Enhancement of triclocarban biodegradation: Metabolic division of labor in co-culture of Rhodococcus sp. BX2 and Pseudomonas sp. LY-1.

Triclocarban (TCC) and its metabolite, 3,4-dichloroaniline (DCA), are classified as emerging organic contaminants (EOCs). Significant concerns arise from water and soil contamination with TCC and its metabolites. These concerns are especially pronounced at high concentrations of up to approximately 20 mg/kg dry weight, as observed in wastewater treatment plants (WWTPs). Here, a TCC-degrading co-culture system comprising Rhodococcus rhodochrous BX2 and Pseudomonas sp. LY-1 was utilized to degrade TCC (14.5 mg/L) by 85.9% in 7 days, showing improved degradation efficiency compared with monocultures. A combination of high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), genome sequencing, transcriptomic analysis, and quantitative reverse transcription-PCR (qRT-PCR) was performed. Meanwhile, through the combination of further experiments involving heterologous expression and gene knockout, we proposed three TCC metabolic pathways and identified four key genes (tccG, tccS, phB, phL) involved in the TCC degradation process. Moreover, we revealed the internal labor division patterns and connections in the co-culture system, indicating that TCC hydrolysis products were exchanged between co-cultured strains. Additionally, mutualistic cooperation between BX2 and LY-1 enhances TCC degradation efficiency. Finally, phytotoxicity assays confirmed a significant reduction in the plant toxicity of TCC following synergistic degradation by two strains. The in-depth understanding of the TCC biotransformation mechanisms and microbial interactions provides useful information for elucidating the mechanism of the collaborative biodegradation of various contaminants.

First report of leaf spot disease caused by Pseudopithomyces chartarum on Lonicera caerulea L. in Heilongjiang Province, China.

Blue honeysuckle (Lonicera caerulea L.) cultivation has gradually expanded in China but continues to be limited by challenges such as leaf spot disease. Between September 2022 and September 2023, a leaf spot disease was observed on approximately 30% of 'Lanjingling' blue honeysuckles grown in a 2.66 ha field (a total of about 11,000 plants) in Jiamusi city (130.47°E, 46.16°N), Heilongjiang Province, China. Affected plants displayed brown necrotic lesions on their leaves that gradually expanded in area until the leaves fell off the plant entirely. Small, 3 to 4 mm segments of infected tissue from 50 randomly selected leaves were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried on paper towels, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA) (Yan et al. 2022). Five pathogens (LD-232, LD-233, LD-234, LD-235, and LD-236) were isolated on PDA and displayed a conidia morphology consistent with Pseudopithomyces spp. (Perelló et al. 2017). The fungal colonies on PDA were villiform, white, and whorled and had sparse aerial mycelium on the surface with black conidiomata. The conidia were obpyriform and dark brown, had 0 to 3 transverse and 0 to 1 longitudinal septa, and measured 9.00 to 15.30 µm × 5.70 to 9.30 µm in size (n = 50). Genomic DNA was extracted from a representative isolate, LD-232, for molecular verification and PCR amplification was performed with ITS1/ITS4 (White et al. 1990), LROR/LR7 (Carbone and Kohn 1999), and RPB2-5F2/RPB2-7CR (Liu et al. 1999) primers. Sequences of LD-232 ITS (OR835654), LSU (OR835652), and RPB2 (OR859769) revealed 99.8% (530/531 nt), 98.8% (639/647 nt), and 99.8% (1015/1017 nt) shared identity with Pseudopithomyces chartarum sequences (OP269600, OP237014, and MK434892), respectively (Wu et al. 2023). Bayesian inference (BI) was used to construct the phylogenies using Mr. Bayes v. 3.2.7 to confirm the identity of the isolates (Ariyawansa et al. 2015). Phylogenetic trees cannot be constructed based on the genes' concatenated sequences because selective strains do not have complete rDNA-ITS, LSU, and RPB2 sequences. Therefore, based on the morphological characteristics and molecular phylogeny, LD-232 was identified as P. chartarum (Perelló et al. 2017; Wu et al. 2023). A pathogenicity test was performed with six healthy, two-year-old 'Lanjingling' blue honeysuckle plants. Three plants were inoculated by spraying the LD-232 conidial suspension (1 × 106 spores/ml) or clean water as an experimental control condition (Wu et al. 2023; Yan et al. 2023). All plants were cultured in a greenhouse at 28℃ under a 12-h light/dark cycle, and each experiment was replicated three times. Typical leaf spot symptoms were observed on inoculated leaves after 10 days. The same pathogens were reisolated from infected leaves, displayed the same morphological and molecular traits, and were again identified as P. chartarum, confirming Koch's postulate. P. chartarum previously caused leaf spot disease on Tetrapanax papyrifer in China (Wu et al. 2023). To our knowledge, this is the first report of blue honeysuckle leaf spot caused by P. chartarum in China. Identification of P. chartarum as a disease agent on blue honeysuckle will help guide future management of leaf diseases for this economically important small fruit tree.

