Characterization of the Positive Transcription Regulator PfaR for Improving Eicosapentaenoic Acid Production in Shewanella putrefaciens W3-18-1.

The biosynthetic pathway of eicosapentaenoic acid (EPA) has previously been reported in marine bacteria, while the regulatory mechanism remains poorly understood. In this study, a putative transcriptional regulator PfaR encoded adjacent to the PFA biosynthesis gene cluster (pfaEABCD) was computationally and experimentally characterized. Comparative analyses on the wild type (WT) strain, in-frame deletion, and overexpression mutants revealed that PfaR positively regulated EPA synthesis at low temperature. RNA-Seq and real-time quantitative PCR analyses demonstrated that PfaR stimulated the transcription of pfaABCD. The transcription start site of pfaR was mapped by using primer extension and highly conserved promoter motifs bound by the housekeeping Sigma 70 factor that were identified in the upstream of pfaR. Moreover, overexpression of PfaR in WT strain W3-18-1 at low temperature could improve EPA productivity from 0.07% to 0.13% (percentage of EPA to dry weight, mg/mg) of dry weight. Taken together, these findings could provide important implications into the transcriptional control and metabolic engineering in terms of EPA productivity for industrial strains. IMPORTANCE We have experimentally confirmed that PfaR is a positive transcription regulator that promotes EPA synthesis at low temperature in Shewanella putrefaciens W3-18-1. Overexpression of PfaR in WT strain W3-18-1 could lead to a 1.8-fold increase in EPA productivity at low temperature. It is further shown that PfaR may be regulated by housekeeping Sigma 70 factor at low temperature.

Pseudaquidulcibacter saccharophilus gen. nov., sp. nov., a novel member of family Caulobacteraceae, isolated from a water purification facility with supplement of starch as a carbon source.

During a survey of microbial communities in the influent (ambient water) and effluent of a water purification facility with aeration and supplement of starch as carbon source, a novel bacterial strain, designated SZ9T, was isolated from the effluent sample. Colonies of strain SZ9T were small (approximately 0.5-1.0 mm in diameter), creamy-white, circular, smooth, translucent and convex. Cells were facultative anaerobic, motile by means of a single polar flagellum, rod-shaped, multiplied by binary fission, Gram-stain-negative, oxidase-positive and catalase-negative. Growth occurred at 10-40 °C (optimum, 28 °C) and pH 5.5-8.0 (optimum, pH 7.5). The range of NaCl concentration for growth was 0-1.0 % (w/v), with an optimum of 0-0.5 % (w/v). Phylogenetic analysis based on 16S rRNA gene sequences suggested that strain SZ9T formed a lineage within the family Caulobacteraceae of the class Alphaproteobacteria and showed the highest 16S rRNA gene sequence similarities to Aquidulcibacter paucihalophilus TH1-2T (92.44%), followed by Vitreimonas flagellata SYSU XM001T (89.61 %), Asprobacter aquaticus DRW22-8T (89.49 %) and Hyphobacterium vulgare WM6T (89.49%). The predominant fatty acids (>10 % of the total fatty acids) of strain SZ9T was summed feature 3 (comprising C16 : 1 ω6c and/or C16 : 1 ω7c), summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) and C16 : 0. The sole respiratory quinone was ubiquinone-10, and the major polar lipids were phosphatidylcholine and two unidentified glycolipids. The whole genome of strain SZ9T was 2 842 140 bp in size, including 2769 protein-coding genes, 37 tRNA genes and two rRNA genes, and the genomic G+C content was 41.4 mol%. The orthologous average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain SZ9T and other genera within the family Caulobacteraceae were 64.50-66.62 %, 46.96-54.17 % and 27.70-31.70 %, respectively. Therefore, based on the results of phenotypic, chemotaxonomic and phylogenetic analyses, the isolated strain SZ9T could be distinguished from other genera, suggesting that it represents a novel species of a novel genus in the family Caulobacteraceae, for which the name Pseudaquidulcibacter saccharophilus gen. nov., sp. nov is proposed. The type strain is SZ9T (=CCTCC AB2021029T=KCTC 82788T).

