CAT and MDH improve the germination and alleviate the oxidative stress of cryopreserved <Emphasis Type="Italic">Paeonia</Emphasis> and <Emphasis Type="Italic">Magnolia</Emphasis> pollen

Researches have reported that reactive oxygen species (ROS)-induced oxidative stress plays an important role in cell cryodamage during cryopreservation. In the current study, pollen from Magnolia denudata and Paeonia lactiflora ‘Zi Feng Chao Yang’ was cryopreserved and incubated with exogenous catalase (CAT) and malate dehydrogenase (MDH) immediately after thawing. The effect of CAT and MDH on the germination of cryopreserved pollen was measured. Based on that, the ROS level, lipid peroxidation and antioxidants activities in fresh pollen, cryopreserved pollen added with or without CAT or MDH were determined to investigate their relationship with oxidative stress. Pollen from Magnolia and Paeonia showed a significant loss of germination, but marked increase of ROS and malondialdehyde (MDA) production after cryostorage. Antioxidant profiles in them were also enhanced. CAT and MDH addition increased the post-LN pollen germination of Magnolia and Paeonia significantly. Their germination rate achieved the highest with 100 IU ml^?1 MDH and 400 IU ml^?1 CAT application, respectively. Compared to their untreated controls, ROS and MDA accumulation reduced significantly in cryopreserved Magnolia pollen treated with 100 IU ml^?1 MDH, while superoxide dismutase (SOD) activity improved markedly. In the case of Paeonia, significantly lower level of ROS and MDA, but higher activity of CAT and SOD were observed in cryopreserved pollen treated with 400 IU ml^?1 CAT. In conclusion, pollen deterioration after cryopreservation is associated with ROS-induced oxidative stress. Exogenous CAT and MDH can reduce the oxidative damage through the activity stimulation of antioxidant enzymes, and play a protective role in the pollen during cryopreservation.     

Effect of photodynamic inactivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> by hypericin

The present study has focused on the effects of hypericin (Hyp) based photodynamic inactivation (PDI) of Escherichia coli (E. coli). To evaluate the efficiency of Hyp based PDI of E. coli, single factor experiments and response surface optimization experiment were conducted to obtain the optimum parameter values (36 µM Hyp, 5.9 J cm^?2 light dose: 16.4 mW cm^?2, 60 W, 260 s, 590 nm and 68 min incubation time) and finally achieved a 4.1 log CFU mL^?1 decrease of E. coli. Cell-Hyp interaction and intracellular reactive oxygen species (ROS) level were detected by fluorescence spectrometric photometer. Data indicated that Hyp possessed a strong ability to bind with cells. In addition, a significant increase was observed in intracellular ROS level after Hyp-based photosensitization treatment. Therefore, Hyp-based photosensitization seems to be a promising method to efficiently inactivate E. coli. It is expected to be a safe, efficient, low cost and practical method which can be applied in the field of food safety.     

Phycoremediation potential,physiological, and biochemical response of <Emphasis Type="Italic">Amphora subtropica</Emphasis> and <Emphasis Type="Italic">Dunaliella</Emphasis> sp. to nickel pollution

Metal pollution can produce many biological effects on aquatic environments. The marine diatom Amphora subtropica and the green alga Dunaliella sp. possess a high metal absorption capacity. Nickel (Ni) removal by living cells of A. subtropica and Dunaliella sp. was tested in cultures exposed to different Ni concentrations (100, 200, 300, and 500 mg L^?1). The amount of Ni removed by the microalgae increased with the time of exposure and the initial Ni concentration in the medium. The metal, which was mainly removed by bioadsorption to Dunaliella sp. cell surfaces (93.63% of total Ni (for 500 mg Ni L^?1) and by bioaccumulation (80.82% of total Ni (for 300 mg Ni L^?1) into Amphora subtropica cells, also inhibited growth. Exposure to Ni drastically reduced the carbohydrate and protein concentrations and increased total lipids from 6.3 to 43.1 pg cell^?1, phenolics 0.092 to 0.257 mg GAE g^?1 (Fw), and carotenoid content, from 0.08 to 0.59 mg g^?1 (Fw), in A. subtropica. In Dunaliella sp., total lipids increased from 26.1 to 65.3 pg cell^?1, phenolics from 0.084 to 0.289 mg GAE g^?1 (Fw), and carotenoid content from 0.41 to 0.97 mg g^?1 (Fw). These compounds had an important role in protecting the algae against ROS generated by Ni. In order to cope with Ni stress shown by the increase of TBARS level, enzymatic (SOD, CAT, and GPx) ROS scavenging mechanisms were induced.     