First report of leaf spot disease caused by Neopestalotiopsis rosae on Lonicera caerulea L. in Heilongjiang Province, China.

Recently, interest in cultivating blue honeysuckle (Lonicera caerulea L.) for horticulture and medicinal uses has grown (Sharma and Lee 2021). Between September 2022 and September 2023, a leaf spot disease (Fig. S1) was observed on approximately 20% of 'Lanjingling' blue honeysuckles grown in a 0.18 ha field in Qiqihar city (123.43°E, 47.92°N), Heilongjiang Province, China. Infected plants displayed black leaf spots that expanded to cover the entire leaf. Small, 3 to 4 mm segments of infected tissue were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried on paper towels, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA) (Ma et al. 2023). To induce sporulation, nine purified cultures (Fig. S2) with similar culture characteristics were finally obtained from ten infected plants and they displayed a conidia morphology consistent with Neopestalotiopsis spp., no other fungi were isolated, and the isolation frequency was 90%. Conidiomata (Fig. S3) were brown to black and distributed in concentric rings with an average size of 261.98 (60.30-451.80) µm (n = 50). The conidia (Fig. S3) were fusoid and had four septa, straight to slightly curved, with an average size of 23.48 (13.50-30.30) × 5.42 (4.50-9.30) µm(n = 50), while basal and apical cells were hyaline and the three middle cells were brown with darker septa. PCR amplification was performed with ITS1/ITS4 (White et al. 1990), EFl-728F/EF1-986R (Carbone and Kohn 1999), and Btub2Fd/Btub4Rd (Glass and Donaldson 1995) primers from the genomic DNA of the LD-330. Sequences of ITS (PP033584), TEF (PP048757), and TUB (PP048758) revealed 99 to 100% (499/500, 255/255, and 481/486) shared identity with Neopestalotiopsis rosae sequences (NR145243, KM199524, and KM199430) (Rebollar-Alviter et al. 2020). Therefore, based on morphological characteristics and molecular phylogeny, LD-330 was identified as N. rosae. Six two-year-old healthy plants of the 'Lanjingling' cultivar were selected for a pathogenicity test (Yan et al. 2023). The leaves were surface disinfected with 75% alcohol and then wiped with sterilized water three times. Three plants were inoculated with 10 ml of LD-330 conidial suspension (1 × 106 spores/ml) or with sterile water as an experimental control, respectively. All plants were in closed plastic bag, incubated in a greenhouse at 28 ℃ and 75% relative humidity (RH) under a 12-h light/dark cycle, and each experiment was performed three times (Rebollar-Alviter et al. 2020). Typical leaf spot symptoms were observed on inoculated leaves after 14 days (Fig. S4), whereas no symptoms were detected on water-treated leaves. The same pathogen was reisolated from infected leaves, displayed the same morphological and molecular traits, and was again identified as N. rosae, confirming Koch's postulate. Neopestalotiopsis rosae was previously reported on pecan (Gao et al. 2022), causing black leaf spot disease in China. To our knowledge, this is the first report of a blue honeysuckle leaf spot caused by N. rosae in China and specifically in the Heilongjiang province which has the largest blue honeysuckle cultivation area in the country. Future research should be directed toward developing comprehensive management measures.

Synergistic detoxification efficiency and mechanism of triclocarban degradation by a bacterial consortium in the liver-gut-microbiota axis of zebrafish (Danio rerio).

Triclocarban (TCC), an emerging organic contaminant, poses a potential threat to human health with long-term exposure. Here, Rhodococcus rhodochrous BX2 and Pseudomonas sp. LY-1 were utilized to degrade TCC at environmental related concentrations for enhancing TCC biodegradation and investigating whether the toxicity of intermediate metabolites is lower than that of the parent compound. The results demonstrated that the bacterial consortium could degrade TCC by 82.0% within 7 days. The calculated 96 h LC50 for TCC, as well as its main degradation product 3,4-Dichloroaniline (DCA) were 0.134 mg/L and 1.318 mg/L respectively. Biodegradation also alleviated histopathological lesions induced by TCC in zebrafish liver and gut tissues. Liver transcriptome analysis revealed that biodegradation weakened differential expression of genes involved in disrupted immune regulation and lipid metabolism caused by TCC, verified through RT-qPCR analysis and measurement of related enzyme activities and protein contents. 16 S rRNA sequencing indicated that exposure to TCC led to gut microbial dysbiosis, which was efficiently improved through TCC biodegradation, resulting in decreased relative abundances of major pathogens. Overall, this study evaluated potential environmental risks associated with biodegradation of TCC and explored possible biodetoxification mechanisms, providing a theoretical foundation for efficient and harmless bioremediation of environmental pollutants.

