Phosphorus recycling from waste activated sludge using the hydrothermal platform: Recovery, solubility and phytoavailability.

To address the grand challenge of increasing the sustainability of wastewater treatment plants, hydrothermal carbonization was studied as a nutrient recovery platform, transforming sludge into a valuable hydrochar. Carbonization was achieved at different temperatures (200-300 °C) and durations (30-120 min). The highest mass recovery (73%) was observed in the lowest temperature, while the lowest (49%) was obsereved at the highest temperature. Under all reaction conditions, phosphorus recovery values exceeded 80%, with the dominated fraction of inorganic-P in the hydrochar being HCl-extractable. Although HCl-extractable P is considered a moderately labile P fraction, P phytoavailability assays indicate that sewage sludge hydrochar is an excellent source for P, surpassing soluble P, likely due to its slow-release nature. We postulate that polyphosphates constitute a significant portion of this P pool. Overall, we emphasize the benefits of using HTC as a circular economy approach to convert sludge into a valuable hydrochar.

Subcritical water extraction as a circular economy approach to recover energy and agrochemicals from sewage sludge.

Global population growth is creating severe pressure on wastewater treatment plants, and specifically on sludge management. Meanwhile, the global challenge of achieving food-security requires the development of green pest control practices that maximize crop productivity. The hydrothermal technology, using subcritical water as the conversion media has been intensively studied, mostly for energy recovery purposes. Here, we focused on the aqueous phase by-product of this process and studied the subcritical water extraction of sewage sludge to recover valuable agrochemicals, with high potential of pre-emergent herbicidal activity. Full characterization of hydrothermal extracts from different reaction temperatures (200-300 °C) and times (30-120 min) highlighted the formation of pyrazine derivatives. Seed germination bioassays with three different species suggested a positive correlation between reaction temperature and extract herbicidal activity. Moreover, differences in seed viability and final root elongation between the tested crop (Trriticum aestivum) and weeds (Lapidium sativum and Amaranthus palmeri) may indicate the competitive abilities that can play a key role in weed management. Our results suggest that subcritical water can be applied as a green solvent for extracting a valuable agrochemical from sewage sludge and improving the circular economy for wastewater treatment.

Transcriptome Profiling of Ornithogalum dubium Leaves and Flowers to Identify Key Carotenoid Genes for CRISPR Gene Editing.

Ornithogalum dubium is a popular ornamental monocot native to South Africa with flower colors ranging from pure white to deep orange. Gene editing based on the CRISPR/Cas9 system has recently been shown to hold potential for color improvement in ornamental flower crops. To apply this approach to Ornithogalum color manipulation, genomic or transcriptomic data must first be collected. Here, cDNA libraries of O. dubium leaves and flowers were constructed and sequenced using the Illumina HiSeq 2500. Over 155 million 100-bp paired-end reads were assembled into a transcriptome database of 360,689 contigs, of which 18,660 contigs were differentially expressed between leaves and flowers. Carotenoids are the main pigment imparting spectrum of orange hues to O. dubium flowers. By querying our database, we identified a total of 16 unique transcripts (unigenes) predicted to be involved in the carotenoid biosynthesis pathway of Ornithogalum. Combining carotenoid profiles, we further inferred several key unigenes responsible for floral coloration and accumulation in O. dubium, of which the gene LCYB/comp146645_c0 was found as a suitable target to generate potentially red flower varieties of O. dubium. Our research thus provides a framework for the application of CRISPR/Cas9 technology to improve this ornamental crop.

Analysis of apocarotenoid volatiles during the development of Ficus carica fruits and characterization of carotenoid cleavage dioxygenase genes.

In plants the oxidative cleavage of carotenoid substrates produces volatile apocarotenoids, including ß-ionone, 6-methyl-5-hepten-2-ol, and α-ionone; these compounds are important in herbivore-plant communication. Combined chemical, biochemical, and molecular studies were conducted to evaluate the differential accumulation of carotenoids and volatile apocarotenoids during the development of pollinated and parthenocarpic fig fruits. Pollinated fig fruits showed less emission of apocarotenoid volatiles than the parthenocarpic figs, while in the case of carotenoid pigments, pollinated figs manifested higher accumulation. The apocarotenoids, 6-methyl-5-hepten-2-ol and ß-cyclogeraniol, showed a marked increase after the two weeks of hand-pollination in pollinated and parthenocarpic figs; but afterwards these volatile levels decreased during further fruit development. In addition, we report a transcriptome-based identification and functional characterization of the carotenoid cleavage dioxygenase (FcCCD) genes. These genes were overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant FcCCD1A enzyme showed specificity for the 9,10 (9',10') double bond position of cyclic carotenoids to generate α-ionone and ß-ionone, while FcCCD1B cleaved lycopene and an acyclic moiety of δ-carotene, producing 6-methyl-5-hepten-2-one. The qRT-PCR analysis of FcCCD genes revealed differential gene expression during fig fruit development. Our results suggest a role for the FcCCD1genes in apocarotenoid biosynthesis in fig fruits.

Biochemical and Molecular Factors Governing Peel-Color Development in 'Ora' and 'Shani' Mandarins.

To identify factors governing peel-color development in mandarins, we examined carotenoid content and composition and the expression of carotenoid-related genes during four stages of ripening (i.e., green, breaker, yellow, and orange) in two varieties: 'Ora', which has orange fruit, and 'Shani', which has orange-reddish fruit. The two varieties had different carotenoid compositions, and 'Shani' had a significantly higher level of total carotenoid pigments. 'Shani' was rich in the deep orange ß-cryptoxanthin and the orange-reddish ß-citraurin, whereas 'Ora' was rich in the orange violaxanthin. RNA-Seq analysis revealed significantly greater expression of the carotenoid-biosynthesis genes PSY, ßLCY, ßCHX, and CCD4b, as well as MEP-pathway genes and several ethylene-biosynthesis and -signaling genes in 'Shani' fruit. In contrast, the expression levels of genes involved in the synthesis of α-branch carotenoids (i.e., εLCY and εCHX) and ZEP, which is involved in the formation of violaxanthin, were significantly higher in the 'Ora' fruit.

Global and local perturbation of the tomato microRNA pathway by a trans-activated DICER-LIKE 1 mutant.

DICER-like 1 (DCL1) is a major player in microRNA (miRNA) biogenesis and accordingly, its few known loss-of-function mutants are either lethal or display arrested development. Consequently, generation of dcl1 mutants by reverse genetics and functional analysis of DCL1 in late-developing organs are challenging. Here, these challenges were resolved through the unique use of trans-activated RNA interference. Global, as well as organ-specific tomato DCL1 (SlDCL1) silencing was induced by crossing the generated responder line (OP:SlDCL1IR) with the appropriate driver line. Constitutive trans-activation knocked down SlDCL1 levels by ~95%, resulting in severe abnormalities including post-germination growth arrest accompanied by decreased miRNA and 21-nucleotide small RNA levels, but prominently elevated levels of 22-nucleotide small RNAs. The increase in the 22-nucleotide small RNAs was correlated with specific up-regulation of SlDCL2b and SlDCL2d, which are probably involved in their biogenesis. Leaf- and flower-specific OP:SlDCL1IR trans-activation inhibited blade outgrowth, induced premature bud senescence and produced pale petals, respectively, emphasizing the importance of SlDCL1-dependent small RNAs in these processes. Together, these results establish OP:SlDCL1IR as an efficient tool for analysing processes regulated by SlDCL1-mediated gene regulation in tomato.