Dissimilatory Nitrate Reduction to Ammonium (DNRA) and Denitrification Pathways Are Leveraged by Cyclic AMP Receptor Protein (CRP) Paralogues Based on Electron Donor/Acceptor Limitation in Shewanella loihica PV-4.

Under anoxic conditions, many bacteria, including Shewanella loihica strain PV-4, could use nitrate as an electron acceptor for dissimilatory nitrate reduction to ammonium (DNRA) and/or denitrification. Previous and current studies have shown that DNRA is favored under higher ambient carbon-to-nitrogen (C/N) ratios, whereas denitrification is upregulated under lower C/N ratios, which is consistent with our bioenergetics calculations. Interestingly, computational analyses indicate that the common cyclic AMP receptor protein (designated CRP1) and its paralogue CRP2 might both be involved in the regulation of two competing dissimilatory nitrate reduction pathways, DNRA and denitrification, in S. loihica PV-4 and several other denitrifying Shewanella species. To explore the regulatory mechanism underlying the dissimilatory nitrate reduction (DNR) pathways, nitrate reduction of a series of in-frame deletion mutants was analyzed under different C/N ratios. Deletion of crp1 could accelerate the reduction of nitrite to NO under both low and high C/N ratios. CRP1 is not required for denitrification and actually suppresses production of NO and N2O gases. Deletion of either of the NO-forming nitrite reductase genes nirK or crp2 blocked production of NO gas. Furthermore, real-time PCR and electrophoretic mobility shift assays (EMSAs) demonstrated that the transcription levels of DNRA-relevant genes such as nap-ß (napDABGH), nrfA, and cymA were upregulated by CRP1, while nirK transcription was dependent on CRP2. There are tradeoffs between the different physiological roles of nitrate/lactate, as nitrogen nutrient/carbon source and electron acceptor/donor and CRPs may leverage dissimilatory nitrate reduction pathways for maximizing energy yield and bacterial survival under ambient environmental conditions.IMPORTANCE Some microbes utilize different dissimilatory nitrate reduction (DNR) pathways, including DNR to ammonia (DNRA) and denitrification pathways, for anaerobic respiration in response to ambient carbon/nitrogen ratio changes. Large-scale industrial nitrogen fixation and fertilizer application raise the concern of emission of N2O, a stable gas with potent global warming potential, as consequence of microbial respiration, thereby aggravating global warming and climate change. However, little is known about the molecular mechanism underlying the choice of two competing DNR pathways. We demonstrate that the global regulator CRP1, which is widely encoded in bacteria, is required for DNRA in S. loihica PV-4 strain, while the CRP2 paralogue is required for transcription of the nitrite reductase gene nirK for denitrification. Sufficient carbon source lead to the predominance of DNRA, while carbon source/electron donor deficiency may result in an incomplete denitrification process, raising the concern of high levels of N2O emission from nitrate-rich and carbon source-poor waters and soils.

Tropical and temperate wastewater treatment plants assemble different and diverse microbiomes.

