Enhanced biodegradation of phenanthrene using different inoculum types in a microbial fuel cell

Environmental pollution by petroleum hydrocarbons from contaminated groundwater and soils is a serious threat to human health. Microbial fuel cells (MFCs) could be employed in the treatment of these recalcitrant pollutants with concomitant bioelectricity generation. In this study, the use of MFCs in biodegradation of phenanthrene, a model hydrocarbon, was investigated with respect to its biodegradation rate, biodegradation efficiency, and power production using a range of inocula (Shewanella oneidensis MR1 14063, Pseudomonas aeruginosa NCTC 10662, mixed cultures, and combinations thereof). All the inocula showed high potentials for phenanthrene degradation with a minimum degradation efficiency of 97%. The best overall performing inoculum was anaerobically digested sludge supplemented with P. aeruginosa NCTC 10662, having a degradation rate, maximum power density and chemical oxygen demand removal efficiency of 27.30 μM/d, 1.25 mW/m² and 65.6%, respectively. Adsorption of phenanthrene on the carbon anode was also investigated; it conformed to a Type II adsorption isotherm and could be modelled using a modified Brunauer, Emmett and Teller model with a maximum monolayer capacity of 0.088 mg/cm². This work highlights the possibility of using MFCs to achieve high degradation rates of phenanthrene through co‐metabolism and could potentially be used as a replacement of permeable reactive barriers for remediation of hydrocarbon‐contaminated groundwater.     

Biodegradation of phenanthrene and analysis of degrading cultures in the presence of a model organo-mineral matrix and of a simulated NAPL phase

Two mixed bacterial cultures (C_B-BT and C_I-AT) degraded phenanthrene when it was: (i) in the presence of either hexadecane as a non aqueous phase liquid or a montmorillonite–Al(OH)x-humic acid complex as a model organo-mineral matrix; (ii) sorbed to the complex, either alone or in the presence of hexadecane. The cultures had different kinetic behaviours towards phenanthrene with or without hexadecane. The degradation of Phe alone as well as that of Phe in hexadecane ended in 8 and 15 days with C_B-BT and C_I-AT cultures, respectively. Hexadecane increased Phe bioavailability for C_I-AT bacteria which degraded Phe according to first-order kinetics. The same effect was observed for C_B-BT bacteria, but with an initial 2 days lag phase and in accordance with zero-order kinetics. The presence of hexadecane did not affect the degradation of phenanthrene sorbed and aged on the complex by C_I-AT culture. This capability was exhibited also after experimental aging of 30 days. The dynamics of the bacterial community composition was investigated through PCR-DGGE (denaturing gradient gel electrophoresis) of 16S rRNA gene fragments. Individual bands changed their intensity during the incubation time, implying that particular microbe’s relative abundance changed according to the culture conditions. Isolation of phenanthrene and/or hexadecane degraders was in accord with cultivation-independent data. Growth-dependent changes in the cell surface hydrophobicity of the two cultures and of the isolates suggested that modulation of cell surface hydrophobicity probably played an important role for an efficient phenanthrene assimilation/uptake.     

多环芳烃降解菌的筛选、鉴定及降解特性

【目的】多环芳烃(PAHs)是一类普遍存在于环境中且具有高毒性的持久性有机污染物,高效降解菌的筛选对利用生物修复技术有效去除环境中的多环芳烃具有重要意义。研究拟从供试菌株中筛选多环芳烃高效降解菌,并分析其降解特性,为多环芳烃污染环境的微生物修复提供资源保障和科学依据。【方法】采用平板法从25株供试菌株中筛选出以菲和芘为唯一碳源和能源的高效降解菌,经16S rRNA基因序列进行初步鉴定,通过单因素实验法分析其在液体培养基中的降解特性。【结果】筛选出的3株多环芳烃高效降解菌SL-1、02173和02830经16S rRNA基因序列分析,02173和02830分别与假单胞菌属中的Pseudomonas alcaliphila和Pseudomonas corrugate同源性最近,SL-1为本课题组发表新类群Rhizobium petrolearium的模式菌株;降解实验表明,菌株SL-1 3 d内对单一多环芳烃菲(100 mg/L)和芘(50 mg/L)的降解率分别达到100%和48%,5 d后能够降解74%的芘;而其3 d内对混合PAHs中菲和芘的降解率分别为75.89%和81.98%。菌株02173和02830 3 d内对混合多环芳烃中萘(200 mg/L)、芴(50 mg/L)、菲(100 mg/L)和芘(50 mg/L)的降解率均分别超过97%。【结论】筛选出的3株PAHs降解菌SL-1、02173和02830不仅可以高效降解低分子量PAHs,还对高分子量PAHs具有很好的降解潜力。研究表明,由于共代谢作用低分子量多环芳烃可促进高分子量多环芳烃的降解,而此时低分子量多环芳烃的降解将受到抑制。     