First Report of Postharvest Fruit Rot Caused by Botrytis cinerea on Blue Honeysuckle (Lonicera caerulea L.) Fruit in China.

Blue honeysuckle (Lonicera caerulea L.) fruit is growing in popularity as a natural, functional 'super fruit', but its storage is challenged by pathogen infection. In June 2022, approximately 30% of 100 kg of blue honeysuckle fruits (cv. Lanjingling) obtained in Harbin, China (128.70°E, 44.87°N) showed postharvest fruit rot symptoms after 15 d of storage at 4°C, leading to whole fruit rotting with gray fungal growth (Fig.1 A). Small (1-2 mm) segments of infected tissue were obtained from 20 randomly selected fruits which were surface sterilized with 75% ethanol for 30 s and 5% sodium hypochlorite (NaOCl) for 3 min, rinsed three times with sterile distilled water, dried in paper towel, and plated in 9 cm Petri dishes containing potato dextrose agar (PDA). Five purified cultures were obtained and their front colonies were dark brown (Fig.1 C) on the PDA plates after 5 d at 25°C (Alam et al. 2019; Riquelme-Toledo et al. 2020). The conidia (n = 50) were single-celled, hyaline, either ellipsoid or ovoid, and measured 7.5-15.0 µm (11.7 µm average) × 6.0-11.4 µm (8.3 µm average). The conidiophores (Fig.1 E) were branched at the apex bearing bunches of conidia resembling grape clusters (Ellis 1971). For molecular confirmation, genomic DNA was extracted from a representative isolate LDGS-3 using the Ezup Column Fungi Genomic DNA Purification kit (Sangon Biotech, Shanghai, China). The internal transcribed spacer region (ITS, GenBank ON952502), heat shock protein (HSP60, GenBank OP039103), the second-largest subunit of RNA polymerase II (RPB2, GenBank OP186114) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH, GenBank OQ658508) genes were partially amplified with the respective primers ITS1/ITS4, HSP60f/HSP60r, RPB2f/RPB2r, and G3PDH-F/G3PDH-R (Staats et al. 2005; White et al. 1990). BLAST analysis revealed that the sequences of the four genes showed 100% homology with the MH782039, MH796663, MN448501 and MH796662 sequences for isolates of Botrytis cinerea. Based on morphology and molecular characteristics, the isolate LDGS-3 was identified as B. cinerea. For pathogenicity, twenty healthy blue honeysuckle fruits (cv. Lanjingling) were superficially sterilized with 75% ethanol and washed with distilled water. Ten inoculated blue honeysuckle fruits, which were injected with 10 µL conidial suspension of isolate LDGS-3 (106 spores/mL) displayed fruit rot symptoms (Fig.1 B) inside 9 cm Petri dishes after 10 d at 4°C, while no symptoms were detected on ten fruits inoculated with sterile distilled water (Alam et al. 2019). The same isolate that was reisolated from infected fruits with the same morphological and molecular traits was also identified as B. cinerea, confirming Koch's postulates. B. cinerea was previously reported in Henan Province, China in hawthorn (Zhang et al. 2018). To our knowledge, this is the first report of postharvest fruit rot caused by B. cinerea on blue honeysuckle fruit in China, which will aid future management of this emerging postharvest disease.

First report of Didymella glomerata causing leaf spot on Lonicera caerulea L. in Heilongjiang province, China.