The diversity and assembly of activated sludge microbiomes play a key role in the performances of municipal wastewater treatment plants (WWTPs), which are the most widely applied biotechnological process systems. In this study, we investigated the microbiomes of municipal WWTPs in Bangkok, Wuhan, and Beijing that respectively represent tropical, subtropical, and temperate climate regions, and also explored how microbiomes assembled in these municipal WWTPs. Our results showed that the microbiomes from these municipal WWTPs were significantly different. The assembly of microbiomes in municipal WWTPs followed deterministic and stochastic processes governed by geographical location, temperature, and nutrients. We found that both taxonomic and phylogenetic α-diversities of tropical Bangkok municipal WWTPs were the highest and were rich in yet-to-be-identified microbial taxa. Nitrospirae and ß-Proteobacteria were more abundant in tropical municipal WWTPs, but did not result in better removal efficiencies of ammonium and total nitrogen. Overall, these results suggest that tropical and temperate municipal WWTPs harbored diverse and unique microbial resources, and the municipal WWTP microbiomes were assembled with different processes. Implications of these findings for designing and running tropical municipal WWTPs were discussed. KEY POINTS: • Six WWTPs of tropical Thailand and subtropical and temperate China were investigated. • Tropical Bangkok WWTPs had more diverse and yet-to-be-identified microbial taxa. • Microbiome assembly processes were associated with geographical location.

Azoarcus halotolerans sp. nov., a novel member of Rhodocyclaceae isolated from activated sludge collected in Hong Kong.

A floc-forming bacterial strain, designated HKLI-1T, was isolated from the activated sludge of a municipal sewage treatment plant in Hong Kong SAR, PR China. Cells of this strain were Gram-stain-negative, strictly aerobic, catalase- and oxidase-positive, rod-shaped and motile by means of a single polar flagellum. Growth occurred at 18-37 °C (optimum, 28 °C), pH 5.5-9.0 (optimum, pH 7.5) and with 0-8.0 % (w/v) NaCl (optimum, 1-1.5 %) concentration. The major fatty acids of strain HKLI-1T were C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and three unidentified lipids. The DNA G+C content was 63.5 mol% from whole genomic sequence analysis. Based on the results of 16S rRNA gene sequences analysis, this strain should be assigned to the genus Azoarcus and is closely related to Azoarcus olearius DQS-4T (94.93 % 16S rRNA gene sequence pairwise similarity), Azoarcus toluclasticus MF63T (94.91 %) and Azoarcus communis SWub3T (94.01 %), but separate from them by large distances in different phylogenetic trees. Based on whole genome analysis, the orthologous average nucleotide identity and in silico DNA-DNA hybridization values against four of the closest relatives were 73.03-74.83 and 17.2-23.0 %, respectively. The phylogenetic, genotypic, phenotypic and chemotaxonomic data demonstrated that strain HKLI-1T could be distinguished from its phylogenetically related species, and that this strain represented a novel species within the genus Azoarcus, for which the name Azoarcus halotolerans sp. nov. is proposed. The type strain is HKLI-1T (= 72659T=CCTCC AB 2019312T).

Differential gene content and gene expression for bacterial evolution and speciation of Shewanella in terms of biosynthesis of heme and heme-requiring proteins.

BACKGROUND: Most species of Shewanella harbor two ferrochelatase paralogues for the biosynthesis of c-type cytochromes, which are crucial for their respiratory versatility. In our previous study of the Shewanella loihica PV-4 strain, we found that the disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), but it is different in Shewanella oneidensis MR-1. Hence, the function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, are investigated in S. oneidensis MR-1. RESULT: In the present study, deletion of either hemH1 or hemH2 in S. oneidensis MR-1 did not lead to overproduction of extracellular protoporphyrin IX (PPIX) as previously described in the hemH1 mutants of S. loihica PV-4. Moreover, supplement of exogenous hemins made it possible to generate the hemH1 and hemH2 double mutant in MR-1, but not in PV-4. Under aerobic condition, exogenous hemins were required for the growth of MR-1ΔhemH1ΔhemH2, which also overproduced extracellular PPIX. These results suggest that heme is essential for aerobic growth of Shewanella species and MR-1 could also uptake hemin for biosynthesis of essential cytochrome(s) and respiration. Besides, the exogenous hemin mediated CymA cytochrome maturation and the cellular KatB catalase activity. Both hemH paralogues were transcribed in wild-type MR-1, and the hemH2 transcription was remarkably up-regulated in MR-1ΔhemH1 mutant to compensate for the loss of hemH1. The periplasmic glutathione peroxidase gene pgpD, located in the same operon with hemH2, and a large gene cluster coding for iron, heme (hemin) uptake systems are absent in the PV-4 genome. CONCLUSION: Our results indicate that the genetic divergence in gene content and gene expression between these Shewanella species, accounting for the phenotypic difference described here, might be due to their speciation and adaptation to the specific habitats (iron-rich deep-sea vent versus iron-poor freshwater) in which they evolved and the generated mutants could potentially be utilized for commercial production of PPIX.

