黄土高原石油污染土壤微生物群落结构及其代谢特征

针对污染胁迫下土壤微生物群落变化和代谢变异等问题,基于平板稀释法和Biolog微平板分析方法,研究了陕北黄土高原石油污染土壤微生物群落结构、代谢特征及其功能多样性。结果表明,不同类群的土壤微生物对石油污染胁迫的响应不同,污染土壤细菌和真菌数量高出清洁土壤1个数量级,而污染土壤的放线菌数量极显著减少(P0.01);污染土壤和清洁土壤微生物对糖类和多聚物类碳源较易利用,污染土壤微生物总体上代谢碳源的种类和活性均低于清洁土壤。微生物群落主成分分析(PCA)表明,石油污染土壤和清洁土壤的微生物群落存在显著差异(P0.01),起分异作用的碳源主要为糖类,其次是羧酸类和氨基酸类;随着土壤石油含量增加,典型变量值变异(离散)增大,土壤微生物群落结构稳定性降低。微生物群落多样性分析表明,Shannon丰富度指数(H)、McIntosh均一度指数(U)和Simpson优势度指数(1/D)均达到极显著差异(P0.01),污染土壤微生物群落H和U低于清洁土壤,但是一定浓度的石油污染可以刺激土壤微生物群落中优势种群的生长,1/D增高。研究结果为陕北黄土高原石油污染区土壤微生物修复提供理论基础。     

News & Notes: Bioremediation of Crude Oil Contamination with Acinetobacter sp. A3

Acinetobacter sp. A3 is able to extensively degrade Bombay High Crude Oil (BHCO) and utilize it as the sole source of carbon. A total degradation of 70% BHCO was noted by the end of 120 h of growth of Acinetobacter sp. A3 under shake flask condition, 60% of which was due to biodegradation. In crude oil-contaminated soil (5%) amended with Acinetobacter sp. A3, there was both an increase in colony-forming units (CFU) and crude oil degradation. This is in contrast to a decrease in CFU of the indigenous microorganisms and lower degradation in unamended soil within the same 30-day period. Also, Acinetobacter sp. A3-treated soil permitted better germination of Mung beans (Phaseolus aureus) and growth as evidenced by better length and weight of the plants and chlorophyll content of its leaves, which was attributed to the reduction in phytotoxicity of the crude oil owing to its degradation. This crude oil degradative capability of Acinetobacter sp. A3 could be exploited for bioremediation purposes. Received: 16 December 1996 / Accepted: 10 February 1997     

Biodegradation of wood in crude oil-polluted soil

A field investigation (April–November) in Nigeria showed that biodegradation of obeche (Triplochiton scleroxylon) wood blocks was initially retarded in crude oil-contaminated soil but later became enhanced as indicated by loss of compression resistance. Further indication of this pattern was the detection of soft-rot cavities and basidiomycete fungi after 2–3 months exposure when compared to control blocks in uncontaminated soil. Laboratory tests with Pleurotus sp., Trametes sp., Gloeophyllum sp. (basidiomycetes) and Chaetomium sp. (soft-rot fungus) confirmed that degradation of crude oil-coated obeche blocks was markedly retarded without the presence of hydrocarbon-degrading bacteria. The filtrate of hydrocarbon-degrading Pseudomonas sp. grown in mineral salt/crude oil medium for 3–4 weeks supported growth of the test fungi better than in carboxymethyl cellulose medium but less than in potato dextrose broth. Similarly, wood blocks immersed in the filtrate became significantly more susceptible to fungal degradation. Pseudomonas sp. from stationary phase growth in crude oil medium depleted residual sugar in basidiomycete-degraded sawdust with a concomitant marked increase in its population. It may be concluded that readily metabolizable products of crude oil degradation by soil organisms and the removal of residual sugar which may have prevented catabolite repression of cellulases, culminated in increased attack on the wood by soil-borne wood-decomposing organisms.     

