromis niloticus and Tilapia zillii. The biochemical and molecular potential effects of ZnNPs (500 and 2000 μg L⁻¹) on the antioxidant system in the brain tissue of O. niloticus and T. zillii were investigated. Four hundred fish were used for acute and sub-acute studies. ZnNP LC₅₀ concentrations were investigated in O. niloticus and T. zillii. The effect of 500 and 2000 μg L⁻¹ ZnNPs on brain antioxidants of O. niloticus and T. zillii was investigated. The result indicated that 69 h LC₅₀ was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. Fish exposed to 500 μg L⁻¹ ZnNPs showed a significant increase in reduced glutathione (GSH), total glutathione (tGSH) levels, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activity and gene expression. On the contrary, malondialdehyde (MDA) levels significantly decreased. Meanwhile, fish exposed to 2000 μg L⁻¹ ZnNPs showed a significant decrease of GSH, tGSH levels, SOD, CAT, GR, GPx and GST activity and gene expression. On the contrary, MDA levels significantly increased. It was concluded that, the 96 h LC₅₀ of ZnNPs was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. ZnNPs in exposure concentrations of 2000 μg/L induced a deleterious effect on the brain antioxidant system of O. nilotica and T. zillii. In contrast, ZnNPs in exposure concentrations of 500 μg L⁻¹ produced an inductive effect on the brain antioxidant system of O. nilotica and T. zillii.     

Alexandrium pacificum Litaker sp. nov (Group IV): Resting cyst distribution and toxin profile of vegetative cells in Bizerte Lagoon (Tunisia,Southern Mediterranean Sea)

A high spatial resolution sampling of Alexandrium pacificum cysts, along with sediment characteristics (% H₂O, % organic matter (OM), granulometry), vegetative cell abundance and environmental factors were investigated at 123 study stations in Bizerte Lagoon (Tunisia). Morphological examination and ribotyping of cells obtained from a culture called ABZ1 obtained from a cyst isolated in lagoon sediment confirmed that the species was A. pacificum. The toxin profile from the ABZ1 culture harvested during exponential growth phase was simple and composed of the N-sulfocarbamoyl toxins C1 (9.82 pg toxin cell⁻¹), the GTX6 (3.26 pg toxin cell⁻¹) and the carbamoyl toxin Neo-STX (0.38 pg toxin cell⁻¹). The latter represented only 2.8% of the total toxins in this strain.High abundance of A. pacificum cysts correlated with enhanced percentages of water and organic matter in the sediment. In addition, sediment fractions of less than 63 μm were examined as a favorable potential seedbed for initiation of future blooms and outbreaks of A. pacificum in the lagoon. A significant difference in the cyst distribution pattern was recorded among the lagoon's different zones, with the higher cyst abundance occurring in the inner waters. Also, no correlation due to the specific hydrodynamics of the lagoon was observed in the spatial distribution of A. pacificum cysts and vegetative cells.     

Improved β-carotene production of Rhodotorula glutinis in fermented radish brine by continuous cultivation

Fermentation kinetics of growth and β-carotene production by Rhodotorula glutinis DM28 in batch and continuous cultures using fermented radish brine, a waste generated from fermented vegetable industry, as a cultivation medium were investigated. The suitable brine concentration for β-carotene production by R. glutinis DM28 was 30 g l^?1. Its growth and β-carotene production obtained by batch culture in shake flasks were 2.2 g l^?1 and 87 μg l^?1, respectively, while, in a bioreactor were 2.6 g l^?1 and 186 μg l^?1, respectively. Furthermore, its maximum growth rate and β-carotene productivity in continuous culture obtained at the dilution rate of 0.24 h^?1 were 0.3 g l^?1 h^?1 and 19 μg l^?1 h^?1, respectively, which were significantly higher than those in the batch. Therefore, improved growth rate and β-carotene productivity of R. glutinis in fermented radish brine could be accomplished by continuous cultivation.     

