一株苯酚降解菌的筛选、鉴定及其降解特性

本研究采用逐量分批驯化的方法,从造纸废水中分离得到一株能够以苯酚为唯一碳源生长的苯酚降解菌株F5-1.经形态观察、生理生化特性鉴定及16S rDNA序列分析,将该菌株鉴定为克雷伯菌(Klebsie-lla sp.).该菌株能够在7 h时完全降解初始浓度为100 mg/L的苯酚,降解苯酚主要发生在生长对数期;在pH 5.0～9.0,NaCl浓度0～80 g/L,温度20～40℃范围内,菌株F5-1均可有效降解初始浓度为100～1 200 mg/L的苯酚;能够耐受的最大苯酚浓度为1 500 mg/L.本研究结果表明,F5-1菌株对处理环境条件复杂的含酚废水具有潜在的应用前景.     

盐胁迫条件下古菌超氧化物歧化酶增强细菌降解硝基酚

【背景】Burkholderia sp. SJ98利用对硝基酚和2-氯-4-硝基酚为唯一碳源和能源进行生长,通过异源表达嗜盐古菌Haloferax sp. D1227中的超氧化物歧化酶SodA,使菌株SJ98在500 mmol/L NaCl条件下仍具有降解对硝基酚的能力。然而该重组细菌在普通和高盐条件下其降解基因的转录和降解酶比活力的高低,以及该菌在高盐条件下是否还能降解对硝基酚衍生物尚未知晓。【目的】研究Burkholderia sp. SJ98的耐盐上限,观察含有sodA的细菌SJ98在普通和高盐条件下降解对硝基酚和2-氯-4-硝基酚的能力,检测重组菌中pnpA基因的转录和硝基酚单加氧酶的活力。【方法】在添加葡萄糖、对硝基酚或2-氯-4-硝基酚的无机盐培养基(分别含400-800 mmol/L NaCl)或M9培养基(含0和500 mmol/L NaCl)中培养细菌SJ98及其重组菌。通过紫外分光光度计和高效液相色谱法检测菌株生长和底物降解。通过实时荧光定量PCR分别以两种硝基酚为诱导物,检测未添加和添加500 mmol/L NaCl时,硝基酚单加氧酶编码基因pnpA的转录量变化。利用紫外分光光度计分别以两种硝基酚为底物,检测在添加500 mmol/L NaCl时,重组菌和空载体菌的粗酶液中硝基酚单加氧酶对两种底物的活力变化。【结果】野生型菌株SJ98以葡萄糖为碳源生长的NaCl耐受浓度是600mmol/L。未添加NaCl时,重组菌SJ98[pCM-pnpR-PpnpA-sodA-rfp]生长和降解对硝基酚的能力远优于野生菌。添加500 mmol/L NaCl时,重组菌SJ98[pBBR-sodA]仍保持了利用2-氯-4-硝基苯酚底物生长和降解该底物的能力,而空载体菌SJ98[pBBR1MCS-2]的生长和降解能力完全丧失;重组菌SJ98[pBBR-sodA]粗酶液中单加氧酶对于对硝基酚和2-氯-4-硝基酚的活力均约为野生菌的1/3。分别以两种硝基酚为诱导物时,无论是否添加NaCl,重组菌SJ98[pBBR-sodA]中硝基酚单加氧酶编码基因pnpA的转录量比野生型中高出约17-25倍;但添加500 mmol/L NaCl时,pnpA的转录均受到部分抑制。【结论】本研究为利用古菌超氧化物歧化酶对细菌进行改造以提高普通环境和高盐环境中细菌降解硝基芳烃污染物能力的应用提供了潜在的可行性。     