石油污染土壤自然衰减过程多环芳烃降解基因动态特征

新疆石油污染土壤中微生物多环芳烃(polycyclic aromatic hydrocarbons,PAHs)降解功能基因研究甚少,且环境因子和功能基因之间相关性仍不清楚。【目的】揭示新疆石油污染砂质土壤自然衰减过程中多环芳烃降解关键基因结构和变化规律。【方法】以新疆准东油田为研究区,分析同一采油区不同石油污染年限土壤理化因子和多环芳烃含量变化,采用扩增子测序研究石油自然衰减过程中多环芳烃降解酶基因结构变化规律,利用Mental检验探讨其环境驱动因子。【结果】石油污染时间1年和3年的土壤中有多项理化指标与背景土存在显著性差异,而污染5年土壤与背景土之间仅2项指标具有显著性差异,随石油自然衰减逐渐恢复至正常。石油污染1年的土壤中16种多环芳烃除苊烯和?以外,其余14种多环芳烃均高于石油污染3年和5年土壤,多环芳烃总量和含油率污染1年土壤均显著高于污染3年和5年的土壤,多环芳烃会在污染后短时间内迅速被降解。扩增子测序结果显示,萘双加氧酶基因分类操作单元(operational taxonomic units,OTUs)序列随污染年限延长逐渐增多;芳环羟化双加氧酶基因OTUs序列BLAST(...     

Phytoremediation of Light Crude Oil by Maize (Zea mays L.) Bio-Augmented with Plant Growth Promoting Bacteria

The aim of this study was to evaluate the converged effect of maize and plant growth promoting bacteria on degradation of petroleum hydrocarbons under axenic conditions. Artificially spiked sand with 10 g kg^?1 light crude oil was planted with maize alone and in combination with eight bacterial isolates having plant growth promotion and bioremediation potential to observe the dissipation of petroleum hydrocarbons. Results showed remarkable suppression of maize growth and biomass production due to phytotoxicity of the crude oil contamination. However, bio-augmentation of plants with bacteria having ACC-deaminase activity significantly compensated the reduction in plant growth compared to uninoculated plants. The results revealed that plants bio-augmented with PM32Y exhibited significant increase in root length (75%), plant height (74%), and biomass (67%) as compared to uninoculated plants after 60 days of planting. The same bacterium in convergence with maize caused 43% degradation of petroleum hydrocarbons as compared to the unplanted and uninoculated control. Amplification, sequencing and phylogenetic analysis of 16S rRNA gene sequence identified PM32Y bacterium as Bacillus subtilis strain. It is concluded that bio-augmentation of plants with plant growth promoting bacteria having bioremediation potential and ACC-deaminase activity can successfully be used in phytoremediation of petroleum hydrocarbons.     

Screening of plants for phytoremediation of oil-contaminated soil

Several species of ornamental flowering plants were evaluated regarding their phytoremediation ability for the cleanup of oil-contaminated soil in Japanese environmental conditions. Thirty-three species of plants were grown in oil-contaminated soil, and Mimosa, Zinnia, Gazania, and cypress vine were selected for further assessment on the basis of their favorable initial growth. No significant difference was observed in the above-ground and under-ground dry matter weight of Gazania 180 days after sowing between contaminated and non-contaminated plots. However, the other 3 species of plants died by the 180^th day, indicating that Gazania has an especially strong tolerance for oil-contaminated soil. The total petroleum hydrocarbon concentration of the soils in which the 4 species of plants were grown decreased by 45–49% by the 180^th day. Compared to an irrigated plot, the dehydrogenase activity of the contaminated soil also increased significantly, indicating a phytoremediation effect by the 4 tested plants. Mimosa, Zinnia, and cypress vine all died by the 180^th day after seeding, but the roots themselves became a source of nutrients for the soil microorganisms, which led to a phytoremediation effect by increase in the oil degradation activity. It has been indicated that Gazania is most appropriate for phytoremediation of oil-contaminated soil.     

