两株高效苯酚降解菌的筛选、鉴定及生物强化-DTRO组合工艺初步验证

【目的】从煤化工废水中分离、筛选苯酚高效降解微生物,初步考察微生物与DTRO技术联用,构建含酚废水生物强化处理工艺的可行性。【方法】采用苯酚浓度梯度培养基对苯酚降解微生物进行分离和筛选;根据菌体形态电子显微镜观察、菌株生理生化特性考察和16S r RNA基因系统发育树构建,对菌株进行初步生物学鉴定;将筛选出的高效苯酚降解菌制备成相应的菌剂与碟管式反渗透(DTRO)技术组合形成"生物强化-DTRO"工艺,并试用于含酚废水的处理。【结果】共获得7株纯化细菌,其中Phe-03和Phe-05为高效苯酚降解菌;该2株菌均可以苯酚为唯一碳源生长。经鉴定Phe-03为壤霉菌属(Agromyces)菌株;Phe-05为棒杆菌属(Corynebacterium)菌株。到目前为止,壤霉菌属(Agromyces)菌株降解苯酚尚未见报道。在初始苯酚浓度达到1 300 mg/L条件下,Phe-03和Phe-05菌株44 h内对苯酚降解率均达到70%以上;76 h后苯酚降解率均超过90%。组合形成的"生物强化-DTRO"工艺不仅可以有效去除废水中的酚类化合物,而且还能减少反渗透膜污染,以及增加膜的通透性。【结论】研究表明微生物技术可与DTRO技术联用,构建含酚废水生物强化处理工艺,可为含酚废水处理技术研究提供一种选择思路。     

高效苯酚降解菌细胞固定化方法与条件的研究

含酚废水是一种难降解有机废水,对环境污染非常严重。目前常利用细菌处理含酚废水。但利用细菌处理含酚废水存在一些缺点,为此将1株高效苯酚降解菌进行细胞固定化。采用正交实验设计方法确定了该菌株固定化的最佳条件,并且考察了该固定化细胞降解苯酚的最佳条件。实验表明:该菌株的固定化细胞降解苯酚能力和耐受苯酚能力均大于游离细胞,经36 h可将1 800 mg/L苯酚降解完全。其降解苯酚的最适温度为30℃,最佳pH值为5~9。     

N+离子注入诱变选育高效降酚菌的研究

以合肥钢铁厂焦化废水处理厂的活性污泥为对象,从中驯化筛选得到降酚菌株并利用离子束对出发菌株进行诱变筛选,获得高效降解菌株KE2,它与出发菌株KE相比较,降解苯酚的能力显著提高。在温度为30℃左右,pH值为7时,KE2菌株生长和降解苯酚情况最佳,适量的外加碳源(葡萄糖)对KE2的生长和降酚率有促进作用。     

一株高效苯酚降解真菌的分离鉴定及其菌剂的制备

【背景】含酚废水是普遍存在的有毒、难降解的有机污染物之一,生物法处理含酚废水因成本低、无二次污染而具有广阔的应用前景。可降解苯酚的微生物中,真菌比细菌对恶劣环境的适应性更好。针对液态菌液保存时间较短和运输困难的瓶颈,制备固体菌剂可以提高菌体存活率和储藏稳定性。【目的】筛选一株能够高效降解苯酚的真菌,优化其降酚性能并选择合适的载体制备菌剂。【方法】通过逐级驯化和纯化分离降酚菌,筛选得到降酚性能较强的真菌并通过ITS r DNA基因测序进行种属鉴定,通过参数优化进一步提高菌株降解苯酚的性能;以不同材料为载体制备菌剂,通过稀释平板计数法和苯酚降解实验探究菌剂在不同温度下的保存效果。【结果】分离筛选得到一株高效降解苯酚真菌QWD1,通过鉴定证明其属于Magnusiomyces capitatus,其最适降解条件:(NH_4)_2SO_4为氮源,接种量为15%,pH为7.0,温度为35°C,氮源浓度为14 mmol/L。在此条件下,28 d内对1 600 mg/L苯酚去除率可以达到97.15%;制备菌剂最合适载体为谷糠,适宜保存温度为4°C,保存时间可达到90 d甚至更长,活菌数高达2.5×10~8 CFU/g左右,降解苯酚效果良好。【结论】筛选得到了一株高效降解苯酚真菌,优化其降解性能并将其制备成菌剂,为处理含酚废水提供了新菌种和理论支持。     

