Microbial Oxidation of the Isoprenoid Alkane Pristane

Microorganisms capable of utilizing pristane (2,6,10,14-tetramethylpentadecane) as a sole source of carbon and energy were isolated from soil specimens. A strain BPM 1613, tentatively classified in the genus Nocardia, accumulated several oxidation products of pristane in the culture fluid. Silica gel chromatography of the ethyl ether extract from the culture fluid yielded pristanol and pristanic acid as major products and pristyl pristanate and pristyl aldehyde as minor products. The confirmations on the estimated structures of these oxidation products are described in detail.     

Microbial Oxidation of Isoprenoid Alkanes,Phytane, Norpristane and Farnesane

Rhodococcus sp. BPM 1613, a pristane oxidizing microorganism, grows on isoprenoid hydrocarbons such as phytane (2,6,10,14-tetramethylhexadecane), norpristane (2,6,10-trimethylpentadecane) and farnesane (2,6,10-trimethyldodecane) as the sole carbon source, resulting in accumulation of oxidation products in the culture broth. The oxidation products of phytane, norpristane and farnesane in the respective culture broth were isolated and purified by the use of silica gel column chromatography. Their chemical structures were determined by instrumental analyses such as IR, NMR and mass spectrometry. The oxidation products of phytane were identified as 2,6,10,14-tetramethyl-1-hexadecanol and 2,6,10,14-tetramethylhexadecanoic acid, the product of norpristane as 2,6,10-trimethyl-1-pentadecanol, and that of farnesane as 2,6,10-trimethyl-1-dodecanol. All these oxidation products were either monoalcohols or monocarboxylic acids derived through oxidation of the isopropyl terminus of each alkane.

In addition, the relationship between the terminal structure of isoprenoid hydrocarbons and microbial oxidation was explored on the basis of these results.     

Anaerobic, Nitrate-Dependent Microbial Oxidation of Ferrous Iron

Enrichment and pure cultures of nitrate-reducing bacteria were shown to grow anaerobically with ferrous iron as the only electron donor or as the additional electron donor in the presence of acetate. The newly observed bacterial process may significantly contribute to ferric iron formation in the suboxic zone of aquatic sediments.     

Microbial Oxidation of Allylic Alcohols

A new method for the oxidation of primary ad secondary allylic alcohols to the corresponding aldehydes/acids and ketones respectively is described utilizing Nocardia corallina B-276. In contrast, Pseudomonas oleovorans TF4-1L oxidized only the primary allylic alcohols but not the secondary allylic alcohols.     

Microbial Hydrocarbon Co-oxidation: I. Oxidation of Mono- and Dicyclic Hydrocarbons by Soil Isolates of the Genus Nocardia

Nocardia cultures, isolated from soil by use of n-paraffins as the sole carbon source, have been shown to bring about significant oxidation of several methyl-substituted mono- and dicyclic aromatic hydrocarbons. Oxygen uptake by washed cell suspensions was not a reliable indicator of oxidation. Under co-oxidation conditions in shaken flasks, o- and p-xylenes were oxidized to their respective mono-aromatic acids, o-toluic and p-toluic acids. In addition, a new fermentation product, 2, 3-dihydroxy-p-toluic acid, was found in the p-xylene oxidation system. Of 10 methyl-substituted naphthalenes tested (1-methyl, 2-methyl, 1, 3-dimethyl, 1, 4-dimethyl, 1, 5-dimethyl, 1, 8-dimethyl, 1, 6-dimethyl, 2, 3-dimethyl, 2, 6-dimethyl, 2, 7-dimethyl), only those containing a methyl group in the β position were oxidized at this position to the mono acid.     

Microbial Oxidation of sec-Alcohols to Ketones

The major anthocyanin compound in buckwheat sprouts was determined to be cyanidin 3-O-rutinoside (C3R), based on HPLC data and MS/MS spectra. Investigation of the content of phenolic compounds in commercial buckwheat sprouts indicated that hypocotyls are abundant in C3R and rutin, whereas all of the detected flavonoids are abundant in cotyledons. The superoxide anion radical-scavenging activities (SOD-like activities) of phenolic compounds in buckwheat sprouts and their contents indicated that rutin, isoorientin, and orientin contributed mainly to the SOD-like activity of the extract from buckwheat sprouts. In contrast, the contribution of C3R was substantially lower than that of flavonoids.     