Plant and fertiliser effects on rhizodegradation of crude oil in two soils with different nutrient status

Soils and sediments polluted with crude oil are of major environmental concern on various contaminated sites. Outdoors pot experiments were conducted to test the phytodegradation potential of common reed (Phragmites australis) and poplar (Populus nigra × maximowiczii) in fertilised and non-fertilised control treatments. Two topsoils (E, G) of different texture were mixed with crude oil. Soil analysis included hydrocarbon (HC) measurements, detection of labile phosphorus and mineralised nitrogen as well as dehydrogenase activity. Increased HC degradation by native soil biota was clearly related to higher P availability in soil G and to fertilisation in soil E. Except of the non-fertilised common reed treatment, plants did not enhance crude oil degradation. We found even inhibited degradation of high molecular weight HC in the presence of plants together with declining labile phosphorous concentrations due to planting on soil E. Native soil biota were able to use the whole range of crude oil compounds (C₁₀ to C₆₀) as a carbon source in the presence of sufficient nutrient concentrations in soil. This study is the first to show that reduced HC degradation in the higher molecular weight crude oil fraction (C₂₀ to C₄₀) is likely to be a consequence of decreased phosphorus availability for microorganisms in the plant rhizosphere.     

石油烃污染土壤的生物修复

从中原油田污染土壤中通过实验室驯化培养分离到一组能以中原原油为碳源的快速生长的石油烃降解菌.用该组降解菌接种原油污染土壤,研究其原位生物联合修复实验,接种降解菌的各区分别种植大豆、施有机肥料、施有机肥料和锯末,与空白试样作对比.经过120d的联合修复,各区石油降解菌的总数(lgcfu/g)由接种时的5.25分别变为7.79、4.96、5.15、4.67.石油烃降解率分别达到89.4%、72.5%、76.7%、49.2%.表明分离的该组石油烃降解菌是一组高效降解菌且其与植物联合修复石油污染土壤能显著提高修复效果.     

Isolation and characterization of long-chain alkane–degrading Acinetobacter sp. BT1A from oil-contaminated soil in Diyarbakır,in the Southeast of Turkey

A strain of long-chain alkane–degrading bacteria, BT1A, was isolated from oil-contaminated soil in Diyarbak?r, in the southeast of Turkey. Morphological, biochemical, and physiological characterization and 16S rRNA gene sequence analysis showed that the strain BT1A was a member of Acinetobacter genus, and it was found to be closely related to Acinetobacter baumannii. The strain BT1A was able to utilize crude petroleum as carbon and energy sources in order to grow. Among the aliphatic hydrocarbons, growth was observed only in the medium containing long-chain alkanes (tridecane, pentadecane, and hexadecane) and squalene. Hexadecane was the most preferred hydrocarbon among the long-chain alkanes. Gas chromatography–mass spectrometry (GC-MS) analysis showed that BT1A degraded 83% of n-alkanes of 1% crude oil in 7 days. The present study indicates that the isolated strain can well be used for biodegradation of hydrocarbons in oil-contaminated sites.     

Bioremediation of oil by marine microbial mats

Cyanobacterial mats developing in oil-contaminated sabkhas along the African coasts of the Gulf of Suez and in the pristine Solar Lake, Sinai, were collected for laboratory studies. Samples of both mats showed efficient degradation of crude oil in the light, followed by development of an intense bloom of Phormidium spp. and Oscillatoria spp. Isolated cyanobacterial strains, however, did not degrade crude oil in axenic cultures. Strains of sulfate-reducing bacteria and aerobic heterotrophs were capable of degrading model compounds of aliphatic and aromatic hydrocarbons. Results indicate that degradation of oil was done primarily by aerobic heterotrophic bacteria. The oxygenic photosynthesis of oil-insensitive cyanobacteria supplied the molecular oxygen for the efficient aerobic metabolism of organisms, such as Marinobacter sp. The diurnal shifts in environmental conditions at the mat surface, from highly oxic conditions in the light to anaerobic sulfide-rich habitat in the dark, may allow the combined aerobic and anaerobic degradation of crude oil at the mat surface. Hence, coastal cyanobacterial mats may be used for the degradation of coastline oil spills. Oxygen microelectrodes detected a significant inhibition of photosynthetic activity subsequent to oil addition. This prevailed for a few hours and then rapidly recovered. In addition, shifts in bacterial community structure following exposure to oil were determined by denaturing gradient gel electrophoresis of PCR-amplified fractions of 16S rRNA from eubacteria, cyanobacteria and sulfate-reducing bacteria. Since the mats used for the present study were obtained from oil-contaminated environments, they were believed to be preequilibrated for petroleum remediation. The mesocosm system at Eilat provided a unique opportunity to study petroleum degradation by mats formed under different salinities (up to 21%). These mats, dominated by cyanobacteria, can serve as close analogues to the sabkhas contaminated during the Gulf War in Kuwait and Saudi Arabia. Electronic Publication     

