Strategies for the efficient synthesis of peptide macrocycles have been a long‐standing goal. In this paper, we demonstrate the use of the peptide ligase termed omniligase‐1 as a versatile and ...broadly applicable enzymatic tool for peptide cyclization. Several head‐to‐tail (multi)cyclic peptides have been synthesized, including the cyclotide MCoTI‐II. Cyclization and oxidative folding of the cyclotide MCoTI‐II were efficiently performed in a one‐pot reaction on a 1‐gram scale. The native cyclotide was isolated and the correct disulfide bonding pattern was confirmed by NMR structure determination. Furthermore, compatibility of chemo‐enzymatic peptide synthesis (CEPS) using omniligase‐1 with methods such as chemical ligation of peptides onto scaffolds (CLIPS) was successfully demonstrated by synthesizing a kinase‐inhibitor derived tricyclic peptide. Our studies indicate that the minimal ring size for omniligase‐1 mediated cyclization is 11 amino acids, whereas the cyclization of peptides longer than 12 amino acids proceeds with remarkable efficiency. In addition, several macrocycles containing non‐peptidic backbones (e.g., polyethylene glycol), isopeptide bonds (amino acid side‐chain attachment) as well as d‐amino acids could be efficiently cyclized.
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•Organics and P recovery from PACl-sludge via alkaline fermentation was studied.•43.3% of organics and 36.49% of P can be released to fermentation liquid at pH 11.•24.9% of organics ...and 27.9% of P from wastewater can be formed to valuable product.•Alkaline fermentation promotes organics and phosphorus recovery from PACl sludge.
In this study, alkaline fermentation was applied to promote organics and P recovery from polyaluminum chloride (PACl)-enhanced primary sedimentation sludge. Coagulant results demonstrated that the optimum PACl dosage of 100 mg/L resulted in the effective concentration of 73% of organic matter and 90% of P from wastewater into sludge. Batch fermentation results highlighted the ability of alkaline fermentation in improving the biodegradability of PACl sludge. More specifically, at pH 11, 43.3% of soluble organics and 36.49% of P were released to the fermentation supernatant. Furthermore, P fractionation fermented sludge results revealed that partial Al-P dissolution and organic phosphorus hydrolysis were the main drivers of the released P. Finally, at pH 11, 85% of P was recovered as magnesium ammonium phosphate from the fermentation supernatant at the 2:1 Mg/P molar ratio. In conclusion, 24.9% of organics and 27.9% of P from raw wastewater were converted to valuable products via alkaline fermentation.
Primary sludge from wastewater treatment is of rich organic contents that can be resource for recovery. However, the sludge derived from the chemically enhanced primary sedimentation (CEPS), ...especially using Al-based coagulants, features a rather low biodegradability and treatability due to the agglomeration of solid matters by the coagulant. In the present study, alkali pretreatments by three chemicals (NaHCO3, Na2CO3 and NaOH) were applied to disintegrate Al-based CEPS sludge (Al-sludge) aggregates and improve its hydrolysis and biodegradability for fermentation and organic recovery. Results showed that alkali pretreatment significantly reduced the Al-sludge particle size and accelerated the release of the enmeshed organics. NaHCO3 exhibited the best pretreatment result. At ambient temperature, the yield of volatile fatty acids (VFAs) of the Al-sludge pretreated by Na2CO3 (initial pH 10) was improved by four times compared to the untreated sludge, resulting from both the improved abiotic and biotic hydrolysis effects. The effect of alkali pretreatment on Al-sludge was found to be mainly attributable to the pH adjustment rather than the alkalinity increase. Illumina MiSeq sequencing revealed that protein-fermenting bacteria Treponema and Bacteroides became dominant during fermentation of the alkali pretreated sludge. By PACl-based CEPS and enhanced sludge acidogenesis, around 17% of organics in municipal wastewater can be recovered, mainly in the form of VFAs, which is significantly greater than 6% by the conventional primary sedimentation and sludge fermentation.
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•PACl dosing for chemically enhanced primary sedimentation (CEPS) produced Al-rich sludge.•PACl coagulant in Al-sludge inhibited the hydrolysis and biodegradability of the sludge.•Alkali pretreatment disintegrated Al-sludge particles and enhanced organic hydrolysis.•About 17% of organics in wastewater was recovered by enhanced sludge fermentation.