China has the largest blue honeysuckle (Lonicera caerulea L.) cultivation area globally. In June 2022, leaf spots were observed on approximately 10% of blue honeysuckle (cv. 'Lanjingling') leaves in a 0.03-ha field in Harbin (127.66°E, 45.61°N), Heilongjiang Province, China. The leaves of the affected plants displayed chlorotic to tan dieback with a darker brown margin along the leaftip and leave margins. Cross-sectional segments of approximately 3 mm were cut from 50 typical infected plant leaves. Their surfaces were sterilized with 75% ethanol for 30 s followed by 3 min in 5% sodium hypochlorite (NaOCl), rinsed three times with sterile water, and transferred to 9-cm Petri dishes containing 15 ml of sterile PDA growth medium. Five purified cultures with similar culture characteristics were finally obtained and their colonies were dark brown on the PDA plates. The pycnidia were subglobular and deep black and measured avg. 215.48 (135.30-331.20) µm × avg. 170.28 (99.90-282.90) µm (n = 50) (Chen et al., 2015; Huang et al., 2018). Conidia were single-celled, hyaline, and ellipsoidal and measured avg. 6.22 (5.40-7.20) µm × avg. 3.42 (2.70-3.90) µm (n = 50). For molecular verification, genomic DNA was extracted from a representative isolate, LD-75. The internal transcribed spacer region (ITS), the second-largest subunit of RNA polymerase II (rpb2), the partial 28S large subunit rDNA (LSU), beta-tubulin (TUB), and actin (ACT) genes were amplified with the primers ITS1/ITS4, RPB2f/RPB2r, LROR/LR7, TUB2Fd/TUB4Rd, and ACT512f/ACT783R, respectively (White et al. 1990; Carbone and Kohn, 1999; Staats et al., 2005; de Gruyter et al., 2009; Chen et al., 2015). BLAST results indicated that the genes of LD-75 (GenBank OP218870, OP264863, OQ561448, OQ597233, and OQ597232) shared 99%-100% identity with those of Didymella glomerata (OK485138, GU371781, EU754185, MZ073910, and MW963190, respectively). Therefore, based on morphological characteristics and molecular phylogeny, LD-75 was identified as D. glomerata. Six two-year-old healthy plants from the 'Lanjingling' cultivar were selected for a pathogenicity test. The leaves were surface disinfested with 75% ethanol and then wiped with sterilized water three times. All plants were cultured in a greenhouse at 28℃ under a 12-h light/dark cycle. Whole plants sprayed with conidial suspension of isolate LD-75 (106 spores/mL) (n = 3) displayed leaf spot symptoms after 14 d, while no symptoms were detected on whole plants sprayed with sterile water (n = 3). The same isolate, reisolated from infected leaves and with the same morphological and molecular traits, was also identified as D. glomerata, confirming Koch's postulate. The fungus was previously reported in Cornus officinalis in Nanyang City, China (Huang et al., 2018). To our knowledge, this is the first report of blue honeysuckle leaf spot caused by D. glomerata in China. Reducing blue honeysuckle production losses caused by leaf spots is crucial for growers, and we hope that researchers will develop efficient control strategies for managing this emerging plant disease.

First report of leaf spot disease caused by Alternaria tenuissima on Lonicera caerulea L. in Heilongjiang Province, China.

Blue honeysuckle (Lonicera caerulea L.) is a perennial plant of the Caprifoliaceae family and Lonicera genus, the largest genus in the plant kingdom. Between September 2021 and September 2022, a leaf spot disease was observed on ~20% of blue honeysuckles of the 'Lanjingling' cultivar grown in a 3.33 ha field at the Xiangyang base (126.96°E, 45.77°N) of the Northeast Agricultural University, Harbin (Heilongjiang Province, China). Leaf spots first presented black mildew centers, gradually covering large areas of the leaf until it eventually fell off. Small 3-4 mm segments of infected tissue from 50 randomly selected leaves were surface sterilized with 75% ethanol and 5% sodium hypochlorite, rinsed in sterile distilled water, and transferred to 9 cm Petri dishes containing potato dextrose agar (PDA) after drying. Finally, two isolated pathogens were obtained through single spore culture on PDA; they appeared as gray-black colonies and were named LD-12 and LD-121. The observed LD-12 and LD-121 conidia displayed a morphology consistent with Alternaria spp. They were obpyriform and dark brown, with 0-6 transverse and 0-3 longitudinal septa, measuring 6.00-17.70 µm × 9.30-42.30 µm and 5.70-20.70 µm × 8.40-47.70 µm for LD-12 and LD-121, respectively (n = 50). Genomic DNA was extracted from the two isolates for molecular verification, and PCR amplification was performed with ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Woudenberg et al. 2015), EFl-728F/EF1-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-7CR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005) primers. Sequences of LD-12 ITS (OQ607743), GPD (OQ623200), TEF (OQ623201), RPB2 (OQ658509), and ALT (OQ623199) revealed 99-100% of identity with Alternaria tenuissima sequences (KC584567, MK451973, LT707524, MK391051, and ON357632). Sequences of LD-121 ITS (OQ629881), GPD (OQ850078), TEF (OQ850075), RPB2 (OQ850076), and ALT (OQ850077) revealed 99-100% identity with A. alternata sequences (MN826219, ON055384, KY094927, MK637444, and OM849255). Nine two-year-old healthy plants from the 'Lanjingling' cultivar were selected for a pathogenicity test. Three plants were inoculated with either the LD-12 or LD-121 conidial suspension (1 × 106 spores/ml) or with clean water as an experimental control condition (Mirzwa-Mróz et al., 2018; Liu et al., 2021). All plants were cultured in a greenhouse at 28℃ under a 12-h light/dark cycle, and each experiment was performed three times. Typical leaf spot symptoms were observed on inoculated leaves after 10 d. The same pathogens reisolated from infected leaves displayed the same morphological and molecular traits. They were again identified as A. tenuissima and A. alternata, confirming Koch's postulate. A. tenuissima and A. alternata were previously reported on Orychophragmus violaceus (Liu et al., 2021) and L. caerulea (Yan et al., 2022) in China. This study is the first report of a blue honeysuckle leaf spot caused by A. tenuissima in China. In the future, effective biological and chemical control should be used to prevent blue honeysuckle leaf spots in China.

