Estimation of the Hydrocarbon-Oxidizing Activity of Microorganisms

A method allowing microorganisms growing on substrates with low water solubility (oil, fuel oil, resins, and asphalthenes) to be isolated and counted was developed. The method makes it possible to estimate the oil-utilizing activity of each strain visually according to the decolorized zones formed during its growth on oil products. The sizes of these zones indicate which oil-degrading strains are most active.     

高效降解石油外生菌根真菌的室内筛选

对7个外生菌根真菌菌株在不同石油浓度培养基中的生长及其对石油的降解作用进行了研究。结果表明:(1)不同菌株在同一石油浓度培养基中生长速度不同,同一菌株在不同石油浓度培养基中的生长速度亦不同。菌株010和菌株025在不同石油浓度培养基中生长速度均较其他菌株快。菌株010的菌丝干重随着石油浓度的增加而增加,当石油质量浓度为500 mg/L时,菌丝干重达到最大值,高于对照5.27%,石油对其生长产生了促进作用;当石油质量浓度为700 mg/L时,菌丝干重低于对照,随着石油质量浓度的升高,菌株生长呈下降趋势。石油质量浓度为100 mg/L时,菌株025生长最慢,菌丝干重低于对照9.1%。随着石油浓度的增加,菌株生长加快,当石油质量浓度为500 mg/L时,菌丝干重高于对照;当石油质量浓度为700 mg/L时,菌株025菌丝干重最高,高于对照25.65%。随着石油浓度的再度升高,菌株生长呈下降趋势。(2)不同菌株在同一石油浓度下对石油的降解能力不同,同一菌株在不同石油浓度下对石油的降解能力亦不同。菌株025对石油的降解能力最强,对石油的降解能力随着石油浓度的升高而提高。当培养基中石油质量浓度为900 mg/L时,菌株025对石油的降解率最高,达到73.65%。(3)菌株0100、09、035、LH004的菌丝生物量与对石油的降解能力呈正相关。     

Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation

Among 25 crude oil-degrading bacteria isolated from a marine environment, four strains, which grew well on crude oil, were selected for more study. All the four isolated had maximum growth on 2.5% of crude oil and strain BC (Pseudomonas) could remove crude oil by 83%. The drop collapse method and microtiter assay show that this strain produces more biosurfactant, and its biofilm formation is higher compared to other strains. Bacterial adhesions to crude oil for strains CS-2 (Pseudomonas), BC, PG-5 (Rhodococcus) and H (Bacillus) were 30%, 46%, 10% and 1%, respectively. Therefore, strain H with a low production of biosurfactant and biofilm formation had showed the least growth on these compounds. PCR analysis of these four strains showed that all isolates had alk-B genes from group (III) alkane hydroxylase. All isolate strains could utilize cyclohexan, octane, hexadecane, octadecan and diesel fuel oil; however, the microtiter plate assay showed that strain BC had more growth, respiration and biofilm formation on octadecan.     

Rapid selection of microorganisms producing extracellular lipases]

A method for rapid selection of microorganisms producing extracellular lipases is described. The method is based on diffusion of the enzyme into agar containing olive oil. The hydrolysis zones revcal by 0,05--0,25% OSO4 solution.     

Sphingobacterium bambusaue及其紫外诱变菌株的石油降解功能

【目的】研究Sphingobacterium bambusaue及其紫外诱变菌株的石油降解功能。【方法】紫外诱变后筛选石油降解高效菌株; 以不同石油浓度、pH值及盐浓度优化培养条件, 用重量法检测高效菌株石油降解率。【结果】发现菌株S. bambusaue在石油降解培养基中培养5 d的石油降解率为25.86%, UV诱变高效菌株IBFC2009-S3培养5 d的石油降解为42.85%, 比始发菌株提高65.7%; UV诱变菌株IBFC2009-S3的优化培养条件为石油浓度0.5 g/L、pH值7.0以及NaCl质量浓度为10 g/L, 其石油降解率可达50.51%。【结论】首次报道S. bambusaue具有石油降解功能; 紫外诱变获得的菌株S3的石油降解能力较强。     

蒜油对MRSA菌株的抗菌作用及作用机制研究

目的研究蒜油对MRSA菌株的抗菌作用及其作用机制,为治疗MRSA菌株感染提供实验依据。方法纸片法药敏试验测抑菌环直径,微量稀释法测其最低抑菌浓度(MIC),酶标仪测定不同时间MRSA菌株生长A640值,革兰染色镜检观察蒜油对MRSA菌株细胞壁的影响,SDS-PAGE凝胶电泳分析蒜油对MRSA菌株蛋白的影响。结果蒜油对MRSA菌株具有明显抗菌作用,其MIC和MBC分别为10mg/mL和20 mg/mL;1/2MIC和1MIC的蒜油作用MRSA菌株后,菌株生长曲线平缓,无对数期;且其对对数期细菌有明显杀菌作用;蒜油对MRSA菌株细胞壁无明显影响,凝胶电泳分析有特征条带出现。结论蒜油对MRSA菌株具有明显抗菌作用,其杀菌机制与蛋白合成有关。     