Challenges in enzymatic hydrolysis and fermentation of pretreated Arundo donax revealed by a comparison between SHF and SSF

The perennial herbaceous crop Arundo donax is a potential feedstock for second-generation bioethanol production. In the present work, two different process options were investigated for the conversion of two differently steam-pretreated batches of A. donax. The pretreated raw material was converted to ethanol with a xylose-consuming Saccharomyces cerevisiae strain, VTT C-10880, by applying either separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF). The highest overall ethanol yield and final ethanol concentration were achieved using SHF (0.27 g g^?1 and 20.6 g L^?1 compared to 0.24 g g^?1 and 19.0 g L^?1 when SSF was used). The performance of both SHF and SSF was improved by complementing the cellulolytic enzymes with hemicellulases. The higher amount of acetic acid in one of the batches was shown to strongly affect xylose consumption in the fermentation. Only half of the xylose was consumed when batch 1 (high acetic acid) was fermented, compared to that 94% of the xylose was consumed in fermentation of batch 2 (lower acetic acid). Furthermore, the high amount of xylooligomers present in the pretreated materials considerably inhibited the enzymatic hydrolysis. Both the formation of xylooligomers and acetic acid thus need to be considered in the pretreatment process in order to achieve efficient conversion of A. donax to ethanol.     

Relationship between microbial activity and microbial community structure in six full-scale anaerobic digesters

High activity levels and balanced anaerobic microbial communities are necessary to attain proper anaerobic digestion performance. Therefore, this work was focused on the kinetic performance and the microbial community structure of six full-scale anaerobic digesters and one lab-scale co-digester. Hydrolytic (0.6–3.5 g COD g^?1 VSS d^?1) and methanogenic (0.01–0.84 g COD g^?1 VSS d^?1) activities depended on the type of biomass, whereas no significant differences were observed among the acidogenic activities (1.5–2.2 g COD g^?1 VSS d^?1). In most cases, the higher the hydrolytic and the methanogenic activity, the higher the Bacteroidetes and Archaea percentages, respectively, in the biomasses. Hydrogenotrophic methanogenic activity was always higher than acetoclastic methanogenic activity, and the highest values were achieved in those biomasses with lower percentages of Methanosaeta. In sum, the combination of molecular tools with activity tests seems to be essential for a better characterization of anaerobic biomasses.     

Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 × His tag was found to be 22.6 U mg^?1 of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 °C, respectively and the inulinase was stable in the pH range of 3–8 and in the temperature range of 0–50 °C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g^?1 of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10 h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L^?1 citric acid and 5.3 g L^?1 iso-citric acid from inulin while 68.9 g L^?1 of citric acid and 4.1 g L^?1 iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.     

Methanol vapor biofiltration coupled with continuous production of recombinant endochitinase Ech42 by Pichia Pastoris

Methanol biofiltration using methylotrophic microorganisms has been previously reported by various authors. In a previous study, a modified strain of Pichia pastoris was tested for the ability to produce endochitinase (Ech42) when coupled with methanol vapor biodegradation in batch tests. The next challenge was to validate the process in a continuous system. Thus, in the present study, a biofilter packed with perlite and inoculated with P. pastoris transformed with the plasmid pPIC-ech42 was used for methanol vapor biofiltration and the continuous production of recombinant endochitinase (Ech42) for 60 days. The maximum elimination capacity (EC) of methanol obtained was 1320 g m^?3 h^?1 at a loading rate of 1465 g m^?3 h^?1. The extracellular protein production rate in the leachate was 2360 μg h^?1 with a chitinase enzymatic activity of 123 U L^?1. The protein content on the biofilm samples was negligible, indicating the effectiveness of the overall process and of P. pastoris to excrete proteins. The carbon balance indicated that 81% of the consumed methanol was mineralized and 5.8% was incorporated into biomass. The results of this study and the economic balance underscore the promising application of linking methanol vapor biofiltration to the continuous production of recombinant proteins.     

