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Rice-shrimp rotation systems are one of the widespread farming practices in the Vietnamese Mekong Delta coastal areas. However, greenhouse gas (GHG) emissions in the system have remained unclear. This study aimed to examine methane (CH4) and nitrous oxide (N2O) emissions from the system, including (i) land-based versus high-density polyethylene-lined (HDPE) nursery ponds and (ii) conventional versus improved grow-out ponds inoculated with effective microorganisms (EM) bioproducts. The results showed that CH4 flux in land-based and HDPE-lined nursery ponds were 1.04 and 0.25 mgCH4 m-2 h-1, respectively, while the N2O flux was 8.37 and 6.62 µgN2O m-2 h-1, respectively. Global warming potential (GWP) from land-based nursery ponds (18.3 g CO2eq m-2) was approximately 3 folds higher than that of the HDPE-lined nursery pond (6.1 g CO2eq m-2). Similarly, the mean CH4 and N2O fluxes were 15.84 mg CH4 m-2 h-1 and 7.17 µg N2O m-2 h-1 for the conventional ponds, and 10.51 mg CH4 m-2 h-1 and 7.72 µg N2O m-2 h-1 for the improved grow-out ponds. Conventional practices (2388 g CO2eq m-2) had a higher 1.5-fold GWP compared to the improved grow-out pond (1635 g CO2eq m-2). The continuation of the land-based nursery pond and conventional aquacultural farming practices increase CH4 emission and GWP, while applying HDPE-lined nursery ponds combined with improved grow-out ponds could be a promising approach for reducing GHG emissions in rice-shrimp rotation systems. This study recommends further works in the rice-shrimp rotation systems, including (i) an examination of the effects of remaining rice stubbles in the platform on the availability of TOC levels and GHG emissions and (ii) ameliorating dissolved oxygen (DO) concentration on the effectiveness of GHG emission reduction.

RESUMEN

Halophilic bacteria have adapted to survive in high-salinity environments by accumulating amino acids and their derivatives as organic osmolytes. L-Proline (Pro) is one such osmolyte that is also being used as a feed stimulant in the aquaculture industry. Halomonas elongata OUT30018 is a moderately halophilic bacterium that accumulates ectoine (Ect), but not Pro, as an osmolyte. Due to its ability to utilize diverse biomass-derived carbon and nitrogen sources for growth, H. elongata OUT30018 is used in this work to create a strain that overproduces Pro, which could be used as a sustainable Pro-rich feed additive. To achieve this, we replaced the coding region of H. elongata OUT30018's Ect biosynthetic operon with the artificial self-cloned proBm1AC gene cluster that encodes the Pro biosynthetic enzymes: feedback-inhibition insensitive mutant γ-glutamate kinase (γ-GKD118N/D119N), γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase. Additionally, the putA gene, which encodes the key enzyme of Pro catabolism, was deleted from the genome to generate H. elongata HN6. While the Ect-deficient H. elongata KA1 could not grow in minimal media containing more than 4% NaCl, H. elongata HN6 thrived in the medium containing 8% NaCl by accumulating Pro in the cell instead of Ect, reaching a concentration of 353.1 ± 40.5 µmol/g cell fresh weight, comparable to the Ect accumulated in H. elongata OUT30018 in response to salt stress. With its genetic background, H. elongata HN6 has the potential to be developed into a Pro-rich cell factory for upcycling biomass waste into single-cell feed additives, contributing to a more sustainable aquaculture industry.IMPORTANCEWe report here the evidence for de novo biosynthesis of Pro to be used as a major osmolyte in an ectoine-deficient Halomonas elongata. Remarkably, the concentration of Pro accumulated in H. elongata HN6 (∆ectABC::mCherry-proBm1AC ∆putA) is comparable to that of ectoine accumulated in H. elongata OUT30018 in response to high-salinity stress. We also found that among the two γ-glutamate kinase mutants (γ-GKD118N/D119N and γ-GKD154A/E155A) designed to resemble the two known Escherichia coli feedback-inhibition insensitive γ-GKD107N and γ-GKE143A, the γ-GKD118N/D119N mutant is the only one that became insensitive to feedback inhibition by Pro in H. elongata. As Pro is one of the essential feed additives for the poultry and aquaculture industries, the genetic makeup of the engineered H. elongata HN6 would allow for the sustainable upcycling of high-salinity waste biomass into a Pro-rich single-cell eco-feed.

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RESUMEN

L-Proline (Pro) is an essential amino acid additive in livestock and aquaculture feeds. Previously, we created a Pro overproducing Halomonas elongata HN6 by introducing an engineered salt-inducible Pro biosynthetic mCherry-proBm1AC operon and deleting a putA gene that encoded a Pro catabolic enzyme in the genome of H. elongata OUT30018. Here, we report a generation of a novel Pro overproducing H. elongata HN10 strain with improved salt tolerance and higher Pro yield by expressing the mCherry-proBm1AC operon and deleting the putA gene in the genome of a spontaneous mutant H. elongata Glutamic acid Over-Producing, which overproduces glutamic acid (Glu) that is a precursor for Pro biosynthesis. The optimal salt concentration for growth of H. elongata HN10 was found to be 7% to 8% w/v NaCl, and the average Pro yield of 166 mg/L was achieved when H. elongata HN10 was cultivated in M63 minimal medium containing 4% w/v glucose and 8% w/v NaCl.

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A passive sampling device, called Monitor of NICotine (MoNIC), was constructed and evaluated by the laboratory of the Institute for Work and Health to determine nicotine in Environmental Tobacco Smoke (ETS). To support a large campaign on the subject of passive smoking, the CIPRET Valais gracefully distributed more than 1500 MoNIC badges to the Swiss population in order to measure ETS. Non-stimulated saliva was also collected to determine nicotine/cotinine levels of participating volunteers. The inhaled Cigarette Equivalents (CE) by non-smokers was calculated for non-smokers, based on a reference of 0.2 mg of nicotine per cigarette. Using the detected CE on the badge for non-smokers, and comparing it to the nicotine/cotinine levels in saliva, we could confirm the use of the CE concept for estimating exposure to ETS.

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