Both widespread PEP-CTERM proteins and exopolysaccharides are required for floc formation of Zoogloea resiniphila and other activated sludge bacteria.

Bacterial floc formation plays a central role in the activated sludge (AS) process, which has been widely utilized for sewage and wastewater treatment. The formation of AS flocs has long been known to require exopolysaccharide biosynthesis. This study demonstrates an additional requirement for a PEP-CTERM protein in Zoogloea resiniphila, a dominant AS bacterium harboring a large exopolysaccharide biosynthesis gene cluster. Two members of a wide-spread family of high copy number-per-genome PEP-CTERM genes, transcriptionally regulated by the RpoN sigma factor and accessory PrsK-PrsR two-component system and at least one of these, pepA, must be expressed for Zoogloea to build the floc structures that allow gravitational sludge settling and recycling. Without PrsK or PrsR, Zoogloea cells were planktonic rather than flocculated and secreted exopolysaccharides were released into the growth broth in soluble form. Overexpression of PepA could circumvent the requirement of rpoN, prsK and prsR for the floc-forming phenotype by fixing the exopolysaccharides to bacterial cells. However, overexpression of PepA, which underwent post-translational modifications, could not rescue the long-rod morphology of the rpoN mutant. Consistently, PEP-CTERM genes and exopolysaccharide biosynthesis gene cluster are present in the genome of the floc-forming Nitrospira comammox and Mitsuaria strain as well as many other AS bacteria.

Bacterial Communities and Their Predicted Functions Explain the Sediment Nitrogen Changes Along with Submerged Macrophyte Restoration.

Submerged vegetation biomass fluctuation usually occurs during the preliminary stage of vegetation restoration in shallow lakes, which impacts the final status and duration for achieving a macrophyte-dominant state. This study uncovered the sediment N characteristics and the sediment bacterial community and their predicted functions during the preliminary stage of vegetation recovery in the West Lake, a typical subtropical degenerated shallow lake in China. Results showed increased amounts of sediment TN and NH4-N, reaching 3425.76 and 345.5 mg kg-1, respectively, when the vegetation biomass decreased from its maximum to its minimum. The maximum concentration of sediment NH4-N reached 508.60 mg kg-1 with the decline in vegetation, which might restrict further growth of the submerged macrophytes. The bacterial community structure during the high macrophyte biomass (HMB) period was distinct from that observed during the low macrophyte biomass (LMB) period. Specific taxa such as the phyla Chloroflexi and Acidobacteria and the genus Anaerolineaceae that are related to organic carbon degradation were significantly higher during the LMB period. Potential denitrifiers, such as Lactococcus and Bacillus genera decreased during the LMB period. Accumulation of sediment ammonia could be attributed to the enhanced production by assimilatory nitrate reduction, organic N degradation, and/or the decreased consumption by nitrification. Our findings highlight that the unstable preliminary stage of vegetation restoration brings drastic fluctuation of sediment N loading, of which NH4-N accumulation caused by bacterial communities prevents further growth of the submerged macrophytes. Therefore, extra management measures for the vegetation recovery areas should be taken to avoid excess NH4-N accumulation in sediments.

Effects of Planted Versus Naturally Growing Vallisneria natans on the Sediment Microbial Community in West Lake, China.

