高效降解烷烃的无色杆菌X_L株的分离鉴定及其降解特性

从山东东营胜利油田附近被石油污染的土壤中,通过逐步驯化筛选原油降解菌株,并对其降解特性进行研究。结果表明:①分离到一株以原油为唯一碳源,且摇瓶培养对原油降解率高达72.6%的降解菌XL。②通过形态、生理生化和16 SrDNA序列分析,XL与木糖氧化无色杆菌(Achromobacter xylosoxidans)的同源性达99.0%,最终确定为木糖氧化无色杆菌(Achromobacter xylosoxidans)。③通过GC-MS分析表明,菌株对C12-C23和C27-C43的烷烃几乎能彻底降解,说明菌株具有高效较宽的烷烃降解谱。④XL菌株发酵液溶血圈和排油圈直径较大,分别为3.6 cm和4.7 cm,说明其能产生表面活性剂。⑤通过油污土壤的室内外培养降解试验发现,当土壤中原油含量为10%时,锯末、秸秆、菌剂及N、P营养物协同处理60 d后,土壤原油降解率为79.5%(室内)和71.0%(室外),明显高于仅加菌剂或加菌剂+N、P营养物的处理组。⑥室外堆制试验中,XL菌株对石油污染物的降解速度、能力均低于室内的。     

功夫菊酯降解菌 GF-1 的分离鉴定及其降解特性研究

从农药厂废水处理池的活性污泥中分离到一株功夫菊酯高效降解菌 GF-1,根据表型特征、生理生化特性和 16S rDNA 序列同源性分析,将 GF-1 鉴定为芽孢杆菌属(Bacillus sp.).GF-1 能以功夫菊酯为唯一 C 源进行生长,72 h 对100 mg/L的功夫菊酯的降解率达 85%.GF-1 降解功夫菊酯的最适温度为 30℃,最适 pH 为 7.0,该菌降解功夫菊酯的速率与初始接种量呈正相关.GF-1 投加土壤,可以提高土壤中功夫菊酯的降解速率.通过吖啶橙与升温法对质粒消除,结果表明 GF-1 的降解功能与质粒相关.     

人工湿地高效好氧反硝化菌的分离鉴定及反硝化特性研究

从增氧型复合垂直流人工湿地中采集样品,利用间歇曝气法富集好氧反硝化菌,并进行分离纯化,共得到10株好氧反硝化菌。其中编号为B13的菌株在初始硝态氮含量为277.23mg·L-1、碳氮比为5的条件下,24h的硝态氮去除率达92.80%,亚硝态氮积累只有12.57mg·L-1,脱氮速率达到20.58mg·L-·1h-1。16S rDNA序列分析表明,该菌与Pseudomonas stutzeri同源性达100%。选用四因素三水平L（934）正交试验表设计实验,通过测定对硝态氮去除能力和亚硝态氮的积累量,研究碳源、碳氮比（C/N）、pH以及溶解氧含量（DO）4种不同因素对B13号菌株好氧反硝化性能的影响。结果表明,该菌株对硝态氮的去除率最大可达99.88%,几乎没有亚硝态氮积累。对硝态氮去除率影响最大的因素为碳氮比,其次为pH,溶解氧含量和碳源。对应的最优条件是碳源为葡萄糖,碳氮比为10,pH为9,溶解氧含量为1.84～3.57mg·L-1。     

猪毛角蛋白降解菌的分离筛选及其降解特性研究

为了筛选能高效降解猪毛角蛋白的微生物,该研究利用猪毛角蛋白为唯一碳氮源的培养基从土壤中分离角蛋白降解菌,并对其菌种分类及降解角蛋白的特性进行了研究。通过驯化共筛选到6株能够降解猪毛角蛋白的菌株,经过7 d发酵培养,菌株X-3对猪毛角蛋白的降解率达77.3%,降解效果最佳。结合菌株形态特征、生理生化特征及16S rDNA发育进化树分析,初步鉴定该菌株为凝结芽孢杆菌(Bacillus coagulans)。菌株X-3是一株广谱的角蛋白降解菌,对猪毛、羊毛、鸡毛、鹅毛、鸭毛的降解率均大于60%。该菌株在角蛋白底物作用下可产生角蛋白酶和二硫键还原酶,且酶活与猪毛角蛋白降解率变化存在显著的正相关性,表明菌株X-3产生的角蛋白酶和二硫键还原酶在角蛋白降解中起到非常关键的作用。在降解过程中,产生了大量的可溶性蛋白,二硫键中的硫也随之转化为硫酸盐、亚硫酸盐和巯基化合物,其中硫酸盐是主要的转化形式。该菌株的分离筛选丰富了猪毛角蛋白降解菌的微生物资源库,在氨基酸饲料生产中具有潜在的应用价值。     

