脂肽生物表面活性剂作用下多菌种石油生物降解影响因素优化

探索多种菌种降解石油过程中菌种和脂肽生物表面活性剂的作用,筛选石油降解的主要因素及最佳合,并为石油污染物的降解机理研究和石油污染修复提供指导。基于正交实验筛选主要影响因素,采用Box-Behnken实验探讨各因素最佳水平。正交实验中脂肽生物表面活性剂是多菌种降解石油过程中最主要的影响因素,在Box-Behnken实验中,其能显著地影响石油降解率。菌种降解能力是石油饱和烃组分生物降解的最主要影响因素,但脂肽生物表面活性剂是芳烃、胶质和沥青质组分降解的最主要的影响因素。研究所用菌种中,解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和假单胞菌(Pseudomonas aeruginosa)在石油降解过程最重要,是本实验的石油降解最优菌。菌种和脂肽生物表面活性剂的添加浓度配比对于石油降解具有重要的影响。解淀粉芽孢杆菌和假单胞菌添加量5%,脂肽生物表面活性剂粗品添加量200 mg·L~(-1)的降解效果最优,理论上,最高降解率可达63.78%,验证降解率达到了53.89%,相对于多菌种正交实验最高降解率提高了5.54%。利用正交实验和Box-Behnken实验筛选最优降解菌和最优菌种组合的方法,具有分析因素多、实验量少等优点,具有较好的应用前景。     

用于石油污染土壤降解的高效降解菌的筛选及其降解条件优化

经过富集、分离优选出高效石油降解菌L-1,根据形态观察和生理生化特征初步鉴定为琼氏不动杆菌;采用单因素花盆实验模拟微生物原位修复并对其降解条件进行优化。结果表明,将高效石油降解菌应用于修复石油污染土壤,适宜接种量、表面活性剂浓度、CNP比、翻耕频率分别为15%、0.1%、100∶10∶1和1 d 1次;在该降解条件下修复28 d,可达到16.80%的石油降解率,远远高于土著微生物6.92%的降解率。     

生物炭添加对餐厨废弃物堆肥腐殖酸形成及微生物演替的影响

针对餐厨废弃物堆肥腐殖酸不稳定易降解的问题,研究生物炭添加对餐厨废弃物堆肥腐殖酸形成的影响。一共设置两个处理(CK,不添加生物炭;BC,按照堆体干质量添加5%生物炭),整个堆肥周期持续15 d。堆肥结束时,BC的还原糖、氨基酸、多酚分别为2.05、1.57、2.25 mg/g,分别比CK低1.97、0.68、0.43 mg/g; BC的胡敏酸(HA)、HA/富里酸(FA)(质量比)分别为17.04 mg/g、1.85,较CK提升了61.21%、110.23%。BC的气单胞菌(Aerococcus)、土地芽孢杆菌(Terribacillus)、蜡样芽孢杆菌(Cerasibacillus)、巨球菌(Macrococcus)、Tepidanaerobacter相对丰度比CK高0.30、0.04、0.01、0.01、0.03百分点,土芽孢杆菌(Geobacillus)、小多孢菌(Micropolyspora)相对丰度比CK分别低0.79、0.16百分点。结果表明添加生物炭有利于：(1)还原糖、氨基酸、多酚向腐殖酸的转化;(2)促腐微生物(气单胞菌、土地芽孢杆菌、蜡样芽孢杆菌、巨球菌、Tepid...     

Tween-80和H2O2对Burkholderia cepacia降解油脂的影响

油脂废水是一种重要的水污染源.研究了表面活性剂和H2O2对洋葱伯克霍尔德氏菌(Burkholderia cepacia)降解油脂的影响.结果表明:适量的表面活性剂和H2O2促进油脂的降解.表面活性剂可以增强油脂在水相中的溶解度,提高油脂在水中的乳化程度从而增加其生物可利用性.当Tween-80浓度600mg/L时,油脂降解率达到91%.低浓度H2O2不仅能刺激微生物生长,而且使培养液中的pH值保持相对稳定,提高了微生物对油脂的降解率.H2O2在浓度为400mg/L时,能使油脂降解率达到90%.     

