ALBERT

Description: The sesquiterpene β-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, β-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of β-caryophyllene. In this study, a full-length cDNA of a new duplicated β-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce β-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt–nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg−1 fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce β-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography–mass spectrometry (GC–MS). GC–MS results revealed that the AaCPS1 produced maximum 26.5 ± 1 mg of β-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.

Description: The Baltic Sea is the largest area suffering from eutrophication-driven hypoxia. Low oxygen levels are threatening its biodiversity and ecosystem functioning. The main causes for eutrophication-driven hypoxia are high nutrient loadings and global warming. Wastewater treatment plants (WWTP) contribute to eutrophication as they are important sources of nitrogen to coastal areas. Here, we evaluated the effects of wastewater treatment plant effluent inputs on Baltic Sea planktonic communities in 4 experiments. We tested for effects of effluent inputs on chlorophyll a content, on bacterial community composition, and on metabolic rates: gross primary production (GPP), net community production (NCP), community respiration (CR) and bacterial production (BP). Nitrogen-rich dissolved organic matter (DOM) inputs from effluents increased bacterial production and decreased primary production and community respiration. Nutrient amendments and seasonally variable environmental conditions lead to lower alpha-diversity and shifts in bacterial community composition (e.g. increased abundance of a few cyanobacterial populations in the summer experiment), concomitant with changes in metabolic rates. An increase in BP and decrease in CR could be caused by high lability of the DOM that can support secondary bacterial production, without an increase in respiration. Increases in bacterial production and simultaneous decreases of primary production lead to more carbon being consumed in the microbial loop, and shifts the ecosystem towards heterotrophy.

Description: The Baltic Sea is the world's largest area suffering from eutrophication-driven hypoxia. Low oxygen levels are threatening its biodiversity and ecosystem functioning. The main causes for eutrophication-driven hypoxia are high nutrient loadings and global warming. Wastewater treatment plants (WWTP) contribute to eutrophication as they are important sources of nitrogen to coastal areas. Here, we evaluated the effects of wastewater treatment plant effluent inputs on Baltic Sea planktonic communities in four experiments. We tested for effects of effluent inputs on chlorophyll a content, bacterial community composition, and metabolic rates: gross primary production (GPP), net community production (NCP), community respiration (CR) and bacterial production (BP). Nitrogen-rich dissolved organic matter (DOM) inputs from effluents increased bacterial production and decreased primary production and community respiration. Nutrient amendments and seasonally variable environmental conditions lead to lower alpha-diversity and shifts in bacterial community composition (e.g. increased abundance of a few cyanobacterial populations in the summer experiment), concomitant with changes in metabolic rates. An increase in BP and decrease in CR could be caused by high lability of the DOM that can support secondary bacterial production, without an increase in respiration. Increases in bacterial production and simultaneous decreases of primary production lead to more carbon being consumed in the microbial loop, and may shift the ecosystem towards heterotrophy.