Adsorption of dirhamnolipid on four microorganisms and the effect on cell surface hydrophobicity

In this study, adsorption of dirhamnolipid biosurfactant on a Gram-negative Pseudomonas aeruginosa, two Gram-positive Bacillus subtilis, and a yeast, Candida lipolytica, was investigated, and the causality between the adsorption and change of cell surface hydrophobicity was discussed. The adsorption was not only specific to the microorganisms but also depended on the physiological status of the cells. Components of the biosurfactant with different rhamnosyl number or aliphatic chain length also exhibited slight difference in adsorption manner. The adsorption indeed caused the cell surface hydrophobicity to change regularly; however, the changes depended on both the concentrations of rhamnolipid solutions applied and the adsorbent physiological conditions. Orientation of rhamnolipid monomers on cell surface and micelle deposition are supposed to be the basic means of adsorption to change cell hydrophobicity at low and high rhamnolipid concentrations, respectively. This study proposed the possibility to modify cell surface hydrophobicity with biosurfactant of low concentrations, which may be of importance in in situ soil remediation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Taxonomic identification,phenanthrene uptake activity,and membrane lipid alterations of the PAH degrading <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. strain Sphe3

This report describes phenanthrene uptake as well as the effect of phenanthrene on the membrane phospholipid and fatty acid composition in a newly isolated bacterial strain, Sphe3, that we taxonomically identified as Arthrobacter sp. Strain Sphe3 is able to utilize phenanthrene as a carbon source at high rates and appears to internalize phenanthrene with two mechanisms: a passive diffusion when cells are grown on glucose, and an inducible active transport system when cells are grown on phenanthrene as a sole carbon source. Active transport followed Michaelis-Menten kinetics, and it was amenable to inhibition by 2,4-dinitrophenol and sodium azide. Evidence provided here indicates that apart from inducing an active PAH uptake, the presence of phenanthrene elicits significant changes in membrane fluidity.     

Bacterial cell hydrophobicity is modified during the biodegradation of anionic surfactants

Abstract A biphasic increase in surface hydrophobicity of the surfactant-biodegrading bacterium Pseudomonas C12B has been correlated with biodegradation of the primary alkyl sulphate, sodium dodecyl sulphate. Using both hydrophobic interaction chromatography and microbial adhesion to hydrocarbon to measure surface hydrophobicity, it was shown that the first phase coincides with production of the primary metabolite dodecan-1-ol. The direct addition of dodecan-1-ol to Pseudomonas C12B resulted in the instantaneous increase in surface hydrophobicity, with a subsequent decrease which coincided with dodecan-1-ol biodegradation. In contrast, incubation of Pseudomonas C12B with sodium dodecane sulphonate, a non-metabolizable surfactant analogue of SDS, or the growth-supporting carbon source sodium pyruvate did not alter the surface hydrophobicity. These data are interpreted in terms of a model in which the hydrophobic metabolite dodecan-1-ol enters the bacterial membranes, thus increasing surface hydrophobicity and that these surfactant-biodegradation-dependent changes in bacterial surface hydrophobicity are correlated with reversible attachment of the bacteria to sediment surfaces.     

Isolation of phenanthrene-degrading bacteria and characterization of phenanthrene metabolites

Three aerobic bacterial consortia GY2, GS3 and GM2 were enriched from polycyclic aromatic hydrocarbon-contaminated soils with water-silicone oil biphasic systems. An aerobic bacterial strain utilizing phenanthrene as the sole carbon and energy source was isolated from bacterial consortium GY2 and identified as Sphingomonas sp. strain GY2B. Within 48 h and at 30°C the strain metabolized 99.1% of phenanthrene (100 mg/l) added to batch culture in mineral salts medium and the cell number increased by about 40-fold. Three metabolites 1-hydroxy-2-naphthoic acid, 1-naphthol and salicylic acid, were identified by gas chromatographic mass spectrometry and UV–visible spectroscopy analysis. A degradation pathway was proposed based on the identified metabolites. In addition to phenanthrene, strain GY2B could use other aromatic compounds such as naphthalene, 2-naphthol, salicylic acid, catechol, phenol, benzene and toluene as a sole source of carbon and energy.     