Enhanced bioremediation of triclocarban-contaminated soil by Rhodococcus rhodochrous BX2 and Pseudomonas sp. LY-1 immobilized on biochar and microbial community response.

Triclocarban (TCC), an emerging organic contaminant (EOC), has become a severe threat to soil microbial communities and ecological security. Here, the TCC-degrading strain Rhodococcus rhodochrous BX2 and DCA-degrading strain Pseudomonas sp. LY-1 (together referred to as TC1) were immobilized on biochar to remove TCC and its intermediates in TCC-contaminated soil. High-throughput sequencing was used to investigate the microbial community structure in TCC-contaminated soil. Analysis of co-occurrence networks was used to explore the mutual relationships among soil microbiome members. The results showed that the immobilized TC1 significantly increased the removal efficiency of TCC from 84.7 to 92.7% compared to CK (no TC1 cells on biochar) in 10 mg/L TCC liquid medium. The utilization of immobilized TC1 also significantly accelerated the removal of TCC from contaminated soil. Microbial community analysis revealed the crucial microorganisms and their functional enzymes participating in TCC degradation in soil. Moreover, the internal labor division patterns and connections of TCC-degrading microbes, with a focus on strains BX2 and LY-1, were unraveled by co-occurrence networks analysis. This work provides a promising strategy to facilitate the bioremediation of TCC in soil, which has potential application value for sustainable biobased economies.

A Novel Estrone Degradation Gene Cluster and Catabolic Mechanism in Microbacterium oxydans ML-6.

Global-scale estrone (E1) contamination of soil and aquatic environments results from the widespread use of animal manure as fertilizer, threatening both human health and environmental security. A detailed understanding of the degradation of E1 by microorganisms and the associated catabolic mechanism remains a key challenge for the bioremediation of E1-contaminated soil. Here, Microbacterium oxydans ML-6, isolated from estrogen-contaminated soil, was shown to efficiently degrade E1. A complete catabolic pathway for E1 was proposed via liquid chromatography-tandem mass spectrometry (LC-MS/MS), genome sequencing, transcriptomic analysis, and quantitative reverse transcription-PCR (qRT-PCR). In particular, a novel gene cluster (moc) associated with E1 catabolism was predicted. The combination of heterologous expression, gene knockout, and complementation experiments demonstrated that the 3-hydroxybenzoate 4-monooxygenase (MocA; a single-component flavoprotein monooxygenase) encoded by the mocA gene was responsible for the initial hydroxylation of E1. Furthermore, to demonstrate the detoxification of E1 by strain ML-6, phytotoxicity tests were performed. Overall, our findings provide new insight into the molecular mechanism underlying the diversity of E1 catabolism in microorganisms and suggest that M. oxydans ML-6 and its enzymes have potential applications in E1 bioremediation to reduce or eliminate E1-related environmental pollution. IMPORTANCE Steroidal estrogens (SEs) are mainly produced by animals, while bacteria are major consumers of SEs in the biosphere. However, the understanding of the gene clusters that participate in E1 degradation is still limited, and the enzymes involved in the biodegradation of E1 have not been well characterized. The present study reports that M. oxydans ML-6 has effective SE degradation capacity, which facilitates the development of strain ML-6 as a broad-spectrum biocatalyst for the production of certain desired compounds. A novel gene cluster (moc) associated with E1 catabolism was predicted. The 3-hydroxybenzoate 4-monooxygenase (MocA; a single-component flavoprotein monooxygenase) identified in the moc cluster was found to be necessary and specific for the initial hydroxylation of E1 to generate 4-OHE1, providing new insight into the biological role of flavoprotein monooxygenase.

Biodegradation characteristics of lignin in pulping wastewater by the thermophilic Serratia sp. AXJ-M: Performance, genetic background, metabolic pathway and toxicity assessment.

The key to the efficient removal of pulping wastewater lies in the effective degradation of lignin at high temperature. There is thus an urgent need to seek effective eco-environmental techniques to overcome this environmental limit for lignin degradation. The soil isolate thermophilic Serratia sp. AXJ-M efficiently metabolizes lignin. Nevertheless, the underlying comprehensive molecular mechanism of lignin degradation by thermophilic AXJ-M is poorly understood. Here, strain AXJ-M showed excellent degradation ability toward diverse lignin-related aromatic compounds. Functional genome analysis and RNA-Seq disclosed several traits which in joint consideration suggest a high efficiency of AXJ-M representative to the lignin degradation and environmental adaptation. Multiomics analyses combined with GC-MS revealed seven potential lignin biodegradation pathways. DyP was predicted to be involved in the breakdown of the ß-O-4 ether bond, Cα-Cß bond and Cα oxidation of lignin by prokaryotic expression and gene knockout and complementation. Molecular docking deepens the understanding of the interaction between DyP and lignin. Toxicity assessment experiments clearly indicated that AXJ-M significantly reduced the toxicity of the metabolites. This work expands the knowledge about the degradation mechanism of thermophilic lignin-degrading bacteria, most importantly, offers a new perspective on potential applications in utilizing this strain in pulping wastewater bioremediation.