嗜盐古菌Haloferax sp. D1227中超氧化物歧化酶功能鉴定及其增强细菌耐盐性的研究

【背景】细菌、酵母或植物来源的超氧化物歧化酶(Superoxide dismutase,SOD)编码基因在异源宿主中表达并提高宿主耐盐性的研究已有一些报道,其异源宿主也多为植物,而古菌来源的超氧化物歧化酶编码基因在细菌中成功表达并提高其耐盐性的研究尚无报道。【目的】寻找嗜盐古菌Haloferax sp.D1227中的超氧化物歧化酶编码基因并鉴定其功能,将其在4-硝基苯酚降解细菌Burkholderia sp.SJ98中表达,研究该古菌的超氧化物歧化酶对菌株SJ98耐盐性和降解4-硝基苯酚功能的影响。【方法】通过生物信息学方法寻找嗜盐古菌D1227中潜在的超氧化物歧化酶编码基因,利用表达载体p ET-28a和广泛宿主载体p BBR1MCS-2将其分别在E.coli BL21(DE3)和4-硝基苯酚的降解菌株SJ98中异源表达,检测细胞抽提液和纯化蛋白的超氧化物歧化酶比活力。分别以葡萄糖和4-硝基苯酚为碳源,在M9培养基和添加500 mmol/L Na Cl(Na Cl含量约3%)的M9培养基中分别培养细菌SJ98的重组菌株和空载体重组菌株,利用全自动生长曲线分析仪和高效液相色谱等方法检测重组菌株的生长能力和对4-硝基苯酚的降解能力。【结果】通过生物信息学分析,在嗜盐古菌D1227基因组中发现了潜在的超氧化物歧化酶编码基因sod A,其在E.coli BL21(DE3)和菌株SJ98中分别异源表达均具有超氧化物歧化酶活力[细胞抽提液的比活力分别为21.07±0.02 U/mg和84.56±0.16 U/mg,从BL21(DE3)菌株纯化的蛋白Sod AD1227比活力为179.46±3.43 U/mg]。在添加500 mmol/L Na Cl的M9培养基中培养时,以葡萄糖为碳源,重组菌株SJ98[p BBR-sod A]仍可正常生长,而空载体对照菌株SJ98[p BBR1MCS-2]几乎丧失了生长能力;以4-硝基苯酚为碳源,菌株SJ98[p BBR-sod A]保持了利用底物生长和降解底物的能力,而菌株SJ98[p BBR1MCS-2]的生长和降解能力几乎丧失。用软件Phyre2模拟分析Sod AD1227的单体结构,该蛋白拥有Fe/Mn-SOD家族的典型结构特征,推测其属于Fe/Mn-SOD家族。【结论】本研究为利用古菌SOD对细菌进行改造以适应高盐环境中降解有机污染物的应用提供了潜在的可行性。     

苯酚高效降解菌的筛选和降解特性的研究

从天津市煤气厂的活性污泥中筛选、分离得到一株高效苯酚降解菌。经BIOLOG细菌自动鉴定系统及16SrDNA鉴定,该菌株为粪产碱杆菌(Alcaligenesfaecalis)。苯酚降解实验证实,该菌能在76h内完全降解1600mg·L-1的苯酚,并且随着苯酚浓度的增加,底物抑制作用增强,细胞得率下降。     

苯酚降解菌F6的筛选鉴定及降解特性

本研究利用逐级驯化的方法,从广西某工厂的废水中分离得到一株能利用苯酚作为唯一碳源生长的高效苯酚降解菌F6。采用16S r DNA序列分析的方法,将菌株F6鉴定为芽孢杆菌(Bacillus sp.)。F6菌株在8 h内几乎完全降解100 mg/L的苯酚,降解率达99.9%,该菌株的菌体生长与苯酚降解呈同步趋势,主要在对数生长期降解苯酚。F6最高耐受苯酚浓度为1 800 mg/L,在温度25~40℃,p H值6.0~9.0,盐度0~40 g/L范围内,F6菌株均能保持对苯酚良好的降解能力。菌株F6的降解底物具有广谱性,除了能够利用苯酚作为唯一碳源,还可以利用邻苯二酚、间苯二酚、对苯二酚、连苯三酚、甲苯、氯苯等酚类化合物为其生长代谢提供碳源和能源。综上所述,菌株F6在应用于处理成分复杂、含酚浓度较高的废水中将具有很大的潜力。     

