Dynamic changes in fungal communities and functions in different air-curing stages of cigar tobacco leaves.

Introduction: Air curing (AC) plays a crucial role in cigar tobacco leaf production. The AC environment is relatively mild, contributing to a diverse microbiome. Fungi are important components of the tobacco and environmental microbiota. However, our understanding of the composition and function of fungal communities in AC remains limited. Methods: In this study, changes in the chemical constituents and fungal community composition of cigar tobacco leaves during AC were evaluated using flow analysis and high-throughput sequencing. Results: The moisture, water-soluble sugar, starch, total nitrogen, and protein contents of tobacco leaves exhibited decreasing trends, whereas nicotine showed an initial increase, followed by a decline. As determined by high-throughput sequencing, fungal taxa differed among all stages of AC. Functional prediction showed that saprophytic fungi were the most prevalent type during the AC process and that the chemical composition of tobacco leaves is significantly correlated with saprophytic fungi. Conclusion: This study provides a deeper understanding of the dynamic changes in fungal communities during the AC process in cigar tobacco leaves and offers theoretical guidance for the application of microorganisms during the AC process.

Microbial community and chemical composition of cigar tobacco (Nicotiana tabacum L.) leaves altered by tobacco wildfire disease.

Tobacco wildfire disease caused by Pseudomonas syringae pv. tabaci is one of the most destructive foliar bacterial diseases occurring worldwide. However, the effect of wildfire disease on cigar tobacco leaves has not been clarified in detail. In this study, the differences in microbiota and chemical factors between wildfire disease-infected leaves and healthy leaves were characterized using high-throughput Illumina sequencing and a continuous-flow analytical system, respectively. The results demonstrated significant alterations in the structure of the phyllosphere microbial community in response to wildfire disease, and the infection of P. syringae pv. tabaci led to a decrease in bacterial richness and diversity. Furthermore, the content of nicotine, protein, total nitrogen, and Cl- in diseased leaves significantly increased by 47.86%, 17.46%, 20.08%, and 72.77% in comparison to healthy leaves, while the levels of total sugar and reducing sugar decreased by 59.59% and 70.0%, respectively. Notably, the wildfire disease had little effect on the content of starch and K+. Redundancy analysis revealed that Pseudomonas, Staphylococcus, Cladosporium, and Wallemia displayed positive correlations with nicotine, protein, total nitrogen, Cl- and K+ contents, while Pantoea, Erwinia, Sphingomonas, Terrisporobacter, Aspergillus, Alternaria, Sampaiozyma, and Didymella displayed positive correlations with total sugar and reducing sugar contents. Brevibacterium, Brachybacterium, and Janibacter were found to be enriched in diseased leaves, suggesting their potential role in disease suppression. Co-occurrence network analysis indicated that positive correlations were prevalent in microbial networks, and the bacterial network of healthy tobacco leaves exhibited greater complexity compared to diseased tobacco leaves. This study revealed the impact of wildfire disease on the microbial community and chemical compositions of tobacco leaves and provides new insights for the biological control of tobacco wildfire disease.

Fluorescent probes for lighting up ferroptotic cell death: A review.

Ferroptosis is a newly discovered form of regulated cellular demise, characterized by the accumulation of intracellular oxidative stress that is dependent on iron. Ferroptosis plays a crucial role not only in the development and treatment of tumors but also in the pathogenesis of neurodegenerative diseases and illnesses related to ischemia-reperfusion injury. This mode of cell death possesses distinctive properties that differentiate it from other forms of cell death, including unique morphological changes at both the cellular and subcellular levels, as well as molecular features that can be detected using specific methods. The use of fluorescent probes has become an invaluable means of detecting ferroptosis, owing to their high sensitivity, real-time in situ monitoring capabilities, and minimal damage to biological samples. This review comprehensively elucidates the physiological mechanisms underlying ferroptosis, while also detailing the development of fluorescent probes capable of detecting ferroptosis-related active species across various cellular compartments, including organelles, the nucleus, and the cell membrane. Additionally, the review explores how the dynamic changes and location of active species from different cellular compartments can influence the ignition and execution of ferroptotic cell death. Finally, we discuss the future challenges and opportunities for imaging ferroptosis. We believe that this review will not only aid in the elucidation of ferroptosis's physiological mechanisms but also facilitate the identification of novel treatment targets and means of accurately diagnosing and treating ferroptosis-related diseases.

