Microbial Degradation Systems Research Articles

Integrative omics analyses of the ligninolytic Rhodosporidium fluviale LM-2 disclose catabolic pathways for biobased chemical production

BackgroundLignin is an attractive alternative for producing biobased chemicals. It is the second major component of the plant cell wall and is an abundant natural source of aromatic compounds. Lignin degradation using microbial oxidative enzymes that depolymerize lignin and catabolize aromatic compounds into central metabolic intermediates is a promising strategy for lignin valorization. However, the intrinsic heterogeneity and recalcitrance of lignin severely hinder its biocatalytic conversion. In this context, examining microbial degradation systems can provide a fundamental understanding of the pathways and enzymes that are useful for lignin conversion into biotechnologically relevant compounds.ResultsLignin-degrading catabolism of a novel Rhodosporidium fluviale strain LM-2 was characterized using multi-omic strategies. This strain was previously isolated from a ligninolytic microbial consortium and presents a set of enzymes related to lignin depolymerization and aromatic compound catabolism. Furthermore, two catabolic routes for producing 4-vinyl guaiacol and vanillin were identified in R. fluviale LM-2.ConclusionsThe multi-omic analysis of R. fluviale LM-2, the first for this species, elucidated a repertoire of genes, transcripts, and secreted proteins involved in lignin degradation. This study expands the understanding of ligninolytic metabolism in a non-conventional yeast, which has the potential for future genetic manipulation. Moreover, this work unveiled critical pathways and enzymes that can be exported to other systems, including model organisms, for lignin valorization.

Molecular mechanism of zero valent iron-enhanced microbial azo reduction

Zero valent iron (ZVI)–microbe technology has an increasing application on the removal of organic pollution, yet the molecular mechanism of microbe respond to ZVI is still a mystery. Here, we established a successive ZVI-enhanced microbial system to remove azo dye (a typical organic pollutant) by Shewanella decolorationis S12 (S. decolorationis S12, an effective azo dye degradation bacterium) and examined the gene expression time course (10, 30, 60, and 120 min) by whole genome transcriptional analysis. The addition of ZVI to the microbial degradation system increases the rate of azo reduction from ~60% to over 99% in 16 h reaction, suggesting the synergistic effect of ZVI and S12 on azo dye degradation. Comparing with the treatment without ZVI, less filamentous cells were observed in ZVI treated system, and approximately 8% genes affiliated with 10 different gene expression profiles in S. decolorationis S12 were significantly changed in 120 min during the ZVI-enhanced azo reduction. Intriguingly, MarR transcriptional factor might play a vital role in regulating ZVI-enhanced azo reduction in the aspect of energy production, iron homeostasis, and detoxification. Further investigation showed that the induced [Ni–Fe] H2ase genes (hyaABCDEF) and azoreductase genes (mtrABC-omcA) contributed to ZVI-enhanced energy production, while the reduced iron uptake (hmuVCB and feoAB), induced sulfate assimilation (cysPTWA) and cysteine biosynthesis (cysM) related genes were essential to iron homeostasis and detoxification. This study disentangles underlying mechanisms of ZVI-enhanced organic pollution biotreatment in S. decolorationis S12.

Combination of zeolite barrier and bio sparging techniques to enhance efficiency of organic hydrocarbon remediation in a model of shallow groundwater

Adsorption and bioremediation are effective processes for remediation of benzene, toluene, and ethylbenzene (BTE) through Permeable Reactive Barriers (PRBs). A few researches focus on adsorption of natural zeolite because of its hydrophilic property. On the other hand, PRBs need to be replaced by fresh materials after a while when all the possible absorption positions were filled up. We tried to find a way to increase the efficiency of PRB, elongation of its replacement period and of course decreasing the cost of remediation. Equipping of PRB with microbial degradation system was the idea. The present study describes the performances of natural Clinoptilolite-Heulandite Zeolite (CH-Z) and three new strains (safe and low-cost media) utilized in a PRB for removing BTE from contaminated shallow groundwater. First, batch tests were conducted to recognize the optimal removal conditions for utilization of C-HZ and strains to remediate BTE compounds. Then, an aerobic PRB system filled with a natural zeolite was designed and investigated in a continuous flow sand-tank model to assess the efficiency of combined PRBs (zeolite + biosparging), for BTE-contaminated groundwater. Batch experiments showed that the BTE removal of zeolite was 89%, as well as, a consortium of three bacterial strains, Variovorax sp. OT16, Pseudomonas balearica OT17, and Ornithinibacillus sp. OT18 efficiently removed the BTE mixture. The process of BTE removal in the PRB under continuous-flow condition was divided into three phases: Phase I, in which the barrier was made of the only zeolite, and in Phases II and III the reactor was fed by microorganisms. This experiment revealed that in Phases I, the concentrations of BTE decrease (92%) due to zeolite adsorption. In Phase II and III, the degradation process became the principal removal mechanism (68% and 81%, respectively). Consequently, this research showed high ability of C-HZ in the BTE treatment, and a combination of Natural Zeolite, with a biological degradation system (CH-Z –PRB) improves the efficiency of BTE remediation. However, the slow biodegradation rates and the continuous injection of BTE in the model confirmed that longer time was needed for the PRB to function optimally. Finally, the combined method of CH-Z- BIO PRB showed the great potential in the restriction of the BTE migration that can be used at the field-scale after up-scaling.

