Rhamnolipid production using Shewanella seohaensis BS18 and evaluation of its efficiency along with phytoremediation and bioaugmentation for bioremediation of hydrocarbon contaminated soils

Abstract

This study reports the combined use of a rhamnolipid type biosurfactant (BS) along with phytoremediation and bioaugmentation (BA) for bioremediation of hydrocarbon-contaminated soils. Bacterial isolates obtained from hydrocarbon contaminated soil were screened for rhamnolipid production and isolate BS18, identified as Shewanella seohaensis, was selected for bioremediation experiments. Growth of BS18 in mineral salt medium (MSM) with diesel oil as the carbon source showed a maximum biomass of 8.2?g L^?1, rhamnolipid production of 2.2?mg g^?1 cell dry weight, surface tension reduction of 28.6?mN/m and emulsification potential (EI₂₄%) of 65.6. Characterization of rhamnolipid based on Fourier transmittance infrared (FTIR) analysis confirmed the presence of OH, CH₂/CH₃, C=O, and COO stretching vibrations, respectively, which are distinctive features of rhamnolipid type BSs. In bioremediation experiments, the lowest hydrocarbon concentration of 2.1?mg g^?1 of soil for non-sterilized soil and 4.3?mg g^?1 of soil for sterilized soil was recorded in the combined application of rhamnolipid, phytoremediation, and BA. This treatment also yielded the highest hydrocarbon degrading bacterial population (6.4 Log Cfu g^?1 of soil), highest plant biomass (8.3?g dry weight plant^?1), and the highest hydrocarbon uptake (512.3?mg Kg^?1 of plant).     

Laboratory study on the bioremediation of petrochemical sludge-contaminated soil

This study evaluated by biological and chemical analyses the effectiveness of bioremediation of sludge from the petrochemical industry in systems containing artificially contaminated soil. The sludge–soil systems were prepared with three different initial concentrations of sludge, and during bioremediation 86–95% of the hydrocarbons was eliminated. Simultaneously, soil bacterial populations and inhibition of seed germination by aqueous extracts increased in all sludge–soil systems during the first 180 days of treatment. After 1 year of bioremediation, a loss in the catabolic capacity of the Gram-negative bacterial population was observed, but was not dependent on the initial sludge concentration. Furthermore, residual levels of hydrocarbons and seed germination inhibitory effect decreased sharply, but some level of toxicity remained in the systems containing the highest initial sludge concentration. Independent of the initial sludge concentration, the contaminated soils did not re-establish their original features even when residual hydrocarbon concentrations suggested the end of the process.     

Enhanced bioremediation of n-alkane in petroleum sludge using bacterial consortium amended with rhamnolipid and micronutrients

The purpose of the present study was to investigate possible methods to enhance the rate of biodegradation of oil sludge from crude oil tank bottom, thus reducing the time usually required for bioremediation. Enhancement of biodegradation was achieved through bioaugmentation and biostimulation. About 10% and 20% sludge contaminated sterile and non-sterile soil samples were treated with bacterial consortium (BC), rhamnolipid biosurfactant (RL) and nitrogen, phosphorus and potassium (NPK) solution. Maximum n-alkane degradation occurred in the 10% sludge contaminated soil samples. The effects of treatment carried out with the non-sterile soil samples were more pronounced than in the sterile soils. Maximum degradation was achieved after the 56th day of treatment. n-Alkanes in the range of nC8-nC11 were degraded completely followed by nC12-nC21, nC22-nC31 and nC32-nC40 with percentage degradations of 100%, 83-98%, 80-85% and 57-73% respectively. Statistical analysis using analysis of variance and Duncan's multiple range test revealed that the level of amendments, incubation time and combination of amendments significantly influenced bacterial growth, protein concentration and surface tension at a 1% probability level. All tested additives BC, NPK and RL had significant positive effects on the bioremediation of n-alkane in petroleum sludge.     