Ecology of bacterial endophytes associated with wetland plants growing in textile effluent for pollutant-degradation and plant growth-promotion potentials

In this study, 41 culturable endophytic bacteria were isolated from the roots and shoots of three wetland plants, Typha domingensis, Pistia stratiotes and Eichhornia crassipes, and identified by 16S rRNA gene sequencing. Textile effluent-degrading and plant growth-promoting activities of these endophytes were determined. The analysis of endophytic bacterial communities indicated that plant species had a pronounced effect on endophytic bacterial association and maximum endophytes (56.5%) were associated with T. domingensis. These endophytic bacteria mainly belonged to different species of the genera Bacillus (39%), Microbacterium (12%) and Halomonas (12%). Eight of the 41 strains showing maximum efficiency of textile effluent degradation also exhibited plant growth-promoting activities such as production of indole-3-acetic acid and siderophore, presence of 1-amino-cyclopropane-1-carboxylic acid deaminase, and solubilization of inorganic phosphorous. This is the first study describing the diversity and plant-beneficial characteristics of the textile effluent-degrading endophytic bacteria associated with wetland plants. T. domingensis showed better growth in textile effluent and also hosted maximum number of endophytic bacteria in roots and shoots. The interactions between T. domingensis and its associated endophytic bacteria could be exploited to enhance the efficiency of constructed wetlands during the remediation of industrial effluent.     

The Dynamic Change of Microbial Communities in Crude Oil-Contaminated Soils from Oil Fields in China

To study the biodegradability of microbial communities in crude oil contamination, crude oil-contaminated soil samples from different areas of China were collected. Using polyphasic approach, this study explored the dynamic change of the microbial communities during natural accumulation in oil field and how the constructed bioremediation systems reshape the composition of microbial communities. The abundance of oil-degrading microbes was highest when oil content was 3–8%. This oil content is potentially optimal for oil degrading bacteria proliferation. During a ～12 months natural accumulation, the quantity of oil-degrading microbes increased from 10⁵ to 10⁸ cells/g of soil. A typical sample of Liaohe (LH, oil-contaminated site near Liaohe River, Liaoning Province, China) was remediated for 50 days to investigate the dynamic change of microbial communities. The average FDA (a fluorescein diacetate approach) activities reached 0.25 abs/hr·g dry soil in the artificially enhanced repair system, 32% higher than the 0.19 abs/hr·g dry soil in natural circumstances. The abundance of oil-degrading microbes increased steadily from 0.001 to 0.068. During remediation treatment, oil content in the soil sample was reduced from 6.0% to 3.7%. GC–MS analysis indicated up to 67% utilization of C₁₀–C₂₀ normal paraffin hydrocarbons, the typical compounds that undergo microbial degradation.     

Selection of Specific Endophytic Bacterial Genotypes by Plants in Response to Soil Contamination

Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants. At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (ntdAa) and nitrotoluene monooxygenase (ntnM), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of ndoB-positive endophytes in Scirpus pungens, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants. Similarly, the numbers of alkB- or ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not with the numbers of alkB- or ndoB-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.     