一株高效降酚菌的质粒特性及柠檬酸细菌表达的研究

在焦化废水处理厂的活性污泥中筛选的降酚效果较好的菌株H,研究其质粒特性并提取其降解质粒将其转入柠檬酸细菌进行表达。结果表明H菌株具有质粒,且质粒片段较大最大的超过10 kb,最小的2 kb左右。通过SDS和原生质体再生法分别对H菌进行质粒消除,结果发现质粒去除的菌株降酚能力也随之消失。说明H菌株的质粒上有控制酚降解基因存在;提取H菌的质粒将其转入柠檬酸细菌进行表达,获得了转化子。转化子具有较好的降酚效果12 h可达77.34%,但转化子的降解速率较小。另证明了转化子内含有与H菌株相同特性的质粒得到具有降酚能力的柠檬酸细菌表达体系。     

苯酚降解菌红球菌(Rhodococcus sp.) P1的鉴定及其在焦化废水中的应用

摘要:【目的】酚类物质的去除是焦化废水处理的关键问题,目的是从焦化废水中分离高效的苯酚降解细菌。【方法】以苯酚为唯一碳源筛选纯化降解苯酚细菌,菌株鉴定采用菌落形态和16S rRNA 序列分析方法,并研究其苯酚降解特性和在焦化废水中的除酚作用。【结果】菌落形态和16S rRNA序列比对分析表明分离的P1菌株为红球菌属(Rhodococcus sp.)细菌;其耐酚浓度高达1400 mg/L,苯酚降解的最适条件为32℃－42℃、pH 7.0和0－4%盐;苯酚降解动力学曲线符合Haldane动力学模型,qmax=0.517/h,Ks=77.487 mg/L,Ki=709.965 mg/L;不同重金属对红球菌P1菌株的苯酚降解抑制作用不同,Zn2+、Mn2+和低浓度的Pb2+对菌株降酚没有影响,Cu2+、Ni2+、Cd2+均抑制菌株对酚的降解;红球菌P1菌株2d内可完全降解1/3焦化原水中的279.9 mg/L酚类物质。【结论】P1菌株是1株高效的苯酚降解菌,具有生物处理焦化废水酚类物质的潜力。     

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.     

脱除游离棉酚乳酸菌的分离鉴定及益生性能研究

从藜麦茎叶样品中筛选分离出乳酸菌株,测定菌株脱除游离棉酚能力,通过人工胃液、人工肠液耐受实验、抑菌实验、抗生素敏感实验及表面特性研究菌株各项性能,并通过生理生化及16S rDNA法对菌株进行鉴定。结果表明,筛选出1株高效游离棉酚脱除乳酸菌LR002,其游离棉酚脱除率为62.87%,该菌株具有高效的耐人工胃液、肠液效果,且具有广谱抑菌作用,对抗生素不敏感,经鉴定该菌株为植物乳杆菌（Lactobacillus plantarum）。本研究旨在筛选出高效游离棉酚脱除乳酸菌菌株,为棉粕在畜禽生产中的合理利用提供参考。     

含油脂废水中一株栗褐芽胞杆菌的筛选和鉴定

目的从含油脂废水中筛选鉴定出油脂降解菌。方法从含油脂废水中取样,通过分离、培养,筛选出以油脂为唯一碳源和能源并能分解油脂的菌株,对其基因组16S DNA测序,在核酸数据库中进行BLAST比对,并进行生化反应,进行菌种的鉴定。结果从含油脂废水中筛选出8株微生物,最终筛选出1株油脂降解菌,鉴定为栗褐芽胞杆菌。结论从含油脂废水中筛选出1株栗褐芽胞杆菌,为下一步实验证实其分解油脂的作用和机制奠定了基础。     