Selected reactive oxygen species and antioxidant enzymes in common bean after <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">phaseolicola</Emphasis> and <Emphasis Type="Italic">Botrytis cinerea</Emphasis> infection

Phaseolus vulgaris cv. Korona plants were inoculated with the bacteria Pseudomonas syringae pv. phaseolicola (Psp), necrotrophic fungus Botrytis cinerea (Bc) or with both pathogens sequentially. The aim of the experiment was to determine how plants cope with multiple infection with pathogens having different attack strategy. Possible suppression of the non-specific infection with the necrotrophic fungus Bc by earlier Psp inoculation was examined. Concentration of reactive oxygen species (ROS), such as superoxide anion (O₂ ^?) and H₂O₂ and activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were determined 6, 12, 24 and 48 h after inoculation. The measurements were done for ROS cytosolic fraction and enzymatic cytosolic or apoplastic fraction. Infection with Psp caused significant increase in ROS levels since the beginning of experiment. Activity of the apoplastic enzymes also increased remarkably at the beginning of experiment in contrast to the cytosolic ones. Cytosolic SOD and guaiacol peroxidase (GPOD) activities achieved the maximum values 48 h after treatment. Additional forms of the examined enzymes after specific Psp infection were identified; however, they were not present after single Bc inoculation. Subsequent Bc infection resulted only in changes of H₂O₂ and SOD that occurred to be especially important during plant–pathogen interaction. Cultivar Korona of common bean is considered to be resistant to Psp and mobilises its system upon infection with these bacteria. We put forward a hypothesis that the extent of defence reaction was so great that subsequent infection did not trigger significant additional response.     

Enhancement of Skin Permeation and Skin Immunization of Ovalbumin Antigen <Emphasis Type="Italic">via</Emphasis> Microneedles

The purpose of this study was to evaluate the use of different types of microneedles and doses of ovalbumin antigen for in vitro skin permeation and in vivo immunization. In vitro skin permeation experiments and confocal laser scanning microscopy revealed that hollow microneedles had a superior enhancing effect on skin permeation compared with a solid microneedle patch and untreated skin by efficiently delivering ovalbumin-fluorescein conjugate into the deep skin layers. The flux and cumulative amount of ovalbumin-fluorescein conjugate at 8 h after administering with various conditions could be ranked as follows: hollow MN; high dose?>?medium dose?>?low dose?>?MN patch; high dose?>?medium dose?>?low dose?>?untreated skin; high dose?>?medium dose?>?low dose?>?without ovalbumin-fluorescein conjugate. As the dose of ovalbumin-fluorescein conjugate was increased to 500 μg, the antigen accumulated in the skin to a greater extent, as evidenced by the increasing green fluorescence intensity. When the hollow microneedle was used for the delivery of ovalbumin into the skin of mice, it was capable of inducing a stronger immunoglobulin G immune response than conventional subcutaneous injection at the same antigen dose. Immunoglobulin G levels in the hollow MN group were 5.7, 11.6, and 13.3 times higher than those of the subcutaneous injection group for low, medium, and high doses, respectively. Furthermore, the mice immunized using the hollow microneedle showed no signs of skin infection or pinpoint bleeding. The results suggest that the hollow MN is an efficient device for delivering the optimal dose of antigen via the skin for successful immunization.     

Programmed cell death in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> induced by allelopathic effect of submerged macrophyte <Emphasis Type="Italic">Myriophyllum spicatum</Emphasis> in co-culture system

This investigation demonstrates that programmed cell death (PCD) in a cyanobacterium, Microcystis aeruginosa, resulting from allelopathic stress induced by a submerged macrophyte, Myriophyllum spicatum, in a co-culture system. The hallmarks of PCD, caspase-3-like protease activity, DNA fragmentation, and destruction of cell ultrastructure, as well as intracellular PCD signaling radicals, reactive oxygen species (ROS), and nitric oxide (NO), were measured in M. aeruginosa cells co-cultured with M. spicatum for 7 days. The results showed a dose–response relationship between M. spicatum biomass and M. aeruginosa mortality. A caspase-3-like protease was activated and elevated from day 3. Thylakoid disintegration, cytoplasmic vacuolation, and fuzzy nuclear zone were observed by transmission electron microscopy, and distinct DNA fragmentation was detected in M. aeruginosa cells at a M. spicatum biomass of 6.0 g fresh weight (FW) L^?1 during the 7 days. Allelochemicals of total phenolic compounds (TPCs) were determined in co-culture water, and the concentration increased with increasing of M. spicatum biomass and co-culture time. Compared with the level of ROS production in the control group, a significant overproduction of ROS was detected in M. aeruginosa cells in the treatment group, and this was positively correlated with TPC concentration. Furthermore, the level of intracellular NO increased with the percent mortality of M. aeruginosa. The results indicated that a PCD pathway was induced in the cyanobacterium M. aeruginosa when co-cultured with the submerged macrophyte M. spicatum.     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