Phytoremediation potentials of cowpea (Vigina unguiculata) and maize (Zea mays) for hydrocarbon degradation in organic and inorganic manure-amended tropical typic paleustults

A field study on phytoremediation of hydrocarbon contaminated soil was designed to assess the effects of organic manures (poultry droppings and cassava peels) and NPK fertilization on the potentials of cowpea (Vigina unguiculata) and maize (Zea mays) to stimulate hydrocarbon degradation in soil. Cowpea and maize crops were established on the hydrocarbon contaminated soil amended with three rates (0, 4, and 8 ton/ha) of the soil amendments, and arranged in 3 x 3 x 3 factorial in Randomized Complete Block Design. Hydrocarbon was significantly (P < 0.05) reduced in plots treated with the combined forms of the soil amendments. While the treatment combinations of 8 t/ha Poultry Droppings (PD) + 8 t/ha Cassava Peels (CP) + 4 t/ha NPK fertilizer was optimal for hydrocarbon degradation in the cowpea plots, 4 t/ha PD + 8 t/ha CP + 8 t/ha NPK fertilizer was the most preferred in the maize plot. Cowpea showed greater potential for hydrocarbon degradation at the first year. The mean values of hydrocarbon concentrations at the cowpea and maize plots indicated no significant difference at the second year. Grain yield of cowpea increased by 87% at the second year, while maize was unable to grow to maturity in the first year.     

Nutrient Amendments of Inorganic Fertiliser and Oil Palm Empty Fruit Bunch and Their Influence on Bacterial Species Dominance and Degradation of the Associated Crude Oil Constituents

The effects of inorganic commercial fertiliser (N:P:K = 8:8:1) and oil palm empty fruit bunch (EFB) as nutrient amendments for crude oil degradation and microbial population shift by a microbial consortium [Pseudomonas sp. (UKMP-14T), Acinetobacter sp. (UKMP-12T), Trichoderma sp. (TriUKMP-1M and TriUKMP-2M)] were assessed. The bacterial populations present during crude oil degradation were analysed by spread plate method and 16S rRNA sequences, whereas the presence of fungi was assessed by growth on potato dextrose agar. Crude oil degradation analysed using gas chromatography-flame ionisation detection showed total petroleum hydrocarbon reduced between 70 and 100%, depending on the type of amendments compared to control (≈55%) after 30 days of incubation. Nutrient amendments using NPK fertiliser or EFB were found to influence the domination of different bacterial species, which in turn preferentially utilised different hydrocarbons. This study suggested different nutrient amendments could be used to preferentially select bacteria to degrade different components of crude oil, particularly pertaining to the recalcitrant phytane. This information is very useful for application of in situ bioremediation of soil hydrocarbon contamination.     

Soil initial bacterial diversity and nutrient availability determine the rate of xenobiotic biodegradation

Understanding the relative importance of soil microbial diversity, plants and nutrient management is crucial to implement an effective bioremediation approach to xenobiotics-contaminated soils. To date, knowledge on the interactive effects of soil microbiome, plant and nutrient supply on influencing biodegradation potential of soils remains limited. In this study, we evaluated the individual and interactive effects of soil initial bacterial diversity, nutrient amendments (organic and inorganic) and plant presence on the biodegradation rate of pyrene, a polycyclic aromatic hydrocarbon. Initial bacterial diversity had a strong positive impact on soil biodegradation potential, with soil harbouring higher bacterial diversity showing ~ 2 times higher degradation rates than soils with lower bacterial diversity. Both organic and inorganic nutrient amendments consistently improved the degradation rate in lower diversity soils and had negative (inorganic) to neutral (organic) effect in higher diversity soils. Interestingly, plant presence/type did not show any significant effect on the degradation rate in most of the treatments. Structural equation modelling demonstrated that initial bacterial diversity had a prominent role in driving pyrene biodegradation rates. We provide novel evidence that suggests that soil initial microbial diversity, and nutrient amendments should be explicitly considered in the design and employment of bioremediation management strategies for restoring natural habitats disturbed by organic pollutants.     