Submerged macrophytes play an important role in aquatic ecosystems, which has led to an increase in studies on vegetation recovery in polluted lakes from which submerged macrophytes have disappeared. The comparison of microbial communities in sediment cloned with planted and naturally growing submerged macrophytes is an interesting but rarely studied topic. In this investigation, Maojiabu and Xilihu, two adjacent sublakes of West Lake (Hangzhou, China), were selected as aquatic areas with planted and naturally growing macrophytes, respectively. Sediment samples from sites with/without Vallisneria natans were collected from both sublakes. The results showed that sediment total nitrogen and organic matter were significantly lower in the plant-covered sites than that in the non-plant sites in Maojiabu. Additionally, the sediment microbial community characterized by 16S ribosomal RNA (rRNA) sequencing differed more significantly for Maojiabu than for Xilihu. The relative abundances of microbes involved in C, N, and S elemental cycling were significantly higher in the sediments with plants than in those without. Results from both fatty acid methyl ester analysis and 16S rRNA sequencing indicated that vegetation significantly influenced the sulfate-reducing bacteria (SRB). Thus, the gene copies and composition of SRB were explored further. The relative gene abundance of SRB was 66% higher with natural vegetation colonization but was not influenced by artificial colonization. An increase in dominant SRB members from the families Syntrophobacteraceae and Thermodesulfovibrionaceae contributed to the increase of total SRB. Thus, macrophyte planting influences sediment nutrient levels and microbial community more than natural growth does, whereas the latter is more beneficial to sediment SRB.

Pre-meiotic deletion of PEX5 causes spermatogenesis failure and infertility in mice.

Peroxisomes are involved in the regulation of various pathological processes. Peroxisomal biogenesis factor 5 (PEX5), which plays an essential role in peroxisomal biogenesis, is critical for reactive oxygen species (ROS) accumulation. However, its underlying functions in spermatogenesis have not yet been identified. Pex5 was deleted by crossing Stra8-Cre mice with Pex5flox/flox mice before the onset of meiosis. The morphology of testes and epididymides, spermatogenesis function, and fertility in both wild type (WT) and Pex5-/- mice were analysed by haematoxylin and eosin (HE) and immunofluorescent staining. Mechanism of PEX5 affecting peroxisomes and spermatogenesis were validated by Western blot and transmission electron microscopy (TEM). Transcriptome RNA sequencing (RNA-seq) was used to profile the dysregulated genes in testes from WT and Pex5-/- mice on postnatal day (P) 35. The adult Pex5 knockout male mice were completely sterile with no mature sperm production. Loss of Pex5 in spermatocytes resulted in multinucleated giant cell formation, meiotic arrest, abnormal tubulin expression, and deformed acrosome formation. Furthermore, Pex5 deletion led to delayed DNA double-strand break repair and improper crossover at the pachytene stage. Impaired peroxisome function in Pex5 knockout mice induced ROS redundancy, which in turn led to an increase in germ cell apoptosis and a decline in autophagy. Pex5 regulates ROS during meiosis and is essential for spermatogenesis and male fertility in mice.

Environmental Contamination of SARS-CoV-2 Delta VOC by COVID-19 Patients Staying in the Hospital for More Than Two Weeks.

Background: Patients infected with SARS-CoV-2 Delta VOC have a longer course of disease. We detected the air, surfaces, and patient's personal items in the wards of the second hospital of Nanjing during the outbreak of the COVID-19 Delta Variant to identify the environmental contamination, which provides a theoretical basis for the prevention and control of COVID-19 variation beads in the future. Methods: In the cross-sectional study, we collected and analyzed clinical features, demographic and epidemiological data, laboratory and swab test results, and surface and air samples of 144 COVID-19 cases. Results: The time from symptom onset to surface sampling was 25 days (IQR, 21 to 33 days). Positive throat swabs were detected in 52(36.1%) patients, of which only 8(5.6%) patients had N or ORF1a/b genes Ct value <35 on the surface sampling day. Among the 692 environmental surface and air specimens collected from 144 COVID-19 cases, 3 specimens (3/692, 0.4%) related to 5 cases (3.5%, 5/144) were detected positive on RT-PCR. Overall, bedside tables (2/144, 1.4%) were most likely to be contaminated, followed by toilet seats (1/81, 1.2%). Conclusion: The environmental contamination by SARS-CoV-2 Delta VOC-infected cases with disease duration of more than two weeks is limited.