Nickel-Catalyzed Amidation of Aryl Alkynyl Acids with Tetraalkylthiuram Disulfides: A Facile Synthesis of Aryl Alkynyl Amides.

Nickel-catalyzed amidation of aryl alkynyl acids using tetraalkylthiuram disulfides as the amine source is described, affording a series of aryl alkynyl amides in good to excellent yields under mild conditions. This general methodology provides an alternative pathway for the synthesis of useful aryl alkynyl amides in an operationally simple manner, which shows its practical synthetic value in organic synthesis. The mechanism of this transformation was explored through control experiments and DFT calculations.

Preparation of esterified biomass waste hydrogels and their removal of Pb2+, Cu2+ and Cd2+ from aqueous solution.

The treatment of polluted water is a serious environmental problem in the world. Biomass is easily modified and can be prepared into adsorbent materials, which is expected to solve the problem of heavy metal ion adsorption in sewage. In this paper, esterified tobacco straw based hydrogels (ETS-PAA) were synthesized from waste tobacco straw biomass. The structure and thermal stability of these hydrogels were characterized by FTIR, SEM, EDS, XPS and TG. The adsorption of metal ions by the hydrogel was measured by ICP-MS. The effects of initial ion concentration, adsorption time, pH, and temperature on the heavy metal adsorption were investigated. The results showed that ETS-PAA possessed more pores, which led to a better adsorption capacity. The maximum adsorption amounts of Pb2+, Cu2+ and Cd2+ were 2.41 mmol·g-1, 1.93 mmol·g-1 and 1.77 mmol·g-1, respectively. Finally, the adsorption mechanism and kinetics were analyzed. The adsorption was mainly accomplished by ion exchange of -COOK on the monomer chain with heavy metal ions, coordination of -OH and -CONH with heavy metal ions and interaction of ester bond, -COOH with heavy metal ions. The adsorption process was in accordance with the pseudo-second-order kinetic model and Freundlich model. The adsorption process belonged to multilayer chemisorption. This work shows that ETS-PAA was a promising material for the removal of heavy metal pollutants from aqueous solution.

The bacterial succession and its role in flavor compounds formation during the fermentation of cigar tobacco leaves.

Fermentation is the key process required for developing the characteristic properties of cigar tobacco leaves, complex microorganisms are involved in this process. However, the microbial fermentation mechanisms during the fermentation process have not been well-characterized. This study investigated the dynamic changes in conventional chemical composition, flavor compounds, and bacterial community during the fermentation of cigar tobacco leaves from Hainan and Sichuan provinces in China, as well as the potential roles of bacteria. Fermentation resulted in a reduction of conventional chemical components in tobacco leaves, with the exception of a noteworthy increase in insoluble protein content. Furthermore, the levels of 10 organic acids and 19 amino acids showed a significant decrease, whereas the concentration of 30 aromatic substances exhibited a unimodal trend. Before fermentation, the bacterial community structures and dominant bacteria in Hainan and Sichuan tobacco leaves differed significantly. As fermentation progressed, the community structures in the two regions became relatively similar, with Delftia, Ochrobactrum, Rhodococcus, and Stenotrophomonas being dominant. Furthermore, a total of 12 functional bacterial genera were identified in Hainan and Sichuan tobacco leaves using bidirectional orthogonal partial least squares (O2PLS) analysis. Delftia, Ochrobactrum, and Rhodococcus demonstrated a significant negative correlation with oleic acid and linoleic acid, while Stenotrophomonas and Delftia showed a significant negative correlation with undesirable amino acids, such as Ala and Glu. In addition, Bacillus showed a positive correlation with benzaldehyde, while Kocuria displayed a positive correlation with 2-acetylfuran, isophorone, 2, 6-nonadienal, and ß-damascenone. The co-occurrence network analysis of microorganisms revealed a prevalence of positive correlations within the bacterial network, with non-abundant bacteria potentially contributing to the stabilization of the bacterial community. These findings can improve the overall tobacco quality and provide a novel perspective on the utilization of microorganisms in the fermentation of cigar tobacco leaves.

Preparation of biomass-based hydrogels and their efficient heavy metal removal from aqueous solution.