产表面活性剂菌筛选及其对柴油降解影响研究

运用微生物培养方法,从饭店排污口污泥中筛选了7株产表面活性剂菌株。为了研究产表面活性剂菌在柴油降解过程中所产生的影响,分别将这些菌株与柴油降解菌(实验室提取的另一株假单胞菌)共同作用,其中一株(B_(26))不但可以将降解诱导期从6 d缩短至4 d,而且能将降解率由原来的71.1%提高至80.6%,具有较大的研究价值。实验着重对B_(26)的作用机理、柴油降解的影响因素等进行了分析研究。经形态学和生理生化实验鉴定,B_(26)属于芽孢菌属,其代谢过程中产生脂肽类表面活性剂,利用冷冻干燥法提取生物表面活性剂产量为1.57 g/L。     

固定化微生物修复石油污染土壤影响因素研究

针对石油污染土壤修复,利用实验室已筛选的高效石油降解单菌SM-3,以天然有机材料为载体,吸附法制备固定化微生物。将游离与固定化微生物应用于室内花盆模拟修复石油污染土壤,对C/N/P、微生物投加量、石油含量、氧化剂和表面活性剂设计5因素4水平正交实验,探讨不同修复时期各影响因素的重要性顺序,最佳条件下各菌株的修复效果。结果表明,不同微生物在不同降解时期,各影响因素的重要性会发生变化;经过21 d的修复,固定化单菌SM-3石油降解率为22.77%,修复过程中,接种量是最重要的影响因素,营养元素N、P投加影响较大,表面活性剂和氧化剂影响次之。     

表面活性剂强化甲基营养型芽孢杆菌修复柴油污染土壤

使用吐温80(Tween80)、鼠里糖脂(RL)、十二烷基苯磺酸钠(SDBS)和十二烷基苯硫酸钠(SDS)强化甲基营养型芽孢杆菌(Bacillus methylotrophicus)修复柴油污染土壤,研究了表面活性剂对甲基营养型芽孢杆菌的影响。结果表明:甲基营养型芽孢杆菌可以较好地降解柴油,但由于土壤的吸附,土壤中柴油的修复效率较低,仅为22.68%;因此,需要使用表面活性剂进行强化。浓度为3 000 mg·L~(-1)的SDBS和SDS、2 000 mg·L~(-1)的Tween80、500 mg·L~(-1)的RL具有较好的洗脱效果。微生物毒性实验表明,Tween80和RL对甲基营养型芽孢杆菌具有促进作用,可以用于强化修复柴油污染土壤。2 000 mg·L~(-1)Tween80强化甲基营养型芽孢杆菌修复柴油土壤的修复效果(46.11%)优于500 mg·L~(-1)RL的修复效果(45.32%),同时具有较好的经济性,具有较好的应用前景。     

土壤中六六六和滴滴涕的堆肥生物修复研究

采用堆肥生物修复法对土壤中六六六(HCH)和滴滴涕(DDT)的降解效果进行了研究.试验结果表明,六六六和滴滴涕的降解先后经历了快速降解阶段和缓慢降解阶段;堆肥第21天后,六六六和滴滴涕降解率均达60%以上;γ-HCH降解效果最好,其次为β-HCH,α-HCH和δ-HCH的降解效果差别较大;p,p'-DDE的降解效果要好于p,p'-DDD,初始质量浓度为0.03 mg/kg的p,p'-DDT未得到降解;微生物对六六六和滴滴涕的降解作用均较明显.     

共代谢基质强化微生物修复四氯乙烯污染地下水

针对微生物修复地下水中四氯乙烯(tetrachloroethylene,PCE)周期长的问题,通过添加共代谢基质强化微生物修复技术以提高修复速率。以某污水处理厂的厌氧活性污泥为菌种来源,采用振荡培养法进行PCE高效降解菌群的驯化和筛选,对微生物降解PCE的温度、初始pH和PCE初始浓度3种影响因素进行了条件优化;使用甲醇、乙醇、葡萄糖、酵母浸膏以及乳酸钠作为共代谢基质,研究了不同共代谢基质条件下微生物群落对PCE的降解规律,并建立了反应动力学模型。结果表明:在种水平上,梭状芽孢杆菌Clostridium sp. FCB45是优势菌种;PCE初始浓度为1 mg·L-1,pH在中性,温度为30℃,共代谢基质为酵母浸膏时,微生物群落的降解效果最好,PCE降解率可高达96.75%,降解速率常数最高可达0.327 d-1;添加共代谢基质强化的微生物降解过程全部符合一级反应动力学模型。添加共代谢基质的微生物实验结果表明,添加共代谢基质可以有效缩短微生物修复周期,对污染地下水的原位生物修复具有一定的参考价值。     