一株耐盐菌Halomonas sp. A20的分离及降解糖精钠废水的特性

【背景】糖精钠废水是一种难处理的高盐有机工业废水。【目的】为了提高糖精钠废水的生物降解效果,需要研究糖精钠废水降解菌的特性。【方法】采用纯培养技术从处理糖精钠废水的多级生物接触氧化系统内的活性污泥中分离筛选糖精钠废水降解菌,对分离菌株的形态特征、生理生化特性和16S rRNA基因序列进行分析,利用单因素实验和响应面法考察分离菌株降解糖精钠废水的最佳条件。【结果】筛选获得一株糖精钠废水降解菌A20,归属于盐单胞菌属(Halomonas),当糖精钠废水的盐分为5%,菌接种量为15%,pH值为8.0,温度为30°C时,菌株A20对糖精钠废水中的化学需氧量(chemical oxygen demand,CODcr)去除率在60%以上;通过响应面法优化,菌株A20降解糖精钠废水的最佳条件为：pH 8.0,温度为30.3°C,接种量为14.1%,其CODcr去除率为65.4%。【结论】分离到一株能高效降解糖精钠废水中有机物的耐盐菌Halomonas sp. A20,可为高盐、高浓度糖精钠废水的处理提供优良的微生物菌种资源。     

工业废水中降酚菌的分离及ARDRA多态性分析

分别将炼油废水、印染废水、造纸废水样品倍比稀释后涂布平板分离菌株,用苯酚羟化酶基因特异引物检测苯酚降解菌,共分离得到87株降酚菌。经ERIC-PCR指纹图分析,显示15种不同的类型。进一步对显示不同ERIC—PCR指纹图的15种分离物的代表菌株进行ARDRA多态性分析,结果可分为4个OTUs（Operational Taxonomic Unit,OTU）,表明实验分离得到的降酚菌至少存在4个不同的种（species）。     

竹炭固定化施氏假单胞菌Pseudomonas stutzeri ZH-1降解苯酚的研究

通过竹炭固定化以加强施氏假单胞菌Pseudomonas stutzeri ZH-1对苯酚的降解能力。采用正交试验优化竹炭对菌株ZH-1固定化条件,冷场发射扫描电镜（SEM）观察固定化后的菌株在竹炭上的附着情况,对比固定化菌和游离菌的降解性能并对固定化菌做重复利用性能测试。结果显示,竹炭固定化P. stutzeri ZH-1的最适条件为竹炭1 g,接种量5%(体积分数),吸附时间24 h;SEM显示菌附着在竹炭表面和内部空隙中;竹炭固定化后菌株ZH-1对苯酚的降解率显著增加（P<0.05）,处理48 h时降解率增加15%;竹炭固定化菌ZH-1经10轮重复利用后仍有很高的苯酚降解性能,48 h时降解率增加173%（P<0.05）。竹炭固定化菌ZH-1在去除含苯酚类有机废水中具有较好的应用前景。     

Effect of Co‐Substrates on Aerobic Phenol Degradation by Acclimatized and Non‐acclimatized Enrichment Cultures

Aerobic degradation of 7 mmol/L phenol in the presence of alternative carbon sources (7 mmol/L glucose or acetate or 1–2 mmol/L 2‐chlorophenol) was investigated using non‐acclimatized and acclimatized sewage sludges and enrichment cultures. The substrates represented an intermediate of phenol degradation (acetate), an independent substrate (glucose) or a “precursor‐substrate” of phenol degradation (2‐chlorophenol). Bacteria from sewage sludge, not pre‐adapted to phenol (2 mmol/L), rapidly respired acetate and glucose in the presence of phenol, whereas phenol was only bioconverted to any unknown aromatic metabolite after 24 h. In the presence of phenol and 2‐chlorophenol, no removal of both substances was observed when using the unacclimatized sludge. Sludge that was acclimatized to the degradation of phenol showed an initial preference for easily degradable co‐substrates such as glucose or acetate with only a slow concomitant respiration of phenol. Respiration of phenol increased rapidly after the co‐substrates were depleted. The highest phenol degradation rates were 51.6 mmol/L d, when phenol was the sole carbon substrate. Vice versa, phenol was preferentially respired in the presence of a less easily degradable co‐substrate such as 2‐chlorophenol at a rate of around 7 mmol/L d. Further studies with an enrichment culture that was obtained after 7 successive transfers of phenol‐adapted sludge into mineral medium with phenol as the only carbon source indicated that the acetate and glucose‐degrading capabilities were diminished or almost completely lost. In these enrichment cultures, phenol degradation was not affected by the presence of glucose, but glucose was not degraded. In contrary, the presence of acetate slightly slowed down the phenol degradation rate of the enrichment culture. Growth of the microorganisms apparently occurred at the expense of phenol and acetate respiration. The result of this work may be of practical importance in determining the feeding strategy, which is the key factor for most biological wastewater treatment systems. When acetate was present together with phenol in a wastewater, the phenol degradation rates were influenced by acetate, since acetate was an intermediate of phenol degradation. Glucose as an “independent substrate” was apparently degraded by other bacteria via acetate, and in this way it also influenced the phenol degradation rates. Glucose‐degrading bacteria could be “washed out” from the acclimatized sludge during several transfers into mineral medium with phenol as the sole carbon source. If later on, glucose was added again, it remained undegraded and did not influence phenol degradation. 2‐Chlorophenol degradation also requires other bacteria than phenol degraders.     