Bioremediation Potential of Rhodococcus pyridinivorans NT2 in Nitrotoluene-Contaminated Soils: The Effectiveness of Natural Attenuation,Biostimulation and Bioaugmentation Approaches

This work evaluated the effect of bioremediation treatments including natural attenuation, bioaugmentation, biostimulation as well as combined biostimulation and bioaugmentation on degradation of 4-nitrotoluene (4-NT), 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) in soil microcosms. Bioaugmentation with a previously isolated NTs-degrading bacterium, Rhodococcus pyridinivorans NT2, showed an 86–88% decrease in 4-NT, 2,4-DNT or 2,6-DNT after 60 days. Irrespective of the substrate types, least degradation (6–6.5%) was observed in abiotic control. The addition of β-cyclodextrin or rhamnolipid significantly improved NTs degradation efficiency in soil (18.5–74%) than natural attenuation (22–25%). Exogenous addition of preselected bacterial isolate NT2 along with β-cyclodextrin/rhamnolipid resulted in the greatest number (1.8× and 2.5× high) of total heterotrophic aerobic bacteria and NT degraders, respectively, compared to natural attenuation. Irrespective of the treatment types, the population of NT degraders increased steadily in the first 5 weeks of incubation followed by a plateau within the next few weeks. The treatment BABS2 (Soil + rhamnolipid + NT2) yielded highest microbial-C and -N and dehydrogenase activity, consistent with results of NTs degradation and microbial counts in combined bioaugmentation and biostimulation. Thus the results of this study suggest that bioaugmentation by R. pyridinivorans NT2 may be a promising bioremediation strategy for nitroaromatics-contaminated soils.     

Remediation of oil-based drill cuttings through a biosurfactant-based washing followed by a biodegradation treatment

In this study, oil-based drill cuttings were washed by a rhamnolipid solution and then subjected to bioremediation in stainless steel boxes using sawdust as bulking agent. A mixed bacterial culture, mainly containing Pseudomonas, Acinetobacter, Alcaligenes, Agrobacterium, and Comamonas, was used as inoculums. Approximately 83% of organics were removed after washing under optimal conditions (liquid/solid ratio, 3:1; washing time, 20 min; stirring speed, 200 rpm; rhamnolipid concentration, 360 mg/L; temperature, 60 °C), and the total petroleum hydrocarbon concentration of the cuttings dropped from 85,000 to 12,600 mg/kg. In the bioremediation stage, concentrations of saturated and aromatic hydrocarbons decreased to 2140 and 1290 mg/kg, respectively, after 120 days. Ultrahigh-resolution mass spectrometry demonstrated that oxygen- and nitrogen-containing compounds had undergone biodegradation. The results of this study indicate that this two-stage remedial system can reduce treatment time and increase treatment efficiency as compared with a single bioremediation or washing treatment.     

Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials

This study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low-cost raw materials. Two oil-degrading strains, Pseudomonas aeruginosa GS9-119 and DS10-129, were used to optimize a substrate for maximum rhamnolipid production. Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively. The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth. Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha-Rha-C(10)-C(10)). Emulsification activity of the rhamnolipid biosurfactant produced by P. aeruginosa DS10-129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel. P. aeruginosa GS9-119 emulsified only hexane and kerosene to that level.     

Production of rhamnolipids in solid-state cultivation: Characterization,downstream processing and application in the cleaning of contaminated soils

Pseudomonas aeruginosa UFPEDA 614 produced a rhamnolipid biosurfactant when grown on sugarcane bagasse impregnated with a solution containing glycerol. Biosurfactant levels reached 40 g of rhamnolipid per kilogram of dry initial substrate after 12 days. On the basis of the volume of liquid used, the biosurfactant levels were similar to those obtained in submerged liquid culture of a medium identical to the impregnating solution. The properties of the biosurfactant were very similar to those obtained with rhamnolipids produced in submerged culture, with a critical micelle concentration of 46.8 mg/L and an emulsification index at 24 h of over 90% against gasoline. The surface properties were maintained after autoclaving of the fermented solids, meaning that it is possible to minimize safety risks by killing the producing organism with a heat treatment of the solids prior to product extraction. The biosurfactant was used in the washing of soils contaminated with gasoline. An aqueous biosurfactant solution was 3.2-fold more efficient than water in leaching organic material from the soil, demonstrating the viability of application of rhamnolipids in the bioremediation of soils contaminated with gasoline.     

