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1.

Aim

The fine roots of trees may show plastic responses to their resource environment. Several, contrasting hypotheses exist on this plasticity, but empirical evidence for these hypotheses is scattered. This study aims to enhance our understanding of tree root plasticity by examining intra-specific variation in fine-root mass and morphology, fine-root growth and decomposition, and associated mycorrhizal interactions in beech (Fagus sylvatica L.) and spruce (Picea abies (L.) Karst.) forests on soils that differ in resource availability.

Methods

We measured the mass and morphological traits of fine roots (i.e. ≤ 2 mm diameter) sampled to 50 cm depth. Fine-root growth was measured with ingrowth cores, and fine-root decomposition with litter bags. Mycorrhizal fungal biomass was determined using ingrowth mesh bags.

Results

Both tree species showed more than three times higher fine-root mass, and a ten-fold higher fine-root growth rate on sand than on clay, but no or marginal differences in overall fine-root morphology. Within the fine-root category however, beech stands had relatively more root length of their finest roots on clay than on sand. In the spruce stands, ectomycorrhizal mycelium biomass was larger on sand than on clay.

Conclusions

In temperate beech and spruce forests, fine-root mass and mycorrhizal fungal biomass, rather than fine-root morphology, are changed to ensure uptake under different soil resource conditions. Yet enhancing our mechanistic understanding of fine-root trait plasticity and how it affects tree growth requires more attention to fine-root dynamics, the functional diversity within the fine-roots, and mycorrhizal symbiosis as an important belowground uptake strategy.
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2.

Background and aims

Microbial communities and their associated enzyme activities affect the quantity and chemical quality of carbon in soil. We aimed to evaluate the biochemical mechanisms underlying how N2-fixing species influences soil organic carbon chemical composition through soil microbial functional groups and enzyme activities.

Methods

We examined the effects of N2-fixing species mixed with Eucalyptus on soil carbon storage, and the chemical composition of an 8-year-old pure Eucalyptus urophylla plantation (PP) and a mixed E.urophylla and Acacia mangium plantation (MP).

Results

The soil carbon stock and recalcitrant carbon chemical component significantly increased in surface soil in MP. The total PLFAs and bacterial PLFAs increased by 29.1% and 27.0% in cool-dry season, while in the warm-wet season, the total PLFAs and bacterial PLFAs increased by 13.1% and 27.3%, respectively. However, the fungal PLFAs decreased significantly in warm-wet season in MP. The total activity of the cellulose-degrading enzyme β-glucosidase was significantly greater with mixed N2-fixing species in both dry-cool and wet-warm season. The increase in the Alk-C/O-Alk-C ratio and SOC was strongly associated with both C-acquisition activity and bacterial community.

Conclusions

Our findings highlight the importance of N2-fixing species in regulating both soil microbial communities and their functioning in association with soil extracellular enzyme activities, which contribute to the increased soil carbon storage and recalcitrant carbon composition in Eucalyptus plantations.
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3.

Objectives

To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.

Results

Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.

Conclusions

Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.
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4.

Background, aim, and scope

The rise in wood fuel consumption, particularly of charcoal, has been associated with increased deforestation in Ghana. Plantation developments from teak (Tectona grandis), bamboo (Bambusa balcooa), and Acacia auriculiformis are now being promoted to produce sustainable biomass for charcoal production. While all species have comparable charcoal quality, there is limited available data to elucidate the environmental impacts associated with their plantation development and use as biomass sources for producing charcoal. Therefore, this study quantified and compared the cradle-to-gate environmental impacts of producing charcoal from T. grandis, A. auriculiformis, and B. balcooa.