一株苯酚降解菌的分离与鉴定

目的：筛选能高效降解苯酚的微生物,并进行初步鉴定。方法：从某焦化厂排水沟采集污泥,通过逐步驯化筛选苯酚降解菌株;利用形态观察、生理生化检测、16SrDNA序列分析进行初步鉴定。结果：筛选获得1株苯酚降解菌JDM-2—1,该菌能够以苯酚为惟一碳源,耐酚能力高达2200mg／L,在30℃和pH7．0条件下,42h内能将800mg／L的苯酚彻底降解;初步鉴定其为球形芽孢杆菌（Bacillus sphaericus）。结论：菌株JDM-2-1是一株高效降解苯酚的球形芽孢杆菌。     

烟碱降解细菌的分离、鉴定及其降解性能的初步研究

从福建三明地区的土壤中分离得到一株能够高效降解烟碱的菌株 ,编号为DN2。该菌经常规的形态、生理生化分析以及 16SrDNA序列同源性分析 ,鉴定为Ochrobactrumintermedium ,属于α_变形杆细菌纲。该菌在 30℃～4 0℃和pH 6 . 0～ 9. 0范围内具有较高的降解活性 ,其最适值分别为 30℃和 6 5 ,烟碱的耐受浓度在无机盐培养基中可达到 4 0 0 0mg L。该菌能够以烟碱为唯一碳源生长 ,对于 5 0 0mg L烟碱的降解速率为 15mg L·h ,36h烟碱降解率为97. 6 5 %。该菌在烟草工业和环境保护上可能具有应用前景。     

波茨坦短芽孢杆菌降解苯酚特性及动力学研究

从活性污泥中分离筛选出一株高效苯酚降解菌,经形态特征、生理生化试验及16S rDNA鉴定,该菌株为波茨坦短芽孢杆菌。该菌能以苯酚为唯一碳源和能源,最佳降解条件为:温度30℃,初始pH7.0,摇床转速为160 r/min。苯酚降解试验表明,该菌可在72 h内将初始浓度为1 600 mg/L苯酚完全降解。随着苯酚浓度的增加,底物抑制作用增强。应用Haldane方程对菌株的生长过程进行动力学模拟,拟合曲线与试验测定值相关性良好,各参数分别为μmax(最大比增长率)0.334 h-1,Ks(半饱和常数)14.07 mg/L,Ki(抑制常数)196.89 mg/L,且该菌株苯酚降解动力学与其生长动力学表现出相似的趋势。代谢机制研究表明,苯酚可诱导该菌合成邻苯二酚1,2-加氧酶降解苯酚。     

焦化废水中4株苯酚高效降解菌的分离及鉴定

目的:从焦化废水中筛选苯酚高效降解菌并进行鉴定.方法:在100～1000 mg/L的苯酚为惟-碳源的无机盐培养基上分离出单菌落,测定各菌株的生长曲线以及对苯酚的降解效牢;利用 16S rDNA序列分析结合菌株的形态特征确定各菌株的分类地位.结果:筛选获得4株苯酚降解菌,均能够以苯酚为惟一碳源,在30℃、pH7.0、摇床转速130 r/min、2%的接种量条件下,24h内能将1 000mg/L的苯酚降解91%以上;4株菌可初步鉴定为芽孢杆菌属(ZL1)、产碱杆菌属(ZL2、ZL4)、沙雷氏菌属(ZL3).其中,从焦化废水中分离出高效降解苯酚的沙雷氏菌未见报道.结论:从焦化废水中获得4株苯酚高效降解细菌,对高浓度含酚废水的生物降解具有潜在的应用前景.     

高效苯酚降解菌的选育及降酚特性

以苯酚为惟一碳源,采用逐量分批驯化筛选法筛选高效降酚菌并研究其降酚特性.结果筛选出1株可降解高浓度苯酚的菌株,经鉴定为假单孢菌属(Pityrosporum sp.).该菌株可降解 1 800 mg/L的高浓度苯酚,其降酚性能受许多因素影响:降解苯酚的最适环境条件为温度 30 ℃,pH 6～7,振荡速率大于 150 r/min.     