Isolation and characterization of phenol-degrading yeasts from an oil refinery wastewater in Brazil

This study investigated the aerobic degradation of phenol by yeast strains isolated from an oil refinery wastewater from the Northeast of Brazil. The samples displayed low fungal diversity, as only yeast colonies were detected on Sabouraud dextrose agar containing chloramphenicol 0.05% (w/v). Among the isolates, three yeast strains were selected to be evaluated for their potential for degrading high phenol concentrations. These species were identified through morphological and biochemical characteristics as Candida tropicalis, C. rugosa, and Pichia membranaefaciens. Although the strains were able to degrade the phenol concentration present in the wastewater, which was 7 mg l⁻¹, only C. tropicalis was capable of growing at high concentrations of phenol such as 500 mg l⁻¹ and 1,000 mg l⁻¹ in a mineral medium containing this pollutant as the only carbon source. C. rugosa and P. membranaefaciens were inhibited in the presence of 500 mg l⁻¹ of phenol. However, a longer incubation time was needed for C. tropicalis strain to degrade 1,000 mg l⁻¹ of phenol compared to the time required to degrade 500 mg l⁻¹. Moreover, the strain released a significant amount of polysaccharide biosurfactant in the medium probably to minimize the toxic effect of the high phenol concentration. When challenged with 1,500 and 2,000 mg l⁻¹ of phenol, C. tropicalis was unable to grow at the tested conditions. The results indicate that this strain of C. tropicalis can be considered both a good phenol-degrader and biosurfactant-producer. Application of this strain might be useful in bioremediation activities or treatment of phenol-polluted wastewater.     

采用苯酚羟化酶基因特异引物检测苯酚降解菌

根据苯酚羟化酶基因高度保守序列设计了一对该基因的特异PCR引物。采用该特异引物从苯酚降解菌醋酸钙不动杆菌 (Acinetobactercalcoaceticus)PHEA 2的总DNA中扩增到唯一一条大小为 684bp的片段。该DNA片段与已知的A .calcoaceticusNCIB82 50的苯酚羟化酶基因具有高度的同源性 ,其核苷酸序列的同源性为 84% ,推导的氨基酸序列的同源性为 98%。对苯酚和非苯酚降解菌株的PCR扩增结果表明 :所有苯酚降解菌均能扩增出 684bp的特征片段 ,而非苯酚降解菌则无PCR条带。对炼焦废水中的细菌群落进行PCR扩增和生化特性检测表明 :显示 684bp特征片段的菌株均具有苯酚降解特性。上述结果表明 ,利用苯酚羟化酶基因的特异引物可对环境中的苯酚降解菌株进行准确快速的PCR检测。     

低温高效苯酚降解菌的分离与降解特性

从哈尔滨太平污水厂活性污泥中筛选到7株高效苯酚降解菌,可利用苯酚作为唯一碳源和能源。通过对这7株菌在不同温度、pH值、以及不同苯酚浓度下生长和苯酚降解情况的考察,确定了这7株菌的最适生长温度为10°C,最适pH值为7.5,最大可降解苯酚浓度为3000mg/L。通过对这7株苯酚降解菌降解性能的研究表明:其具有较强的苯酚降解能力,在10°C、pH值为7.5、装液量为50mL、接种量15%、摇床振荡速度160r/min的条件下,反应48h后可使500mg/L的苯酚降解率达90%以上。葡萄糖对菌体的生长及苯酚降解能力均有一定的影响,当葡萄糖浓度是500mg/L时,该菌对苯酚的降解率仍在80%以上。该研究对处理含有其它碳源的含酚废水具有一定的意义。通过DGGE图谱条带的分析表明,其亮度可以说明这些菌在各个系统中均表现为优势菌,且在污水环境中表现出较强的活性,其优势地位能够稳定地存在。其中2、4、24、28条带丰富,表现出它们在污水环境系统中的多样性。     