Methods

The study was conducted in accordance with ISO 14040/14044, an international procedural framework for performing life cycle analysis (LCA). For this study, the functional unit of charcoal used was 1 MJ energy produced from three species: T. grandis, A. auriculiformis, and B. balcooa. Data on B. balcooa plantations was collected from a B. balcooa-based intercropping system set up by the International Network for Bamboo and Rattan in Sekyere Central District, Ghana. Input data for A. auriculiformis and T. grandis came from the Forestry Commission of Ghana plantations established within the forest agroecological zone of Ghana. All input data came from primary local sources. Pollutant emissions were also calculated in order to analyze the contribution of all the flow processes to the emissions. The analysis used Simapro version 8, as well as life cycle inventory (LCI) databases of Ecoinvent V3 and Idemat 2015 (a database developed by Delft University of Technology, the Netherlands). The emissions were expressed as eco-costs and used as indicators in an impact assessment.

Results and discussion

The results showed that relative to B. balcooa, the total eco-cost (comprising of human health, ecosystem, resource depletion, and global warming eco-costs) of a cradle-to-gate production of 1 MJ of charcoal will be 140% higher with T. grandis and 113% higher with A. auriculiformis. The increased environmental impacts associated with T. grandis and A. auriculiformis occurred at their biomass production stage. As these species use comparatively large quantities of pesticides, weedicides, and fertilizers with high acidification, ozone depletion, and global warming potentials, their biomass production stage accounted for approximately 85% of their total eco-cost.

Conclusions

The study results suggest that B. balcooa plantations are the most environmentally viable option. In cases where T. grandis or A. auriculiformis plantations are widespread, improvement options at the biomass production stage are required in order to reduce their environmental costs.
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5.

Objectives

To construct an Escherichia coli strain capable of producing riboflavin with high titer and yield.

Results

A low copy number plasmid pLS01 containing a riboflavin operon under the control of a constitutive promoter was constructed and introduced into Escherichia coli MG1655. Subsequently, the pfkA, edd and ead genes were disrupted, and the resulting strain LS02T produced 667 mg riboflavin/l in MSY medium supplied with 10 g glucose/l in flask cultivation. In a fed-batch process, riboflavin production of the strain reached 10.4 g/l with a yield of 56.8 mg riboflavin/g glucose.

Conclusion

To our knowledge, this is the first report of engineered E. coli strains that can produce more than 10 g riboflavin/l in fed-batch cultivation, indicating that E. coli has potential for riboflavin production.
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6.

Objective

A potential thermotolerant l-leucine dehydrogenase from Laceyella sacchari (Ls-LeuDH) was over-expressed in E. coli, purified and characterized.

Results

Ls-LeuDH had excellent thermostability with a specific activity of 183 U/mg at pH 10.5 and 25 °C. It retained a high activity in 200 mM carbonate buffer from pH 9.5 to 11. The optimal temperature for Ls-LeuDH was 60 °C.

Conclusion

It is the first time that a thermostable and highly active LeuDH originating from L. sacchari has been characterized. It may be useful for medical and pharmaceutical applications.
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7.

Background and aims

Invasive plants have been associated with alterations to soil properties, functions, and organisms, with the potential to impact ecosystem processes. An observational study was conducted to determine how the invasive plant Frangula alnus affects soil microbial communities and biogeochemical processes in Wisconsin forests.

Methods

Paired invaded/non-invaded sites (n = 10), including high (n = 5) and low (n = 5) density invasions, were sampled in spring, summer, and fall. Soil was analyzed for extractable and total nitrogen (N), N mineralization rate, total carbon, microbial biomass carbon and N, and microbial community structure using terminal restriction fragment length polymorphisms.

Results

Linear regression analysis with robust variance estimation revealed higher N mineralization rates in invaded sites than non-invaded sites in summer, and in high density invaded sites than non-invaded sites overall (p < 0.05). There was not a corresponding increase in extractable N. No differences between invaded and non-invaded sites were observed for other variables.

Conclusions

Nitrogen-rich F. alnus leaf litter (3.2 % of dry mass) may contribute to elevated N mineralization at these sites, though pre-existing conditions may be responsible. Results suggest that F. alnus alters N cycling but has little impact on soil carbon pools and microbial communities.
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8.

Aims

Forests induce a mechanical reinforcement of soil, generally quantified in terms of additional root cohesion (c r ), which decreases due to root decay after felling. The aim of this work is providing new field data on soil reinforcement by roots after trees cutting.

