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.

[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 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 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] 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.

[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 and identify the plant growth-promoting bacterial strain with saline-alkali tolerance and evaluate its functions. [Methods] The rhizosphere soil of wheat was collected from different saline-alkali regions, and the highly efficient saline-alkali-tolerant bacterial strain was isolated by enrichment under saline-alkali condition and dilution coating. The antifungal spectrum and plant growth-promoting effects of the isolate were evaluated in vitro, and the nitrogenase activity was measured by an ELISA kit. The strain was identified based on the morphological, physiological, biochemical characteristics and phylogenetic analysis. The effect of the strain on wheat growth under salt stress was investigated by a pot experiment. [Results] A highly abundant bacterium with saline-alkali tolerance was enriched from rhizosphere soil and designated as TaRb44, which could grow normally under 3% NaCl and pH 10.0. Strain TaRb44 showed strong antagonism against soil-borne pathogenic fungi such as Fusarium pseudograminearum causing wheat crown rot, Fusarium oxysporum f. sp. niveum causing watermelon Fusarium wilt, and Fusarium oxysporum f. sp. cubense causing banana wilt, as well as Botrytis cinerea causing gray mold of postharvest tomatoes. Further tests showed that strain TaRb44 produced a variety of plant growth-promoting substances such as siderophores, amylase, and cellulase, and it had nitrogen fixation activity with the nitrogenase level of 65.50 U/L. Finally, the strain TaRb44 was identified as Paenibacillus polymyxa, which significantly promoted the growth of wheat seedlings and increased the root biomass under both salt stress and non-salt stress conditions. [Conclusion] In this study, P. polymyxa TaRb44 was obtained with much excellent properties such as disease prevention, plant growth promotion, and saline-alkali tolerance. It serves as an elite strain for developing new microbial fertilizer and soil amendments in the future.

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.

The high salt concentration of Emerald Lake in Mangya, Qinghai Province, makes it an ideal environment for mining halophilic (halotolerant) microorganisms. [Objective] To mine the halophilic (halotolerant) microbial resources in the Emerald Lake, compare the morphological characteristics of halophilic (halotolerant) bacteria exposed to different concentrations of NaCl, and explore the response of halophilic (halotolerant) microorganisms to NaCl stress. [Method] Lake water, lake salt, and lakeshore saline soil samples were collected from the nearshore area of the Emerald Lake. The halophilic (halotolerant) bacteria were isolated by eight different media with three salt concentrations (10%, 15%, and 20%). The colony morphology, cell morphology, and internal structures of three strains of halophilic (halotolerant) bacteria exposed to different concentrations of NaCl were observed by optical microscopy, scanning electron microscopy, and transmission electron microscopy, respectively. [Results] A total of 58 strains of halophilic (halotolerant) bacteria were isolated, including 31 strains of halotolerant bacteria and 27 strains of halophilic bacteria, belonging to 16 genera. Halobacillus (40.7%) and Bacillus (19.3%) were the dominant genera of halophilic bacteria and halotolerant bacteria, respectively. The strains tolerant to Na⁺, Mg²⁺, K⁺, and Ca²⁺ accounted for 84.5%. The cell lengths of Virgibacilluslitoralis TRM 83602 and Piscibacillussalipiscarius TRM 83622 exposed to different concentrations of NaCl were significantly different, while that of Brevibacteriumepidermidis TRM 83610 did not change significantly. [Conclusion] The Emerald Lake harbors abundant halophilic (halotolerant) bacteria. NaCl concentrations can affect the growth, reproduction, colony morphology, cell morphology, and internal structures of halophilic (halotolerant) bacteria. This study enriches the understanding about the microbial resources in the Emerald Lake, providing abundant strain resources for further development and utilization of halophilic (halotolerant) microorganisms.

[Objective] To mine the macrofungal strains capable of efficiently degrading straw at low temperatures, thereby improving the utilization efficiency of straw resources, we measured the lignocellulose degradation abilities of 955 macrofungal strains. [Methods] First, we employed the plate method to screen the strains with carboxymethyl cellulase, xylanase, and laccase activities. Then, we carried out a filter paper degradation test to screen the cellulose-degrading strains. Finally, we performed liquid fermentation with the selected strains and measured their enzyme activities on days three, six, nine, and 12 to identify the dominant strains with strong lignocellulose-degrading abilities. [Results] We identified 11 macrofungal strains exhibiting strong lignocellulose degradation capabilities at a low temperature (15 ℃). The 11 strains were Trametes suaveolens, Irpex lacteus, Crucibulum laeve, Stereum hirsutum, Pleurotus ostreatus, Phlebia acerina, Agaricus xanthodermus, Neofomitella fumosipora, Pholiota multicingulata, Abortiporus biennis, and Armillaria cepistipes. Notably, C. laeve, A. xanthodermus, and P. multicingulata were newly reported for their high lignocellulose-degrading abilities. The maximum activities of carboxymethyl cellulase, xylanase, and laccase in the 11 strains reached 262.31, 91.03, and 196.50 U/mL, respectively. T. suaveolens exhibited carboxymethyl cellulase activity of 168.17 U/mL at 15 ℃, which was significantly higher than that (67.88 U/mL) observed at room temperature. P. ostreatus showed the carboxymethyl cellulase activity of 150.78 U/mL and the laccase activity of 154.32 U/mL. S. hirsutum achieved the laccase activity of 63.27 U/mL at 15 ℃, which was twice the level measured at room temperature. [Conclusion] We successfully identified 11 macrofungal strains with strong lignocellulose-degrading abilities at 15 ℃. The findings provide valuable microbial resources for the degradation of lignocellulose in cold regions and lay a theoretical basis for application of these strains in low-temperature industries.