氧化亚氮还原细菌YSQ030 的筛选、鉴定及其植物促生特性

筛选获得具有氧化亚氮(N₂O)还原活性及植物促生特性的细菌,为新型微生物肥料的研发与应用提供微生物资源。通过固氮培养基筛选以及含硝酸钠和琥珀酸钠的营养肉汤培养基纯化,从水塘芦苇根际土中分离得到一株细菌YSQ030;通过形态观察、16S rRNA基因序列和生理生化特征分析,对菌株进行鉴定;采用无氮反硝化培养基测定菌株的N₂O还原能力;通过纯培养试验测定菌株的促生特征并通过水培试验研究其对水稻的促生效应。结果显示,菌株YSQ030为反硝化无色杆菌(Achromobacter denitrificans),含nosZ基因;在30℃、转速为200r/min、氧气浓度为0%的前提下,还原N₂O的效率为66.3%;分泌吲哚乙酸、具有溶磷和产生1-氨基环丙烷-1-羧酸(ACC)脱氨酶的能力;温室水培试验结果表明,与未接菌的对照组相比,接种YSQ030菌液显著提高了水稻幼苗的地上部鲜重和根鲜重,分别增加了48.3%和37.6%。本研究表明,YSQ030具有明显的N₂O还原能力,同时对水稻生长具有显著的促进作用,可为...     

菲降解菌的分离鉴定及其在污染土壤生物修复中的应用

采用室内培养方法,从吴江市郊长期被多环芳烃污染的土壤中富集到以菲为唯一碳源和能源的菲降解复合微生物菌群,复合菌群在7d内对无机盐液体中菲（含量100mg·L^-1）的降解率达到99%。从复合菌群中分离纯化获得两株菲高效降解菌B1和L2,经过菌体形态特征、生理生化特征和16SrDNA序列分析,鉴定菌株B1为百日咳博行特氏菌（Bordetella petrii）,菌株L2为墨西哥假黄单胞菌（Pseudoxanthomonas mexicana）。这两株菌在菲含量为100mg·L^-1的无机盐培养液中,7d内对菲（含量100mg·L^-1）的降解率大约为80%,9d内的降解率可达到99%。将复合菌群和菲污染土壤混合,在光照培养箱中进行培养修复。结果表明,修复88d后,接种复合菌群的低污染浓度（8.22mg·kg^-1）处理和高污染浓度（39.65mg·kg^-1）处理的菲去除率分别达到95.74%和98.06%。     

一株芽孢杆菌属的苄嘧磺隆降解菌的分离鉴定和降解研究

The objectives of this study were to isolate a bensulfuron-methyl (BSM)-degrading strain of Bacillus spp. and to evaluate its effectiveness in remediation of a BSM-contaminated soil. A BSM-degrading bacterium, strain L1, was successfully isolated in this study. Strain L1 was identified as Bacillus megaterium based on its morphological, physiological, and biochemical properties, G+C content, phylogenetic similarity of 16S rDNA, and fatty acid compositions. Two experiments were used to examine BSM degradation by strain L1. When BSM was used as a sole carbon source in a mineral salt medium, the average degradation rate of BSM by strain L1 was 12.8%, which suggested that the strain was able to utilize BSM as a sole carbon and energy source. In addition, supplement of yeast extract (200 mg L-1) significantly (P ≤ 0.01) accelerated the degradation of BSM by strain L1. Almost complete degradation (97.7%) of BSM could be achieved in 84 h with addition of yeast extract. In addition, when a sterile soil was supplemented with BSM (50 mg L-l), BSM degradation rate was 94.3% in 42 d, indicating the potential of using microbes for the remediation of BSM-contaminated soils in fields.     

