[Objective] To screen out a yeast strain that can efficiently assimilate ammonia nitrogen, optimize the solid-state fermentation conditions based on the nutrient composition, antioxidant activity, and amino acid content of the feed, and provide a scientific basis for the production of single-cell protein feed with this yeast strain. [Methods] Five strains of Candida utilis, four strains of Pichia anomala, five strains of Saccharomyces cerevisiae, and three strains of Issatchenkia orientalis were cultured with (NH₄)₂SO₄ as the sole nitrogen source. The yeast strain with the highest ammonia utilization rate and the highest glutamine synthetase (GS) activity was selected as the test strain, and then the fermentation parameters and fermentation substrates were optimized for this strain. The routine nutrient composition, content of phytate phosphorusand amino acids, and scavenging rates against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline- 6-sulfonicacid) (ABTS) free radicals of the fermented feed were determined. [Results] C. utilis CJ121 showed an ammonia utilization rate of 55.39% and a GS activity of 0.29 μmol/(h·g), which were higher than those of other yeast strains. The optimal fermentation process for C. utilis CJ12 was fermentation with a (NH₄)₂SO₄ addition amount of 2% and an inoculation amount of 8% for 36 h. The optimal fermentation substrate was composed of 94.8% wheat bran, 5% soybean meal, 0.1% protease, and 0.1% cellulase. After fermentation with C. utilis CJ12, the content of crude protein and organic nitrogen increased by 15.95% and 28.46%, respectively (P<0.05), while that of dry matter, crude fiber, crude fat, and phytate phosphorus decreased by 4.19%, 19.41%, 12.29%, and 13.51%, respectively (P<0.05). The total amino acid content increased after fermentation (P<0.05), with glutamine and glutamic acid levels being 111.38 times and 3.02 times those of the control group, respectively. However, the control group exhibited higher levels of tryptophan, asparagine, and 4-aminobutyric acid, which were 13.41, 8.27, and 6.41 times those of the experimental group, respectively. In addition, the scavenging abilities against DPPH and ABTS free radicals increased after fermentation (P<0.05), with the IC₅₀ values decreasing by 73.51% and 6.01%, respectively (P<0.05). [Conclusion] C. utilis CJ12 has high capacities of utilizing ammonia nitrogen and synthesizing glutamine. This strain improves the nutritional value and antioxidant performance of feed after solid-state fermentation, thus demonstrating the potential for producing functional single-cell protein feed.

[Objective] To explore the diversity of halophilic bacteria in the magnesium sulfate-subtype Da Qaidam Salt Lake, compare the effects of different culture conditions on the diversity of halophilic bacteria, and screen the extracellular functional enzymes of halophilic bacteria. [Methods] Illumina MiSeq was used to analyze the diversity of bacteria in the Da Qaidam Salt Lake. Thirteen media, 2 salinities, 8 enrichment culture periods, and 6 dilution gradients were selected to isolate halophilic bacteria, and the taxonomic status of the strains was determined by 16S rRNA gene sequencing and BLAST sequence alignment. According to the sequencing results, 45 representative strains of different species belonging to 18 genera were selected and 7 media were used to screen the strains with activities of functional enzymes including protease, cellulase, amylase, and esterase. [Results] A total of 244 bacterial OTUs were obtained by culture-free high-throughput sequencing in the Da Qaidam Salt Lake. The strains with clear taxonomic status were annotated to 153 genera, 133 families, 92 orders, 53 classes of 19 phyla, with Pseudomonadota and Actinomycetota being the dominant phyla. A total of 593 strains of halophilic bacteria were isolated from the mixed water and mud samples of the Da Qaidam Salt Lake, belonging to 22 genera, 12 families, 8 orders, 5 classes of 4 phyla, of which 11 strains may belong to potential new species. Pseudomonadota and Bacillota were the dominant phyla, and Halomonas, Virgibacillus, and Bacillus were the dominant genera. The number of halophilic bacteria isolated under the culture with 10% NaCl was significantly higher than that with 18% NaCl, indicating that moderately halophilic bacteria were dominant among culturable halophilic bacteria. The media with better isolation performance were 2216E, 1/2 R2A, 1/10 2216E, and 1/10 TSA, all of which are oligotrophic media. The optimal enrichment culture periods ranged from 7 to 30 days. The undiluted samples obtained the best isolation results, followed by dilution gradients of 10^-1 and 10^-2. Among the 45 representative strains, 40.0%, 31.1%, 40.0%, and 82.22% of the strains had activities of protease, cellulase, amylase, and esterase, respectively. [Conclusion] Optimization of the isolation and culture conditions can significantly improve the diversity of halophilic bacteria that can be cultured in salt lakes. The high diversity and high halophilic enzyme activities of the culturable halophilic bacteria from the Da Qaidam Salt Lake provide a basis for further application of these halophilic bacteria.

