[Objective] To establish a methodology assessing the existence and active state of biofilms on mudflat. [Methods] We sampled the biofilms on mudflat and the surface sediments in the nearby regions, and compared the dominant algal species and their abundance between the biofilms and their nearby regions by 18S rRNA gene sequencing. Furthermore, we employed chlorophyll a (Chl-a) assay and flow cytometry to compare the concentration of Chl-a and the amount of Chl-a-containing cells between the biofilms and their nearby regions. [Results] The mudflat biofilms mainly harbored Diatomea, Dinoflagellata, Ochrophyta, Chlorophyta, Cryptophyceae, and Phragmoplastophyta, the relative abundance of which, however, varied significantly in different seasons or geographic locations. There was a significant difference in the concentration of Chl-a between biofilms and their nearby regions. The amount of Chl-a-containing cells in biofilms was significantly higher than that in the nearby regions. A method for assessing biofilms on mudflat was established based on the relative abundance of algae, the concentration of Chl-a, and the amount of Chl-a-containing cells. In brief, a sample is classified as a biofilm in the case of the relative abundance of algae higher than 40%, the Chl-a concentration higher than 500 mg/m³, and the amount of Chl-a-containing cells more than 500 cells/μL. Otherwise, the sample is classified as inexistence of biofilm. Additionally, the amount of Chl-a-containing cells more than 1 500 cells/μL indicates that the biofilm is in the vigorous growth phase, and that between 500 cells/μL and 1 500 cells/μL suggests that the biofilm is in the colonization or recession phase. [Conclusion] We compared the dominant algal species and their relative abundance, the concentration of Chl-a, and the amount of Chl-a-containing cells between biofilms on mudflat and their nearby regions, and established a methodology assessing the existence and active state of biofilms on mudflat based on the above indicators. The findings enriched the knowledge of biofilms on mudflat and provided a theoretical basis for understanding the microbial carbon sequestration capacity of biofilms on mudflat.

[Objective] Poly(butylene adipate-co-butylene 2,5-furandicarboxylate) (PBAF) is a biodegradable furan-based copolyester plastic. In the chemical synthesis of PBAF, the randomness of oligomer polymerization leads to the formation of complex polymers, such as block copolymers, random copolymers, and alternating copolymers. In this study, dimethyl furan-2,5-dicarboxylate (FDME) and 1,4-butanediol (BDO) were used as substrates to synthesize bis-BDO ester by enzymatic reaction to provide the bioplastic precursor for controllable polymerization and avoid complex by-products. [Methods] RgPETase from Rhizobacter gummiphilus was heterogeneously expressed in Escherichia coli BL21(DE3). RgPETase exhibited acyltransferase activity for FDME and BDO. The reaction conditions including pH, temperature, content of BDO (as both substrate and solvent), and amount of enzyme for the synthesis of bis-BDO ester were optimized. [Results] The optimum reaction conditions of RgPETase were pH 8.0, BDO content of 30%, and reaction temperature within the range of 25–30 ℃. Under the optimum conditions (30 ℃ and enzyme concentration of 6 μmol/L), RgPETase can catalyze 10 mmol/L FDME to produce (2.96±0.01) mmol/L bis-BDO ester. [Conclusion] RgPETase exhibits high acyltransferase activity and catalyzes the generation of bis-BDO ester from FDME via acyl transfer reaction under mild conditions, which provides a green and sustainable approach for synthesizing the precursor of PBAF.