石油污染海域的微生物群落及烃的降解

目前世界海洋石油污染问题已经严重威胁到海洋生态环境的安全,生物修复是一种处理石油污染的新方法。综述了海洋环境中烃降解微生物生态学方面的一些研究进展,包括探测未培养细菌的新方法、新的分离方法及主要的烃降解菌株的特性,以便努力改进现有分离石油降解菌及石油乳化细菌的方法,同时发现对于石油降解有益的新菌种。     

鼠李糖脂对微生物降解石油烃废水的影响

目的:研究鼠李糖脂对微生物降解石油烃废水的影响.方法:通过测定生物量和观察菌株表面来研究鼠李糖脂对菌株的影响;通过正交实验设计,确定石油烃降解率影响因素.通过石油烃降解率的测定,探讨鼠李糖脂与H2O2深度氧化协同作用对微生物降解石油烃的影响.结果:菌株对石油烃的降解率达53%,在相同条件下,添加鼠李糖脂的石油烃降解率提高了12%-20%.添加鼠李糖脂后菌株的生物量明显增多,菌株细胞表面疏水.正交设计表明,影响石油烃降解的主导因子是培养温度,其次是培养时间和鼠李糖脂的添加量.正交设计得到最佳组合为A3B2C1,即培养时间为7d;温度为35℃,鼠李糖脂浓度为60mg/L.3个因素的最佳组合下,石油烃降解率为82%.加入200 mg/L的H2O2时,降解率从82%提高到97%.结论:鼠李糖脂能促进菌株的生长.鼠李糖脂与H2O2深度氧化协同作用有助于微生物对石油烃类污染物降解效率的提高.     

Metabolite Profiling Identified Methylerythritol Cyclodiphosphate Efflux as a Limiting Step in Microbial Isoprenoid Production

Isoprenoids are natural products that are all derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These precursors are synthesized either by the mevalonate (MVA) pathway or the 1-Deoxy-D-Xylulose 5-Phosphate (DXP) pathway. Metabolic engineering of microbes has enabled overproduction of various isoprenoid products from the DXP pathway including lycopene, artemisinic acid, taxadiene and levopimaradiene. To date, there is no method to accurately measure all the DXP metabolic intermediates simultaneously so as to enable the identification of potential flux limiting steps. In this study, a solid phase extraction coupled with ultra performance liquid chromatography mass spectrometry (SPE UPLC-MS) method was developed. This method was used to measure the DXP intermediates in genetically engineered E. coli. Unexpectedly, methylerythritol cyclodiphosphate (MEC) was found to efflux when certain enzymes of the pathway were over-expressed, demonstrating the existence of a novel competing pathway branch in the DXP metabolism. Guided by these findings, ispG was overexpressed and was found to effectively reduce the efflux of MEC inside the cells, resulting in a significant increase in downstream isoprenoid production. This study demonstrated the necessity to quantify metabolites enabling the identification of a hitherto unrecognized pathway and provided useful insights into rational design in metabolic engineering.     

Hydrocarbon Leaching, Microbial Population, and Plant Growth in Soil Amended with Petroleum

Two samples of oily waste organics (OWO) from petroleum wells were added to heath soils from Tierra del Fuego, Argentina, and the effects on hydrocarbon leaching, microbial population, and plant growth were studied. These mixtures and a control soil were subjected to four deionized water leachates. For each leachate, total petroleum hydrocarbons (TPH), aliphatic hydrocarbons (ALH), aromatic hydrocarbons (ARH) with three or fewer rings, ARH with more than three rings, and oil and grease (O&G) were measured. After leaching, six Dactylis glomerata L. plants were grown in each soil column. Plant growth and the total number of aerobic and nitrifier microorganisms were measured in soil. The 10% OWO sample increased the TPH in the leachate, but the 1% sample did not. The ALH, ARH, and O&G of each leachate followed patterns similar to that for TPH. Plant growth diminished and the total number of aerobic and nitrifier microorganisms decreased with increasing OWO, especially when the OWO was from a fresh residue rather than an aged residue. The greater inhibitive effect of fresh residue on plant growth was attributed to a higher concentration of light hydrocarbons, which are more toxic than heavy hydrocarbons. For soil with 1% OWO added, the TPH and other organics did not differ from the control soil. This result, combined with the 10-year average annual rainfall and the water table elevation at the site, suggests that the risk of contaminating the water table is relatively low. Thus, a 1% addition of OWO in soil would be appropriate to use in landfarming of OWO.     

Association of Microbial Community Composition and Activity with Lead, Chromium, and Hydrocarbon Contamination

Microbial community composition and activity were characterized in soil contaminated with lead (Pb), chromium (Cr), and hydrocarbons. Contaminant levels were very heterogeneous and ranged from 50 to 16,700 mg of total petroleum hydrocarbons (TPH) kg of soil⁻¹, 3 to 3,300 mg of total Cr kg of soil⁻¹, and 1 to 17,100 mg of Pb kg of soil⁻¹. Microbial community compositions were estimated from the patterns of phospholipid fatty acids (PLFA); these were considerably different among the 14 soil samples. Statistical analyses suggested that the variation in PLFA was more correlated with soil hydrocarbons than with the levels of Cr and Pb. The metal sensitivity of the microbial community was determined by extracting bacteria from soil and measuring [³H]leucine incorporation as a function of metal concentration. Six soil samples collected in the spring of 1999 had IC₅₀ values (the heavy metal concentrations giving 50% reduction of microbial activity) of approximately 2.5 mM for CrO₄²⁻ and 0.01 mM for Pb²⁺. Much higher levels of Pb were required to inhibit [¹⁴C]glucose mineralization directly in soils. In microcosm experiments with these samples, microbial biomass and the ratio of microbial biomass to soil organic C were not correlated with the concentrations of hydrocarbons and heavy metals. However, microbial C respiration in samples with a higher level of hydrocarbons differed from the other soils no matter whether complex organic C (alfalfa) was added or not. The ratios of microbial C respiration to microbial biomass differed significantly among the soil samples (P < 0.05) and were relatively high in soils contaminated with hydrocarbons or heavy metals. Our results suggest that the soil microbial community was predominantly affected by hydrocarbons.     

