A Special Phenotype of Aconidial Aspergillus niger SH2 and Its Mechanism of Formation via CRISPRi.

The complex morphological structure of Aspergillus niger influences its production of proteins, metabolites, etc., making the genetic manipulation and clonal purification of this species increasingly difficult, especially in aconidial Aspergillus niger. In this study, we found that N-acetyl-D-glucosamine (GlcNAc) could induce the formation of spore-like propagules in the aconidial Aspergillus niger SH2 strain. The spore-like propagules possessed life activities such as drug resistance, genetic transformation, and germination. Transcriptomic analysis indicated that the spore-like propagules were resting conidia entering dormancy and becoming more tolerant to environmental stresses. The Dac1 gene and the metabolic pathway of GlcNAc converted to glycolysis are related to the formation of the spore-like propagules, as evidenced by the CRISPRi system, qPCR, and semi-quantitative RT-PCR. Moreover, a method based on the CRISPR-Cas9 tool to rapidly recycle screening tags and recover genes was suitable for Aspergillus niger SH2. To sum up, this suggests that the spore-like propagules are resting conidia and the mechanism of their formation is the metabolic pathway of GlcNAc converted to glycolysis, particularly the Dac1 gene. This study can improve our understanding of the critical factors involved in mechanisms of phenotypic change and provides a good model for researching phenotypic change in filamentous fungi.

Impact of overexpressing NADH kinase on glucoamylase production in Aspergillus niger.

Glucoamylase has a wide range of applications in the production of glucose, antibiotics, amino acids, and other fermentation industries. Fungal glucoamylase, in particular, has attracted much attention because of its wide application in different industries, among which Aspergillus niger is the most popular strain producing glucoamylase. The low availability of NADPH was found to be one of the limiting factors for the overproduction of glucoamylase. In this study, 3 NADH kinases (AN03, AN14, and AN17) and malic enzyme (maeA) were overexpressed in aconidial A. niger by CRISPR/Cas9 technology, significantly increasing the size of the NADPH pool, resulting in the activity of glucoamylase was improved by about 70%, 50%, 90%, and 70%, respectively; the total secreted protein was increased by about 25%, 22%, 52%, and 26%, respectively. Furthermore, the combination of the mitochondrial NADH kinase (AN17) and the malic enzyme (maeA) increased glucoamylase activity by a further 19%. This study provided an effective strategy for enhancing glucoamylase production of A. niger.

Co-metabolic degradation of the antibiotic ciprofloxacin by the enriched bacterial consortium XG and its bacterial community composition.

Ciprofloxacin is a broad spectral and highly refractory antibiotic. It is an emerging pollutant. This study aimed to utilise co-metabolism as a means to degrade ciprofloxacin by a bacterial consortium. The stable bacterial consortium XG capable of efficiently degrading ciprofloxacin was successfully established through successive acclimation of indigenous microorganisms. The consortium XG was primarily consisted of Achromobacter, Bacillus, Lactococcus, Ochrobactrum, and Enterococcus as well as at least other five minor genera. A novel strain YJ17 with CIP-degrading ability was isolated from the consortium and identified as Ochrobactrum sp. The consortium XG utilised amino acids, carbohydrates, and carboxylic acids at a rate approximately 16.6-243-fold greater than the other carbon substrates, but only slow utilisation of ciprofloxacin as a sole carbon source. Ciprofloxacin can be co-metabolized along with many carbon sources, attaining degradation rates up to 63%. Glycyl-l-glutamic acid, d-cellobiose, and itaconic acid are among the substrates most favourable for co-metabolism. The metabolites of ciprofloxacin were identified by LC-QTOF-MS. Co-metabolic degradation of ciprofloxacin by consortium XG led to the removal of essential functional groups from parent compound, thus resulting in formation of metabolites with less bioactive potency. Finally, a possible biochemical pathway for the degradation of ciprofloxacin was proposed. Consortium XG possesses high potential for bioremediation of ciprofloxacin-contaminated environments in the presence of a co-substrate.

Effects of ß-cyclodextrin on phytoremediation of soil co-contaminated with Cd and BDE-209 by arbuscular mycorrhizal amaranth.