     

Glutathione improves survival of cryopreserved embryogenic calli of <Emphasis Type="Italic">Agapanthus praecox</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis>

The effect of supplementation of reduced glutathione (GSH) to cryoprotectant solution on the generation of reactive oxygen species (ROS) (e.g., H₂O₂, OH^·, and O ₂ ^·? ) and antioxidants (e.g., SOD, POD, CAT, AsA, and GSH), as well as membrane lipid peroxidation (i.e., MDA content) mitigation in cryopreserving of embryogenic calli (EC) of Agapanthus praecox subsp. orientalis was investigated. The vitrification-based cryopreservation method was used in this study. The addition of GSH at a final concentration of 0.08 mM to the cryoprotectant solution has significantly improved cryotolerance of A. praecox EC. The EC post-thaw survival rate increased by 68.34 % using the cryoprotectant solution containing 0.08 mM GSH as compared to the control (GSH-free). EC treated with GSH displayed the reduction in  OH^· generation activity and the contents of H₂O₂ and MDA, as well as enhancement in the inhibition of O ₂ ^·? generation and the antioxidant activity. Treatment with exogenous GSH also increased endogenous AsA and GSH contents after dehydration step. Expression of stress-responsive genes, e.g., peroxidase (POD), peroxiredoxin, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione peroxidase (GPX), was also increased during cryopreservation processes. The expression of DAD1 (Defender against apoptotic cell death) was elevated, while cell death-related protease SBT was suppressed. These results demonstrated that the addition of GSH to cryoprotectant solution affects the ROS level and could effectively improve survival of A. praecox EC through enhancing antioxidant enzyme activities and decreasing cell death.     

Development of an efficient process intensification strategy for enhancing <Emphasis Type="Italic">Pfu</Emphasis> DNA polymerase production in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

An efficient induction strategy that consisted of multiple additions of small doses of isopropyl-β-D-thiogalactopyranoside (IPTG) in the early cell growth phase was developed for enhancing Pfu DNA polymerase production in Escherichia coli. In comparison to the most commonly used method of a single induction of 1 mM IPTG, the promising induction strategy resulted in an increase in the Pfu activity of 13.5 % in shake flasks, while simultaneously decreasing the dose of IPTG by nearly half. An analysis of the intracellular IPTG concentrations indicated that the cells need to maintain an optimum intracellular IPTG concentration after 6 h for efficient Pfu DNA polymerase production. A significant increase in the Pfu DNA polymerase activity of 31.5 % under the controlled dissolved oxygen concentration of 30 % in a 5 L fermentor was achieved using the multiple IPTG induction strategy in comparison with the single IPTG induction. The induction strategy using multiple inputs of IPTG also avoided over accumulation of IPTG and reduced the cost of Pfu DNA polymerase production.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> spp. increases root biomass and tropane alkaloid yields in transgenic hairy roots of <Emphasis Type="Italic">Datura</Emphasis> spp.

Transgenic hairy roots of Datura spp., established using strain A4 of Agrobacterium rhizogenes, are genetically stable and produce high levels of tropane alkaloids. To increase biomass and tropane alkaloid content of this plant tissue, four Pseudomonas strains, Pseudomonas fluorescens P64, P66, C7R12, and Pseudomonas putida PP01 were assayed as biotic elicitors on transgenic hairy roots of Datura stramonium, Datura tatula, and Datura innoxia. Alkaloids were extracted from dried biomass, and hyoscyamine and scopolamine were quantified using liquid chromatography-tandem mass spectrometry analysis. D. stramonium and D. innoxia biomass production was stimulated by all Pseudomonas spp. strains after a 5-d treatment. All strains of P. fluorescens increased hyoscyamine yields compared to untreated cultures after both 5 and 10 d of treatment. Hyoscyamine yields were highest in D. tatula cultures exposed to a 5-d treatment with C7R12 (16.633 + 0.456 mg g^?1 dry weight, a 431% increase) although the highest yield increases compared to the control were observed in D. stramonium cultures exposed to strains P64 (511% increase) and C7R12 (583% increase) for 10 d. D. innoxia showed the highest scopolamine yields after elicitation with P. fluorescens strains P64 for 5 d (0.653 + 0.021 mg g^?1 dry weight, a 265% increase) and P66 for 5 and 10 d (5 d, 0.754 + 0.0.031 mg g^?1 dry weight, a 321% increase; 10 d 0.634 + 0.046 mg g^?1 dry weight, a 277% increase). These results show that the Pseudomonas strains studied here can positively and significantly affect biomass and the yields of hyoscyamine and scopolamine from transgenic roots of the three Datura species.     