拟除虫菊酯类农药残留降解菌的筛选及其生理特性研究

从活性污泥的富集培养物中分离得到可降解几种拟除虫菊酯农药的菌株qw5,初步鉴定qw5为地衣芽孢杆菌 (Bacilliuslicheniformis)。菌株qw5在通气、pH7～ 8、温度 3 0℃左右的环境条件下生长较好。培养 5天 ,菌株qw5对氰戊菊酯、氯氰菊酯、溴氰菊酯的降解率分别为 5 3 .8%、41 .2 %和 61 .7%。经质谱分析 ,菌株qw5降解氰戊菊酯产生中间代谢产物 3 -苯氧基苯甲醛。qw5对小白鼠无致病力 ,对几种常用的抗生素敏感。盆钵和小区试验表明 ,菌株qw5对青菜中残留的拟除虫菊酯有明显去除效果。     

阿特拉津降解菌ADH-2的分离、鉴定及其特性研究

从长期施用阿特拉津的玉米地中采集土样,通过富集培养的方法分离出一株能以阿特拉津为唯一碳、氮源生长的细菌ADH-2,结合生理生化特陛及16SrRNA基因的相似性分析将其初步鉴定为节杆菌属（Arthrobacter sp．）。该菌在10h内对100mg·L-1阿特拉津的降解率为99．9％。外加氮源能促进菌株的生长,但对阿特拉津的降解有轻微的抑制作用。外加蔗糖和葡萄糖能显著促进菌株的生长,但对阿特拉津的降解表现出显著的抑制。而淀粉既能促进菌株的生长又能促进阿特拉津的降解。对其降解基因的初步研究显示,该菌含有trzN、atzB和atzC3个阿特拉津降解相关基因。通过与本实验室另外两株阿特拉津降解菌比较,菌株ADH-2具有更好的应用潜力。     

Impact of low-molecular-weight organic acids on the availability of phenanthrene and pyrene in soil

The impact of low-molecular-weight organic acids (LMWOAs) on the availability of phenanthrene and pyrene was investigated using laboratory batch assays. Experiments were conducted with two types of soil with different organic contents. The LMWOAs used were citric and oxalic acid. A mild solvent extraction procedure and a sorption-desorption experiment were used to predict the availability of phenanthrene and pyrene. Results showed that the extractable amounts of phenanthrene and pyrene in both soil types increased with increased citric or oxalic acid concentration. Citric acid addition promoted phenanthrene and pyrene extraction to a greater degree than oxalic acid. Compared with freshly spiked soils, the extractable amounts of phenanthrene and pyrene with the addition of LMWOAs decreased significantly after 60 days of cultivation. Soils with higher values of soil organic carbon content (f_oc) showed decreased phenanthrene and pyrene availability after the addition of LMWOAs. The sorption and desorption results also demonstrated the enhanced availability of PAHs with LMWOA addition. Phenanthrene sorption could be described using a linear model, regardless of the addition of LMWOAs. The simulated distribution constants (K_d) for phenanthrene sorption decreased significantly with the addition of LMWOAs. In contrast, phenanthrene desorption clearly increased with the addition of LMWOAs. These results suggest that the availabilities of phenanthrene and pyrene can be increased with the addition of suitable LMWOAs.     

Determining tolerance limits for restoration and phytoremediation with Spartina patens in crude oil-contaminated sediment in greenhouse