[Objective] Arbuscular mycorrhizal (AM) fungi are crucial components of the plant rhizosphere microbiota, capable of forming symbiotic relationships with 72% of terrestrial plants. However, AM fungi are plant-specific symbiotic fungi in soil, and they are difficult to be enriched for isolation and achieve artificial pure culture. This study aimed to develop a non-plant symbiotic culture system based on the addition of root exudates to solve the problem of difficult invitro culture of AM fungi. [Methods] The “multi-layer sandwich” culture system was used for the in vitro quasi-asymbiotic culture of AM fungal spores from soil. Molecular systematics methods were employed to identify the cultured AM fungi. [Results] A “multi-layer sandwich” culture system was used for the in vitro quasi-asymbiotic culture of AM fungi from soil. It was found that the root exudates of Astragalus sinicus effectively promoted the hyphal growth of AM fungi. A large number (951±45) of secondary spores were produced after 60 days of culture, exceeding those after 30 days and 45 days of culture. Further spore inoculation tests indicated that the secondary spores produced from this culture colonized the roots of A. sinicus seedlings. Two AM fungal species, Funneliformis mosseae and Paraglomus occultum, were identified by molecular characterization as suitable for the “multi-layer sandwich” culture system. Finally, a nutrient solution composed of simulated root exudate components from A. sinicus was used for the “multi-layer sandwich” culture of AM fungal spores. The results showed that the addition of root exudates significantly promoted the hyphal growth of AM fungi. [Conclusion] In the plant-assisted “multi-layer sandwich” culture system, the root exudates of A. sinicus can continually induce AM fungi to produce hyphae and secondary spores capable of colonizing host plants under non-symbiotic conditions. This study provides a new method for solving the problem related to the in vitro culture, isolation, and identification of AM fungi.

[Objective] To screen out a strain that can degrade lignocellulose for the application of lignocellulose deposited by delayed harvest of alfalfa under inappropriate climate and mechanical matching and provide strain resources for the efficient use of alfalfa. [Methods] The primary screening was carried out by culture with alkali lignin and sodium carboxymethyl cellulose. Based on color changes and fading circles, the strain with the ability to secrete ligninase and cellulase was screened out. The target strain was identified by 16S rRNA gene sequencing and whole genome sequencing, and then Kyoto encyclopedia of genes and genomes (KEGG) and carbohydrate-active enzymes (CAZy) were employed to annotate gene functions based on the whole genome sequence. The microstructure of the alfalfa stems degraded by the strain was observed by scanning electron microscopy. The nutrient and microbial community changes in alfalfa hay treated with the strain were evaluated. [Results] A strain S1 producing ligninase and cellulase was identified as Bacillus cereus by whole genome sequencing. A total of 305 genes involved in carbohydrate metabolism were annotated, including 139 genes encoding CAZy. After treatment with this strain, the microstructure of vascular bundles in alfalfa stems changed significantly. The crude protein content in alfalfa hay increased, while the lignin, neutral detergent fiber, and acid detergent fiber content and the alpha-diversity of microorganisms decreased over time. [Conclusion] The screened strain identified as B. cereus demonstrates a robust ability to degrade lignocellulose.