In this work, a porous tobacco straw-based polyacrylic acid hydrogel STS-PAA with high adsorption performance was prepared by polymerizing pretreated waste tobacco straw (TS) with acrylic acid/potassium acrylate by UV radiation initiation. The adsorption performance of metal ions was investigated. The effects of different temperatures (25°C, 35°C, and 45°C), adsorption times (1-420 min), pH values (2.0-6.0) and initial concentrations (0.25-4.0 mmol L-1) of metal ions on the adsorption amount of heavy metal ions were investigated. The results showed that the hydrogel had a high removal rate of Pb2+, Cd2+ and Hg2+ in aqueous solution. The adsorption of Pb2+ was particularly effective. When C0 = 4.0 mmol L-1, pH = 6, the equilibrium adsorption amount of Pb2+, Cd2+ and Hg2+ reached 1.49 mmol g-1, 1.02 mmol L-1 and 0.94 mmol g-1, respectively. The chemical structure and morphology of the hydrogels were characterized by FT-IR, EDS, SEM and XPS. The Langmuir model fits well with the adsorption system. The kinetic data suggest the adsorption of Pb2+, Cd2+ and Hg2+ follow the pseudo-first-order model. This indicates that STS-PAA adsorption of three heavy metal ions is monolayer physical adsorption. Thermodynamic analysis shows that the adsorption of Pb2+, Cd2+ and Hg2+ by STS-PAA is an endothermic (ΔH>0) entropy increase (ΔS>0) non-spontaneous reaction.

Influence of Genotypic and Environmental Factors on Tobacco Leaves Based on Metabolomics.

Environmental factors affect plant metabolites, different climates, cultivation conditions, and biotic stresses and genotypes strongly affect their chemical composition and contents. Our aim is to examine the environmental and genetic interaction effects on tobacco metabolite composition. UPLC-QTOF MS/MS coupled with multivariate data analyses were applied for the metabolomics analysis of three tobacco cultivars from different planting regions in China. Principal component analysis (PCA) revealed that environmental factors have a greater effect on tobacco metabolism compared to genotypes. Twelve biomarkers were screened by orthogonal partial least squares discrimination analysis (OPLS-DA). Univariate analysis indicated that Malate, conjugated chlorogenic acid, chlorogenic acid, quercetin 3-rutinoside-7-glucoside, and unknown compound 5 were only influenced by environmental factors (independent of genotype). Quinate, neochlorogenic acid, and ouabagenin, taxezopidine K1, taxezopidine K2, and taxezopidine K3 in tobacco were influenced by the interaction of environmental factors and the genotype. Our results suggest that metabolomics based on UPLC-QTOF MS/MS could be used to analyze the ecological functions of biomarker metabolites and understand the mechanisms of plant adaption to the environment.

Adenosine triphosphate (ATP) and zinc(II) ions responsive pyrene based turn-on fluorescent probe and its application in live cell imaging.

A novel highly selective and sensitive turn-on fluorescent chemosensor PCE to recognize Zn2+ has been developed. The sensor PCE displays a remarkable fluorescent enhancement at 456 nm (λex = 340 nm) with Zn2+ without the interference of other biologically important relevant metal ions in aqueous acetonitrile solution. Job's plot and mass spectral studies divulge such the interaction of PCE by Zn2+ was 1:1 binding stoichiometry. The association constant and detection limit of PCE to recognize Zn2+ was found to be 0.948 × 104 M-1 and 4.82 × 10-7 M respectively. The nature of turn-on fluorescence sensor was supported by TD-DFT calculations. And the synthesized probe PCE was able to image intracellular Zn2+ in living cells using confocal imaging techniques. PCE-Zn ensemble showed the remarkable fluorescence enhancement with ATP selectively among other biologically important phosphates. 31P NMR experiments suggesting that the triphosphates unit of ATP is intact with the PCEZn. PCE-Zn ensemble can be utilized for monitoring ATP in live cells.

Microbial community and metabolic function analysis of cigar tobacco leaves during fermentation.