Oil spills may detrimentally damage sensitive coastal habitats, such as coastal wetlands. Successful restoration of oiled habitats primarily depends on the tolerance of vegetation transplants to oil. In this study, tolerance limits of the dominant coastal brackish marsh plant Spartina patens to South Louisiana crude (SLC) oil and its phytoremediation effectiveness on petroleum hydrocarbons in wetland sediments were investigated in the greenhouse environment. Spartina patens was transplanted into brackish marsh sediments contaminated with SLC oil at concentrations of 0, 40, 80, 160, 320, 640 and 800 mg SLC oil g^?1 dry sediment. High oil concentrations adversely affected plant stem density, aboveground biomass and belowground biomass even one year after transplantation. At the 320 mg g^?1 oil dosage, plant belowground biomass was significantly lower than the control although aboveground variables were not significantly different from the control. All plant parameters mentioned above at the 640 mg g^?1 oil dosage were less than 50% of the control. Spartina patens did not survive the 800 mg g^?1 oil dosage. The tolerance limit of S. patens to SLC was estimated about 320 mg oil g^?1 dry sediment. In addition, S. patens transplants enhanced oil degradation in the sediment; concentrations of residual total petroleum hydrocarbons (TPH) in the sediments vegetated by S. patens were significantly lower than those of un-vegetated sediments for both the surface and subsurface sediment at the 40 and 160 mg g^?1 SLC oil dosages. Decreases in the concentrations of polycyclic aromatic hydrocarbons (PAHs) further demonstrated the capacity of S. patens to phytoremediate residual oil; residual total targeted PAHs in the phytoremediation treatment were less than 20% of the un-vegetated treatment at the 40 mg g^?1 oil dosage. These results demonstrated the potential of phytoremediation with S. patens to simultaneously restore and remediate petroleum-contaminated coastal marsh habitats.     

Comparison for plant uptake of phenanthrene and pyrene from soil and water

In this study, we evaluated (1) the plant uptake of polycyclic aromatic hydrocarbons (PAHs) from soil and water and (2) the applicability of the partition-limited model on the prediction of plant concentrations with respect to PAH contents in soils and other associated parameters. To accomplish these goals, the plant uptake of PAHs from culture solution and soils were extensively experimented. A steady state was shown for ryegrass kinetic uptake of phenanthrene and pyrene from water after about 48 h. As to the ryegrass uptake from soils, root and shoot concentrations of PAHs generally increased, while root concentration factors (RCFs) and shoot concentration factors (SCFs) tended to decrease with the increasing PAH concentrations in soils after 45 days. One note of interest is that root concentrations and RCFs of phenanthrene and pyrene for ryegrass uptake were larger than shoot concentrations and SCFs, irrespective of soil–plant and water–plant systems. However, root and shoot concentrations, or RCFs and SCFs, for ryegrass uptake from culture solution were always much higher than those for ryegrass uptake from soils at the same PAH concentrations in water or soil interstitial water, indicating that PAHs in culture solution would be more available and susceptible than those in soil interstitial water for uptake by plants. In addition, the partition-limited model showed a high level of model performance on prediction of plant uptake of phenanthrene and pyrene from soils, with the overall differences of the modeled and experimented concentrations in ryegrass roots or shoots less than 187%. This suggests that the partition-limited model might be a potentially useful instrument for vegetation-contamination assessment.     

Changes in Polycyclic Aromatic Hydrocarbons (PAHs) and Soil Biological Parameters in a Revegetated Coal Mine Spoil

Coal mining leads to severe land degradation and creates huge amounts of mine spoil. Coal mine spoil contains toxic polycyclic aromatic hydrocarbons (PAHs) derived from coal, which can be alleviated through revegetation with suitable tree species. The present study was aimed at evaluating the impact of different tree species (Albizia lebbeck , Cassia siamea , Delonix regia , and Dalbergia sissoo ) on the quality of coal mine spoil and changes in PAH concentration. Soil samples were collected from the revegetated coalmine overburden dumps of Jharia coalfield, Dhanbad, India and analysed by GC‐MS for 16 priority PAHs and soil quality parameters were analyzed by standard analytical protocols. Reclamation improved the biological properties of the mine spoil: microbial biomass (+59–176%), dehydrogenase activity (+46–198%), fluorescein diacetate hydrolase activity (+104–127%), phenol oxidase activity (+150–250%), and peroxidase activity (+93–181%). PAH concentration in revegetated mine spoil ranged from 0 · 51 to 1 · 35 mg kg⁻¹, with a significance reduction in total as well as individual PAHs. For individual tree species, total PAH reduction decreased in the order: C. siamea (81 · 6%) > A. lebbeck (55 · 6%) > D. regia (51 · 9%) > D. sissoo (51 · 5%). Correlation analysis showed significant association between the degradation of PAHs and soil biological properties of revegetated site. Microbial biomass carbon and soil enzymes were negatively correlated with PAH content in the mine spoil. But microbial stress indicators like respiration/microbial carbon ratio were not correlated, which revealed no adverse effect of PAH on soil microbes. Principal component analysis revealed that most of the biological parameters were closely associated with the degradation of low molecular weight PAHs. Copyright © 2016 John Wiley & Sons, Ltd.     