Organic fertilizer applications influence on the shoot and root biomass production and plant nutrient of Calopogonium mucunoides from crude oil-contaminated soils

The impact of crude oil-contaminated soil on the shoot and root biomass yield and nutrients uptake of Calopogonium mucunoides Desv. using two types of composted manure (COM) as soil amendments were investigated. This was with a view to assessing the growth response of the test plant under different levels of crude oil soil contamination. Five levels [0, 2.5, 5, 10, and 20% (v/v)] of crude oil, each was replicated thrice to contaminate 3 kg of soil when 12 g pot^?1 COM; 12 g pot^?1 neem-fortified composted manure (NCM) and control, soil without manure application (C) were imposed as manure treatments. The mean fresh shoot biomass yield at zero crude oil soil contamination and with COM application was 2.67 g pot^?1. This value was significantly (p < 0.05) higher than 2.05 g pot^?1 for NCM and 1.67 g pot^?1 for the control. Also, the mean fresh root yield at zero crude oil soil contamination with COM application was 4.02 g pot^?1. This value was significantly (p < 0.05) higher than 2.41 g pot^?1 for NCM and 1.71 g pot^?1 for the control. The dry shoot and root biomass yield followed similar pattern. The shoot and root yield of C. mucunoides significantly (p < 0.05) reduced with increase in crude oil soil contamination. The nutrients uptake of C. mucunoides, particularly N, P, Ca, Mg, and Fe, were enhanced with COM fertilization having higher available P, K, and Na values; and by implication, suggesting the importance of adequately formulated composted manure usage in the rehabilitation studies of crude oil-contaminated soil.     

Identification and characterization of PAH-degrading endophytes isolated from plants growing around a sludge dam

Abstract

This study involved the isolation of bacteria endophytes with PAH-degrading ability from plants growing around a sludge dam. A total of 19 distinct isolates that were morphologically identified were isolated from 4 species of plant with a follow-up confirmatory identification using the molecular technique. Polymerase chain reaction (PCR) of the 16S rRNA gene with specific primers (16S-27F PCR and 16S-1491R PCR) was carried out. The sequence of the PCR products was carried out, compared with similar nucleotides available in GenBank. Results of the phylogenetic analysis of the isolates indicated their belonging to 4 different clades including Proteobacteria, Actinobacteria, Cyanobacteria, and Firmicutes. These were related to the genera Bacillus, Pseudomonas, Terribacillus, Virgibacillus, Stenotrophomonas, Paenibacillus, Brevibacterium, Geobacillus, Acinetobacter. From the result, Pseudomonas demonstrated a high incidence in the plants sampled. The in-vitro degradation study and the presence of dioxygenase genes indicated that these lists of endophytes are able to use the list of PAHs tested as their source of food and energy leading to their breakdown. This means that the bacterial endophytes contributed to the remediation of petroleum hydrocarbons in planta, a situation that may have been phytotoxic to plant alone. Therefore, these bacteria endophytes could be potential organisms for enhanced phytoremediation of PAHs.     

Biodegradation of crude oil by soil microorganisms in the tropic

Five microorganisms, three bacteria and two yeasts, capable of degrading Tapis light crude oil were isolated from oil-contaminated soil in Bangkok, Thailand. Soil enrichment culture was done by inoculating the soil in mineral salt medium with 0.5% v/v Tapis crude oil as the sole carbon source. Crude oil biodegradation was measured by gas chromatography method. Five strains of pure microorganisms with petroleum degrading ability were isolated: three were bacteria and the other two were yeasts. Candida tropicalis strains 7Y and 15Y were identified as efficient oil degraders. Strain 15Y was more efficient, it was able to reduce 87.3% of the total petroleum or 99.6% of n-alkanes within the 7-day incubation period at room temperature of 25 ± 2 °C.     