Mustard seed meal mixtures: management of Meloidogyne incognita on pepper and potential phytotoxicity

Meals produced when oil is extracted from seeds in the Brassicaceae have been shown to suppress weeds and soilborne pathogens. These seed meals are commonly used individually as soil amendments; the goal of this research was to evaluate seed meal mixes of Brassica juncea (Bj) and Sinapis alba (Sa) against Meloidogyne incognita. Seed meals from Bj 'Pacific Gold' and Sa 'IdaGold' were tested alone and in combinations to determine rates and application times that would suppress M. incognita on pepper (Capsicum annuum) without phytotoxicity. Rates of soil application (% w/w) for the phytotoxicity study were: 0.5 Sa, 0.2 Bj, 0.25 Sa + 0.25 Bj, 0.375 Sa + 0.125 Bj, 0.125 Sa + 0.375 Bj, and 0, applied 0 - 5 weeks before transplant. Overall, 0.2% Bj was the least toxic meal to pepper seedlings. By comparison, 0.5% S. alba seed meal did not reduce lettuce (Lactuca sativa) seed germination at week 0, but all seed meal treatments containing B. juncea prevented or significantly reduced germination at week 0. The seed meals did not affect lettuce seed germination at weeks 1-5, but hypocotyl growth was reduced by all except 0.2% Bj at weeks 1, 4 and 5. Brassica juncea and Sa meals were tested for M. incognita suppression at 0.2, 0.15, 0.1 and 0.05%; mixtures were 0.1% Sa + 0.1% Bj, 0.15% Sa + 0.05% Bj, and 0.05% Sa + 0.15% Bj. All treatments were applied 2 weeks before transplant. The 0.2% Bj and 0.05% Sa + 0.15% Bj treatments overall had the longest shoots and highest fresh weights. Eggs per g root were lowest with 0.1 - 0.2% Bj amendments and the seed meal mixtures. The results indicate that Bj and some Bj + Sa mixtures can be applied close to transplant to suppress M. incognita populations on pepper; consequently, a seed meal mixture could be selected to provide activity against more than one pest or pathogen. For pepper, care should be taken in formulating mixtures so that Sa rates are low compared to Bj.     

Efficiency of different types of biochars to mitigate Cd stress and growth of sunflower (Helianthus; L.) in wastewater irrigated agricultural soil

Cadmium contamination in croplands is recognized one of the major threat, seriously affecting soil health and sustainable agriculture around the globe. Cd mobility in wastewater irrigated soils can be curtailed through eco-friendly and cost effective organic soil amendments (biochars) that eventually minimizes its translocation from soil to plant. This study explored the possible effects of various types of plants straw biochar as soil amendments on cadmium (Cd) phytoavailability in wastewater degraded soil and its subsequent accumulation in sunflower tissues. The studied biochars including rice straw (RS), wheat straw (WS), acacia (AC) and sugarcane bagasse (SB) to wastewater irrigated soil containing Cd. Sunflower plant was grown as a test plant and Cd accumulation was recorded in its tissues, antioxidant enzymatic activity chlorophyll contents, plant biomass, yield and soil properties (pH, NPK, OM and Soluble Cd) were also examined. Results revealed that addition of biochar significantly minimized Cd mobility in soil by 53.4%, 44%, 41% and 36% when RS, WS, AC and SB were added at 2% over control. Comparing the control soil, biochar amended soil effectively reduced Cd uptake via plants shoots by 71.7%, 60.6%, 59% and 36.6%, when RS, WS, AC and SB. Among all the biochar, rice husk induced biochar significantly reduced oxidative stress and reduced SOD, POD and CAT activity by 49%, 40.5% and 46.5% respectively over control. In addition, NPK were significantly increased among all the added biochars in soil–plant system as well as improved chlorophyll contents relative to non-bioachar amended soil. Thus, among all the amendments, rice husk and wheat straw biochar performed well and might be considered the suitable approach for sunflower growth in polluted soil.     