[Objective] To reveal the phylogenetic diversity of 12 marine siderophore-producing bacteria isolated from intertidal sediment samples of Naozhou Island in Leizhou Bay of South China Sea, and to unravel the components, functions, genetic diversity and genetic evolution of siderophore biosynthetic gene clusters (BGCs) of the isolates as well as representatives of Microbulbifer. [Methods] The phenotypic characteristics as well as the siderophore-producing activity of the strains were observed by conventional methods. The phylogenetic diversity (including taxon, species, and genetic diversity) of the strains was analyzed based on 16S rRNA gene sequences. Then, the exact phylogenetic status of the representative strain JSM ZJ756 was investigated comprehensively by means of comparative genomics analysis based on whole-genome sequences, including comparisons of G+C content, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) estimated values. We then used multiple bioinformatic stools including antiSMASH 7.0, BLASTn, BLASTp, and MEGA 11 for rapid identification, annotation, and sequence alignment of BGCs, thus exploring the components, functions, genetic diversity, and genetic evolution of siderophore BGCs. [Results] All the 12 isolates were Gram-negative, aerobic, non-sporulating and slightly halophilic rods with siderophore-producing activity. The results of phylogenetic analyses based on 16S rRNA gene sequences as well as whole-genome sequences showed that all the 12 strains belonged to Microbulbifer, representing 6 to 8 species and forming 4 clades with Microbulbifer type strains in the phylogenetic tree. Among them, strain JSM ZJ756 should be a new member of Microbulbifer zhoushanensis. JSM ZJ756 and 9 representative strains of Microbulbifer each carried one copy of NI-siderophore BGCs, and 8 out of the 10 NI-siderophore BGCs shared the similarities ≤40% with known BGCs. According to the types and the similarities of known BGCs with the NI-siderophore BGCs identified in this study, the 10 NI-siderophore BGCs could be categorized into 5 functional subtypes: Ochrobactin (JSM ZJ756 and 2 type strains, JYFX01000060.1, similarity of 28%), Vibrioferrin (M. mangrove DD-13^T, AB082123.1, 100%; M. epialgicus DSM 18651^T, CP005094.1, 85%), Putrebactin (M. agarilyticus GP101, NIBS01000001.1, 40%), Aerobactin (M. echini JCM 30400^T, AB199785.1, 22%), and unknown functional subtype (3 type strains including M. variabilis ATCC 700307^T). The results of BLASTn and BLASTp analyses showed that the core biosynthetic gene sequences of the NI-siderophore BGCs identified were unique, and encoded novel proteins. The results of genetic evolution analysis showed that the core biosynthetic genes of the NI-siderophore BGCs found exhibited high genetic diversity. In the phylogenetic tree based on core biosynthetic gene sequences, 9 out of the 10 NI-siderophore BGCs were grouped to 3 clades, but M. agarilyticus GP101 exhibited an independent evolution path. The comparative analysis revealed the genetic evolution of NI-siderophore BGCs being consistent with that predicted based on the 16S rRNA sequences. [Conclusion] All the 12 siderophore-producing strains isolated from intertidal sediment samples of Naozhou Island in Leizhou Bay of South China Sea belong to Microbulbifer, demonstrating high phylogenetic diversity, and JSM ZJ756 is a new member of M. zhoushanensis. The NI-siderophore BGCs of JSM ZJ756 and representative strains of Microbulbifer are novel and showcase high diversity, which indicates that those strains possess high potential of producing a variety of novel siderophores. Moreover, strong positive correlations exist between the biological functions and genetic evolution of the NI-siderophore BGCs and the phylogeny of JSM ZJ756 and 9 representative strains of Microbulbifer. Therefore, we hypothesize that the 12 marine siderophore-producing bacteria and the representatives of Microbulbifer are typical new-resource microbes, and the taxonomy of Microbulbifer, and the metabolic mechanism and genetic evolution as well as the biotechnological potential of the NI-siderophore BGCs are worth being further explored.