石油降解菌的筛选优化及其对油污土壤的修复特性

分别以牛肉膏蛋白胨-布氏哈斯培养基、蓝色凝胶培养基作为初筛和复筛培养基,从石油污染土壤中筛选出2株可产生微生物表面活性剂的石油烃降解菌。并将菌株投加到油污土壤中进行修复研究,考查了不同影响因素对修复效果的影响。研究结果表明,(1)2株菌对中度石油污染土壤有较好的修复效果,向油污土壤中直接投加菌株修复70 d时对石油烃的去除率为52%;(2)向油污土壤中投加降解菌并同时补充氮营养液,修复70 d时对土壤中总石油烃的去除率可达到75%;对土壤中正构烷烃的去除率为66%;(3)与土壤的含水率及土著菌的降解效果相比,向油污土壤中投加降解菌以及补充氮磷营养液是影响石油污染土壤修复效果的关键因素。     

Bioremediation of diesel-contaminated soil with composting

The major objective of this research was to find the appropriate mix ratio of organic amendments for enhancing diesel oil degradation during contaminated soil composting. Sewage sludge or compost was added as an amendment for supplementing organic matter for composting of contaminated soil. The ratios of contaminated soil to organic amendments were 1:0.1, 1:0.3, 1:0.5, and 1:1 as wet weight basis. Target contaminant of this research was diesel oil, which was spiked at 10,000 mg/kg sample on a dry weight basis. The degradation of diesel oil was significantly enhanced by the addition of these organic amendments relative to straight soil. Degradation rates of total petroleum hydrocarbons (TPH) and n-alkanes were the greatest at the ratio of 1:0.5 of contaminated soil to organic amendments on wet weight basis. Preferential degradation of n-alkanes over TPH was observed regardless of the kind and the amount of organic amendments. The first order degradation constant of n-alkanes was about twice TPH degradation constant. Normal alkanes could be divided in two groups (C10-C15 versus C16-C20) based on the first order kinetic constant. Volatilization loss of TPH was only about 2% of initial TPH. Normal alkanes lost by volatilization were mainly by the compounds of C10 to C16. High correlations (r=0.80-0.86) were found among TPH degradation rate, amount of CO2 evolved, and dehydrogenase activity.     

炼厂"三泥"的生物处理研究

从大港油田筛选和分离出3株以原油为碳源的石油降解菌,初步鉴定为假单胞菌属(Pseudomonas sp.),用这3株菌混合处理某炼厂的"三泥",在适宜的降解条件下,仅需投加混合菌液,无需添加营养物(N,P),可使含油污泥A的残油量从67g/kg降至7.7g/kg,混合油泥试样AB的残油量从19.7g/kg降至3.1g/kg,平均去除率为84%以上,可以实现含油污泥的较为经济的、有效的处理.经生物处理后含油污泥的恶臭味完全消失.     

Slurry-phase biodegradation of weathered oily sludge waste

We assessed the biodegradation of a typical oily sludge waste (PB401) in Mexico using several regimes of indigenous microbial consortium and relevant bioremediation strategies in slurry-phase system. Abiotic loss of total petroleum hydrocarbons (TPH) in the PB401 was insignificant, and degradation rates under the various treatment conditions ranged between 666.9 and 2168.7 mg kg(-1) day(-1) over a 15 days reaction period, while viable cell count peaked at between log(10)5.7 and log(10)7.4 cfu g(-1). Biostimulation with a commercial fertilizer resulted in 24% biodegradation of the TPH in the oily waste and a corresponding peak cell density of log(10)7.4 cfu g(-1). Addition of non-indigenous adapted consortium did not appear to enhance the removal of TPH from the oily waste. It would appear that the complexities of the components of the alkylaromatic fraction of the waste limited biodegradation rate even in a slurry system.     