Identifying polycyclic aromatic hydrocarbon-degrading bacteria in oil-contaminated surface waters at Deepwater Horizon by cultivation, stable isotope probing and pyrosequencing

Polycyclic aromatic hydrocarbons (PAHs) are an important class of chemical pollutants that constitute a major component of total hydrocarbons in crude oils. Based on their poor water solubility, toxicity, persistence and potential to bioaccumulate, these compounds are recognized as high-priority pollutants in the environment and are of significant concern for human health. At oil-contaminated sites, PAH-degrading bacteria perform a critical role in the degradation and ultimate removal of these compounds. In April 2010, enormous quantities of PAHs entered the Gulf of Mexico from the thousands of tons of oil that were released from the ill-fated drilling rig Deepwater Horizon. In the ensuing months after the spill, intense research efforts were devoted to characterizing the microorganisms responsible for degrading the oil, particularly in deep waters where a large oil plume, enriched with aliphatic and low molecular-weight aromatic hydrocarbons, was found in the range of 1,000–1,300 m. PAHs, however, were found mainly confined to surface waters. This paper discusses efforts utilizing DNA-based stable isotope probing, cultivation-based techniques and metagenomics to characterize the bacterial guild associated with PAH degradation in oil-contaminated surface waters at Deepwater Horizon.     

ENDOPHYTIC BACTERIA FROM SEEDS OF NICOTIANA TABACUM CAN REDUCE CADMIUM PHYTOTOXICITY

Although endophytic bacteria seem to have a close association with their host plant, little is known about the influence of seed endophytic bacteria on initial plant development and on their interactions with plants under conditions of metal toxicity. In order to further elucidate this close relationship, we isolated endophytic bacteria from surface sterilized Nicotiana tabacum seeds that were collected from plants cultivated on a cadmium-(Cd) and zinc-enriched soil. Many of the isolated strains showed Cd tolerance. Sterilely grown tobacco plants were inoculated with either the endogenous microbial consortium, composed of cultivable and noncultivable strains; single strains; or defined consortia of the most representative cultivable strains. Subsequently, the effects of inoculation of endophytic bacteria on plant development and on metal and nutrient uptake were explored under conditions with and without exposure to Cd. In general, seed endophytes were found to have a positive effect on plant growth, as was illustrated by an increase in biomass production under conditions without Cd. In several cases, inoculation with endophytes resulted in improved biomass production under conditions of Cd stress, as well as in a higher plant Cd concentration and total plant Cd content compared to noninoculated plants. These results demonstrate the beneficial effects of seed endophytes on metal toxicity and accumulation, and suggest practical applications using inoculated seeds as a vector for plant beneficial bacteria.     

Enhancement of the efficiency of Cd phytoextraction using bacterial endophytes isolated from Chromolaena odorata,a Cd hyperaccumulator

Phytoextraction is a technique using a hyperaccumulator to remove heavy metals from soil. The efficiency of heavy metal uptake can be enhanced by the inoculation of endophytes. In this study, we isolated and identified 23 endophytes from Chromolaena odorata, a cadmium (Cd) hyperaccumulator that consisted of 19 bacteria, 2 actinomycetes and 2 fungi. All bacteria and fungi could produce at least 1 plant growth promoting factors. However, only 4 bacterial isolates; Paenibacillus sp. SB12, Bacillus sp. SB31, Bacillus sp. LB51, and Alcaligenes sp. RB54 showed the highest minimum inhibitory concentration (MIC) value (2.9 mM), followed by Exiguobacterium sp.RB51 (1.7 mM). Then, these 5 high-MIC bacteria and 1 low-MIC bacterium, Bacillus sp. LB15 were inoculated onto sunflower grown in soil supplemented with 250 mg/kg of Cd. After 60 days, all inoculated plants accumulated significantly higher Cd concentration than the non-inoculated counterparts, and those inoculated with strain LB51 showed the highest Cd accumulation and growth. Interestingly, strain LB15 with low MIC also enhanced Cd accumulation in plants. The results suggest that these bacteria, particularly strain LB51, could be applied to improve Cd accumulation in plants, and that bacteria with low MIC also have the potential to enhance the efficiency of phytoextraction.