The distinct influences of cephalosporins (CEPs, i.e., cefamandole nafate and cefpirome sulfate) affiliated to different generations on the volatile fatty acids (VFAs) production and antibiotic ...resistance genes (ARGs) fates during waste activated sludge (WAS) fermentation were unveiled. The presence of CEPs mainly exhibited negative effects on the total VFAs production (5%–15% reduction), especially the cefamandole nafate, which is quite different to previous understanding. Further investigation revealed that the CEPs contributed to the solubilization and hydrolysis but inhibited the acidification process by affecting the functional microbial populations (i.e., Tissierella) and general microbial metabolic activities (i.e., pyruvate metabolism and VFAs biosynthesis). In addition, CEPs (especially the cefpirome sulfate) caused the propagation of ARGs (i.e., blaTEM, tetX and mexF) during WAS fermentation. CEPs enhanced the cell membrane permeability to promote the antibiotics mechanism of efflux pump and the horizontal transfer of ARGs. Also, the CEPs altered the regulatory systems (i.e., two component system) and microbial populations associated with ARGs, resulting in the proliferation of specific ARGs. Overall, the dissimilarity of different CEPs impacts on the WAS fermentation for VFAs production and ARGs variations enlightened the diverse environmental behaviors of anthropogenic pollutants and evoked the caution of ecological risks.
The dissimilarity of different CEPs impacts on the WAS fermentation for VFAs production and ARGs variations enlightened the diverse environmental behaviors of anthropogenic pollutants and evoked the caution of ecological risks.
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Chemically enhanced primary sedimentation (CEPS) technology has been widely applied in Hong Kong, exhibiting excellent performance in contaminants removal from sewage. The generated CEPS sludge ...contains abundance of organics which could be recovered as volatile fatty acids (VFAs) by fermentation for further utilization. In this work, the effect of calcium peroxide (CaO2) on the fermentation of FeCl3 based CEPS sludge was investigated. The feasibility of utilizing the fermentation liquor as substrate for polyhydroxyalkanoates (PHAs) biosynthesis was also evaluated. Results demonstrated that CaO2 addition facilitated the disintegration of CEPS sludge and enhanced VFAs production. The maximum VFAs yield of 455.8 mg COD/g VSS was obtained with the dosage of 0.1 g CaO2/g SS, improving by 44.7% compared with the control sludge. Acetic and propionic acid were the predominant components of the VFAs. Microbial analysis indicated that CaO2 induced microbial reduction of Fe(III), accelerating the initial disintegration of FeCl3 based CEPS sludge. Microbial communities with hydrolysis and acidogenesis functions were enriched effectively. CaO2 treatment had no significant influence on the release of ammonia nitrogen (NH4+-N), while reduced the concentration of orthophosphate (PO43−-P) and ferrous (Fe2+) in fermentation liquor, that was beneficial to the further utilization as substrate for PHAs biosynthesis. The VFA-rich fermentation liquor was proved to be a suitable substrate for PHAs biosynthesis. After cultivation, the PHAs content in activated sludge reached 22.3%, which was comparable to those obtained using waste materials as carbon source. This integrated technology could be a superior alternative of realizing sludge disposal and bioplastic production simultaneously.
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•Fe3+ based CEPS was applied for organics recovery from sewage.•CaO2 enhanced VFAs accumulation during fermentation of CEPS sludge.•The highest VFAs yield of 455.8 mg COD/g VSS was obtained.•PHAs was biosynthesized using CEPS sludge fermentation liquor as sole substrate.
Metarhizium anisopliae, the type species of the anamorph entomopathogenic genus Metarhizium, is currently composed of four varieties, including the type variety, and had been demonstrated to be ...closely related to M. taii, M. pingshaense and M. guizhouense. In this study we evaluate phylogenetic relationships within the M. anisopliae complex, identify monophyletic lineages and clarify the species taxonomy. To this end we have employed a multigene phylogenetic approach using near-complete sequences from nuclear encoded EF-1α, RPB1, RPB2 and β-tubulin gene regions and evaluated the morphology of these taxa, including ex-type isolates whenever possible. The phylogenetic and in some cases morphological evidence supports the monophyly of nine terminal taxa in the M. anisopliae complex that we recognize as species. We propose to recognize at species rank M. anisopliae, M. guizhouense, M. pingshaense, M. acridum stat. nov., M. lepidiotae stat. nov. and M. majus stat. nov. In addition we describe the new species M. globosum and M. robertsii, resurrect the name M. brunneum and show that M. taii is a later synonym of M. guizhouense.