超声波/紫外线-Fenton反应联用去除克拉玛依土壤中石油类污染物

研究了超声波(ultrasonic)和紫外线(ultraviolet)-Fenton反应联用处理干旱区老化石油污染土壤。土壤TPH含量为30 470 mg/kg,pH值为3,H2O2与Fe比例为50∶1时,H2O2浓度为0.37%、0.74%、1.11%和1.85%在超声波处理6 h土壤TPH去除量分别为4 495、11 983、15 470和19 800 mg/kg;TPH去除量随H2O2/Fe2+增大而增大,H2O2/Fe2+为100∶1时,TPH去除量为12 699 mg/kg。溶液pH值接近中性,H2O2浓度为0.74%,H2O2/Fe2+为50∶1,超声波与UV共同作用2 h和4 h,TPH去除量分别达到14 824和21 821 mg/kg;UV单独作用2 h、4 h对土壤TPH去除量为9 253和12 845 mg/kg。超声波-Fenton反应对1,2-二甲苯降解效果最好,其次为C17-C28的直链及支链烷烃,最低为烃类衍生物。     

Bioremediation of acidic oily sludge-contaminated soil by the novel yeast strain Candida digboiensis TERI ASN6

Background, aim, and scope

Primitive wax refining techniques had resulted in almost 50,000 tonnes of acidic oily sludge (pH 1–3) being accumulated inside the Digboi refinery premises in Assam state, northeast India. A novel yeast species Candida digboiensis TERI ASN6 was obtained that could degrade the acidic petroleum hydrocarbons at pH 3 under laboratory conditions. The aim of this study was to evaluate the degradation potential of this strain under laboratory and field conditions.

Materials and methods

The ability of TERI ASN6 to degrade the hydrocarbons found in the acidic oily sludge was established by gravimetry and gas chromatography–mass spectroscopy. Following this, a feasibility study was done, on site, to study various treatments for the remediation of the acidic sludge. Among the treatments, the application of C. digboiensis TERI ASN6 with nutrients showed the highest degradation of the acidic oily sludge. This treatment was then selected for the full-scale bioremediation study conducted on site, inside the refinery premises.

Results

The novel yeast strain TERI ASN6 could degrade 40 mg of eicosane in 50 ml of minimal salts medium in 10 days and 72% of heneicosane in 192 h at pH 3. The degradation of alkanes yielded monocarboxylic acid intermediates while the polycyclic aromatic hydrocarbon pyrene found in the acidic oily sludge yielded the oxygenated intermediate pyrenol. In the feasibility study, the application of TERI ASN6 with nutrients showed a reduction of solvent extractable total petroleum hydrocarbon (TPH) from 160 to 28.81 g kg^?1 soil as compared to a TPH reduction from 183.85 to 151.10 g kg^?1 soil in the untreated control in 135 days. The full-scale bioremediation study in a 3,280-m² area in the refinery showed a reduction of TPH from 184.06 to 7.96 g kg^?1 soil in 175 days.

Discussion

Degradation of petroleum hydrocarbons by microbes is a well-known phenomenon, but most microbes are unable to withstand the low pH conditions found in Digboi refinery. The strain C. digboiensis could efficiently degrade the acidic oily sludge on site because of its robust nature, probably acquired by prolonged exposure to the contaminants.

Conclusions

This study establishes the potential of novel yeast strain to bioremediate hydrocarbons at low pH under field conditions.

Recommendations and perspectives

Acidic oily sludge is a potential environmental hazard. The components of the oily sludge are toxic and carcinogenic, and the acidity of the sludge further increases this problem. These results establish that the novel yeast strain C. digboiensis was able to degrade hydrocarbons at low pH and can therefore be used for bioremediating soils that have been contaminated by acidic hydrocarbon wastes generated by other methods as well.     

Isolation, identification and characterization of Bacillus amyloliquefaciens BZ-6, a bacterial isolate for enhancing oil recovery from oily sludge

Over 100 biosurfactant-producing microorganisms were isolated from oily sludge and petroleum-contaminated soil from Shengli oil field in north China. Sixteen of the bacterial isolates produced biosurfactants and reduced the surface tension of the growth medium from 71 to <30 mN m⁻¹ after 72 h of growth. These bacteria were used to treat oily sludge and the recovery efficiencies of oil from oily sludge were determined. The oil recovery efficiencies of different isolates ranged from 39% to 88%. Bacterial isolate BZ-6 was found to be the most efficient strain and the three phases (oil, water and sediment) were separated automatically after the sludge was treated with the culture medium of BZ-6. Based on morphological, physiological characteristics and molecular identification, isolate BZ-6 was identified as Bacillus amyloliquefaciens. The biosurfactant produced by isolate BZ-6 was purified and analyzed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry. There were four ion peaks representing four different fengycin A homologues.     