Structural analysis of β‐L‐arabinobiose‐binding protein in the metabolic pathway of hydroxyproline‐rich glycoproteins in Bifidobacterium longum

Bifidobacterium longum is a symbiotic human gut bacterium that has a degradation system for β-arabinooligosaccharides, which are present in the hydroxyproline-rich glycoproteins of edible plants. Whereas microbial degradation systems for α-linked arabinofuranosyl carbohydrates have been extensively studied, little is understood about the degradation systems targeting β-linked arabinofuranosyl carbohydrates. We functionally and structurally analyzed a substrate-binding protein (SBP) of a putative ABC transporter (BLLJ_0208) in the β-arabinooligosaccharide degradation system. Thermal shift assays and isothermal titration calorimetry revealed that the SBP specifically bound Araf-β1,2-Araf (β-Ara2 ) with a Kd of 0.150μm, but did not bind L-arabinose or methyl-β-Ara2 . Therefore, the SBP was termed β-arabinobiose-binding protein (BABP). Crystal structures of BABP complexed with β-Ara2 were determined at resolutions of up to 1.78Å. The findings showed that β-Ara2 was bound to BABP within a short tunnel between two lobes as an α-anomeric form at its reducing end. BABP forms extensive interactions with β-Ara2 , and its binding mode was unique among SBPs. A molecular dynamics simulation revealed that the closed conformation of substrate-bound BABP is stable, whereas substrate-free form can adopt a fully open and two distinct semi-open states. The importer system specific for β-Ara2 may contribute to microbial survival in biological niches with limited amounts of digestible carbohydrates. DATABASE: Atomic coordinates and structure factors (codes 6LCE and 6LCF) have been deposited in the Protein Data Bank (http://wwpdb.org/).

Compatibility between weak gel and microorganisms in weak gel-assisted microbial enhanced oil recovery

To investigate weak gel-assisted microbial flooding in Block Wang Long Zhuang in the Jiangsu Oilfield, the compatibility of weak gel and microbe was evaluated using laboratory experiments. Bacillus sp. W5 was isolated from the formation water in Block Wang Long Zhuang. The rate of oil degradation reached 178mg/day, and the rate of viscosity reduction reached 75.3%. Strain W5 could produce lipopeptide with a yield of 1254mg/L. Emulsified crude oil was dispersed in the microbial degradation system, and the average diameter of the emulsified oil particles was 18.54μm. Bacillus sp. W5 did not affect the rheological properties of the weak gel, and the presence of the weak gel did not significantly affect bacterial reproduction (as indicated by an unchanged microbial biomass), emulsification (surface tension is 35.56mN/m and average oil particles size is 21.38μm), oil degradation (162mg/day) and oil viscosity reduction (72.7%). Core-flooding experiments indicated oil recovery of 23.6% when both weak gel and Bacillus sp. W5 were injected into the system, 14.76% when only the weak gel was injected, and 9.78% with strain W5 was injected without the weak gel. The results demonstrate good compatibility between strains W5 and the weak gel and highlight the application potential of weak gel-assisted microbial flooding.

Simultaneous Determination of β-Cypermethrin and Its Metabolite 3-Phenoxybenzoic Acid in Microbial Degradation Systems by HPLC-UV.