Ferric iron-based chemically enhanced primary sedimentation (CEPS) can remove over 70% of organics from municipal wastewater into sludge, and acidogenic fermentation converts 27.9% of solid organic ...substances in the CEPS sludge into soluble ones, mostly in the form of volatile fatty acids. In this study, the CEPS sludge supernatant after fermentation (CEPS-FL) was studied as an organic carbon source for biological denitrification, in comparison with the common external carbon sources of methanol (CH3OH), sodium acetate (NaAc), and sodium propionate (NaPr). The specific denitrification rates of NaAc, NaPr, and CEPS-FL were 701.3, 449.8, and 410.3 g N·kg VSS−1·d−1, respectively, obviously higher than the value of CH3OH (237.2 g N·kg VSS−1·d−1). During heterotrophic denitrification, lower N2O yields were observed in VFAs-based denitrification reactors (0.53% of NaAc, 1.11% of NaPr, and 1.14% of CEPS-FL) than in the reactor fed with CH3OH (3.42%). The bacterial community of the denitrification sludge was significantly affected by the type of carbon sources. The sludge fed with CEPS-FL had a greater alpha diversity than that fed with pure organic carbons, with the dominance of Zoogloea (14.7%), Alicycliphilus (7.6%), and Thauera (6.2%). Based on the mass balance of CEPS-fermentation-denitrification combination, the ammonium-nitrogen in the CEPS-FL would not affect the overall nitrogen removal performance. In comparison with the municipal wastewater treatment supplemented with CH3OH or NaAc as the external carbon source for improved nitrogen removal, the operating cost of this combined system can be considerably lower. Overall, a small modification in primary sedimentation by CEPS in combination with the sludge fermentation would not only decrease the organic load on the subsequent biological treatment and significantly improve the nitrogen removal performance, but also provide an effective, chemical-saving, and energy-efficient way for wastewater treatment.
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•CEPS improved the organic recovery for denitrification in wastewater treatment.•CEPS-FL had a higher denitrification rate and a lower N2O yield than methanol.•NH4+-N from CEPS-FL would not significantly affect the overall N removal.
Nitrate is the main source of nitrogen for plants but often distributed heterogeneously in soil. Plants have evolved sophisticated strategies to achieve adequate nitrate by modulating the root system ...architecture. The nitrate acquisition system is triggered by the short mobile peptides C‐TERMINALLY ENCODED PEPTIDES (CEPs) that are synthesized on the nitrate‐starved roots, but induce the expression of nitrate transporters on the other nitrate‐rich roots through an unclear signal transduction pathway. Here, we demonstrate that the transcription factors HBI1 and TCP20 play important roles in plant growth and development in response to fluctuating nitrate supply. HBI1 physically interacts with TCP20, and this interaction was enhanced by the nitrate starvation. HBI1 and TCP20 directly bind to the promoters of CEPs and cooperatively induce their expression. Mutation in HBIs and/or TCP20 resulted in impaired systemic nitrate acquisition response. Our solid genetic and molecular evidence strongly indicate that the HBI1‐TCP20 module positively regulates the CEPs‐mediated systemic nitrate acquisition.
Nitrate starvation on one side of the root enhances the interaction between the transcription factors HBI1 and TCP20 to induce the expression of genes encoding the short, mobile peptides C‐TERMINALLY ENCODED PEPTIDES (CEPs), which activate systemic nitrate signal transduction and lead to compensatory nitrate uptake on the other side.
Disulfide‐rich macrocyclic peptides—cyclotides, for example—represent a promising class of molecules with potential therapeutic use. Despite their potential their efficient synthesis at large scale ...still represents a major challenge. Here we report new chemoenzymatic strategies using peptide ligase variants—inter alia, omniligase‐1—for the efficient and scalable one‐pot cyclization and folding of the native cyclotides MCoTI‐II, kalata B1 and variants thereof, as well as of the θ‐defensin RTD‐1. The synthesis of the kB1 variant T20K was successfully demonstrated at multi‐gram scale. The existence of several ligation sites for each macrocycle makes this approach highly flexible and facilitates both the larger‐scale manufacture and the engineering of bioactive, grafted cyclotide variants, therefore clearly offering a valuable and powerful extension of the existing toolbox of enzymes for peptide head‐to‐tail cyclization.
Expanding the toolbox of enzymes for peptide head‐to‐tail cyclization: We successfully employed peptiligase variants such as omniligase‐1 for the efficient and scalable one‐pot cyclization/folding assembly of disulfide‐rich macrocyclic peptides, including the cyclotides MCoTI‐II and kalata B1 as well as the θ‐defensin RTD‐1. Multiple ligation sites allow a highly modular synthesis and facilitate cyclotide grafting studies.
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