Biological saccharification coupled with anaerobic digestion using corn straw for sustainable methane production.

In this study, accumulated fermentable sugars from biosaccharified corn straw were used to generate methane through anaerobic digestion (AD). The results showed that reducing sugars from biosaccharification expanded corn straw (BECS) treated with Clostridium thermocellum XF811 accumulated with yields of 94.9 mg/g. The BECS used for AD was converted into a high methane yield (7436 mL), which was 49.3 % higher than that of expanded corn straw (ECS). High-throughput microbial analysis suggested that Methanoculleus and Methanobacterium greatly contributed to the high methane yield. Industrial experiments demonstrated that the methane production from BECS by AD was 72,955 m3, which increased by 13.2 % compared to that from ECS. Biosaccharification pretreatment accelerated ECS destruction and accumulated sugars, thereby increasing methane yields. This study provides a strategy for producing clean energy from lignocellulose biomass.

Genome Functional Analysis of the Psychrotrophic Lignin-Degrading Bacterium Arthrobacter sp. C2 and the Role of DyP in Catalyzing Lignin Degradation.

In the cold regions of China, lignin-rich corn straw accumulates at high levels due to low temperatures. The application of psychrotrophic lignin-degrading bacteria should be an effective means of overcoming the low-temperature limit for lignin degradation and promoting the utilization of corn straw. However, this application is limited by the lack of suitable strains for decomposition of lignin; furthermore, the metabolic mechanism of psychrotrophic lignin-degrading bacteria is unclear. Here, the whole genome of the psychrotrophic lignin-degrading bacterium Arthrobacter sp. C2, isolated in our previous work, was sequenced. Comparative genomics revealed that C2 contained unique genes related to lignin degradation and low-temperature adaptability. DyP may participate in lignin degradation and may be a cold-adapted enzyme. Moreover, DyP was proven to catalyze lignin Cα-Cß bond cleavage. Deletion and complementation of the DyP gene verified its ability to catalyze the first-step reaction of lignin degradation. Comparative transcriptomic analysis revealed that the transcriptional expression of the DyP gene was upregulated, and the genetic compensation mechanism allowed C2ΔDyP to degrade lignin, which provided novel insights into the survival strategy of the psychrotrophic mutant strain C2ΔdyP. This study improved our understanding of the metabolic mechanism of psychrotrophic lignin-degrading bacteria and provided potential application options for energy-saving production using cold-adapted lignin-degrading enzymes.

Insight into biofilm-forming patterns: biofilm-forming conditions and dynamic changes in extracellular polymer substances.

The microbial biofilm adheres to the surface of the carrier, which protects the pollutant-degrading bacteria and resists harsh environments; thus, research on biofilm-forming patterns will help promote the application of biofilms in wastewater treatment. Herein, univariate analysis and response surface methodology (RSM) confirmed that glucose and mannose at 3-5 g/L promoted biofilm formation. Notably, the microplate method demonstrated that compared to trivalent cations, divalent cations could more greatly enhance the activity (especially magnesium) of the biofilm matrix, and the period of biofilm formation in the three strains was divided into the following stages: initial attachment (0-10 h), microcolony (10-24 h), maturation (24-48 h), and dispersion (36-72 h). During maturation, large amounts of extracellular polysaccharides (EPs) and extracellular DNA (eDNA) were distributed in the extracellular and intracellular spaces, respectively, as observed by super-resolution structured illumination microscopy (SR-SIM). This study enhances the understanding of the characteristics and patterns of biofilm formation and can facilitate the application of biofilms in wastewater treatment.

Potential and mechanism for bioremediation of papermaking black liquor by a psychrotrophic lignin-degrading bacterium, Arthrobacter sp. C2.