The wide use of pesticides in agriculture is necessary to guarantee adequate food production worldwide. However, pesticide residues have caused global concern because of their potential health risk to consumers. In this study, we could identify β-cypermethrin (β-CY) and its degradation product 3-phenoxybenzoic acid (3-PBA) by liquid chromatograph-mass spectrometry. Few studies on the simultaneous determination of β-CY and its metabolites have been carried out so far; hence, we established a high-performance liquid chromatography method to determine the concentrations of both β-CY and 3-PBA simultaneously in microbial degradation systems. In this study, a novel β-CY degrading strain, Bacillus licheniformis B-1, was isolated from a tea garden soil, utilizing β-CY as a growth substrate. Good linear relationships between β-CY and 3-PBA were observed and the concentrations of reference solutions were between 0.50 and 60.00 µg/mL. Satisfactory stability and intra- and interday precision were obtained. The limits of detection were 0.06 and 0.13 µg/mL for β-CY and 3-PBA, respectively, and the corresponding limits of quantification were 0.21 and 0.34 µg/mL, respectively. Spiking recoveries for β-CY varied from 98.38 to 105.80%, with relative standard deviations (RSDs) varying from 1.49 to 3.93%. Spiking recoveries for 3-PBA varied from 99.59 to 101.20%, with RSDs varying from 0.58 to 3.64%. The proposed method has advantages of simplicity, rapidity, high accuracy, good separation and reproducibility; thus, it is ideally suitable for simultaneous determination of β-CY and 3-PBA in microbial degradation systems.

Separation of cis- and trans-cypermethrin by reversed-phase high-performance liquid chromatography.

As a pyrethroid insecticide, cypermethrin (CP) possesses three chiral centers and thus consists of eight optical isomers. High-performance liquid chromatography (HPLC) methods have been established for the separation and determination of pyrethroid insecticides. In this article, we report a new reversed-phase high-performance liquid chromatography (RP-HPLC) method of separating the diastereomers of CP. Our method was performed on a C18 column with the following specifications: mobile phase, methanol/acetonitrile/water (58:18:24, v/v/v); flow rate, 1.0 mL/min; column temperature, 20°C and UV-detection wavelength, 235 nm. The results showed that the applied procedure was linear based on calibration curves with equation (y = 16.595x + 1.7892) within the CP concentration range of 5-100 mg/L. The limit of detection and limit of quantification of CP were 0.2 and 0.7 mg/L, respectively. CP or β-CP extracted from the microbial degradation system was determined, and the method was proved to be accurate and applicable. Therefore, the proposed RP-HPLC method was simple, inexpensive and can be used to study stability and determine CP residues in pharmaceutical preparations or in the environment.

A Pretreatment Method for HPLC Analysis of Cypermethrin in Microbial Degradation Systems

In this paper, a pretreatment method for high-performance liquid chromatography (HPLC) determination of cypermethrin (CY) in microbial degradation systems was systemically studied, primarily to solve the problem of inaccurate determination of CY concentration caused by its uneven distribution in the systems. A suitable pretreatment method was established, including sampling, extraction and dehydration of CY. Partial sampling could be taken for bacterial and yeast systems in which CY was uniformly dispersed by an emulsifying agent, while total sampling was only suitable for mold systems with or without an emulsifying agent. CY could be fully extracted from the samples in which microbial cells were disrupted by ultrasonic treatment with acetonitrile under ultrasonic condition. The extract could be effectively dehydrated and purified by passing it through an anhydrous Na(2)SO(4) column followed by an elution with acetonitrile. The determination of CY in the pretreated sample by HPLC showed a high precision [relative standard deviation (RSD) = 1.14%, n = 5] and a good stability over a period of five days (RSD = 1.57%, n = 5). The recoveries of CY in microbial degradation systems at three different spiked levels ranged from 95.68 to 108.09% (RSD = 0.50-5.87%, n = 5).

Employment of the human estrogen receptor beta ligand-binding domain and co-activator SRC1 nuclear receptor-binding domain for the construction of a yeast two-hybrid detection system for endocrine disrupters.

To screen a wide variety of chemicals for endocrine disrupters, and to develop an effective microbial degradation system for them, a good system is needed for the rapid and accurate evaluation of the endocrine-disrupting activities of suspected chemicals and their degradation products. We constructed two-hybrid systems that co-express the Gal4p DNA binding domain/ligand-binding domain of human estrogen receptor (hER) alpha or beta and the Gal4p transactivation domain/nuclear receptor-binding domain of co-activator SRC1, TIF2, or AIB1 in Saccharomyces cerevisiae with a chromosome-integrated lacZ reporter gene under the control of Gal4p-binding sites. We found that the combination of the hERbeta ligand-binding domain and SRC1 nuclear receptor-binding domain was most effective for the xenoestrogen-dependent induction of reporter activity. The extent of transcriptional activation by known xenoestrogens and phytoestrogens was found to correlate well with their estrogenic activities as measured by the previous system with rat ERalpha. This system detects estrogenic activity in some chemicals that have not been suspected of being positive. We also applied this assay system to test the microbial degradation products of gamma-hexachlorocyclohexane (gamma-HCH) by Sphingomonas paucimobilis. Among the gamma-HCH metabolites, 2,5-dichlorohydroquinone and chlorohydroquinone had estrogenic activities similar to the original chemical, while hydroquinone, a later stage metabolite, showed no activity, suggesting the necessity of evaluating intermediate metabolites in microbial degradation systems.