Influence of organic and inorganic soil amendments on plant growth in crude oil-contaminated soil

Phytoremediation can be a viable alternative to traditional, more costly remediation techniques. Three greenhouse studies were conducted to evaluate plant growth with different soil amendments in crude oil-contaminated soil. Growth of alfalfa (Medicago sativa L., cultivar: Riley), bermudagrass (Cynodon dactylon L., cultivar: Common), crabgrass (Digitaria sanguinalis, cultivar: Large), fescue (Lolium arundinaceum Schreb., cultivar: Kentucky 31), and ryegrass (Lolium multiflorum Lam., cultivar: Marshall) was determined in crude oil-contaminated soil amended with either inorganic fertilizer, hardwood sawdust, papermill sludge, broiler litter or unamended (control). In the first study, the addition of broiler litter reduced seed germination for ryegrass, fescue, and alfalfa. In the second study, bermudagrass grown in broiler litter-amended soil produced the most shoot biomass, bermudagrass produced the most root biomass, and crabgrass and bermudagrass produced the most root length. In the third study, soil amended with broiler litter resulted in the greatest reduction in gravimetric total petroleum hydrocarbon (TPH) levels across the six plant treatments following the 14-wk study. Ryegrass produced more root biomass than any other species when grown in inorganic fertilizer- or hardwood sawdust + inorganic fertilizer-amended soil. The studies demonstrated that soil amendments and plant species selection were important considerations for phytoremediation of crude oil-contaminated soil.     

九里香种子脱水耐性和萌发生理的研究

九里香种子自花后 42～ 77d,含水量和电导率逐渐降低 ,种子干重、发芽率、发芽指数和活力指数逐渐增加。硅胶脱水 1～ 6d后 ,种子含水量下降 1 0 %～ 3 5 % ,发芽率、发芽指数和活力指数均有不同程度的降低 ,不同发育时期九里香种子的脱水耐性有别 ,花后 42～ 70d不断增强 ,77d有所减弱。花后 70d的种子含水量降至 1 0 % ,种子发芽率无明显降低 ;含水量为 9%的种子在 4°C和 2 0°C的低温条件贮存 3 0d和 42d ,多数种子仍能萌发 ,这表明九里香种子是一种正常型种子。光照能促进种子的萌发 ;在 2 0～ 3 0°C、室温和 2 0 /3 0°C变温条件下种子萌发较好 ;光照和温度对种子萌发有单独影响 ,但又相互作用 ,同时光照对萌发的影响还与种子含水量有关。     

Fertilization,soil and plant community characteristics determine soil microbial activity in managed temperate grasslands

Background and aims

Contaminated soils can impede germination and growth of selected plant species, restricting effective phytoremediation strategies. The purpose of the present study was to enhance the germination and growth of saltgrass [Distichlis spicata (L.) Greene] by evaluating the efficacy of certain seed pretreatments and soil amendments.

Methods

Ten seed pretreatment methods, two amendments, three soil depths and five saline levels were tested under greenhouse conditions.

Results

Saltgrass germination and growth were negatively correlated with increasing salinity levels when NaCl > 85.6 mM. Among ten seed pretreatments (stratification + Proxy 24 h, hot water + Proxy 24 h, stratification, hot water + Proxy 48 h, Proxy 48 h, Proxy 24 h, hot water, scarification, gibberellins, and KMnO₄), the two best methods were stratification + Proxy 24 h and hot water + Proxy 24 h for enhancing saltgrass germination, with the latter pretreatment being especially useful because of its shorter preparation time and high germination rates. Proxy is a commercial ethephon product. Potting soil (5.0 cm depth) was found to be the best amendment for saltgrass germination and growth in hydrocarbon-contaminated soils.