[Objective] Pseudomonas as one of the dominant spoilage bacteria highly form biofilms in chilled meat products and processing environment when contaminating single or mixed with other species. This study aims to investigate the antibiofilm properties of the cell-free supernatants (CFSs) of three Bacillus species isolated from fermented food and rice seeds on Pseudomonas lundensis (PL) or and Acinetobacter johnsonii (AJ) as mono- or dual-species. [Methods] Biofilm biomass, extracellular polymeric substances (EPSs), and biofilm structure were measured by crystal violet staining, spectrophotometry, confocal laser scanning microscopy (CLSM), respectively, as well as transcription of biofilm-related genes determined by qPCR. [Results] The CFSs of Bacillus amyloliquefaciens ZG08, B. velezensis B5, and B. subtilis YB11 inhibited the biofilm formation of PL and AJ without affecting their growth. The treatment with 50% CFSs of ZG08 and B5 decreased the cell viability of two biofilms by 12.73%–21.04%, which was higher than that of YB11 (0.15%–4.38%). The inhibition rates of 50% CFSs of the three strains were 59.75%–79.59% against the PL biofilm and 63.62%–78.57% against the biofilm of PL+AJ, in which the CFS of YB11 had weaker activity. The content of exopolysaccharides and exoprotein in the two biofilms treated with these CFSs were reduced by 53.77%–73.30% and 54.84%–62.38%, respectively. The treatment with the three CFSs also reduced the adhesive cells, loosened biofilm structures, and thinned their thickness by 57.63%–74.49% and 60.43%–64.64%, respectively. Moreover, the CFSs of ZG08 and B5 effectively eradicated by 41.77%–69.79% against the mature biofilms of PL and PL+AJ, compared to weak activity of YB11. In addition, the antibiofilm activities of the three CFSs were stable under four enzyme digestion and heating conditions. Compared with the control, the CFSs of ZG08 and B5 significantly down-regulated the expression of six biofilm-related genes, lapA, alg44, pelG, luxR, wspR, and rpoS. [Conclusion] The CFSs of ZG08 and B5 have strong antibiofilm activities against PL and AJ as mono- or dual-species.

[Objective] We isolated the aerobic bacteria capable of effectively degrading polylactic acid (PLA) and characterized the bacterial growth and degradation, aiming to lay a theoretical foundation for the bioremediation of PLA contaminated environment. [Methods] The degrading bacterium was identified by 16S rRNA gene sequencing. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were employed to analyze the morphological and chemical changes of PLA films before and after degradation. [Results] A strain of Bacillus sp. JA-4 was screened from activated sludge, and it caused the PLA weight loss of 10.6% after 30 days. The weight loss of PLA reached 5.6% after incubation with the strain at an inoculation amount of 20%, pH 8.0, and 30 ℃ for 7 days. Gelatin significantly enhanced the biodegradation of PLA. In the presence of 3% gelatin, the weight loss of PLA reached 23.1% after 10 days of degradation, and the degradation rate was greatly increased. FTIR results indicated that Bacillus sp. JA-4 degraded PLA by hydrolyzing the ester bonds. [Conclusion] This study enriched the microbial resources for the biodegradation of PLA and provided technical support for the effective degradation of PLA waste in the environment.

Blastocystis sp. is a widespread unicellular protozoan mainly parasitizing the intestines of humans and animals. This parasite has rich genetic diversity, with 42 subtypes (ST1–ST17, ST21, ST23–ST46) reported, of which ST1, ST2, and ST3 are prevalent. However, the pathogenicity levels and the pathogenicity differences among different subtypes have been controversial. [Objective] To determine the pathogenicity of ST1, ST2, and ST3 and explore whether there are differences in the pathogenicity between the three subtypes of Blastocystis sp. [Methods] Blastocystis sp. strains were isolated from fresh human and macaque faeces and identified by morphological observation, 18S rRNA gene amplification and sequencing, and cluster analysis. After BALB/c mice were infected with strains of ST1, ST2, and ST3, the distribution characteristics of the parasites in vivo were observed. In addition, the clinical signs, weight gain, feed conversion rate, and mortality of the infected mice were recorded to compare the pathogenicity of the three subtypes. Finally, the pathological changes in the intestines of mice were observed. [Results] Three subtypes of Blastocystis sp., ST1, ST2 and ST3, were successfully isolated and identified. Animal challenge results showed that the parasites predominantly colonized the intestine and negatively affected the health of mice. A high dose of ST3 resulted in death in mice, while ST1, ST2, and low doses of ST3 did not affect the survival of mice. Blastocystis sp. damaged the intestinal tissue of challenged mice. [Conclusion] Blastocystis sp. causes harm to the host by damaging the intestine. The pathogenicity varies among different subtypes and ST3 has stronger pathogenicity. This study lays a foundation for further studying the pathogenicity mechanism and provides data for assessing the threat of Blastocystis sp. to public health.