一株海草沉积物菲降解菌的筛选、鉴定和降解特性

【背景】多环芳烃(Polycyclic Aromatic Hydrocarbons,PAHs)是一类高毒性的有机污染物,在海洋环境尤其是沿海环境中广泛分布。海草床生态系统作为沿海环境的重要组成部分,深受环境污染等人类活动的影响而处于严重衰退的状态。微生物修复是修复环境中多环芳烃污染的重要途径,具有经济简便、环境友好和无二次污染等特点。【目的】从深圳市大亚湾的海草床沉积物中筛选获得高效多环芳烃降解菌,并分析其降解特性,从而探究海草床生态系统中多环芳烃污染物的微生物修复可行性。【方法】以多环芳烃菲为唯一碳源从海草床沉积物样品中筛选菌株,再通过形态学观察、生理生化实验和16SrRNA基因序列对筛选的菌株进行鉴定,并利用特定引物扩增多环芳烃降解的功能基因——双加氧酶(nidA)基因,最后通过培养实验分析该菌株对菲的降解特性。【结果】筛选出一株高效降解菲的菌株SCSIO 43702,经鉴定为玫瑰杆菌属(Roseovarius)的潜在新菌,并成功扩增得到双加氧酶相似(nidA like)基因;培养实验结果表明,玫瑰杆菌SCSIO 43702在10 d内对100 mg/L菲的降解率最高可达96%,而且其对菲的最适降解条件为:温度30°C、pH值7.5和8.0、盐度3%。【结论】玫瑰杆菌SCSIO 43702凭借其良好的菲降解能力和较强的环境适应性,具有进一步被开发为微生物菌剂以用于多环芳烃污染修复的巨大潜力,为海草床生态系统中多环芳烃污染的微生物修复研究提供了理论依据和可利用的微生物资源。     

细菌降解萘、菲的代谢途径及相关基因的研究进展

多环芳烃(Polycyclic aromatic hydrocarbons,PAHs)是一类在环境中广泛存在的具有毒性的污染物,微生物降解是其在自然界中降解的主要途径,因而尤为重要。随着研究的深入,关于微生物降解PAHs的分子降解机制、途径等的认识逐渐积累。以下对细菌降解萘、菲的研究进展进行了概述,介绍了萘的水杨酸降解途径,菲的水杨酸、邻苯二甲酸及其他降解途径,同时也包括降解过程中涉及的降解基因簇,如nah-like、phn、phd、nid和nag等以及细菌在PAHs胁迫条件下其他相关基因的表达与调节等方面的最新进展。这些进展可为降解菌株的分子及遗传机制研究提供理论依据,将促进通过基因工程优化降解菌、更有效地检测PAHs环境污染及实现PAHs污染的生物修复。     

Biodegradation of phenanthrene in soil-slurry systems with different mass transfer regimes and soil contents

The effect of soil contents and mass transfer rates on soil bioremediation was investigated. Phenanthrene, a 3-ring polycyclic aromatic hydrocarbon (PAH), was chosen as a model target compound. The biodegradation tests were performed in soil-slurry systems at two distinct mass transfer rates: fast in flasks tests at 150 rpm and slow in roller-bottle tests at 2 rpm. The rate of phenanthrene biodegradation was similar at low soil content (2 wt.%) in both slurry systems, but the rates at high soil contents (6 and 18 wt.%) were higher in the roller-bottle tests. The maximum utilization rate constant for sorbed-phase biodegradation obtained from curve fitting using a mathematical model was decreased in the flask tests with increasing soil content, while not decreased in the roller-bottle tests.     

Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp. strain P2

Sphingomonas sp. strain P2, which is capable of utilizing phenanthrene as a sole carbon and energy source, was isolated from petroleum-contaminated soil in Thailand. Gas chromatography-mass spectrometry and (1)H and (13)C nuclear magnetic resonance analyses revealed two novel metabolites from the phenanthrene degradation pathway. One was identified as 5,6-benzocoumarin, which was derived by dioxygenation at the 1- and 2-positions of phenanthrene, and the other was determined to be 1,5-dihydroxy-2-naphthoic acid. Other metabolites from phenanthrene degradation were identified as 7, 8-benzocoumarin, 1-hydroxy-2-naphthoic acid and coumarin. From these results, it is suggested that strain P2 can degrade phenanthrene via dioxygenation at both 1,2- and 3,4-positions followed by meta-cleavage.     

Biodegradation of phenanthrene and pyrene by Ganoderma lucidum

Biodegradation of two polycyclic aromatic hydrocarbons (PAHs), phenanthrene and pyrene, by a white rot fungus, Ganoderma lucidum, in broth cultures was investigated. It was found that the biomass of the organism decreased with the increase of PAH concentration in the cultures. In the cultures with 2 to 50 mg l⁻¹ PAHs, the degradation rate constants (k₁) increased with the PAH concentration, whereas, at the level of 100 mg l⁻¹, the degradation rate constants decreased. In the presence of 20 mg l⁻¹ PAHs, the highest degradation rates of both PAHs occurred in cultures with an initial pH of 4.0 at 30 °C. The addition of CuSO₄, citric acid, gallic acid, tartaric acid, veratryl alcohol, guaiacol, 2,2′-azino-bis-(3- ethylbenzothazoline-6-sulfonate) (ABTS) enhanced the degradation of both PAHs and laccase activities; whereas the supplement of oxalate, di-n-butyl phthalate (DBP), and nonylphenol (NP) decreased the degradation of both PAHs and inhibited laccase production. In conclusion, G. lucidum is a promising white rot fungus to degrade PAHs such as phenanthrene and pyrene in the environment.     