To meet the challenge of bioremediation of black liquor in pulp and paper mills at low temperatures, a psychrotrophic lignin-degrading bacterium was employed in black liquor treatment for the first time. In this study, Arthrobacter sp. C2 exhibited excellent cold adaptability and lignin degradation ability, with a lignin degradation rate of 65.5% and a mineralization rate of 43.9% for 3 g/L lignin at 15 °C. Bioinformatics analysis and multiple experiments confirmed that cold shock protein 1 (Csp1) was the dominant cold regulator of strain C2, and dye-decolorizing peroxidase (DyP) played a crucial role in lignin degradation. Moreover, structural equation modeling (SEM), mRNA monitoring, and phenotypic variation analysis demonstrated that Csp1 not only mediated cold adaptation but also modulated DyP activity by controlling dyp gene expression, thus driving lignin depolymerization for strain C2 at low temperatures. Furthermore, 96.4% of color, 64.2% of chemical oxygen demand (COD), and 100% of nitrate nitrogen (NO3--N) were removed from papermaking black liquor by strain C2 within 15 days at 15 °C. This study provides insights into the association between the cold regulator and catalytic enzyme of psychrotrophic bacteria and offers a feasible alternative strategy for the bioremediation of papermaking black liquor in cold regions.

Efficient conversion of hemicellulose into 2, 3-butanediol by engineered psychrotrophic Raoultella terrigena: mechanism and efficiency.

Low-temperature biorefineries inhibit the multiplication of undesired microorganisms, improve product purity and reduce economic costs. Herein, to improve the 2,3-butanediol (2,3-BD) bioconversion efficiency from hemicellulose, a psychrotrophic hemicellulose-degrading strain Raoultella terrigena HC6 with high ß-xylosidase activity 1520 U/mL was isolated and genetically modified. Xylan (hemicellulose replacement) was depolymerized into xylooligosaccharides (XOS) and xylose by HC6, which were further converted into 2,3-BD. Transcriptomic analysis revealed that ß-xylosidase gene (xynB) and xylose isomerase gene (xylA), which are beneficial for increasing the carbon flux from xylan to 2,3-BD, were significantly upregulated 56.9-fold and 234-fold, respectively. A recombinant strain was constructed by overexpressing xynB in HC6, which obtained 0.389 g/g yield of 2,3-BD from hemicellulose extracted from corn straw at 15 °C. This study proposed a promised strategy for the bioconversion of agricultural waste into 2,3-BD at low temperatures and provides a basis for future efforts in the achievement of carbon neutrality.

First report of leaf spot disease caused by Alternaria alternata on Lonicera caerulea L. in China.

Relatively few studies investigating plant diseases in blue honeysuckle (Lonicera caerulea L.) have been conducted in China. In September 2021, a leaf spot disease was observed on approximately 10% of blue honeysuckle 'Lanjingling' leaves in a 0.02 ha field plantation in Tiekuang Street (124.36°E, 40.12°N) in Dandong City, Liaoning Province, China. The main symptom consisted of leaf spots with black mildew centers typically surrounded by yellow halos. Small pieces (3-4 mm) of the infected leaves were plated onto potato dextrose agar (PDA) medium as described by Wang et al. (2020) and six purified cultures were obtained through single spore culture on PDA. The observed conidia, consistent with the morphology of Alternaria alternata, were obpyriform and dark brown, measuring 5.8 to 15.3 µm × 7.9 to 42.5 µm, with 1-6 transverse septa and 0-3 longitudinal septa (n = 50) (Simmons 2007). For molecular verification, genomic DNA was extracted from a representative isolate LD-8. The ITS (GenBank OL454815), GPD (GenBank OL601993), TEF (GenBank OL538256), RPB2 (GenBank OL601966), and Alt (GenBank OL538257) genes were partially amplified with the respective primers ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Woudenberg et al. 2015), EFl-728F/EFI-986R (Carbone and Kohn 1999), RPB2-5F2/RPB2-7CR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005). BLAST analysis revealed that these genes shared 99%-100% identity with OK345332, MK451977, MN756011, KU933459, and MN655781, respectively. A greenhouse experiment was conducted using six, healthy two-year-old blue honeysuckle 'Lanjingling' plants to observe disease development (Mirzwa-Mróz et al. 2018). After 10 d, we noted typical leaf spot symptoms on inoculated leaves sprayed with a conidial suspension (106 spores/mL) while no symptoms were detected on uninoculated leaves. The same isolate, reisolated from infected leaves with the same morphological and molecular traits, was also identified as A. alternata, confirming Koch's postulates. The fungus was previously reported in cockscomb plants in Heilongjiang Province, China (Wang et al. 2020). To our knowledge, this is the first report of leaf spot disease caused by A. alternata in blue honeysuckle grown in China. This study will provide a basis for future development of effective protection strategies against blue honeysuckle leaf spot in China.

An amidase and a novel phenol hydroxylase catalyze the degradation of the antibacterial agent triclocarban by Rhodococcus rhodochrous.