Methods

The present work investigated c r decay in a mixed Silver Fir-Norway Spruce (Abies alba Mill. Picea abies (L.) Karst.) stand in the Italian Alps over a period of 3 years after felling by monitoring the two c r driving variables: root tensile resistance and root density.

Results

Results showed that a significant difference in root resistance occurred only 3 years after felling, whereas the decrease in the number of roots was significant in the second year. The degradation process was more rapid in shallower layers and for thinner roots, as a consequence of the pattern of biological activity rate. The reduction of c r after felling was, for a reference profile depth of 70 cm, 55 % in the first 2 years and another 16 % in the third year.

Conclusions

The findings of this study, providing new data on the decrease of c r after felling, can be introduced into geotechnical models allowing a better estimation of the stability of forest hillslopes.
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9.

Objectives

To find new metabolic engineering strategies to improve the yield of acetone in Escherichia coli.

Results

Results of flux balance analysis from a modified Escherichia coli genome-scale metabolic network suggested that the introduction of a non-oxidative glycolysis (NOG) pathway would improve the theoretical acetone yield from 1 to 1.5 mol acetone/mol glucose. By inserting the fxpk gene encoding phosphoketolase from Bifidobacterium adolescentis into the genome, we constructed a NOG pathway in E.coli. The resulting strain produced 47 mM acetone from glucose under aerobic conditions in shake-flasks. The yield of acetone was improved from 0.38 to 0.47 mol acetone/mol glucose which is a significant over the parent strain.

Conclusions

Guided by computational analysis of metabolic networks, we introduced a NOG pathway into E. coli and increased the yield of acetone, which demonstrates the importance of modeling analysis for the novel metabolic engineering strategies.
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10.

Background and aims

Low nitrogen negatively affects soil fertility and plant productivity. Glucose-6-phosphate dehydrogenase (G6PDH) and Epichloë gansuensis endophytes are two factors that are associated with tolerance of Achnatherum inebrians to abiotic stress. However, the possibility that E. gansuensis interacts with G6PDH in enhancing low nitrogen tolerance of host grasses has not been examined.

Methods

A. inebrians plants with (E+) and without E. gansuensis (E?) were subjected to different nitrogen concentration treatments (0.1, 1, and 7.5 mM). After 90 days, physiological studies were carried out to investigate the participation of G6PDH in the adaption of host plants to low nitrogen availability.

Results

Low nitrogen retarded the growth of A. inebrians. E+ plants had higher total dry weight, chlorophyll a and b contents, net photosynthesis rate, G6PDH activity, and GSH content, while having lower plasma membrane (PM) NADPH oxidase activity, NADPH/NADP+ ratios, and MDA and H2O2 than in E? A. inebrians plants under low nitrogen concentration.

Conclusions

The presence of E. gansuensis played a key role in maintaining the growth of the A. inebrians plants under low nitrogen concentration by regulating G6PDH activity and the NADPH/NADP+ ratio and improving net photosynthesis rate.
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11.

Objectives

To develop preventive canine oral health bio-materials consisting of probiotics and glucanase to reduce insoluble glucan and volatile sulfur compound formation.

Results

Co-cultivation of Enterococcus faecium T7 with Streptococcus mutans at inoculation ratio of 3:1 (v/v) resulted in 25% reduction in the growth of Streptococcus mutans. Amounts of soluble and insoluble glucans produced by S. mutans were decreased to 70 and 55%, respectively. Insoluble glucan was decreased from 0.6 µg/ml in S. mutans culture to 0.03 µg/ml in S. mutans co-cultivated with E. faecium T7 in the presence of Lipomyces starkeyi glucanase. Volatile sulfur compound, a main component of halitosis produced by Fusobacteria nucleatum, was decreased by co-cultivating F. nucleatum with E. faecium.

Conclusion

E. faecium and glucanase can be combined as potentially active ingredients of oral care products for pets by reducing plaque-forming bacteria growth and their by-products that cause cavity and periodontal disease.
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12.