Conclusion

We conclude that direct seeding of saline soils contaminated with petroleum hydrocarbons is a feasible phytoremediation strategy provided that appropriate seed pretreatments and amendments are utilized.

     

矮沙冬青种子特性和萌发影响因素的研究

 对矮沙冬青(Ammopiptanthus nanus)种子的特性和萌发影响因素进行了初步研究,结果表明：种子不易传播;虫蛀率高,室温贮藏60 d的种子虫害率为38%;易形成硬实,含水量为7.68%的种子在30 ℃温水中浸泡90 h,只有33.33%能吸水膨胀。种子萌发时不需光,在15～30 ℃和室温（18～32 ℃）条件下,经9 d的萌发,发芽率均可达80%以上,30℃时萌发最快;在1～2 cm深的沙壤中,种子出苗率可达75%以上,超过3 cm显著降低,超过6 cm则低于20%;种子在沙壤中萌发时,沙壤的适宜湿度为19.35%～28.75%,高于32.43%或低于3.85%,很少有种子萌发;含水量分别为19.36%、10.64% 和7.68%的种子发芽率无显著差异,在-10 ℃和5 ℃下贮藏7个月,发芽率也无显著降低,但在室温和35 ℃下贮藏7个月则显著下降,发芽率下降的速度与种子本身的含水量和贮藏温度正相关;在湿度分别为7.41%、13.79%和28.57%的沙壤中播种育苗,幼苗死亡率高达77.49%、81.25%和89.49%,即使用三唑酮拌种,死亡率亦高达50.27%、69.53%和76.03%,幼苗死亡率与沙壤湿度正相关。     

Monitoring of microbial diversity and activity during bioremediation of crude oil-contaminated soil with different treatments

The present study compared the microbial diversity and activity during the application of various bioremediation processes to crude oil-contaminated soil. Five different treatments, including natural attenuation (NA), biostimulation (BS), biosurfactant addition (BE), bioaugmentation (BA), and a combined treatment (CT) of biostimulation, biosurfactant addition, and bioaugmentation, were used to analyze the degradation rate and microbial communities. After 120 days, the level of remaining hydrocarbons after all the treatments was similar, however, the highest rate (k) of total petroleum hydrocarbon (TPH) degradatioN was observed with the CT treatment (P < 0.05). The total bacterial counts increased during the first 2 weeks with all the treatments, and then remained stable. The bacterial communities and alkane monooxygenase gene fragment, alkB, were compared by denaturing gradient gel electrophoresis (DGGE). The DGGE analyses of the BA and CT treatments, which included Nocardia sp. H17-1, revealed a simple dominant population structure, compared with the other treatments. The Shannon-Weaver diversity index (H') and Simpson dominance index (D), calculated from the DGGE profiles using 16S rDNA, showed considerable qualitative differences in the community structure before and after the bioremediation treatment as well as between treatment conditions.     

Biodegradation of gasoline by gellan gum-encapsulated bacterial cells

Encapsulated cell bioaugmentation is a novel alternative solution to in situ bioremediation of contaminated aquifers. This study was conducted to evaluate the feasibility of such a remediation strategy based on the performance of encapsulated cells in the biodegradation of gasoline, a major groundwater contaminant. An enriched bacterial consortium, isolated from a gasoline-polluted site, was encapsulated in gellan gum microbeads (16-53 microm diameter). The capacity of the encapsulated cells to degrade gasoline under aerobic conditions was evaluated in comparison with free (non-encapsulated) cells. Encapsulated cells (2.6 mg(cells) x g(-1) bead) degraded over 90% gasoline hydrocarbons (initial concentration 50-600 mg x L(-1)) within 5-10 days at 10 degrees C. Equivalent levels of free cells removed comparable amounts of gasoline (initial concentration 50-400 mg x L(-1)) within the same period but required up to 30 days to degrade the highest level of gasoline tested (600 mg x L(-1)). Free cells exhibited a lag phase in biodegradation, which increased from 1 to 5 days with an increase in gasoline concentration (200-600 x mg L(-1)). Encapsulation provided cells with a protective barrier against toxic hydrocarbons, eliminating the adaptation period required by free cells. The reduction of encapsulated cell mass loading from 2.6 to 1.0 mg(cells) x g(-1) bead caused a substantial decrease in the extent of biodegradation within a 30-day incubation period. Encapsulated cells dispersed within the porous soil matrix of saturated soil microcosms demonstrated a reduced performance in the removal of gasoline (initial concentrations of 400 and 600 mg x L(-1)), removing 30-50% gasoline hydrocarbons compared to 40-60% by free cells within 21 days of incubation. The results of this study suggest that gellan gum-encapsulated bacterial cells have the potential to be used for biodegradation of gasoline hydrocarbons in aqueous systems.     