<Emphasis Type="Italic">Plectosphaerella cucumerina</Emphasis> as a bioherbicide for <Emphasis Type="Italic">Cirsium arvense</Emphasis>: proof of concept

Plectosphaerella cucumerina (Lindf.) W. Gams was evaluated as a bioherbicide for Cirsium arvense L. (Scop.) using a Canadian and a New Zealand isolate. Both isolates defoliated C. arvense when applied at 10¹³ conidia ha^?1 in water volumes ranging from 250 to 6400 l ha^?1 with a rapid decline in effect with declining conidial dose. Repeat application and the addition of the adjuvant Pulse^® penetrant to the conidial suspension increased the disease severity in C. arvense. Maximum disease occurred at 20 °C with a 48 h post-application dew period. The experiments demonstrate that P. cucumerina can defoliate C. arvense under the environmental conditions of temperate pastures where the weed is problematic. The results also show that modifications to formulation and strategic application may reduce the 48 h dew period requirement and risk to non-target species respectively, supporting the conclusion that the fungus has potential as a bioherbicide for C. arvense.     

Dietary Administration of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Enhanced Growth Performance and Innate Immune Response of Siberian Sturgeon, <Emphasis Type="Italic">Acipenser baerii</Emphasis>

We investigated the effects of Lactobacillus plantarum used as a dietary supplement on the growth performance and innate immune response in juvenile Siberian sturgeon Acipenser baerii. Juvenile fish (14.6 ± 2.3 g) were fed three experimental diets prepared by supplementing a basal diet with L. plantarum at different concentrations [1 × 10⁷, 1 × 10⁸ and 1 × 10⁹ colony-forming units (cfu) g^?1] and a control (non-supplemented basal) diet for 8 weeks. Growth performance indices were increased in fish fed the 1 × 10⁸ cfu g^?1 L. plantarum diet compared to the other groups. There was an increased innate immune response in fish fed the experimental diets. The highest levels of lysozyme activity, total immunoglobulin (IgM) and complement component 3 (C3) were observed in fish fed the diet containing L. plantarum at a concentration of 1 × 10⁸ cfu g^?1, but there was no significant difference in the level of complement component 4 (C4) in fish fed the experimental diets or the control diet. The present study underlying some positive effects (growth performance and immune indices) of dietary administration of L. plantarum at a concentration of 1 × 10⁸ cfu g^?1 in the Siberian sturgeon.     

Desiccation-induced ROS accumulation and lipid catabolism in recalcitrant <Emphasis Type="Italic">Madhuca latifolia</Emphasis> seeds

Loss of viability in desiccation-sensitive seeds of Madhuca latifolia (Roxb.) J. F. Macbr., an important multipurpose tropical tree, was correlated with seed water content (WC). WC declined from 0.59 to 0.19 g g^?1 fresh mass, 35 days after harvest from mother plant, at ambient conditions (temperature 25 ± 2 °C, relative humidity 50 ± 2%). The desiccation-induced reduction in viability was related with an accumulation of reactive oxygen species (ROS) that promoted lipid peroxidation associated loss of membrane integrity. Conducted study revealed 1.6–19 folds rise in lipid peroxidized products in desiccated M. latifolia seeds, and was found to be linked inversely with WC and germination percentage. Additionally, increased activities (7 and 13 folds) of lipid hydrolyzing enzymes; lipase (EC 3.1.1.3) and lipoxygenase (EC 1.13.11.12) respectively, were discernible in desiccating M. latifolia seeds. In summary, increased ROS, lipid oxidation, lipase and lipoxygenase were strongly correlated with viability loss in desiccating M. latifolia seeds.     