多环芳烃长期污染土壤的微生物强化修复初步研究

本研究通过室内模拟试验,以急性毒性较强的菲(Phe)和遗传毒性较强的苯并[a]芘(B[a]P)为代表性多环芳烃(PAHs)污染物,以不同C源、通气状况和水分条件为调控因子,对PAHs长期污染土壤的土著微生物强化修复进行初步研究。结果表明,搅动处理使污染土壤中Phe和B[a]P的降解率分别达59.44%和26.14%,而淹水处理使两者降解率分别达46.48%和13.27%。添加C源(淀粉和葡萄糖)处理提高了土壤中PAHs的降解率,且随着C源的施用量而增加。同时也发现污染土壤中PAHs降解菌和微生物总量呈正相关,并随着PAHs降解菌数量的增加,土壤中PAHs降解率也随之提高。可见,土壤中PAHs降解速率主要决定于PAHs的降解菌数量。     

淀粉对无芒雀麦修复煤矿区多环芳烃长期污染土壤的影响

采用温室盆栽试验,以无芒雀麦(W)为修复植物,研究淀粉(D)不同剂量与无芒雀麦组合对煤矿区多环芳烃(PAHs)长期污染农田土壤修复的影响。结果表明,在土壤中添加淀粉剂量D1、D2培养3个月后,显著促进了土著微生物对煤矿区长期污染农田土壤PAHs的降解。D1、D2处理土壤中16种PAHs总量降解率(16.82%,19.06%)分别比对照CK(11.12%)提高了51.26%和71.40%,尤其对6环PAHs的降解增效最为突出。D2处理对6环PAHs的降解达最高,为37.05%,比CK处理提高了241.01%,且为D1处理的1.77倍。在污染土壤中添加淀粉并种植无芒雀麦(D1+W,D2+W)后,与对照CK、淀粉(D)和种植无芒雀麦单一处理(W)相比,土壤中16种PAHs总量降解率有了明显增加,D1+W与D2+W处理下16种PAHs降解率分别为26.26%和28.39%;在PAHs不同环数中对5,6环PAHs的修复效果提升最为明显,对其它环数PAHs降解效果提升不显著,其中5环和6环PAHs降解率在D2+W组合处理下达到最高,为48.63%和58.32%,比CK处理提高了110.36%和436.82%,比无芒雀麦单一处理提高了27.50%和47.77%。从土壤酶活性角度,淀粉、无芒雀麦单一处理及其组合下的土壤过氧化氢酶活性差异不明显,但均显著高于对照CK处理;无芒雀麦对多酚氧化酶活性有明显的激活作用,且与淀粉组合进一步显著提升了该酶的活性,与D1+W与D2+W组合下的5,6环PAHs降解率达最高相一致。综上,利用淀粉与无芒雀麦的优化组合形式能明显提升煤矿区5,6环PAHs长期污染农田土壤的修复效果,可以作为该区污染土壤修复治理的一种选择。     

丛枝菌根作用下土壤中多环芳烃的残留及形态研究

采用盆栽试验方法,以苊为多环芳烃(PAHs)代表物,研究了丛枝菌根(AM)作用下土壤中 PAHs 的残留及形态.供试污染土壤中苊的起始浓度为 35.0 mg/kg.结果表明,AM作用下土壤中苊总残留量明显降低;接种摩西球囊霉菌Glomus mosseae或幼套球囊霉菌Glomus etunicatum后,供试两个污染土样中苊总残留降解率达32.7%～45.2%,比未接种对照高 6.8%～9.8%.有机溶剂提取态是土壤中苊残留的主要部分,AM 作用促进了苊各形态之间的转化;接种AM后土壤 1、2 中苊可脱附态和有机溶剂提取态残留量分别比对照降低了17.0%～37.8% 和 5.4%～26.6%,而结合态残留量比对照增加了12.2%～89.5%.AM 作用能降低土壤中苊可提取态残留含量;但培养55 天后土壤中仍有 65.7%～81.7% 苊属于可提取态残留,对生物有毒害风险.     