Cigar tobacco leaves (CTLs) contain abundant bacteria and fungi that are vital to leaf quality during fermentation. In this study, artificial fermentation was used for the fermentation of CTLs since it was more controllable and efficient than natural aging. The bacterial and fungal community structure and composition in unfermented and fermented CTLs were determined to understand the effects of microbes on the characteristics of CTLs during artificial fermentation. The relationship between the chemical contents and alterations in the microbial composition was evaluated, and the functions of bacteria and fungi in fermented CTLs were predicted to determine the possible metabolic pathways. After artificial fermentation, the bacterial and fungal community structure significantly changed in CTLs. The total nitrate and nicotine contents were most readily affected by the bacterial and fungal communities, respectively. FAPROTAX software predictions of the bacterial community revealed increases in functions related to compound transformation after fermentation. FUNGuild predictions of the fungal community revealed an increase in the content of saprotrophic fungi after fermentation. These data provide information regarding the artificial fermentation mechanism of CTLs and will inform safety and quality improvements.

Transcriptome divergence between developmental senescence and premature senescence in Nicotiana tabacum L.

Senescence is a degenerative process triggered by intricate and coordinated regulatory networks, and the mechanisms of age-dependent senescence and stress-induced premature senescence still remain largely elusive. Thus we selected leaf samples of developmental senescence (DS) and premature senescence (PS) to reveal the regulatory divergence. Senescent leaves were confirmed by yellowing symptom and physiological measurement. A total of 1171 and 309 genes (DEGs) were significantly expressed respectively in the whole process of DS and PS. Up-regulated DEGs in PS were mostly related to ion transport, while the down-regulated DEGs were mainly associated with oxidoreductase activity and sesquiterpenoid and triterpenoid biosynthesis. In DS, photosynthesis, precursor metabolites and energy, protein processing in endoplasmic reticulum, flavonoid biosynthesis were notable. Moreover, we found the vital pathways shared by DS and PS, of which the DEGs were analyzed further via protein-protein interaction (PPI) network analysis to explore the alteration responding to two types of senescence. In addition, plant hormone transduction pathway was mapped by related DEGs, suggesting that ABA and ethylene signaling played pivotal roles in formulating the distinction of DS and PS. Finally, we conducted a model containing oxidative stress and ABA signaling as two hub points, which highlighted the major difference and predicted the possible mechanism under DS and PS. This work gained new insight into molecular divergence of developmental senescence and premature senescence and would provide reference on potential mechanism initiating and motivating senescence for further study.

Biosorption of sterols from tobacco waste extract using living and dead of newly isolated fungus Aspergillus fumigatus strain LSD-1.

Sterols are verified to be able to produce polycyclic aromatic hydrocarbons during its pyrolysis. In this study, a kind of Aspergillus fumigatus (LSD-1) was isolated from cigar leaves, and the biosorption effects on the stigmasterol, ß-sitosterol, campesterol, cholesterol, and ergosterol by using living and dead biomass of LSD-1 were investigated. The results showed that both living and dead biomass could efficiently remove these sterols in aqueous solution and tobacco waste extract (TWE). Interestingly, compared with the living biomass of LSD-1, the dead biomass of LSD-1 not only kept a high adsorption efficiency but also did not produce ergosterol. Overall, dead biomass of LSD-1 was a more suitable biosorbent to sterols in TWE. Furthermore, Brunner-Emmet-Teller (BET), Fourier transformed infrared spectrometer (FTIR) and scanning electron microscope (SEM) analysis were used to explore the biosorption process of living and dead biomass and their differences, suggesting that the biosorption of sterols was a physical process.

Gas Chromatography-Mass Spectrometry Method for Simultaneous Detection of Nine Alkaloids in Tobacco and Tobacco Products by QuEChERS Sample Preparation.

One method based on QuEChERS sample preparation is presented in this study, which leads to simultaneously detect nine alkaloids in tobacco and tobacco products. Nicotine, nornicotine, myosmine, N-methyl anabasine, ß-nicotyrine, anabasine, anatabine, isonicotenine and cotinine can all be found in fresh tobacco leaves, cigars, Virginia-type and blended-type cigarettes. The samples were purified via a certain proportion of adsorbents consisting of anhydrous magnesium sulfate, PSA and carbon after extracting, then centrifuged and filtered before analyzing by GC-MS. The matrix effects were all among 88 - 105%. The limit of detection of all were within the range of 0.0065 - 0.1509 µg/g and limit of quantification were among 0.0217 - 0.5031 µg/g. The recovery rates were higher than 89%. This is the first time that the QuEChERS sample preparation method has been applied for tobacco alkaloids, where more varieties of alkaloids could be quantified regarding sensitivity and reproducibility.