Isolation and characterization of trehalose tetraester biosurfactants from a soil strain Micrococcus luteus BN56

The bacterium Micrococcus luteus BN56, isolated from soil, was found to produce glycolipid biosurfactants when grown on n-hexadecane as the sole carbon source. The purified glycolipids were characterized using ¹H, ¹³C, ¹H COSY NMR-spectroscopy and ESI-MS spectrometry analyses. The two main products were identified as trehalose tetraesters with molecular mass of 876 and 848 g mol^?1. The purified products reduced the surface tension of water from 72 to 24.1 mN m^?1 and the interfacial tension between water and hexadecane from 43.0 to 1.7 mN m^?1. The CMC of these biosurfactants was found to be 25 mg l^?1. The strain formed stable emulsions with hydrocarbon substrates and was suggested that the hydrophobic cells acted as emulsion-stabilizing agents. The results demonstrate that the strain M. luteus BN56 may be well suited for bioremediation of oil-contaminated environments.     

Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae

Xylose is the main pentose and second most abundant sugar in lignocellulosic feedstocks. To improve xylose utilization, necessary for the cost-effective bioconversion of lignocellulose, several metabolic engineering approaches have been employed in the yeast Saccharomyces cerevisiae. In this study, we describe the rational metabolic engineering of a S. cerevisiae strain, including overexpression of the Piromyces xylose isomerase gene (XYLA), Pichia stipitis xylulose kinase (XYL3) and genes of the non-oxidative pentose phosphate pathway (PPP). This engineered strain (H131-A3) was used to initialize a three-stage process of evolutionary engineering, through first aerobic and anaerobic sequential batch cultivation followed by growth in a xylose-limited chemostat. The evolved strain H131-A3-AL^CS displayed significantly increased anaerobic growth rate (0.203±0.006 h^?1) and xylose consumption rate (1.866 g g^?1 h^?1) along with high ethanol conversion yield (0.41 g/g). These figures exceed by a significant margin any other performance metrics on xylose utilization and ethanol production by S. cerevisiae reported to-date. Further inverse metabolic engineering based on functional complementation suggested that efficient xylose assimilation is attributed, in part, to the elevated expression level of xylose isomerase, which was accomplished through the multiple-copy integration of XYLA in the chromosome of the evolved strain.     

Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol

Polyhydroxyalkanoates (PHAs) have been recognized as good substitutes for the non-biodegradable petrochemically produced polymers. However, their high (real or estimated) current production cost limits their industrial applications. This work exploits two strategies to enhance PHAs substitution potential: the increase in PHA volumetric productivity in high density cultures and the use of waste glycerol (GRP), a by-product from the biodiesel industry, as primary carbon source for cell growth and polymer synthesis. Cupriavidus necator DSM 545 was used to accumulate poly(3-hydroxybutyrate) (P(3HB)) from GRP and from commercial glycerol (PG) as control substrate. On PG, productivities between 0.6 g_PHB L^?1 h^?1 and 1.5 g_PHB L^?1 h^?1 were attained. The maximum cell DW was 82.5 g_DW L^?1, the P(3HB) content being 62%. When GRP was used, 68.8 g_DW L^?1 with a P(3HB) accumulation of 38% resulting in a final productivity of 0.84 g_PHB L^?1 h^?1 was obtained. By decreasing the biomass concentration at which accumulation was triggered, a productivity of 1.1 g_PHB L^?1 h^?1 (50% P(3HB), w/w) was attained using GRP. P(3HB) molecular weights (M_w) ranged from 7.9 × 10⁵ to 9.6 × 10⁵ Da.     