新疆艾丁湖中度嗜盐苯酚降解菌多样性研究

高盐含酚废水属于极难处理的废水之一,筛选具有生物学降解能力的嗜盐菌有助于解决这一难题。从新疆艾丁湖盐湖中分离筛选能够降解苯酚的中度嗜盐菌,了解盐湖中度嗜盐苯酚降解菌的多样性组成和降解能力。研究结果表明,10%(质量分数)的盐浓度条件下,分离得到166株嗜盐菌,通过以苯酚为唯一碳源的培养基进行降解活性筛选后得到45株阳性菌,根据细菌16S rRNA基因序列系统进化分析,这45株菌分别归类到3个门,5个科,9个属。其中拟诺卡氏菌属(Nocardiopsis)是优势菌,占总量的68.8%,其余菌分布于Bacillus、Gracilibacillus、Pontibacillus、Halobacillus、Marinococcus和Halomonas属。在含100 mg/L苯酚的液体培养基,经过10 d培养后,这45株菌降解效率为1%~17%。本研究为工业应用提供了嗜盐微生物种质资源,极具进一步发掘和研究价值。     

Meta-pathway degradation of phenolics by thermophilic Bacilli

Thermophilic bacteria capable of degrading phenol as the sole carbon source were isolated from sewage effluent. The isolates were aerobic, sporulating, motile rod-shaped bacteria characterized as Bacillus species with growth temperature optima of 50–60°C. The enzyme catalyzing the second step in the phenol degradation meta-cleavage pathway, catechol-2,3-dioxygenase, was detected in all isolates grown in the presence of phenol. One strain, designated Bacillus strain Cro3.2, was capable of degrading phenol, o-, m-, and p-cresol via the meta-pathway and tolerated phenol at concentrations up to 0.1% (w/v) without apparent inhibition of growth. Phenol degradation activities in strain Cro3.2 were induced 3–5 h after supplementation by phenol, orcinol, and the cresols but not by halo- or nitro-substituted phenols. Maximal rates of phenol degradation in stirred bioreactors (10 μmol/min⁻¹/g⁻¹ cells) were achieved at an O₂ delivery rate of 1.0 vvm and temperatures of 45–60°C; however, catechol-2,3-dioxygenase (but not 2-hydroxymuconic semialdehyde dehydrogenase) was rapidly inactivated at high oxygen concentrations. Whole cells of Bacillus strain Cro3.2 entrapped in calcium alginate, polyacrylamide, and agarose gels showed widely different rates of phenol degradation. In calcium alginate gels, rapid loss of phenol-degrading activity was attributed to calcium-induced inactivation of catechol-2,3-dioxygenase. No stabilization with respect to oxygen-induced inactivation was observed under any of the immobilization conditions. It is concluded that the counteractive effects of oxygen limitation at low dO₂ and inactivation of catechol-2,3-dioxygenase at high dO₂ levels pose a significant impediment to the use of resting thermophile cells in the treatment of phenolic waste streams.     

Isolation and Characterization of Phenol-catabolizing Bacteria from a Coking Plant

New phenol degrading bacteria with high biodegradation activity and high tolerance were isolated as Burkholderia cepacia PW3 and Pseudomonas aeruginosa AT2. Both isolates could grow aerobically on phenol as a sole carbon source even at 3 g/l. The whole-cell kinetic properties for phenol degradation by strains PW3 and AT2 showed a V_max of 0.321 and 0.253 mg/l/min/(mg protein), respectively. The metabolic pathways for phenol biodegradation in both strains were assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.     