[Objective] Within the framework of global climate governance and considering the pivotal role of the Yellow River basin in China's ecological progress, this study focused on the Inner Mongolia segment of the Yellow River in July 2023. [Methods] We collected the surface sediment, water, and gas samples, with the aim of investigating the dissolved nitrous oxide (N₂O) concentration in the water, the N₂O emission flux (FN₂O) at the water-air interface, and the microbial community composition and diversity in surface sediments. [Results] The results revealed that the dissolved nitrous oxide (N₂O) concentration in the water of this segment varied between 0.547 8 mg/m³ and 0.598 2 mg/m³, with an average of 0.574 1 mg/m³. The FN₂O at the water-air interface ranged from −3.645 3 mg/(m²·d) to 4.392 5 mg/(m²·d), averaging 1.086 1 mg/(m²·d), which suggested this area was a net source of atmospheric N₂O. FN₂O showed a significantly positive correlation with pH and a significantly negative correlation with potential. The surface sediments harbored 7 784 operational taxonomic units (OTUs), with Proteobacteria (average abundance of 35.13%) being dominant. Ammonia-oxidizing archaea (AOA), a category of nitrifying bacteria, presented low abundance, with 116 OTUs among which unclassified_d__Unclassified (average abundance of 31.69%) was the dominant genus. For denitrifying bacteria, 3 660 OTUs were identified, with unclassified_k__norank_d__Bacteria (average abundance of 63.12%) being the dominant genus. In view of the scarcity of N₂O data for the Inner Mongolia section of the Yellow River, the findings of this study enrich the N₂O data repository of the Yellow River. [Conclusion] This study not only augments our understanding of the microbial community structure and functionality in sediments but also supports the conservation and purification of the Yellow River.

[Objective] To screen microbial strains with high production of exopolysaccharides (EPS) and provide strain resources for the development of soil improvement agents. [Methods] The string test with a LB-aniline blue plate was employed to qualitatively screen the strains of plant growth-promoting rhizobacteria (PGPR) with EPS production. After fermentation with each strain in four media, the EPS content in the fermentation liquid was determined by low temperature alcohol precipitation and the sulfate-anthranone colorimetric method, on the basis of which the PGPR strain with high EPS production was screened out. The fermentation conditions of the strain screened out were optimized by orthogonal test with EPS content in fermentation liquid as the indicator. The influence of the fermentation liquid of strain F1 on the content of macro-aggregates in sandy loam soil was analyzed by the petri dish culture experiment. [Results] Eight EPS-producing PGPR strains were primarily screened out, among which strain F1 had the highest EPS production. PDA was the best medium for F1 to produce EPS, with the EPS content of 867.54 μg/mL. Based on morphological, physiological and biochemical characteristics, phylogenetic analysis based on 16S rRNA gene sequence, and also average nucleotide identity analysis, F1 was identified as a strain of Bacillus megaterium. The optimal culture conditions for F1 to produce EPS were 28 ℃ and 180 r/min for 24 h, under which the EPS yield reached 1 123.39 μg/mL. After F1 was incubated in sandy loam soil for 40 days, the content of water-stable macro-aggregates with the grain diameter > 0.25 mm in the soil increased by 4.44 times compared with that of the control. [Conclusion] Strain F1 with high EPS production can promote the formation of water-stable macro-aggregates in sandy loam soil. The optimal conditions for F1 to produce of EPS was incubation in PDA at 28 ℃ and 180 r/min for 24 h.