Pot experiments were conducted to investigate the effects of a series of ß-cyclodextrin (ß-CD) on phytoremediation of soil co-contaminated with Cd and BDE-209 by amaranth (Amaranthus hypochondriacus L.) inoculated with arbuscular mycorrhizal fungus (AMF) - Rhizophagus intraradices. Results showed that the combination of mycorrhizal amaranth and 0.4% ß-CD (RI+ß0.4) significantly enhanced Cd concentrations and contents in shoots, total PBDEs concentration in roots, and BDE-209 dissipation in soil. Moreover, the RI+ß0.4 treatment exerted the highest removal efficiency of both Cd and BDE-209. On the contrary, the xylem area, shoot Cd and BDE-209 concentrations and contents, and removal efficiency of Cd were markedly reduced in mycorrhizal amaranth with 0.8% or 1.2% ß-CD treatments (RI+ß0.8, RI+ß1.2), compared with single inoculation treatment. The well-organized chloroplast and well-defined root anatomical structure were also observed in the treatment of RI+ß0.4. Positive correlation was found between shoot biomass and chlorophyll concentrations. Shoot Cd or BDE-209 concentrations were positively correlated with xylem areas. In conclusion, mycorrhizal amaranth added with 0.4% ß-CD could be used for the decontamination of soil polluted with mixture of Cd and BDE-209 due to the higher chlorophyll concentration and the larger xylem area.

Novel phosphate-solubilising bacteria isolated from sewage sludge and the mechanism of phosphate solubilisation.

A great amount of insoluble phosphate in agricultural soils is not available for crops. Three strains of bacteria (Bacillus megaterium YLYP1, Pseudomonas prosekii YLYP6 and Pseudomonas sp. YLYP29) isolated from activated sludge and soil could efficiently solubilise tricalcium phosphate. In particular, the novel strain P. prosekii YLYP6 produced 716 mg L-1 of available phosphate within 6 days under the optimal culture conditions [20 °C, pH 7.9, inoculum size of 0.5% (v:v)] determined by response surface methodology. P. prosekii YLYP6 demonstrated efficient phosphate solubilisation in response to broad variations in pH (5-9) and temperature (15-35 °C). The phosphate solubilisation curves of the strains fit well with a first-order kinetic model (R2 > 0.939), with a half-life of 1.51-5.94 d for 5.0 g L-1 calcium phosphate. Continuous culture experiments combined with scanning electron microscopic observations and gas chromatography-mass spectrometry analysis revealed that 2,3-dimethylfumaric acid, gluconic and N-butyl-tert-butylamine that were produced by P. prosekii YLYP6 were responsible for phosphate solubilisation by supplying H+ ions and organic anions. Efficient phosphate solubilisation in actual soil by P. prosekii YLYP6 demonstrated the strong application potential to reduce the use of chemical P fertilisers and the resulting agricultural nonpoint pollution.

Genotypic variation and mechanism in uptake and translocation of perfluorooctanoic acid (PFOA) in lettuce (Lactuca sativa L.) cultivars grown in PFOA-polluted soils.

The cultivation of crop cultivars with low pollutant accumulation is an important strategy to reduce the potential health risks of food produced from polluted soils. In this study, we identified three loose-leaf lettuce cultivars with low accumulation of perfluorooctanoic acid (PFOA), a highly toxic and persistent organic pollutant. PFOA concentrations in the shoots of low-PFOA cultivars were 3.7-5.5-fold lower than those of high-PFOA cultivars. The identification of low-PFOA cultivars could contribute to ensuring food safety despite cultivation in highly polluted soils (1 mg/kg) based on the tolerable daily PFOA intake (1.5 µg/kg/d). We detected lower desorbing fractions of PFOA in rhizosphere soil, lower bioconcentration factors, and higher distribution in the cell walls and organelles of roots in low-PFOA cultivars, all of which are key factors in limiting PFOA uptake and translocation from soil to shoots, than in high-PFOA cultivars. This study reveals the mechanism of PFOA uptake from soil to crop and lays a foundation for establishing a cost-effective strategy to plant crops in polluted soil and reduce exposure risk due to persistent organic pollutants in crops.