Triclocarban (TCC) is an emerging and intractable environmental contaminant due to its hydrophobicity and chemical stability. However, the antibacterial property of TCC limits its biodegradation, and only the functional enzyme TccA involved in TCC degradation has been characterized to date. In this study, we report a highly efficient TCC-degrading bacterium, Rhodococcus rhodochrous BX2, that could degrade and mineralize TCC (10 mg/L) by 76.8% and 56.5%, respectively, within 5 days. Subsequently, the TCC biodegradation pathway was predicted based on the detection of metabolites using modern mass spectrometry techniques. Furthermore, an amidase (TccS) and a novel phenol hydroxylase (PHIND) encoded by the tccS and PHIND genes, respectively, were identified by genomic and transcriptomic analyses of strain BX2, and these enzymes were further unequivocally proven to be the key enzymes responsible for the metabolism of TCC and its intermediate 4-chloroaniline (4-CA) by using a combination of heterologous expression and gene knockout. Our results shed new light on the mechanism of TCC biodegradation and better utilization of microbes to remediate TCC contamination.

Efficient vanillin biosynthesis by recombinant lignin-degrading bacterium Arthrobacter sp. C2 and its environmental profile via life cycle assessment.

Vanillin is a natural flavoring agent that is widely used in the bioengineering industry. To enable sustainable development, joint consideration of bacterial performance and negative environmental impacts are critical to vanillin biosynthesis. In this study, a cold shock protein (csp) gene was upregulated for maintaining stable growth in Arthrobacter sp. C2 responding to vanillin and cold stress. Furthermore, the recombinant strain C2 was constructed by simultaneously deleting the xylC gene encoding benzaldehyde dehydrase and overexpressing the pchF gene encoding vanillyl alcohol oxidase and achieved a maximum vanillin productivity of 0.85 mg/g DCW/h with alkaline lignin as the substrate. Finally, this process generated an environmental impact value of 25.05, which was the lowest environmental impact achieved according to life cycle assessment (LCA). Improvement strategies included reducing electricity consumption and replacing chemicals. This study achieved the development of an effective strategy, and future studies should focus on precise vanillin biosynthesis methods for large-scale application.

Impact of microbial inoculants combined with humic acid on the fate of estrogens during pig manure composting under low-temperature conditions.

To investigate the efficiency of psychrotrophic cellulose-degrading fungal strains (PCDFSs) and estrogen-degrading bacteria (EDBs) combined with humic acid (HA) on estrone (E1) and 17-ß-estradiol (E2) degradation, five compost groups (T, HA, EDB, PCDFS, and CK) were prepared and composted for 32 days at 11-14°C. The results indicated that inoculation increased the temperature to 62.2°C and promoted E1 degradation to the lowest level of 100.1 ng/kg, while E2 was undetected from day 16. Metagenomic analysis revealed that inoculation altered the microbial community structure by increasing the abundance of cellulose-degrading fungi, especially Meyerozyma (16.7%) (among PCDFSs), and of estrogen-degrading bacteria, particularly Microbacterium (13.4%) (involved in EDBs). Moreover, inoculation increased the levels (>0.500%) of Gene Ontology (GO) associated with estrogen degradation, like 3-ß-hydroxy-delta 5-steroid dehydrogenase and monooxygenase. Redundancy analysis demonstrated that temperature and Microbacterium were positively correlated with estrogen degradation. Structural equation model indicated that temperature and estrogen-degrading bacterial genera exhibited positive, significant (p < 0.001) and direct impacts on estrogen degradation. This is the first study to suggest that applying microbial inoculants and HA could accelerate estrogen degradation during composting in cold regions. The research outcomes offer a practical reference for managing compost safety, thereby decreasing its potential environmental and human health impacts.

A novel biodemulsifier of Bacillus mojavensis XH1 - Oxalate decarboxylase with the potential for demulsification of oilfield emulsion.

In recent years, special attention has been devoted to biodemulsifiers as a new type of environment-friendly demulsifiers. A novel biodemulsifying oxalate decarboxylase (OxdC) secreted by Bacillus mojavensis XH1 is reported in the present study. A genome-wide comparison showed that strains with high demulsification efficiencies all possess alkane degradation genes. An analysis of the differentially expressed genes and proteins induced by different substrates showed that OxdC secreted by XH1 was an effective demulsifier. Moreover, the demulsification ability was verified by prokaryotic gene expression, knockout and complementation analyses. OxdC from XH1 exhibited a strong demulsification capacity and significantly outperformed the model protein Bacillus subtilis 168 OxdC (Yvrk), which shared a high amino acid similarity but showed limited demulsification ability. Based on a comparison of the structural characteristics, the hydrophobic amino acids on the surface of OxdC were identified as a key factor driving the favorable demulsification activity of XH1. The metabolic pathways of XH1 used liquid paraffin and glucose as substrates, illustrating that hydrocarbons are necessary for biodemulsifier secretion. The present study provides new insight into the application of OxdC as an additional genetic resource in biodemulsification.