Characterization of a field-grown transgenic pineapple clone containing the genes <Emphasis Type="Italic">chitinase</Emphasis>, <Emphasis Type="Italic">AP24</Emphasis>, and <Emphasis Type="Italic">bar</Emphasis>

We previously introduced the bar gene, along with chitinase and AP24 genes, into the pineapple genome. The present report focuses on the evaluation of the first vegetative generation of a transgenic clone containing these genes. Three materials were compared: macropropagated controls (non-transformed), micropropagated controls (non-transformed), and micropropagated transformed plants. From each group, 50% of the plants were sprayed with FINALE^® 3 mo after the experiment initiation. The characterization was performed after 1 yr of field growth. FINALE^® killed all non-transgenic plants. Plants that survived the herbicide application showed 2n?=?50 chromosomes in their roots after 1 yr in the field. Micropropagated transformed plants sprayed with FINALE^® did not show phenotype differences from micropropagated transformed plants not sprayed with the herbicide. Between the micropropagated transformed plants sprayed with FINALE^® and the micropropagated control plants, the following differences were observed: modifications in levels of cell wall-linked, free and total phenolics, and total proteins. Moreover, changes of the fruit mass without crown were also recorded. Between the micropropagated transformed plants sprayed with FINALE^® and the macropropagated control plants, levels of chlorophyll b, total chlorophyll pigments, and proteins were different. Furthermore, activities of phenylalanine ammonia-lyase, superoxide dismutase, and glutamine synthetase were dissimilar. The plant height and diameter, and the crown height were also different. Until now, we have evaluated transformed pineapple plants during hardening and field growth. Although some unexpected variations were recorded, we believe they are not relevant enough to justify rejection of transgenesis as an important tool for pineapple genetic improvement.     

<Emphasis Type="Italic">Pedobacter panacis</Emphasis> sp. nov., isolated from <Emphasis Type="Italic">Panax ginseng</Emphasis> soil

A novel strain, DCY108^T was isolated from soil of a Panax ginseng field, Yeoncheon province (38°04′N 126°57′E), Republic of Korea. Strain DCY108^T is Gram-negative, non-motile, non-flagellate, rod-shaped, and aerobic. The bacterium grows optimally at 25–30 °C, pH 6.5–7.0 and 1 % NaCl. Phylogenetically, strain DCY108^T is closely related to Pedobacter jejuensis JCM 18824^T, Pedobacter aquatilis JCM 13454^T, Pedobacter kyungheensis LMG 26577^T and the type strain of the genus Pedobacter heparinus DSM 2366^T. The DNA–DNA relatedness values between strain DCY108^T and its close phylogenetic neighbors were below 30.0 %. The DNA G+C content of strain DCY108^T was determined to be 45.1 mol%. The predominant quinone was menaquinone 7 (MK-7). The major polar lipids were identified as phosphatidylethanolamine and three unidentified aminolipids AL1, AL13 and AL17. Iso-C_15:00, iso-C_17:03OH and summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) were identified as the major fatty acids present in strain DCY108^T. The results of physiological and biochemical tests allowed strain DCY108^T to be differentiated phenotypically from other recognized species belonging to the genus Pedobacter. Therefore, it is suggested that the newly isolated organism represents a novel species, for which the name Pedobacter panacis sp. nov is proposed with the type strain designated as DCY108^T (=CCTCCAB 2015196^T = KCTC 42748^T).     

Perturbed Synaptosomal Calcium Homeostasis and Behavioral Deficits Following Carbofuran Exposure: Neuroprotection by <Emphasis Type="Italic">N</Emphasis>-Acetylcysteine

The protective effects of N-acetylcysteine (NAC) on carbofuran-induced alterations in calcium homeostasis and neurobehavioral functions were investigated in rats. Rats were exposed to carbofuran at a dose of 1 mg/kg body weight, orally for a period of 28 days. A significant decrease in Ca²⁺ATPase activity was observed following carbofuran exposure with a concomitant increase in K⁺-induced ⁴⁵Ca²⁺ uptake through voltage operated calcium channels. This was accompanied with a marked accumulation of intracellular free calcium in synaptosomes. The increase in intracellular calcium levels were associated with an increased lipid peroxidation and decreased glutathione content in carbofuran exposed animals. NAC administration (200 mg/kg body weight, orally) to the carbofuran exposed animals had a beneficial effect on carbofuran-induced alterations in calcium homeostasis and resulted in repletion in glutathione levels and resulted in lowering the extent of lipid peroxidation. Marked impairment in the motor functions were seen following carbofuran exposure, which were evident by the significant decrease in the locomotor activity and reduction in the retention time of the rats on rotating rods. Cognitive deficits were also seen as indicated by the significant decrease in active and passive avoidance response. NAC treatment, on the other hand, protected the animals against carbofuran-induced neurobehavioral deficits. The results support the hypothesis that carbofuran exerts its toxic effects by disrupting calcium homeostasis, which may have serious consequences on neuronal functioning, and clearly show the potential beneficial effects of N-acetylcysteine on carbofuran induced alterations in synaptosomal calcium homeostasis.     