Sediment-associated retene bioavailability of sediment-associated retene to an oligochaete wormlumbriculus variegatus

The bioavailability of retene spiked to two sediment concentrations (50 and 200 μg/g dry weight) was measured in long-term (28-day) bioaccumulation tests performed on the oligochaeteLumbriculus variegatus Müller. Three sediment organic carbon (SOC) concentrations (1.1, 3.0, 23.0% of dry weight) were used. Growth and reproduction, used as endpoints, indicated that retene was chronically nontoxic at the concentrations employed. Retene was clearly bioavailable, but bioaccumulated about 100 times less in the SOC-rich (23.0%) sediment than in the other two (SOC <3.0%). At the lower retene concentration, retene in the worms was undetectable in the high-SOC sediment. Based on final retene present at the end of exposure, the bioaccumulation factors (BAFs) ranged between 1.1–28.2. Normalization to organism lipid and SOC content diminished the variation, so that the biota-sediment accumulation factors (BSAFs) varied from 0.8 to 13.5. When calculated from initial retene concentrations, BAFs within the range of 0.04 to 5.3 and BSAFs from 0.1 to 2.5 were obtained. Results demonstrate that high levels of SOC diminish the bioavailability of sediment-associated retene inL. variegatus considerably. However, the remainder variation in the BSAFs suggests that besides of SOC and worm lipid contents there are also other factors that can affect the bioaccumulation dynamics of sediment-associated retene and the final outcome of its bioavailability.     

产漆酶真菌筛选及其对PAHs污染土壤修复的初步研究

真菌漆酶可以高效转化多环芳烃(PAHs),因此,产漆酶真菌在PAHs污染土壤修复中极具应用前景。根据漆酶可将愈创木酚氧化为红色物质的特性,成功从土壤中筛选出一株能够分泌漆酶的真菌菌株F-1,初步鉴定该菌为疣孢漆斑菌(Myrothecium verrucaria)。通过Plackett-Burman试验对菌株F-1的产酶能力进行了分析,发现特定培养条件组合可将其酶活提高近300倍,达5628 U L-1,表明F-1的漆酶活性受到环境条件的显著影响。应用菌株F-1对PAHs污染土壤进行了初步修复研究,结果表明,接种F-1对菲、荧蒽、芘、苯并(a)蒽、屈、苯并(b)荧蒽、苯并(k)荧蒽、苯并(a)芘、二苯并(a,h)蒽、苯并(g,h,i)苝、茚苯(1,2,3-cd)芘等11种PAHs均有不同程度的降解,提示产漆酶真菌在PAHs污染土壤修复中的应用潜力。     

丛枝菌根对芘污染土壤修复及植物吸收的影响

采用温室盆栽试验方法,研究了两种丛枝菌根真菌Glomus mosseae和 Glomus etunicatum对三叶草(Trifolium subterraneum L.)和辣椒（Capsicum annuum L.）修复芘污染土壤的影响。供试土样中芘初始浓度为0 ~ 75.18 mg/kg。结果表明,接种AMF可促进供试植物对土壤中芘的吸收,并且显著提高三叶草根的芘含量、根系富集系数、根和茎叶的芘积累量,但对辣椒根和茎叶芘含量、根系富集系数的影响不显著,这主要与植物的菌根侵染率和“菌根依赖度”不同有关。接种AMF土壤中芘的削减率高于普通植物修复,但植物吸收积累对修复的贡献率小于0.2%;因此推测,AM作用下良好的根际环境对土壤微生物数量和活性的提高、进而对土壤中芘降解的促进可能是菌根修复的主要机理。