Bioaugmentation with a novel alkali-tolerant Pseudomonas strain for alkaline phenol wastewater treatment in sequencing batch reactor

A novel alkali-tolerant strain JY-2, which could utilize phenol as sole source of carbon and energy, was isolated from activated sludge. It was identified as Pseudomonas sp. by 16S rDNA sequencing analysis. The appropriate conditions for strain growth and phenol biodegradation were as follows: pH 8.0–10.0 and temperature 23–30°C. With initial phenol concentrations of 225, 400, 550 and 750 mg/l, the degradation efficiencies were 94.9, 93.3, 89.3 and 48.2% within 40 h at pH 10.0 and 30°C, respectively. The alkaline phenol-containing wastewater treatment augmented with strain JY-2 in sequencing batch reactor (SBR) system was investigated, which suggested that the bioaugmented (BA) system exhibited the better performance for adjusting high pH to neutral value than the non-bioaugmented (non-BA) one. Also, the BA system showed strong abilities for phenol degradation and maintaining good sedimentation coefficient (SV₃₀). The microbial community dynamics of both sequencing batch reactor (SBR) systems were analyzed by Denaturing Gradient Gel Electrophoresis (DGGE) technique, which showed substantial changes between the two systems. This study suggests that it is feasible to treat alkaline phenol-containing wastewater augmented with strain JY-2.     

不同富集和分离培养条件下的含酚废水处理生物膜微生物群落结构与活性比较分析

采用Biolog和变性梯度凝胶电泳(DGGE)技术研究了不同苯酚浓度培养对焦化废水处理厂反硝化池生物膜样品中微生物群落结构和代谢类型的影响。DGGE结果表明, 不同浓度苯酚和不同培养方式富集培养后, 细菌16S rDNA的部分条带分布谱形发生改变, 还有部分条带只受到了苯酚浓度变化的影响; 富集培养过程中由于碳源组成相对焦化废水简单, DGGE条带所代表的优势微生物多样性有所降低。Biolog试验结果表明, 生物膜样本的细菌群落代谢能力最强; 低浓度苯酚富集后的样品能利用的底物碳源类型最丰富。对Biolog试验结果的主成分分析显示, 相同浓度苯酚富集培养后的细菌群落代谢功能多样性相似, 但从DGGE结果看出其结构组成产生了变化。富集培养使样品微生物群落的代谢功能发生改变, 低浓度的苯酚富集增加了群落中微生物的代谢类型。而不同条件获得的分离物其苯酚降解能力的初步分析也表明, 富集与分离条件对苯酚降解菌的分离能力和得到的菌株特性具有差别。     

Hydrocarbon degradation and enzyme activities of cold-adapted bacteria and yeasts

The potential of 89 culturable cold-adapted isolates from uncontaminated habitats, including 61 bacterial and 28 yeast strains, to utilize representative fractions of petroleum hydrocarbons (n-alkanes, monoaromatic and polycyclic aromatic hydrocarbons) for growth and to produce various enzymes at 10°C was investigated. The efficiency of bacterial and yeast strains was compared. The growth temperature range of the yeast strains was significantly smaller than that of the bacterial strains. Sixty percent of the yeasts but only 8% of the bacteria could be classified as true psychrophiles, showing no growth above 20°C. A high percentage (89%) of the yeast strains showed lipase activity. More than one-third of the 61 bacterial strains produced amylase, -lactamase, -galactosidase or lipase; more than two-thirds were protease producers. Only 6% of the bacterial strains but 79% of the yeast strains utilized n-hexadecane for growth; 13% of the bacterial strains and 21–32% of the yeast strains utilized phenol, phenanthrene or anthracene for growth. Only four yeast strains but none of the bacterial strains could grow with all hydrocarbons tested. The biodegradation of phenol was investigated in fed-batch cultures at 10°C. Three yeast strains degraded phenol concentrations as high as 10 mm (one strain) or 12.5 mm (two strains). Of eight bacterial strains, two strains degraded up to 10 mm phenol. The optimum temperature for phenol degradation was 20°C for all eight bacterial strains and for two yeast strains. Biodegradation by five yeast strains was optimal at 10°C and faster at 1°C than at 20°C. All phenol-degrading strains produced catechol 1,2 dioxygenase activity.Communicated by K. Horikoshi