Objective

To develop a safe and effective oral vaccine against Helicobacter pylori using its HpaA protein expressed in Lactococcus lactis.

Results

The gene encoding HpaA was obtained by PCR and ligated to pNZ8110-lysM following digestion with NaeI + SphI. The recombinant plasmid was transferred into E. coli for multiplication, and then into L. lactis. The recombinant L. lactis was induced to express HpaA, resulting in two products of 29 and 25 kDa, both of which yielded positive immunoreaction with mouse antisera against H. pylori, as confirmed by immunoblot assays. The 29 kDa product constituted 12% of the cell lysates. Oral inoculation with the engineered L. lactis evoked significantly elevated serum IgG level in mice (P < 0.05).

Conclusions

A novel engineered L. lactis strain was developed that efficiently produces whole HpaA protein with desired antigenicity and potent immunogenicity. It provides a basis for approaches to L. lactis-delivered anti-H. pylori vaccination.
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13.

Aims

Plants interact by modifying soil conditions in plant-soil feedback processes. Foliar endophytes of grasses exert multiple effects on host rhizosphere with potential consequences on plant-soil feedback. Here, we hypothesize that the grass-endophyte symbiosis impairs soil symbiotic potential, and in turn influences legume performance and nitrogen acquisition.

Methods

Soil was conditioned in pots, growing Lolium multiflorum with or without the fungal endophyte Epichloë and with or without arbuscular mycorrhizal fungi (AMF). Then, Trifolium repens grew in all types of conditioned soils with high or low rhizobia availability.

Results

Endophyte soil conditioning reduced AMF spores number and rhizobial nodules (?27 % and ?38 %, respectively). Seedling survival was lower in endophyte-conditioned soil and higher in mycorrhizal soils (?27 % and +24 %, respectively). High rhizobia-availability allowed greater growth and nitrogen acquisition, independent of soil conditioning. Low rhizobia-availability allowed both effects only in endophyte-conditioned soil.

Conclusion

Endophyte-induced changes in soil (i) hindered symbiotic potential by reducing AMF spore availability or rhizobia nodulation, (ii) impaired legume survival irrespective of belowground symbionts presence, but (iii) mimicked rhizobia effects, enhancing growth and nitrogen fixation in poorly nodulated plants. Our results show that shoot and root symbionts can be interactively involved in interspecific plant-soil feedback.
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14.

Objectives

To discover novel ketoreductases (KRED) from soil metagenome preparation of chiral alcohols.

Results

Three putative KRED were cloned, heterologously expressed in Eschericha coli and characterized based on the sequence analysis of soil metagenome. All the three enzymes (KRED424, KRED432, and KRED433) had maximum activity at 55 °C and pH 7. KRED424 had a broader substrate spectrum compared with the other two. Three prochiral carbonyl compounds were used to evaluate the abilities of enantioselective reductions of the KRED. For N-Boc-3-pyrrolidone, all enzymes produced an (S)-type alcohol in enantiomeric excess (>99 % ee). For ethyl 2-oxo-4-phenylbutyrate, KRED424 showed a higher conversion (91.5 %) and enantioselectivity (S-type, >99 % ee) than KRED432 and KRED433. For ethyl 4-chloroacetoacetate (COBE), both of KRED424 and KRED433 completely converted 20 mM substrate and KRED433 could obtain an (R)-alcohol with 94 % ee.

Conclusions

The three ketoreductases have potential in the preparation of pharmaceuticals and fine chemicals.
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15.

Objectives

To engineer Escherichia coli for the heterologous production of di-rhamnolipids, which are important biosurfactants but mainly produced by opportunistic pathogen Pseudomonas aeruginosa.

Results

The codon-optimized rhlAB and rhlC genes originating from P. aeruginosa and Burkholderia pseudomallei were combinatorially expressed in E. coli to produce di-rhamnolipids with varied congeners compositions. Genes involved in endogenous upstream pathways (rhamnose and fatty acids synthesis) were co-overexpressed with rhlABrhlC, resulting in variations of rhamnolipids production and congeners compositions. Under the shake-flask condition, co-overexpression of rfbD with rhlABrhlC increased rhamnolipids production (0.64 ± 0.02 g l?1) than that in strain only expressing rhlABrhlC (0.446 ± 0.009 g l?1), which was mainly composed of di-rhamnolipids congeners Rha–Rha–C10–C10.