Microbial degradation and fate in the environment of methyl tert-butyl ether and related fuel oxygenates.

Oxygenates, mainly methyl tert-butyl ether (MTBE), are commonly added to gasoline to enhance octane index and improve combustion efficiency. Other oxygenates used as gasoline additives are ethers such as ethyl tert-butyl ether (ETBE), tert-amyl methyl ether (TAME), and alcohols such as tert-butyl alcohol (TBA). As a result of its wide use, MTBE has been detected, mainly in the USA, in groundwater and surface waters, and is a cause of concern because of its possible health effects and other undesirable consequences. MTBE is a water-soluble and mobile compound that generates long pollution plumes in aquifers impacted by gasoline releases from leaking tanks. Field observations concur in estimating that, because of recalcitrance to biodegradation, natural attenuation is slow (half-life of at least 2 years). However, quite significant advances have been made in recent years concerning the microbiology of the degradation of MTBE and other oxygenated gasoline additives. The recalcitrance of these compounds results from the presence in their structure of an ether bond and of a tertiary carbon structure. For the most part, only aerobic microbial degradation systems have been reported so far. Consortia capable of mineralizing MTBE have been selected. Multiple instances of the cometabolism of MTBE with pure strains or with microflorae, growing on n-alkanes, isoalkanes, cyclohexane or ethers (diethyl ether, ETBE), have been described. MTBE was converted into TBA in all cases and was sometimes further degraded, but it was not used as a carbon source by the pure strains. However, mineralization of MTBE and TBA by several pure bacterial strains using these compounds as sole carbon and energy source has recently been reported. The pathways of metabolism of MTBE involve the initial attack by a monooxygenase. In several cases, the enzyme was characterized as a cytochrome P-450. After oxygenation, the release of a C -unit as formaldehyde or formate leads to the production of TBA, which can be converted to 2-hydroxyisobutyric acid and further metabolized. Developments in microbiology make biological treatment of water contaminated with MTBE and other oxygenates an attractive possibility. Work concerning ex situ treatment in biofilters by consortia and by pure strains, and involving or not cometabolism, is under way. Furthermore, the development of in situ treatment processes is a promisinggoal.

Analysis of Biodesulfurization Products Using Solid-Phase Extraction

Alkylated dibenzothiophenes (DBTs) are not efficiently desulfurized by the conventional hydrodesulfurization (HDS) process, and thus they remain targets for deeper desulfurization of HDS-treated oil. Microbial degradation systems are expected to convert these heterocyclic sulfur compounds to inorganic sulfur (sulfate or sulfite). For screening and characterization of the potentially useful desulfurizing microbes, it is very important to analyze microbial metabolites of these DBTs. For this purpose, highly sensitive methods of analysis and efficient concentration are required because there would be only small amounts of the metabolites in microbial culture media. A sample preparation technique based on solid-phase extraction (SPE) was studied to separate DBT metabolites from an organic matrix such as gas oil. Our results demonstrate that the metabolites with polar functional groups such as 2-hydroxybiphenyl or ο,ο'-biphenol produced by a desulfurizing microbe (Rodococcus erythropolis) can be efficiently separated from the organic matrix and concentrated quantitatively by SPE. Moreover it has been proven that the combination of SPE with gas chromatography-mass spectrometry or gas chromatography-atomic emission detection analysis can be successfully used for detailed characterization of gas oil treated by desulfurizing biocatalysts. gency of sulfur emission standards is increasing around the world. In this respect, biological desulfurization systems have recently attracted attention for their potential advantages com­ pared with HDS. There are several reasons for this: they work at low temperatures and under low pressure, require no addi­ tion of hydrogen gas, and possibly degrade organosulfur com­ pounds, which are considerably resistant to HDS treatment (3-5). Dibenzothiophene (DBT) has been used as the model compound to represent the organosulfur material present in

環境微生物分解系遺伝子のダイナミズム４ 脱ハロゲン化酵素遺伝子の多様性

環境微生物分解系遺伝子のダイナミズム４ 脱ハロゲン化酵素遺伝子の多様性