Seasonal variation of phenol/benzoate-degrading iron-reducing bacteria in irrigated tropical paddy soils as affected by some management practices

The study provides the first evidence of the presence and abundance of bacterial population that coupled ferric iron reduction to aromatic compounds degradation in tropical irrigated paddy soils in the Philippines. Culturable phenol/benzoate degrading iron-reducing bacteria was enumerated by the most probable number (MPN) counts using phenol or benzoate as sole carbon source, and ferric oxide [Fe(OH)(3)] as the sole electron acceptor. Population density of phenol degrading iron-reducing bacteria (P-IRB) in irrigated paddy soil ranged from 10(2) to 10(8)g(-1) dry soil, and increased with the progressive rice growth in rice cropping seasons; the study also revealed a significant rhizosphere effect on population of P-IRB. However, high enumeration of benzoate degrading iron-reducing bacteria (B-IRB) was obtained in all the tested soil samples averaging at 1.2 x 10(6)g(-1) dry soil, and did not fluctuate significantly over the rice cropping seasons. Statistical data showed that less cropping density with aerated fallow and high nitrogen rate favored the population growth of P-IRB. However, results showed that population size of B-IRB was relatively insensitive to the effect of either seasonal or extrinsic factors tested in this study.     

Dynamics of phenol degradation by Pseudomonas putida

Pure cultures of Pseudomonas putida (ATCC 17484) were grown in continuous culture on phenol at dilution rates of 0.074-0.085 h(-1) and subjected to step increases in phenol feed concentration. Three distinct patterns of dynamic response were obtained depending on the size of the step change used: low level, moderate level, or high level. During low level responses no accumulations of phenol or non-phenol, non-glucose-dissolved organic carbon, DOC(NGP), were observed. Moderate level responses were characterized by the transient accumulation of DOC(NGP) with a significant delay prior to phenol leakage. High level responses demonstrated a rapid onset of phenol leakage and no apparent accumulations of DOC(NGP). The addition of phenol to a continuous culture of the same organism on glucose did not result in transient DOC(NGP) accumulations, although transient phenol levels exceeded 90 mg l(-1). These results were consistent with intermediate metabolite production during phenol step tests coupled with substrate-inhibited phenol uptake and suggested that traditional kinetic models based on the Haldane equation may be inadequate for describing the dynamics of phenol degrading systems. (c) 1993 John Wiley & Sons, Inc.     

Silica-immobilized Methylobacterium sp. NP3 and Acinetobacter sp. PK1 degrade high concentrations of phenol

Aims: To immobilize Methylobacterium sp. NP3 and Acinetobacter sp. PK1 to silica and determine the ability of the immobilized bacteria to degrade high concentrations of phenol. Methods and Results: The phenol degradation activity of suspended and immobilized Methylobacterium sp. NP3 and Acinetobacter sp. PK1 bacteria was investigated in batch experiments with various concentrations of phenol. The bacterial cells were immobilized by attachment to or encapsulation in silica. The encapsulated bacteria had the highest phenol degradation rate, especially at initial phenol concentrations between 7500 and 10 000 mg l^?1. Additionally, the immobilized cells could continuously degrade phenol for up to 55 days. Conclusions: The encapsulation of a mixed culture of Methylobacterium sp. NP3 and Acinetobacter sp. PK1 is an effective and easy technique that can be used to improve bacterial stability and phenol degradation. Significance and Impact of the Study: Wastewater from various industries contains high concentrations of phenol, which can cause wastewater treatment failure. Silica‐immobilized bacteria could be applied in bioreactors to initially remove the phenol, thereby preventing phenol shock loads to the wastewater treatment system.     

Enhancement of phenol degradation by soil bioaugmentation with Pseudomonas sp. JS150