Conclusion

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically engineered E. coli strains were achieved via combiniations of mono-/di-rhamnolipids synthesis modules and endogenous upstream modules.
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16.

Objectives

A new solvent-tolerant species, Staphylococcus aureus, was isolated and characterized during the screening of butanol-tolerant microorganisms.

Results

Three isolates of S. aureus were obtained as contaminants during improvement of butanol tolerance of E. coli K12. Their cell dry weights were 135 % that of K12 in the absence of butanol stress. S. aureus had a growth advantage over K12 when cultured with various concentrations of butanol. It can tolerate up to 3 % (v/v) butanol, while most solventogenic bacteria can tolerate only 2 % (v/v) butanol. The addition of 10–20 g glucose/l enhanced its butanol tolerance. The relative cell biomass of the S. aureus was 71–306 % that of E. coli under 5.5–10 % (v/v) ethanol stress, indicating ethanol resistance.

Conclusions

This is the first study to observe butanol-tolerant S. aureus. As this organism can be genetically manipulated, it could have a wide array of applications.
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17.

Objectives

To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli.

Results

We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coliE. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L.

Conclusions

Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.
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18.

Objectives

To characterize a novel feruloyl esterase from Escherichia coli BL21 DE3.

Results

The gene encoding BioH was cloned and overexpressed in E. coli. The protein was purified and its catalytic activity was assessed. BioH exhibited feruloyl esterase activity toward a broad range of substrates, and the corresponding kinetic constants for the methyl ferulate, ethyl ferulate, and methyl p-coumarate substrates were: K m values of 0.48, 6.3, and 1.9 mM, respectively, and k cat /K m values of 9.3, 3.8, and 3.8 mM?1 s?1, respectively.

Conclusions

Feruloyl esterase from E. coli was expressed for the first time. BioH was confirmed to be a feruloyl esterase.
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19.

Background

This study is aimed at investigating myocardial multi-directional systolic deformation in hypertensive with different left ventricular ejection fraction (LVEF), and exploring its contribution to LVEF.

Methods

One hundred and twenty-three patients with primary hypertension (HT) were divided into group A (LVEF ≥ 55%), group B (45% ≤ LVEF < 50%, or 50% ≤ LVEF < 55% + LVEDVI ≥ 97 ml/m2), and group C (LVEF < 45%). Two-dimensional strain echocardiography (2DSE) including LV longitudinal strain (SL), radial strain (SR) and circumferential strain (SC) were measured.

Results

SL decreased gradually from group A, B to C (all p < 0.05) while SR and SC were reduced only in group B and C (all p < 0.05). All strain measurements correlated to LVEF, with the strongest correlation in SC (r = ?0.82, p < 0.01) and the second in SL (r = ?0.76). The diastolic E/e increased from group A, B to C.

Conclusions

Left ventricular multi-directional deformation correlated well to LVEF in hypertension and particularly SC, indicating that it was SC, not SL or SR, that makes the prominent contribution to left ventricular pump function.
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20.

Objective

To improve the production of welan gum and obtain a carotenoid-free strain while reducing the fermentation and post-treatment costs.

Results

The vitreoscilla globin (vgb) gene combined with the β-galactosidase (lacZ) promoter was inserted into the phytoene synthase (crtB) gene region of the chromosome in Alcaligenes sp. ATCC31555. When the recombinant strain was grown in a 5 l fermentor, welan gum was produced at 24 ± 0.4 g l?1 compared to 21 g ± 0.4 g l?1 in the wild type. Furthermore, the carotenoid-free welan gum produced using Alcaligenes sp. ATCC31555 VHb strain was less expensive with improved properties.

Conclusions

Alcaligenes sp. ATCC31555 VHb strain was a better neutral welan-producing strain with a higher production than the wild-type strain.
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