Conditional deletion of Hspa5 leads to spermatogenesis failure and male infertility in mice.

Heat shock proteins (HSPs) have important roles in different developmental stages of spermatogenesis. The heat shock 70 kDa protein 5 (HSPA5) is an important component of the unfolded protein response that promotes cell survival under endoplasmic reticulum (ER) stress conditions. In this study, we explored the function of HSPA5 in spermatogenesis, by generating a germ cell-specific deletion mutant of the Hspa5 gene (conditional knockout of the Hspa5 gene, Hspa5-cKO) using CRISPR/Cas9 technology and the Cre/Loxp system. Hspa5 knockout resulted in severe germ cell loss and vacuolar degeneration of seminiferous tubules, leading to complete arrest of spermatogenesis, testicular atrophy, and male infertility in adult mice. Furthermore, defects occurred in the spermatogenic epithelium of Hspa5-cKO mice as early as Cre recombinase expression. Germ cell ablation of Hspa5 impaired spermatogonia proliferation and differentiation from post-natal day 7 (P7) to P10, which led to a dramatic reduction of differentiated spermatogonia, compromised meiosis, and led to impairment of testis development and the disruption of the first wave of spermatogenesis. Consistent with these results, single-cell RNA sequencing (scRNA-seq) analysis showed that germ cells, especially differentiated spermatogonia, were dramatically reduced in Hspa5-cKO testes compared with controls at P10, further confirming that HSPA5 is crucial for germ cell development. These results suggest that HSPA5 is indispensable for normal spermatogenesis and male reproduction in mice.

Spatial distribution and influencing mechanism of CO2, N2O and CH4 in the Pearl River Estuary in summer.

Estuaries, considered as the important carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4) sources to the atmosphere, are increasingly affected by near-bottom hypoxia. However, the impact of estuarine hypoxic zone development on GHGs production and discharge remains poorly understood due to the seasonal and spatially distributed heterogeneity of estuarine hypoxia occurrence and the lack of simultaneous monitoring of the distribution of bottom hypoxic waters and the vertical distribution of GHGs. Here, we conducted high spatial resolution vertical stratification sampling and analysis of water column GHGs in the Pearl River Estuary (PRE), a large estuary with frequent hypoxia in recent years. Our results showed that Pearl River runoff is the main source of GHGs in the PRE. Strong nitrification is an important N2O production mechanism in the PRE. In situ generation of water and resuspension of surface sediments were the main sources of CH4 in bottom water, while massive organic matter (OM) mineralization is the main driver of CO2 in bottom water. The development of a hypoxic zone in the PRE significantly increased the concentration of N2O and CH4 in the bottom water and thus increased air-water fluxes. The air-water fluxes of N2O, CH4 and CO2 of PRE in summer were 31.9 ± 7.5 µmol m-2 d-1, 192.5 ± 229.4 µmol m-2 d-1 and 51.9 ± 14.1 mmol m-2 d-1, respectively. This study reveals that GHGs fluxes from estuarine waters to the atmosphere will increase significantly with increasing eutrophication caused by human activities and the expansion of hypoxic zones in estuarine waters.

Environmental sampling of the severe acute respiratory syndrome coronavirus 2 Delta variant in the inpatient wards of a hospital in Nanjing.