Manipulation of pyrite colonization and leaching by iron-oxidizing <Emphasis Type="Italic">Acidithiobacillus</Emphasis> species

In this study, the process of pyrite colonization and leaching by three iron-oxidizing Acidithiobacillus species was investigated by fluorescence microscopy, bacterial attachment, and leaching assays. Within the first 4–5 days, only the biofilm subpopulation was responsible for pyrite dissolution. Pyrite-grown cells, in contrast to iron-grown cells, were able to oxidize iron(II) ions or pyrite after 24 h iron starvation and incubation with 1 mM H₂O₂, indicating that these cells were adapted to the presence of enhanced levels of reactive oxygen species (ROS), which are generated on metal sulfide surfaces. Acidithiobacillus ferrivorans SS3 and Acidithiobacillus ferrooxidans R1 showed enhanced pyrite colonization and biofilm formation compared to A. ferrooxidans ^T. A broad range of factors influencing the biofilm formation on pyrite were also identified, some of them were strain-specific. Cultivation at non-optimum growth temperatures or increased ionic strength led to a decreased colonization of pyrite. The presence of iron(III) ions increased pyrite colonization, especially when pyrite-grown cells were used, while the addition of 20 mM copper(II) ions resulted in reduced biofilm formation on pyrite. This observation correlated with a different extracellular polymeric substance (EPS) composition of copper-exposed cells. Interestingly, the addition of 1 mM sodium glucuronate in combination with iron(III) ions led to a 5-fold and 7-fold increased cell attachment after 1 and 8 days of incubation, respectively, in A. ferrooxidans ^T. In addition, sodium glucuronate addition enhanced pyrite dissolution by 25 %.     

Raw starch conversion by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> amylases

Background

Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol.

Results

The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l^-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l^-1 (0.038 and 0.028 g l^-1 h^-1), respectively, after 10 days. With a substrate load of 200 g l^-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l^-1 ethanol (0.58 g l^-1 h^-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l^-1 ethanol (0.36 g l^-1 h^-1).

Conclusions

In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l^-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.

     

<Emphasis Type="Italic">Streptomyces ginkgonis</Emphasis> sp. nov., an endophyte from <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

A novel endophytic actinomycete strain, designated KM-1-2^T, was isolated from seeds of Ginkgo biloba at Yangling, China. A polyphasic approach was used to study the taxonomy of strain KM-1-2^T and it was found to show a range of phylogenetic and chemotaxonomic properties consistent with those of members of the genus Streptomyces. The diamino acid of the cell wall peptidoglycan was identified as LL-diaminopimelic acid. No diagnostic sugars were detected in whole cell hydrolysates. The predominant menaquinones were identified as MK-9(H₆) and MK-9(H₈). The diagnostic phospholipids were found to be phosphatidylethanolamine and phosphatidylcholine. The DNA G + C content of the novel strain was determined to be 72.9 mol%. The predominant cellular fatty acids (> 10.0?%) were identified as iso-C_14?:?0, iso-C_16?:?0, C_16?:?0 and C_17?:?0 cyclo. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain is closely related to Streptomyces carpaticus JCM 6915^T (99.3%), Streptomyces harbinensis DSM 42076^T (98.9%) and Streptomyces cheonanensis JCM 14549^T (98.5%). DNA-DNA hybridizations with these three close relatives gave similarity values of 39.1 ± 1.9, 35.8 ± 2.3, and 47.4 ± 2.7%, respectively, which indicated that strain KM-1-2^T represents a novel species of the genus Streptomyces. This is consistent with the morphological, physiological and chemotaxonomic data. Cumulatively, these data suggest that strain KM-1-2^T represents a novel Streptomyces species, for which the name Streptomyces ginkgonis sp. nov. is proposed, with the type strain KM-1-2^T (= CCTCC AA2016004^T = KCTC 39801^T).