Amidst the escalating issues of water eutrophication and water resource scarcity, the development of high-efficiency wastewater treatment technologies has become increasingly imperative. Traditional nitrogen and phosphorus removal processes face challenges in achieving efficient simultaneous elimination of nitrogen and phosphorus due to the disparities in sludge age and the competition for carbon sources among microorganisms. Denitrifying phosphorus-accumulating organisms (DPAOs) possess the capability to remove both nitrogen and phosphorus, demonstrating significant potential in wastewater treatment. However, population-level studies often overlook cellular heterogeneity, leading to an inadequate understanding of the nitrogen and phosphorus removal mechanisms of DPAOs. Moreover, the “traditional culture-first, screen-second” method yields a limited number of efficient DPAO strains, the stability and adaptability of which face challenges in actual wastewater treatment environments. Single-cell analysis technologies provide new perspectives for a deeper understanding of microbial ecological niches and metabolic mechanisms. Coupling non-destructive single-cell phenotypic identification technologies, such as single-cell Raman spectroscopy (SCRS), with the culture method paves new avenues for the exploration of DPAO strains. This review summarizes the research status and progress in the exploration of DPAO strain resources and their metabolic mechanisms, focusing on the potential of single-cell technologies in revealing the mechanisms of nitrogen and phosphorus removal by DPAOs and in the exploration of DPAO resources. The aim is to provide a new theoretical foundation and technical support for the research and application of DPAOs, thereby promoting the development of efficient wastewater treatment technologies.

[Objective] This study isolated and identified an algicidal bacterium from the East China Sea near Wenzhou and investigated its algicidal characteristics and mechanisms, aiming to contribute a solid scientific basis to the microbial control against red tides. [Methods] The strain was identified by morphological observation, physiological and biochemical tests, and 16S rRNA gene sequence analysis. The algicidal characteristics such as algicidal activity, influences of environmental factors on the algicidal activity, and algicidal specificity were assessed. The algicidal mechanism was explored by electron microscopy, measurements of photosynthetic parameters, and determination of reactive oxygen species (ROS) and malondialdehyde levels and antioxidant enzyme activities. [Results] The algicidal strain J75 was identified as Pseudoalteromonas sp. The algicidal rate of strain J75 against Skeletonemacostatum reached 95.97% within 36 h. Strain J75 induced lysis of algal cells indirectly by secreting extracellular algicidal substances, maintaining the algicidal activity across broad ranges of temperatures (-20 °C to 80 °C) and pH levels (5.0 to 9.0). In addition, strain J75 demonstrated algicidal activities against other harmful microalgae, including Kareniamikimotoi, Prorocentrumminimum, and Phaeocystisglobosa. Under the stress of J75 cell-free supernatant, the algal cells showed morphological structure damage, a significant decrease in the photosynthetic activity, elevations in the levels of ROS, membrane lipid peroxidation, activities of superoxide dismutase, catalase, and peroxidase, and the level of glutathione. These results indicated that strain J75 caused oxidative damage to algal cells, ultimately leading to algal death. [Conclusion] The marine algicidal bacterium Pseudoalteromonas sp. J75, which holds significant potential for the control of red tides, demonstrates its efficacy by secreting algicidal compounds that inhibit the growth of diverse red tide microalgae. The exploration of the algicidal characteristics and mechanisms of this strain provides a theoretical basis for advancing red tide management strategies.

[Objective] To explore the effects of endophytic bacteria in soybean nodules on the growth of soybean seedlings under salt stress and provide reference for the high-quality development of crops in the Yellow River Basin. [Methods] A pot experiment was conducted with the soybean cultivar ‘Xudou 20’ under artificial climate conditions. Three groups were designed: control, salt stress, and salt stress inoculated with endophytic bacteria. The growth of soybean seedlings in each group was measured. [Results] Regarding strain 72, in the case of inoculation with the suspension at OD₆₀₀=0.75 (2:1) and the salt concentration of 200 mmol/L, the activity of catalase (CAT) in the seedlings of 21 days was the highest (2.010 0 U/g FW), which was 54.02% higher than that in the salt stress group; in the case of inoculation with the suspension at OD₆₀₀=0.33 (1:2) and the salt concentration of 300 mmol/L, the proline content was the highest (0.028 6 mg/g); in the case of inoculation with the suspension at OD₆₀₀=0.75 (2:1) and the salt concentration of 100 mmol/L, the proline content was 64.38% higher than that in the salt stress group. Regarding strain 146, the seedlings of 21 days in the case of inoculation with the suspension at OD₆₀₀=0.50 (1:1) and the salt concentration of 50 mmol/L had the highest CAT activity (1.350 0 U/g FW); the seedlings of 28 days in the case of inoculation with the suspension at OD₆₀₀=0.75 (2:1) and the salt concentration of 100 mmol/L had the CAT activity 272.73% higher than that in the salt stress group; the seedlings in the case of inoculation with the suspension at OD₆₀₀=0.75 (2:1) and the salt concentration of 150 mmol/L had the highest proline content of 0.147 0 mg/g, which was 860.78% higher than that in the salt stress group. The results indicated that inoculation with the endophytic bacterial strain 72 or 146 had a repairing effect. The increases in the CAT activity and proline content in the soybean seedlings inoculated with strain 146 over those in the salt stress group were generally higher than those in the case of inoculation with strain 72, indicating that the repairing effect of strain 146 was more obvious. The phylogenetic tree built based on 16S rRNA gene sequences showed that strains 72 and 146 had the highest similarity with Bacillus subtilis (99.45%) and B. protolyticus (100%), respectively. [Conclusion] Inoculating endophytic bacteria was of great significance for alleviating the effect of saline-alkali stress in the Yellow River Basin on the growth of soybean seedlings and developing biological repairing agents.