Microbial Enzymes for Oxidation of Organic Molecules

Abstract

Enzymatic systems employed by microorganisms for oxidative transformation of various organic molecules include laccases, ligninases, tyrosinases, monooxygenases, and dioxygenases. Reactions performed by these enzymes play a significant role in maintaining the global carbon cycle through either transformation or complete mineralization of organic molecules. Additionally, oxidative enzymes are instrumental in modification or degradation of the ever-increasing man-made chemicals constantly released into our environment. Due to their inherent stereo- and regioselectivity and high efficiency, oxidative enzymes have attracted attention as potential biocatalysts for various biotechnological processes. Successful commercial application of these enzymes will be possible through employing new methodologies, such as use of organic solvents in the reaction mixtures, immobilization of either the intact microorganisms or isolated enzyme preparations on various supports, and genetic engineering technology.     

Microbial Oxidation of Cephalosporins to Cephalosporin Sulfoxides

The highest inhibition rate of conidial germination of Pyricularia oryzae was shown by extracts of rice plant leaves inoculated by a pathogen after treatment with probenazole, a rice blast controlling agent. Four anti-conidial germination substances were isolated from these extracts. Substances A, C and D inhibited the germination of the conidia at concentrations between 100 and 200 mcg/ml, and substance B caused morphological changes in the germination tubes of the conidia with a little inhibition of germination. These substances were differentiated from momilactone A, B and the degraded or metabolized products of probenazole. Besides anti-conidial germination activity, they showed antimicrobial activities against several kinds of phytopathogenic bacteria of fungi on agar plates by diffusion method.     

微生物砷氧化调控研究进展

微生物砷氧化是指微生物通过砷氧化酶AioAB将毒性强的亚砷酸盐[As(Ⅲ)]氧化为毒性较弱的砷酸盐[As(Ⅴ)]的过程。该过程一方面有利于微生物自身和环境的修复,另一方面能够提供能量供给部分砷氧化菌生长。介绍微生物砷氧化调控机制的最新研究进展。     

Microbial Hydrocarbon Co-oxidation. II. Use of Ion-Exchange Resins

Anion-exchange resins, a weakly basic polystyrene-polyamine type and a macro-reticular type, IR-45 and IRA-93, respectively, were shown to significantly increase yields of acidic products in co-oxidation systems. p-Toluic, 2,3-dihydroxy-p-toluic, and alpha,alpha-cis,cis dimethylmuconic acids, resulting from the oxidation of p-xylene by three cultures of Nocardia, accumulated on the resin in shaken flasks or agar plates during the cultivation. Final product concentration increased with increasing resin concentration. Mineral balances were not affected if the resin was properly conditioned before use.     

Microbial Oxidation of Vinyl Sulfides to Chiral sulfoxides

A variety of bacteria, yeasts and fungi were screened for the preparation of enantiomers of chiral sulfoxides from aryl-aryl or alkyl-aryl (E) and (Z) vinyl sulfides 1. The asymmetric oxidation was studied on a model substrate, (E)-methyl-(2-phenyl)-vinylsulfide la. Along with various amounts of sulfone, all strains tested gave the corresponding sulfoxide, optically active, albeit with different chemical and optical yields. The stereochemical outcome of the reaction appears to be quite invariant and both enantiomers can be obtained. The high sensitivity towards substrate structure observed in a variety of sulfides is related to the toxicity of the substrates and the microbial oxidation is therefore assumed to be a detoxication reaction.     

Microbial Dioxygenase Gene Population Shifts during Polycyclic Aromatic Hydrocarbon Biodegradation

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.     

New Insights into Microbial Oxidation of Antimony and Arsenic

Sb(III) oxidation was documented in an Agrobacterium tumefaciens isolate that can also oxidize As(III). Equivalent Sb(III) oxidation rates were observed in the parental wild-type organism and in two well-characterized mutants that cannot oxidize As(III) for fundamentally different reasons. Therefore, despite the literature suggesting that Sb(III) and As(III) may be biochemical analogs, Sb(III) oxidation is catalyzed by a pathway different than that used for As(III). Sb(III) and As(III) oxidation was also observed for an eukaryotic acidothermophilic alga belonging to the order Cyanidiales, implying that the ability to oxidize metalloids may be phylogenetically widespread.