一株油脂降解菌的筛选鉴定及降解效果分析

为了筛选分离得到一株具有油脂降解能力的菌株,同时探究菌株的特性和降解能力。以屠宰场污染土作为菌源,通过梯度驯化法最终筛选分离得到能够将橄榄油作为单一碳源生长的降解菌。随后通过形态特征观察、Biolog生理生化测试以及16S rRNA基因序列比对分析鉴定,实验菌株为革兰氏阴性菌,属于无色杆菌属（Achromobacter sp.）,在构建的系统发育树上与Achromobacter pulmonis聚为一支。综合运用紫外分光光度法和高效液相色谱法检测,测得实验菌株培养4~5 d时对橄榄油的降解率可以达到90%,同时测得菌株降解油脂的最适pH和最适温度分别为7.5和35 ℃,该菌株在盐浓度低于40 g·L-1环境中降解率较高。此外实验结果表明,实验菌株对各类型油脂均具有较高的降解效率,具有广泛的应用前景。     

一株表面活性剂产生菌的分离及抑菌活性

【目的】研究分离得到的表面活性剂产生菌的产表面活性剂能力、分类地位和抑菌活性。【方法】采用血平板、油平板进行表面活性剂产生菌的分离,以排油圈法进行表面活性的测定;通过生理生化特性和16S rDNA序列相似性分析对BS1菌株进行初步鉴定;利用对峙培养法和菌丝生长、孢子囊形成、孢子萌发的抑制率测定研究其抑菌活性。【结果】从石油污染土壤中分离到的BS1菌株可产生表面活性剂,在分类学地位上属于假单胞菌属(Pseudomonas sp.)。BS1菌体、发酵上清液、挥发性物质对12种供试病原真菌均表现出一定的抑制作用。BS1菌体、发酵上清液对大豆疫霉菌(Phytophthora sojae)的抑制率最大,分别达到65.31%和95.93%。发酵上清液通过影响大豆疫霉菌菌丝生长、孢子囊形成、孢子萌发等方式抑制病原菌的正常生长,稀释20倍的发酵上清液依然具有明显的抑制作用。BS1菌株产生的挥发性物质对大豆菌核菌(Sclerotinia sclerotiorum)的抑菌效果最好,抑制率达到84.25%。【结论】BS1菌株在产生表面活性剂的同时,还具有生物防治作用潜力。     

Effect of storage conditions on population variability and activity of Streptomyces recifensis var. lyticus

The results of the study on survival and variation of Streptomyces recifensis var. lyticus 2435 producing lytic enzymes are presented. The culture was maintained for 2.5 years under a layer of vaseline oil, at a temperature of -20 degrees C and in lyophilized state. It was shown that irrespective of the storage method strain 2435 preserved its viability. However, the most intensive growth was observed in the lyophilized cultures. During the storage the content of the productive colonies characteristic of the morphological type culture in the population decreased while the number of the low active variants increased. Lyophilization of the strain spores in the sucrose-gelatine medium provided insignificant morphological variation of the culture and preservation of the initial level of its lytic activity against a number of test-microbes except S. aureus and M. lysodeikticus. Storage of the culture under vaseline oil and at a temperature of -20 degrees C resulted in lowering of its lytic activity against all the test-microbes used. For long-term maintenance of Streptomyces recifensis var. lyticus 2435 the method of lyophilization in the sucrose-gelatine medium is recommended.     

Large expansion of oil industry in the Ecuadorian Amazon: biodiversity vulnerability and conservation alternatives

Ecuador will experience a significant expansion of the oil industry in its Amazonian region, one of the most biodiverse areas of the world. In view of the changes that are about to come, we explore the conflicts between oil extraction interests and biodiversity protection and apply systematic conservation planning to identify priority areas that should be protected in different oil exploitation scenarios. First, we quantified the current extent of oil blocks and protected zones and their overlap with two biodiversity indicators: 25 ecosystems and 745 species (whose distributions were estimated via species distribution models). With the new scheme of oil exploitation, oil blocks cover 68% (68,196 km²) of the Ecuadorian Amazon; half of it occupied by new blocks open for bids in the southern Amazon. This region is especially vulnerable to biodiversity losses, because peaks of species diversity, 19 ecosystems, and a third of its protected zones coincide spatially with oil blocks. Under these circumstances, we used Marxan software to identify priority areas for conservation outside oil blocks, but their coverage was insufficient to completely represent biodiversity. Instead, priority areas that include southern oil blocks provide a higher representation of biodiversity indicators. Therefore, preserving the southern Amazon becomes essential to improve the protection of Amazonian biodiversity in Ecuador, and avoiding oil exploitation in these areas (33% of the extent of southern oil blocks) should be considered a conservation alternative. Also, it is highly recommended to improve current oil exploitation technology to reduce environmental impacts in the region, especially within five oil blocks that we identified as most valuable for the conservation of biodiversity. The application of these and other recommendations depends heavily on the Ecuadorian government, which needs to find a better balance between the use of the Amazon resources and biodiversity conservation.     