Transcriptome Analysis of Gene Expression Patterns Potentially Associated with Premature Senescence in Nicotiana tabacum L.

Senescence affects the remobilization of nutrients and adaption of the plant to the environment. Combined stresses can result in premature senescence in plants which exist in the field. In this study, transcriptomic analysis was performed on mature leaves and leaves in three stages of premature senescence to understand the molecular mechanism. With progressive premature senescence, a declining chlorophyll (chl) content and an increasing malonaldehyde (MDA) content were observed, while plasmolysis and cell nucleus pyknosis occurred, mitochondria melted, thylakoid lamellae were dilated, starch grains in chloroplast decreased, and osmiophilic granules increased gradually. Moreover, in total 69 common differentially expressed genes (DEGs) in three stages of premature senescing leaves were found, which were significantly enriched in summarized Gene Ontology (GO) terms of membrane-bounded organelle, regulation of cellular component synthesis and metabolic and biosynthetic processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the plant hormone signal transduction pathway was significantly enriched. The common DEGs and four senescence-related pathways, including plant hormone signal transduction, porphyrin and chlorophyll metabolism, carotenoid biosynthesis, and regulation of autophagy were selected to be discussed further. This work aimed to provide potential genes signaling and modulating premature senescence as well as the possible dynamic network of gene expression patterns for further study.

Purification, Characterization, and Antioxidant Activity of Polysaccharides Isolated from Cortex Periplocae.

In this study, crude Cortex Periplocae polysaccharides (CCPPs) were extracted with water. CCPPs were decolored with AB-8 resin and deproteinated using papain-Sevage methods. Then, they were further purified and separated through DEAE-52 anion exchange chromatography and Sephadex G-100 gel filtration chromatography, respectively. Three main fractions-CPP1, CPP2, and CPP3, (CPPs)-were obtained. The average molecular weights, monosaccharide analysis, surface morphology, and chemical compositions of the CPPs were investigated by high-performance gel permeation chromatography (HPGPC), gas chromatography-mass spectrometry (GC/MS), UV-vis spectroscopy, Fourier transform infrared (FT-IR) spectrum, and nuclear magnetic resonance (NMR). In addition, the antioxidant activities of these three polysaccharides were investigated. The results indicated that all of the CPPs were composed of rhamnose, arabinose, mannose, glucose, and galactose. These three polysaccharides exhibited antioxidant activities in four assays including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) radical, reducing power, and total antioxidant activity in vitro. The data indicated that these three polysaccharides could be utilized as potential natural sources of alternative additives in the functional food, cosmetics, and pharmaceutical industries.

Synthesis of graphene/DPA composite for determination of nicotine in tobacco products.

In this contribution, the azo dye (E)-1-(4-((4-(phenylamino)phenyl)diazenyl) phenyl)ethanone (DPA) was combined with reduced graphene oxide (RGO) for the electrochemical modification of a pencil graphite electrode (RGO/DPA/PGE) surface. A series of electrochemical measurements were used for the characterization of the modified electrode surfaces. At the modified electrode, nicotine was irreversibly reduced. An obvious increase was observed in the reductive peak current of nicotine at the modified electrode, indicating the capability of the RGO/DPA composite to increase the electron transfer rate. The current was found proportional to the nicotine concentration in a range of 31 to 1900 µM, and the limit of detection (LOD) was calculated as 7.6 µM.

The influence of light quality on the accumulation of flavonoids in tobacco (Nicotiana tabacum L.) leaves.

Flavonoids are important secondary metabolites in plants regulated by the environment. To analyze the effect of light quality on the accumulation of flavonoids, we performed a rapid analysis of flavonoids in extracts of tobacco leaves using UHPLC-QTOF. A total of 12 flavonoids were detected and identified in tobacco leaves, which were classified into flavonoid methyl derivatives and flavonoid glycoside derivatives according to the groups linked to the flavonoid core. Correlation analysis was further conducted to investigate the effect of different wavelengths of light on their accumulation. The content of flavonoid methyl derivatives was positively correlated with the proportions of far-red light (FR; 716-810nm) and near-infrared light (NIR; 810-2200nm) in the sunlight spectrum and negatively correlated with the proportion of ultraviolet (UV-A; 350-400nm) and the red/far-red ratio (R/FR). By contrast, the content of flavonoid glycoside derivatives was positively correlated with the proportion of UV-A and the R/FR, and negatively correlated with FR and NIR. The results indicated that light quality with higher proportions of FR and NIR increases the activity of flavonoid methyltransferases but suppresses the activity of flavonoid glycoside transferases. While a high proportion of UV-A and a high R/FR can increase flavonoid glycoside transferase activity but suppress flavonoid methyltransferase activity.