Evaluation of inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil

A full-scale study evaluating an inoculum addition to stimulate in situ bioremediation of oily-sludge-contaminated soil was conducted at an oil refinery where the indigenous population of hydrocarbon-degrading bacteria in the soil was very low (10(3) to 10(4) CFU/g of soil). A feasibility study was conducted prior to the full-scale bioremediation study. In this feasibility study, out of six treatments, the application of a bacterial consortium and nutrients resulted in maximum biodegradation of total petroleum hydrocarbon (TPH) in 120 days. Therefore, this treatment was selected for the full-scale study. In the full-scale study, plots A and B were treated with a bacterial consortium and nutrients, which resulted in 92.0 and 89.7% removal of TPH, respectively, in 1 year, compared to 14.0% removal of TPH in the control plot C. In plot A, the alkane fraction of TPH was reduced by 94.2%, the aromatic fraction of TPH was reduced by 91.9%, and NSO (nitrogen-, sulfur-, and oxygen-containing compound) and asphaltene fractions of TPH were reduced by 85.2% in 1 year. Similarly, in plot B the degradation of alkane, aromatic, and NSO plus asphaltene fractions of TPH was 95.1, 94.8, and 63.5%, respectively, in 345 days. However, in plot C, removal of alkane (17.3%), aromatic (12.9%), and NSO plus asphaltene (5.8%) fractions was much less. The population of introduced Acinetobacter baumannii strains in plots A and B was stable even after 1 year. Physical and chemical properties of the soil at the bioremediation site improved significantly in 1 year.     

Induction of defense response against Rhizoctonia solani in cucumber plants by endophytic bacterium Bacillus thuringiensis GS1

An endophytic bacterium, Bacillus thuringiensis GS1, was isolated from bracken (Pteridium aquilinum) and found to have maximal production of chitinase (4.3 units/ml) at 5 days after culture. This study investigated the ability of B. thuringiensis GS1 to induce resistance to Rhizoctonia solani KACC 40111 (RS) in cucumber plants. Chitinase activity was greatest in RS-treated plants at 4 days. beta-1,3- Glucanase activity was highest in GS1-treated plants at 5 days. Guaiacol peroxidase (GPOD) activity increased continuously in all treated plants for 5 days. Ascorbate peroxidase (APX) activity in RS-treated plants was increased 1.5-fold compared with the control at 4 days. Polyphenol oxidase (PPO) activity in RS-treated plants was increased 1.5-fold compared with the control at 3 days. At 5 days after treatment, activity staining revealed three bands with chitinase activity (Ch1, Ch2, and Ch3) on SDSPAGE of cucumber plants treated with GS1+RS, whereas only one band was observed for RS-treated plants (Ch2). One GPOD isozyme (Gp1) was also observed in response to treatment with RS and GS1+RS at 4 days. One APX band (Ap2) was present on the native-PAGE gel of the control, and GS1- and GS1+RS-treated plants at 1 day. PPO bands (Po1 and Po2) from RS- and GS1+RS-treated plants were stronger than in the control and GS1-treated plants upon native-PAGE at 5 days. Taken together, these results indicate that the induction of PR proteins and defense-related enzymes by B. thuringiensis GS1 might have suppressed the damping-off caused by R. solani KACC 40111 in cucumber plants.