Aims: To test whether bioaugmentation with genetically modified Pseudomonas sp. JS150 strain could be used to enhance phenol degradation in contaminated soils. Methods and Results: The efficiency of phenol removal, content of humic carbon, survival of inoculant, number of total culturable autochthonous bacteria and changes in fatty acid methyl esters (FAME) profiling obtained directly from soils were examined. Bioaugmentation significantly accelerated phenol biodegradation rate in tested soils. Phenol applied at the highest concentration (5·0 mg g^?1 soil) was completely degraded in clay soil (FC) within 65 days, whereas in sand soil (FS) within 72 days. In comparison, phenol biodegradation proceeded for 68 and 96 days in nonbioaugmented FC and FS soils, respectively. The content of humic carbon remained at the same level at the beginning and the end of incubation time in all soil treatments. The number of introduced bacteria (2·50 × 10⁹ g^?1 soil) markedly decreased during the first 4 or 8 days depending on contamination level and type of soil; however, inoculant survived over the experimental period of time. Analysis of FAME patterns indicated that changes in the percentages of cyclopropane fatty acids 17:0 cy and 19:0 cyω10c and branched fatty acids might be useful markers for monitoring the progress of phenol removal from soil. Conclusions: It was confirmed that soil bioaugmentation with Pseudomonas sp. JS150 significantly enhanced soil activity towards phenol degradation. Cyclopropane and branched fatty acids were sensitive probes for degree of phenol utilization. Significance and Impact of the Study: In future, genetically modified Pseudomonas sp. JS150 strain could be of use in the bioaugmentation of phenol‐contaminated areas.     

Influence of phenol on the biodegradation of pyridine by freely suspended and immobilized Pseudomonas putida MK1

AIMS: To study the effect of co-contaminants (phenol) on the biodegradation of pyridine by freely suspended and calcium alginate immobilized bacteria. METHODS AND RESULTS: Varying concentrations of phenol were added to free and calcium alginate immobilized Pseudomonas putida MK1 (KCTC 12283) to examine the effect of this pollutant on pyridine degradation. When the concentration of phenol reached 0.38 g l(-1), pyridine degradation by freely suspended bacteria was inhibited. The increased inhibition with the higher phenol levels was apparent in increased lag times. Pyridine degradation was essentially completely inhibited at 0.5 g l(-1) phenol. However, immobilized cells showed tolerance against 0.5 g l(-1) phenol and pyridine degradation by immobilized cell could be achieved. CONCLUSIONS: This works shows that calcium alginate immobilization of microbial cells can effectively increase the tolerance of P. putida MK1 to phenol and results in increased degradation of pyridine. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of wastewater stream can be negatively affected by the presence of co-pollutants. This work demonstrates the potential of calcium alginate immobilization of microbes to protect cells against compound toxicity resulting in an increase in pollutant degradation.     

Enhanced biodegradation of phenol by a microbial consortium in a solid–liquid two phase partitioning bioreactor

Two phase partitioning bioreactors (TPPBs) operate by partitioning toxic substrates to or from an aqueous, cell-containing phase by means of second immiscible phase. Uptake of toxic substrates by the second phase effectively reduces their concentration within the aqueous phase to sub-inhibitory levels, and transfer of molecules between the phases to maintain equilibrium results in the continual feeding of substrate based on the metabolic demand of the microorganisms. Conventionally, a single pure species of microorganism, and a pure organic solvent, have been used in TPPBs. The present work has demonstrated the benefits of using a mixed microbial population for the degradation of phenol in a TPPB that uses solid polymer beads (comprised of ethylene vinyl acetate, or EVA) as the second phase. Polymer modification via an increase in vinyl acetate concentration was also shown to increase phenol uptake. Microbial consortia were isolated from three biological sources and, based on an evaluation of their kinetic performance, a superior consortium was chosen that offered improved degradation when compared to a pure strain of Pseudomonas putida ATCC 11172. The new microbial consortium used within a TPPB was capable of degrading high concentrations of phenol (2000mgl^–1), with decreased lag time (10h) and increased specific rate of phenol degradation (0.71g phenolg^–1cellh). Investigation of the four-member consortium showed that it consisted of two Pseudomonas sp., and two Acinetobacter sp., and tests conducted upon the individual isolates, as well as paired organisms, confirmed the synergistic benefit of their existence within the consortium. The enhanced effects of the use of a microbial consortium now offer improved degradation of phenol, and open the possibility of the degradation of multiple toxic substrates via a polymer-mediated TPPB system.