[Pyrolysates of novel latent fragrant compound 3,6-dimethyl-2,5-pyrazinedicarboxylic acid menthol ester].

In order to develop a new tobacco flavor released at high-temperature, the novel latent fragrant compound 3,6-dimethyl-2,5-pyrazinedicarboxylic acid menthol ester (DPAME) was synthesized by esterification using 2,3,5,6-tetramethylpyrazine and menthol as raw materials. In air atmosphere, the pyrolysis behavior of DPAME was investigated using an on-line pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) method at three temperature levels of 300, 600 and 900 degrees C, separately. The pyrolysis products were directly introduced into GC-MS and were qualitatively and semi-quantitatively analyzed. The results showed that a variety of aroma compounds of aldehydes, 3-p-menthene and menthol were released and identified at 300 degrees C. While at 600 degrees C and 900 degrees C, flavor alkene class, the alkyl pyrazines, menthol and 3-p-menthene were generated. And the types and relative amounts of pyrazines were significantly increased, at these two temperatures. Combined the analytical results of DPAME pyrolysates and the results of sensory evaluation of the cigarette, the possible pyrolysis mechanism was preliminarily speculated. The Py-GC-MS technique for the study of the pyrolysis products of DPAME was convenient and rapid. The investigation provided a reliable theoretical foundation for the perfume reinforcement technology in tobacco products, contributing to the development of cigarette products with better aroma and taste. This method is an accurate and quick way to study the pyrolysis products of latent fragrant substance.

[Identification of dominant and fragrance-enhancing microorganisms of tobacco leaves during ripening].

OBJECTIVE: We screened dominating microbial species isolated from aging flue-cured tobacco and studied their aroma improving effect. METHODS: Total DNA of microorganisms from the fermentation flue-cured tobacco surface of NC89, ZhongYan 100 and ZhongYan 101 were extracted. Under the PCR-DGGE, the diversity of microorganisms on fermentation tobacco leaves were studied and dominating microbial species were screened. We further studied the influence of dominating microbial species on the content of aroma components of the fermentation flue-cured tobacco. RESULTS: 1) By using DGGE analysis, there were 5 dominant bands A, B, C, D and E in all tobacco leaves samples of the three varieties; In further studies, five dominant DGGE bands were isolated, cloned and sequenced. From them we screened a dominant microorganism. 2) The content of most aroma components in tobacco leaves increased when they were sprayed with the dominant microorganism, comparing with the control. CONCLUSION: The dominant microorganism can improve the flavor of tobacco leaves during ripening.

Analysis of bacterial communities on aging flue-cured tobacco leaves by 16S rDNA PCR-DGGE technology.

Many microorganisms, growing on aging flue-cured tobacco leaves, play a part in its fermentation process. These microflora were identified and described by culture-dependent methods earlier. In this study we report the identity of the microflora growing on the tobacco leaf surface by employing culture-independent methods. We have amplified microbial 16S rDNA sequences directly from the leaf surface and used denaturing gradient gel electrophoresis (DGGE) to identify bacterial community on the tobacco leaves. Our culture-independent methods for the study of microbial community on tobacco leaves showed that microbial community structures on leaves of variety Zhongyan 100, NC89 and Zhongyan 101 were similar between 0 and 6 months aging, and between 9 and 12 months aging, while the similarity is low between 0 and 6, and between 9 and 12 months aging, respectively. There were certain similarities of bacterial communities (similarity up to 63%) among the three tobacco varieties for 0 to 6 months aging. Five dominant 16S rDNA DGGE bands A, B, C, D and E were isolated, cloned, and sequenced. They were most similar to two cultured microbial species Bacteriovorax sp. EPC3, Bacillus megaterium, and three uncultured microbial species, respectively.