嗜热解烃菌DM-2产生的生物乳化剂

【目的】生物乳化剂是一类由微生物代谢产生的大分子生物表面活性物质,从胜利油田中1区N3块地层环境中筛选到一株能产生一种生物乳化剂的嗜热解烃菌DM-2,经鉴定为嗜热脂肪地芽孢杆菌(Geobacillus stearothermophilus),研究其产生的生物乳化剂的化学组成和理化性质。【方法】采用化学显色、红外光谱、高效液相色谱和氨基酸自动分析等方法确定乳化剂的化学组成;根据乳化剂在不同条件下的乳化指数(EI-24)确定其理化性质。【结果】菌株DM-2产生的乳化剂主要由多糖(71.4%,质量比)和蛋白(27.75%,质量比)组成,对柴油、苯、二甲苯和煤油等石油烃均有很好的乳化效果。理化性质分析显示它是一种耐高温、耐盐、耐酸碱的高效乳化剂。【结论】菌株DM-2产生的乳化剂是一种新型的生物乳化剂,在石油开采、原油集输、油罐清洗和石油污染治理等领域具有潜在的应用价值。     

Improved method for direct screening of true lipase-producing microorganisms with particular emphasis on alkaline conditions

A rapid and effective method for direct detection, selection and testing of microorganisms able to produce both cell-bound and extracellular true lipases is described. The method is based on formation of clearance zones on turbid solid media with emulsified olive oil around or under the colonies, cell fractions or culture supernatant of lipase-producing organisms. The method was successfully applied for the screening and isolation of microorganisms producing alkaline lipases. The article is published in the original.     

高产不饱和油脂海鞘真菌桔青霉Asc-2-4的诱变选育

诱变育种是获得高产菌株,实现微生物工业化生产油脂的重要措施。以前期获得的高产不饱和油脂菌株桔青霉（Penicillium citrinum）Asc-2-4为出发菌株,利用丙二酸建立快速筛选高产不饱和脂肪酸突变菌的方法,通过紫外线 氯化锂复合诱变得到1株高产油脂突变菌Asc-2-4-1,油脂含量比出发菌株提高了92.98%。经过初步的培养基无机盐优化,其油脂得率和不饱和脂肪酸产量达到了7.10 g/L和3.84 g/L,与Asc-2-4相比,分别提高了84.42%和77.78%。结果表明,通过复合诱变选育技术可选育出高产突变菌株,选育的Asc-2-4-1可望作为产油微生物被开发利用。     

原生质体紫外诱变选育可利用廉价碳源的高产油新菌株

【目的】研究并建立利用原生质体紫外诱变技术选育可利用廉价碳源发酵的高产油新菌株的方法。【方法】采用1.5%蜗牛酶和1.0%纤维素酶混合液水解去除细胞壁得到2A00015(近平滑假丝酵母,Candida parapsilosis)的原生质体,将其放于紫外灯下诱变及再生壁培养,筛选获得可利用廉价碳源发酵的高产油酵母,并采用气相色谱质谱联用法(GC-MS)测定其脂肪酸组成。【结果】突变效果最好的突变菌株2A00015/25用葡萄糖发酵培养7 d后,其生物量、油脂产率和产油量分别为17.77 g/L、58.12%和10.32 g/L,较原始菌株分别提高了12.45%、23.32%和38.68%;利用废糖蜜发酵培养,其生物量、油脂产率和产油量分别为18.54 g/L、49.44%和9.17 g/L,较原始菌株分别提高了9.09%、21.16%和32.18%。利用废糖蜜培养其产油效率虽低于利用葡萄糖培养,但从环境保护及原材料成本的角度考虑,用废糖蜜作为碳源发酵培养产生油脂更具优势。诱变菌株利用废糖蜜发酵后产生油脂经检测含有8种脂肪酸,其脂肪酸组成与植物油近似,其中不饱和脂肪酸含量占脂肪酸总量的82.4%。【结论】通过利用原生质体紫外诱变技术,成功选育出一株新的可利用廉价碳源的高产油海洋菌株,产油率达到49.4%,提高了21.2%。     

Improved Method for Detection of Starch Hydrolysis

A new starch hydrolysis detection method which does not rely on iodine staining or the use of color-complexed starch is described. A linear relationship was obtained with agar-starch plates when net clearing zones around colonies of yeasts were plotted against enzyme levels (semilogarithm scale) produced by the same yeast strains in liquid medium. A similar relationship between starch clearing zones and α-amylase levels from three different sources was observed. These observations suggest that the method is useful in mutant isolations, strain improvement programs, and the prediction of α-amylase activities in culture filtrates or column effluents.     