BACKGROUND: Little is known about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant of concern (VOC)-contaminated environmental surfaces and air in hospital wards admitting COVID-19 cases. Our study was designed to identify high-risk areas of Delta VOC contamination in the hospital and provide suggestions to in-hospital infection control. We analyzed the SARS-CoV-2 Delta VOC contamination in the air and environmental surface samples collected from a hospital in Nanjing, China. METHODS: We collected data on clinical features, laboratory tests, swab tests, and hospital wards, identified the factors associated with environmental contamination, and analyzed patients' hygiene behaviors during hospitalization. RESULTS: A total of 283 environmental surface and air samples were collected from a hospital admitting 36 COVID-19 patients. Twelve swab samples from ten patients were positive. Toilet seats had the highest contamination rate (11.8%), followed by bedside tables (8.2%), garbage bins (5.9%), and bedrails (1.6%). The median time of symptom onset to surface sampling was shorter in the positive environment group than in the negative environment group (11 vs. 18 days; P=0.001). The results indicated that environmental surface contamination was associated with positive anal swabs [odds ratio (OR) 27.183; 95% CI: 2.359-226.063; P=0.003] and the time from symptom onset to surface sampling (OR 0.801; 95% CI: 0.501-0.990; P=0.046). The survey revealed that 33.3% of the patients never cleaned or disinfected their bedside tables or toilets, and 8.3% of them only cleaned their bedside tables or toilets. More than half of the patients often (25%) or always (30.6%) put the used masks on their bedside tables. Only 16.7% of the patients threw the masks into the specific garbage bin for used masks. CONCLUSIONS: The SARS-CoV-2 Delta VOC was detected on environmental surfaces, especially toilet seats and bedside tables, within a median time of 11 days after symptom onset. Our study provided potential predictors for environmental surface contamination, including positive anal swabs and the time from symptom onset to sampling. Disinfecting high-risk environmental surfaces should be emphasized in hospital wards, especially for patients in the early stage of COVID-19.

Production of Highly Active Extracellular Amylase and Cellulase From Bacillus subtilis ZIM3 and a Recombinant Strain With a Potential Application in Tobacco Fermentation.

In this study, a series of bacteria capable of degrading starch and cellulose were isolated from the aging flue-cured tobacco leaves. Remarkably, there was a thermophilic bacterium, Bacillus subtilis ZIM3, that can simultaneously degrade both starch and cellulose at a wide range of temperature and pH values. Genome sequencing, comparative genomics analyses, and enzymatic activity assays showed that the ZIM3 strain expressed a variety of highly active plant biomass-degrading enzymes, such as the amylase AmyE1 and cellulase CelE1. The in vitro and PhoA-fusion assays indicated that these enzymes degrading complex plant biomass into fermentable sugars were secreted into ambient environment to function. Besides, the amylase and cellulase activities were further increased by three- to five-folds by using overexpression. Furthermore, a fermentation strategy was developed and the biodegradation efficiency of the starch and cellulose in the tobacco leaves were improved by 30-48%. These results reveal that B. subtilis ZIM3 and the recombinant strain exhibited high amylase and cellulase activities for efficient biodegradation of starch and cellulose in tobacco and could potentially be applied for industrial tobacco fermentation.

EPS solubilization treatment by applying the biosurfactant rhamnolipid to reduce clogging in constructed wetlands.

Application of extracellular polymeric substances (EPS) solubilization treatment with biosurfactant rhamnolipid (RL) to reduce clogging in constructed wetlands was first conducted in this study. The results showed significant improvement in the solubilization and dispersion of clogging matter following the treatment. And RL dosage of 0.09-0.15g/L altered microbial group make-up and had an overall positive effect on the growth of microorganisms. Moreover, RL was found to enhance EPS dissolution and dispersion, which was beneficial for the release of enzymes embedded in the EPS, and resulted in enhanced pollutant removal. The treatment had no apparent detrimental effect on wetland plants. Our results indicate that the optimum dosage of RL is 0.12g/L, and that the approach provides a promising and moderate option to reverse wetland clogging through RL-mediated solubilization treatment.