[Objective] To establish a theoretical foundation for the application and development of chitinases, this study isolated and screened chitin-degrading bacteria from the intestines of amphibians, optimized their fermentation conditions, characterized their enzymatic properties, and analyzed their whole genomes. [Methods] A strain capable of producing chitinase was isolated from the intestinal contents of Rana kukunoris and identified based on morphological characteristics and molecular biological evidence. The enzyme production conditions of the strain were optimized by single factor and response surface methodology (RSM) experiments, and the enzymatic properties were studied. Whole genome sequencing was carried out for identification of the chitinase gene family. [Results] The chitin-degrading strain JD-3 was identified as Carnobacteriummaltaromaticum. This strain achieved the highest enzyme activity of 12 mU/mL after fermentation with the inoculum amount of 4% at 31.4 ℃ and initial pH 4.9 for 2.47 d. The optimal reaction conditions of the enzyme were 20 ℃ and pH 3.0, and the enzyme maintained good stability at room temperature and under acidic conditions. The genome of JD-3 was 4 195 636 bp long, containing 6 circular contigs, 63 tRNA genes, 19 rRNA genes, and 3 864 protein coding sequences. Two chitinase genes belonging to the glycoside hydrolase family 18 (GH18) were identified and phylogenetically classified into two distinct categories. [Conclusion] We isolated an acid-tolerant chitin-degrading bacterium, C. maltaromaticum JD-3, from the intestines of plateau amphibians. The findings provide new insights into the development and utilization of microbial resources in the digestive systems of animals.

Soybean (Glycine max) is an important cereal and oil crop in the world, and the supply-side structural reform in the agricultural sector requires an increase in the planting area of high-quality edible soybean. Due to the inherent characteristics of arable land resources in China, the domestic production of soybean is far from self-sufficiency, and there is an urgent need to increase the planting area and production of soybean domestically, reducing the dependence on imports. Rhizobia are the earliest developed microbial fertilizer, while their application area is limited in China. [Objective] To select suitable strains from the stored rhizobia to provide germplasm resources for alleviating food issues. [Methods] We reviewed taxonomic status of rhizobia preserved in the Agricultural Culture Collection of China over decades based on the 16S rRNA and recA gene sequences. The hydroponic nodulation test was carried out to assess the nodulating and soybean growth-promoting effects based on comprehensive consideration of the nodulation rate, nodule number, nodule weight, plant height, and dry weight. [Results] A total of 213 strains of soybean rhizobia were activated and identified, including 156 strains of Bradyrhizobium, 48 strains of Sinorhizobium, and 9 strains of Rhizobium. Among them, 149 strains were able to nodulate with soybean cultivar selected in this study, and 43 strains significantly contributed to the growth of soybean plants. [Conclusion] This study further clarifies the taxonomic status of preserved soybean rhizobia and evaluates their nodulating and plant growth-promoting effects, providing abundant microbial resources for the development of soybean rhizobia-based agents.