糖脂类生物表面活性剂的特性及其对白腐真菌降解蒽的强化作用

【目的】对铜绿假单胞菌(Pseudomonas aeruginosa)所产生物表面活性剂的稳定性进行分析,考察该生物表面活性剂对乳白耙齿菌F17(Irpex lacteus F17)降解蒽的强化作用。【方法】采用三氯甲烷萃取的方法从铜绿假单胞菌的发酵液中提取生物表面活性剂,采用表/界面张力仪测定该生物表面活性剂在不同条件下的表面张力值,对其进行稳定性研究。在乳白耙齿菌F17降解蒽的过程中加入适量的生物表面活性剂,测定蒽的降解率,探讨其对蒽生物降解的强化作用。【结果】铜绿假单胞菌所产生物表面活性剂的临界胶束浓度为40 mg/L,在15-150°C及pH 6.0-13.0范围内表现出优良的稳定性,对盐浓度的耐受性也很高。在蒽的生物降解过程中,生物表面活性剂能极大地促进蒽的降解,在生物表面活性剂浓度为50 mg/L时,第15天蒽的降解率达到了82.9%。生物表面活性剂在接种乳白耙齿菌F17前1天加入培养基中,能更好地促进蒽的降解。与化学表面活性剂相比,生物表面活性剂对蒽降解的强化作用更显著。【结论】该生物表面活性剂性能优良、稳定性好,能够显著强化乳白耙齿菌F17对蒽的降解,具有良好的应用前景。     

Effects of phenanthrene on seed germination and some physiological activities of wheat seedling

Polycyclic aromatic hydrocarbons (PAHs) are one of the highly persistent organic pollutants, and they are toxic to plants and other living organisms, including human beings. To analyze the response of higher plant to PAHs, we investigated the effects of phenanthrene (PHE) on seed germination and various physiological changes of wheat seedlings. Specifically, we investigated growth, chlorophyll content, lipid peroxidation (LPO), activities of antioxidant enzymes and H₂O₂ accumulation. The results showed that PHE inhibited seed germination, affected the growth and chlorophyll level of wheat seedlings. Furthermore, PHE elevated the levels of LPO and induced H₂O₂ accumulation in leaf tissues in a dose-dependent manner, accompanied by the changes in the antioxidant status. The activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), displayed a decreasing trend with the increasing of PHE concentration. The results indicated that PHE could exert oxidative damages in the early development stage of wheat and the harmfulness occurred mainly in samples with higher concentrations of PHE.     

Utilization of monocyclic aromatic hydrocarbons individually and in mixture by bacteria isolated from petroleum-contaminated soil

The fate of benzene, ethylbenzene, toluene, xylenes (BTEX) compounds through biodegradation was investigated using two different bacteria, Ralstonia picketti (BP-20) and Alcaligenes piechaudii (CZOR L-1B). These bacteria were isolated from extremely polluted soils contaminated with petroleum hydrocarbons. PCR and Fatty Acid Methyl Ester (FAME) were used to identify the isolates. In this study, BTEX biodegradation, applied as a mixture or as individual compounds by the bacteria was evaluated. Both bacteria were shown to degrade each of the BTEX compounds individually and in mixture. However, Alcaligenes piechaudii was a better degrader of BTEXs both in the mixture and individually. Differences between BTEX biodegradation in the mixture and individually were observed, especially in the case of benzene. The degradation of all BTEXs in the mixture was lower than the degradation of individual compounds for both bacteria tested. In the all experiments, toluene and m + p- xylenes were better removed than the other BTEXs. No intermediates of biodegradation were detected. Biosurfactant production was observed by culture techniques. In addition, 3-hydroxy fatty acids, important in biosurfactant production, were observed by FAME analysis. The test results indicate that the bacteria could contribute to bioremediation of aromatic hydrocarbon pollution.     

Effect of chlorhexidine and phenoxyethanol on cell surface hydrophobicity of Gram-positive and Gram-negative bacteria

The surfaces of mutants of Escherichia coli and Pseudomonas aeruginosa were markedly more hydrophobic than the corresponding wild types, as were the latter when the organisms were pre-treated with chlorhexidine diacetate (CHA) or phenoxyethanol (POE). A combination of CHA and POE demonstrated that only at higher concentrations was there a marked effect on hydrophobicity compared with that of either drug used alone. The three methods used to determine hydrophobicity correlated well as long as constant conditions were employed.