Biological and molecular responses of Chironomus riparius (Diptera,Chironomidae) to herbicide 2,4-D (2,4-dichlorophenoxyacetic acid)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water. It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level. To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Chironomus riparius after long-term exposure to 2,4-D. The larvae were treated with 0.1, 1 or, 10 μg L^? 1 of 2,4-D for short- and long-term exposure periods. The sex ratio was changed in C. riparius exposed to only 10 μg L^? 1 of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C. riparius exposed to 0.1 μg L^? 1 of 2,4-D. Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), ferritins and glutathione S-transferases (GSTs) in C. riparius exposed to 2,4-D for 24 h. The expressions of HSP70, HSP40, HSP90 and GST levels in C. riparius were significantly increased after exposure to a 10 μg L^? 1 concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure. Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C. riparius. Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment.     

A stable immobilized d-psicose 3-epimerase for the production of d-psicose in the presence of borate

Maximal activity of the immobilized d-psicose 3-epimerase from Agrobacterium tumefaciens on Duolite A568 beads was achieved at pH 9.0 and 55 °C with borate, and at pH 8.5 and 50 °C without borate. The half-lives of the immobilized enzyme at 50 °C with and without borate were increased 4.2- and 128-fold compared to that of the free enzyme without borate, respectively. The immobilized enzyme with borate produced 441 g l^?1 psicose from 700 g l^?1 fructose at pH 9.0 and 55 °C, whereas 193 g l^?1 psicose was produced without borate at pH 8.5 and 50 °C after 120 min in a batch reaction. The immobilized enzyme in a packed-bed bioreactor without borate was produced continuously 325 g l^?1 psicose from 500 g l^?1 fructose at a dilution rate of 1.62 h^?1 over a 236 h period with productivity of 527 g l^?1 h^?1 while that without borate produced 146 g l^?1 psicose at 4.15 h^?1 over a 384-h period with productivity of 606 g l^?1 h^?1. The operational half-lives of the enzyme with and without borate in the bioreactor were 601 and 645 h, respectively. In the present study, psicose was produced stably with high productivity using the immobilized d-psicose 3-epimerase in the presence of borate.     

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l^?1. In batch fermentation, 45.5 g l^?1 succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l^?1 of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l^?1 at a rate of 1.21 g l^?1 h^?1 after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.     

Production of yeast chitin–glucan complex from biodiesel industry byproduct

Crude glycerol from the biodiesel industry was used as carbon source for high cell density fed-batch cultivation of Pichia pastoris aiming at producing a chitin–glucan complex (CGC). More than 100 g L^?1 biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g g^?1 during the batch phase and 0.63 g g^?1 during the fed-batch phase. The chitin–glucan complex was recovered from the yeast cell wall by hot alkaline extraction. CGC content in the cell wall was found to be relatively constant throughout the cultivation (18–26%) with a volumetric productivity of 1.28 g L^?1 h^?1 at the end of the fed-batch phase. The molar ratio of chitin:β-glucan in the extracted biopolymer was 16:84, close to other CGC extracted from Aspergillus biomass. The extracted polymer was characterized by Differential Scanning Calorimetry (DCS) and solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and compared with commercial biopolymers, namely, crab shell chitin and/or chitosan, algal β-glucan (laminarin) and fungal chitin–glucan complex (kiOsmetine).     

Enantioselective oxidation of racemic lactic acid to d-lactic acid and pyruvic acid by Pseudomonas stutzeri SDM

d-Lactic acid and pyruvic acid are two important building block intermediates. Production of d-lactic acid and pyruvic acid from racemic lactic acid by biotransformation is economically interesting. Biocatalyst prepared from 9 g dry cell wt l^?1 of Pseudomonas stutzeri SDM could catalyze 45.00 g l^?1 dl-lactic acid into 25.23 g l^?1 d-lactic acid and 19.70 g l^?1 pyruvic acid in 10 h. Using a simple ion exchange process, d-lactic acid and pyruvic acid were effectively separated from the biotransformation system. Co-production of d-lactic acid and pyruvic acid by enantioselective oxidation of racemic lactic acid is technically feasible.