高效堆肥菌种的筛选及在城市污泥堆肥上的应用

分离筛选出了4株猪粪堆肥菌种,初步的分类鉴定表明它们均属于芽孢杆菌属.利用这些菌种制成混合菌剂,并进行城市污泥堆肥对比试验,结果显示,接菌处理在各项堆肥指标上明显优于未接菌的空白处理,自制菌与酵素菌在堆肥过程中具有非常相似的发酵能力,均能加快堆肥的升温速度,提高堆肥的最高温度,加快物料含水率的下降.说明自制菌不但可以应用于猪粪的堆肥发酵,在城市污泥高温堆肥方面也有很好的应用潜力.     

Dynamics of carbon, nitrogen and hydrocarbons in diesel-contaminated soil amended with biosolids and maize

Contamination of soil with hydrocarbons occurs frequently when petroleum ducts are damaged. Restoration of those contaminated soils might be achieved by applying readily available organic material. An uncontaminated clayey soil sampled in the vicinity of a duct carrying diesel which ruptured recently, was contaminated in the laboratory and amended with or without maize or biosolids while production of carbon dioxide (CO(2)), dynamics of ammonia (NH(4)(+)), nitrates (NO(3)(-)), and total petroleum hydrocarbons (TPH) were monitored. The fastest mineralization of diesel, as witnessed by production of CO(2), was found when biosolids were added, but the amount mineralized after 100 days, approximately 88%, was similar in all treatments. Approximately 5 mg of the 48 mg TPH kg(-1) found in the sterilized soil at the beginning of the experiment could not be accounted for after 100 days. The concentration of TPH in the unsterilized soil decreased rapidly in all treatments, but the rate of decrease was different between the treatments. The fastest decrease was found in the soil amended with biosolids and approximately 30 mg TPH kg(-1) or 60% could not be accounted for within 7 days. The decrease in concentration of TPH at the onset of the incubation was similar in the other treatments. After 100 days, the concentration of TPH was similar in all soils and appear to stabilize at 19 mg TPH kg(-1) soil. It was concluded that biosolids accelerated the decomposition of diesel and TPH due to its large nutrient content, but after 100 days the amount of diesel mineralized and the residual concentration of TPH was not affected by the treatment applied.     

A field trial for an ex-situ bioremediation of a drilling mud-polluted site

The remediation of drilling mud-polluted sites in the Southeast of Mexico is a top priority for Mexican oil industry. The objective of this work was to find a technology to remediate these sites. A field trial was performed by composting in biopiles, where four 1ton soil-biopiles were established, one treatment in triplicate and one unamended biopile. Amended biopiles were added with nutrients to get a C/N/P ratio of 100/3/0.5 plus a bulking agent (straw) at a soil/straw ratio of 97/3. Moisture content was maintained around 30-35%. Results showed that, after 180 d, total petroleum hydrocarbon (TPH) concentrations decreased from 99300+/-23000mgTPHkg(-1) soil to 5500+/-770mgTPHkg(-1) for amended biopiles and to 22900+/-7800mgTPHkg(-1) for unamended biopile. An undisturbed soil control showed no change in TPH concentrations. Gas chromatographic analysis showed residual alkyl dibenzothiophene type compounds. Highest bacterial counts were observed during the first 30 d which correlated with highest TPH removal, whereas fungal count increased at the end of the experimentation period. Results suggested an important role of the straw, nutrient addition and water content in stimulating aerobic microbial activity and thus hydrocarbon removal. This finding opens an opportunity to remediate old polluted sites with recalcitrant and high TPH concentration.     

垃圾堆肥高效纤维素分解菌的筛选与培育技术

从堆肥、马粪、果园土、污泥等20个样品中，分离筛选出3株对滤纸分解旺盛的纤维素分解菌：C1、C2、C3，并外购康氏木霉、白腐菌、变色栓菌一起作为出发株，经紫外诱变处理后，在含葡萄糖的产酶培养基平板上筛选到能形成较大透明圈的突变株，并进行CMC酶活、微晶纤维素酶活及天然粗纤维分解能力测定。实验结果证明白腐菌经紫外线照射60s诱变而得的C16不仅透明圈大，CMC酶活高（60.08U/mL）是出发株的2倍，而且其对天然粗纤维分解能力强，10d分解率达35％。