[Objective] To explore the effects of different concentrations of chlortetracycline on the characteristics of microbial communities involved in inorganic phosphorus (Pi) dissolution and organic phosphorus (Po) mineralization in the soil applied with organic fertilizer, focusing on soil P transformation and availability. [Methods] The purple soil collected from Tongnan District of Chongqing was used for a pot experiment with the addition of chicken manure as the organic fertilizer. Three chlortetracycline treatments (No-CTC, Low-CTC, and High-CTC) were designed with the addition levels of 0.0, 0.1, and 4.0 mg/kg, respectively. The soil samples were collected on days 7 (D7) and 30 (D30) after pepper ('Xinxiang 8') was planted. Real-time qPCR and Illumina MiSeq high-throughput sequencing were employed to analyze the community characteristics of the bacteria carrying the key genes (pqqC and phoD) of Pi dissolution and Po mineralization, respectively. Furthermore, the sequencing results and biologically based P (BBP) fractionation were employed to examine the effects of CTC addition on soil P transformation. [Results] High-CTC increased the content of Citrate-P and Enzyme-P by 8.2% and 44.0%, respectively, compared with No-CTC on D7. Low-CTC and High-CTC increased the content of Enzyme-P by 44.0% and 65.6%, respectively, compared with No-CTC on D30. The addition of CTC suppressed alkaline phosphatase (ALP) activity and affected the community structures of pqqC and phoD-harboring bacteria in the soil. The Mantel test results showed that Citrate-P was significantly associated with the dominant pqqC-carrying taxa Pseudomonas, Geodermatophilus, and Saccharothrix on D7. The dominant phoD-carrying taxa Bradyrhizobium, Ensifer, and Skermanella exhibited notable correlations with Enzyme-P on D7, and such correlations weakened over time. The average degree of the community network of the bacteria carrying pqqC increased in the Low-CTC treatment and decreased in the High-CTC treatment on D7. The average degree of this network decreased in High-CTC and Low-CTC treatments on D30. The average degree of the community network of the bacteria carrying phoD decreased with the increase in CTC addition on D7, while this trend was opposite on D30. [Conclusion] The addition of CTC significantly affected soil Enzyme-P by regulating the community structure of pqqC- and phoD-carrying bacteria as well as acid phosphatase (ACP) and ALP activities, thereby affecting the P forms and availability in the soil. This study contributes to a deeper understanding of alterations in microbial communities associated with P cycling in the soil-plant system contaminated by CTC. Moreover, it lays a scientific foundation for enhancing nutrient utilization efficiency in the soil applied with antibiotics.

Lignin, the most abundant aromatic biopolymer resource in the nature, is difficult to be degraded by common microorganisms due to its complexity and highly aggregated aromatic structure. Lignin-degrading microorganisms from extreme environments are considered as suitable candidates for lignin bioprocessing. This review summarizes several types of extremophiles capable of degrading lignin and the extremozymes produced by them and elucidates the properties, catalytic mechanisms, and metabolic pathways of the extremozymes. Furthermore, this article discusses the prospects for the identification of novel extremophiles and extremozymes by multi-omics and makes an outlook on the development and utilization methods of extremophiles, with a view to providing a reference for the subsequent screening and development of more efficient lignin-degrading strains.

[Objective] To realize the de novo biosynthesis of caffeic acid from glucose by reconstruction of its biosynthetic pathway in Escherichia coli. Fine-tuning gene expression allows us to improve caffeic acid production, which paves a way for the high production of caffeic acid and its derivatives in E. coli. [Methods] The biosynthetic pathway of caffeic acid was reconstructed based on FjTAL and EchpaBC, which encoded the tyrosine ammonia lyase in Flavobacterium johnsoniaeu and the 4-hydroxyphenylacetate 3-hydroxylase complex in E. coli, respectively. The reconstructed pathway was then introduced into commonly used E. coli strains. We improved the expression levels of FjTAL and EchpaBC by screening constitutive promoters, utilizing an intermediate-based biosensor, and increasing the copy number of the key gene. Thus, a total of fourteen recombinant strains were obtained, and the production of caffeic acid and the intermediate p-coumaric acid in these strains was quantified by HPLC. Moreover, the effects of different nitrogen sources and substrate concentrations on the production of caffeic acid were investigated. [Results] We realized de novo biosynthesis of caffeic acid from glucose in E. coli. The use of constitutive promoters other than the commonly used T7 promoter contributed to the yield increase of caffeic acid. When glucose was used as the substrate, the yield of caffeic acid was increased from 1.40 mg/L to 96.40 mg/L. When tyrosine was used as the substrate, the yield of caffeic acid was increased from 1.78 mg/L to 123.31 mg/L. Furthermore, the yield of caffeic acid reached 162.73 mg/L when a p-coumaric acid biosensor instead of a constitutive promoter was used to drive the expression of EchpaBC. Moreover, the yield of caffeic acid was improved to 185.15 mg/L in the case of introducing an extra copy of EchpaBC. [Conclusion] We constructed the strains with high production of caffeic acid by promoter engineering, using an intermediate-base biosensor, and increasing copy number of the key gene. Our study laid a solid foundation for the high production of caffeic acid.