Non-uniform strain distribution within rat cartilaginous growth plate under uniaxial compression

Growth plates are highly inhomogeneous in morphology and composition. Mechanical loading can modulate longitudinal bone growth, though the mechanisms underlying this mechanobiology are poorly understood. The proximal tibial growth plates of six rats were tested in vitro under uniaxial compression to 5% strain, and confocal microscopy was used to track and capture images of fluorescently labeled cell nuclei with increasing applied strains. The local strain patterns through the growth plate thickness were quantified using texture correlation analysis. The technique of texture correlation analysis was first validated by comparing theoretical simulated strain maps generated from numerically distorted images. The texture correlation algorithm was sensitive to the grid size superimposed on the original image, but remained insensitive to parameters related to the size of the final image mask, which was searched by the correlation algorithm for each grid point of the original image. Within the growth plate, experimental strain distributions were non-uniform in all six specimens. Growth plates were mostly under compression strains. The strain distributions differed among the histomorphological zones of the growth plate, which was most obvious in specimens with regular growth plate shape: higher compressive strains (4-8 times higher than the applied 5% strain) were located mainly in regions overlapping the reserve and hypertrophic zones with lower compressive strains in the proliferative zone. This study documents the non-uniform mechanical behavior of growth plate across its three histological zones when exposed to compression. Further investigation is required to establish the significance of non-uniform strain fields during growth in vivo.     

Root colonisation of Pseudomonas aeruginosa strain UPMP3 and induction of defence‐related genes in oil palm (Elaeis guineensis)

Pseudomonas aeruginosa strain UPMP3 labelled with β‐glucuronidase (gusA) and green fluorescent protein (gfp) by electrotransformation yielded ca 1 × 10⁷ transformants µg^?1 DNA. The data obtained from the dilution plate count showed that over 28 days both epiphytic and endophytic populations of P. aeruginosa strain UPMP3 increased from 5.76 log₁₀ [colony forming unit (CFU) + 1] g^?1 fresh weight (FW) to 8.19 log₁₀ (CFU + 1) g^?1 FW and 4.10 log₁₀ (CFU + 1) g^?1 FW to 6.23 (CFU + 1) g^?1 FW, respectively. Confocal laser scanning microscopic analysis of oil palm roots treated with gusA:gfp‐tagged P. aeruginosa strain UPMP3 showed intense root colonisation over the sampling period. The root surface colonisation by P. aeruginosa strain UPMP3 was followed by a second stage, characterised by cortical infection, and a third stage, which involves xylem ingression. The colonisation of oil palm roots by the gusA:gfp‐tagged strain was concentrated on root areas potentially rich in nutrients such as the elongation zones, ridges between epidermal cells and points of secondary adventitious root emergence. Different expression levels of defence‐related genes, namely, chitinase and β‐1,3‐glucanase in the strain UPMP3–host interaction recorded over 28 days, suggested the potential role of P. aeruginosa strain UPMP3 in triggering the defence mechanism in oil palm. This is the first report on root colonisation and upregulation of defence‐related genes on oil palm roots by P. aeruginosa strain UPMP3 and shows the potential of this strain to be used as a biocontrol agent in oil palm.     

Surfactant activity of a naphthalene degrading Bacillus pumilus strain isolated from oil sludge

We studied the growth, biosurfactant activities and petroleum hydrocarbon compounds utilisation of strain 28-11 isolated from a solid waste oil. The isolate was identified as Bacillus pumilus. It grew well in the presence of 0.1% (w/v) of crude oil and naphthalene under aerobic conditions and utilised these substances as carbon and energy source. The capacity of strain 28-11 to emulsify crude oil and its ability to remove hydrocarbons looks promising for its application in environmental technologies.     

Genome Sequence of Pseudomonas aeruginosa Strain SJTD-1, a Bacterium Capable of Degrading Long-Chain Alkanes and Crude Oil

Pseudomonas aeruginosa strain SJTD-1 can utilize long-chain alkanes, diesel oil, and crude oil as sole carbon sources. We report the draft genome sequence of strain SJTD-1 (6,074,058 bp, with a GC content of 66.83%) and major findings from its annotation, which could provide insights into its petroleum biodegradation mechanism.     

Importance of malate synthase in the glyoxylate cycle of <Emphasis Type="Italic">Ashbya gossypii</Emphasis> for the efficient production of riboflavin

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption.