Influence of a nonaqueous phase liquid (NAPL) on biodegradation of phenanthrene

A series of batch reactor experiments was carried out to examine the effect of a nonaqueous phase liquid (NAPL) on the biodegradation of a hydrophobic solute. A mathematical program model that describes physical processes of solute solubilization and partitioning between the NAPL and aqueous phases as well as microbial degradation and oxygen utilization was used to analyze the test data. The model calculates the cumulative changes in concentration of substrate, cell mass, carbon dioxide, and dissolved oxygen as a function of time. The equations incorporate the effects of solute solubilization, partitioning, biodegradation, as well as oxygen availability. Hexadecane was used as the model NAPL and was not biodegraded in the timeframe of the experiments performed. The model solute was the polyaromatic hydrocarbon, phenanthrene. In agreement with several previous studies, experimental measurements showed that hexadecane increased rates of mineralization of 15 mg phenanthrene when present at low mass but decreased rates at high mass. Model results suggest that partitioning of the phenanthrene into the hexadecane phase limits bioavailability at high NAPL mass. Further the model suggests that mineralization rates were higher with the low NAPL mass because aqueous phenanthrene concentrations were higher in those treatments from ca. 20 to 40 h than in other treatments. Finally, experiments showed that the presence of hexadecane, at all masses tested, resulted in a lower cell yield, effectively increasing the amount of CO₂ produced during the experiment. Model results suggest that this is due to changes in phenanthrene metabolism that are induced by the presence of the hexadecane phase. Model studies aimed at increasing rates of biodegradation by modifying operating conditions are described along with practical approaches to implementing these modifications.     

Isolation and Characterization of Biosurfactant-Producing Bacteria from Oil-Contaminated Soil

Two biosurfactant-producing Pseudomonas aeruginosa strains (KISR C1 and KISR B1) were isolated from Kuwaiti oil-contaminated soil, which resulted from the Gulf War. The optimum environmental conditions that supported the growth and surfactant production of both isolates were examined. The two isolates differed in their biosurfactant-stimu-lating carbon source, nitrogen concentration, and the pH of the medium. C-1 isolate produced two types of rhamnolipids with a final concentration of 98.4?g/l after spiking the nitrogen-limited medium with 10?mg/ml olive oil. The other isolate (B-1) produced only one type of rhamnolipid (5.9?g/l) after spiking the medium with crude oil. The biosurfactant produced by this strain was found to be very effective in the emulsifica-tion of crude oil. The result suggests that this isolate can potentially be used to enhance bioremediation of oil-contamination and enhanced oil recovery.     

Use of rhamnolipid biosurfactant for membrane biofouling prevention and cleaning

Rhamnolipids were evaluated as biofouling reducing agents in this study. The permeability of the bacterial outer membrane was increased by rhamnolipids while the growth rate of Pseudomonas aeruginosa was not affected. The surface hydrophobicity was increased through the release of lipopolysaccharides and extracellular polymeric substances from the outer cell membrane. Rhamnolipids were evaluated as agents for the prevention and cleaning of biofilms. A high degree of biofilm detachment was observed when the rhamnolipids were used as a cleaning agent. In addition, effective biofilm reduction occurred when rhamnolipids were applied to various species of Gram-negative bacteria isolated from seawater samples. Biofilm reduction using rhamnolipids was comparable to commercially available surfactants. In addition, 20% of the water flux was increased after rhamnolipid treatment (300 μg ml^?1, 6 h exposure time) in a dead-end filtration system. Rhamnolipids appear to have promise as biological agents for reducing membrane biofouling.     

Continuous rhamnolipid production using denitrifying Pseudomonas aeruginosa cells in hollow‐fiber bioreactor

Rhamnolipids are high‐value effective biosurfactants produced by Pseudomonas aeruginosa. Large‐scale production of rhamnolipids is still challenging especially under free‐cell aerobic conditions in which the highly foaming nature of the culture broth reduces the productivity of the process. Immobilized systems relying on oxygen as electron acceptor have been previously investigated but oxygen transfer limitation presents difficulties for continuous rhamnolipid production. A coupled system using immobilized cells and nitrate instead of oxygen as electron acceptor taking advantage of the ability of P. aeruginosa to perform nitrate respiration was evaluated. This denitrification‐based immobilized approach based on a hollow‐fiber setup eliminated the transfer limitation problems and was found suitable for continuous rhamnolipid production in a period longer than 1,500 h. It completely eliminated the foaming difficulties related to aerobic systems with a comparable specific productivity of 0.017 g/(g dry cells)‐h and allowed easy recovery of rhamnolipids from the cell‐free medium. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 346–351, 2013     

发酵碳源对铜绿假单胞菌NY3所产鼠李糖脂结构及性能的影响

为研究发酵碳源对铜绿假单胞菌NY3所产鼠李糖脂结构及性能的影响,从鼠李糖脂的结构组分、性能和应用效果等方面展开研究。薄层实验证明两种鼠李糖脂均含有单糖脂和双糖脂。液质分析发现以橄榄油作碳源时,鼠李糖脂中双糖脂(Rha-Rha-C5-C6:1和Rha-Rha-C8-C8:2)比例更大,约为73.09%。而地沟油作碳源时,单糖脂(Rha-C10-C10和Rha-C16-C16:2)的比例更高,约为76.91%。橄榄油和地沟油为碳源的鼠李糖脂的临界胶束浓度(CMC)分别为55 mg/L和80mg/L。相同投加量时,前者乳化性和乳化稳定性均优于后者。NY3菌降解含油污泥时,投加双糖脂含量高的鼠李糖脂会使C16-C30直链烷烃的去除率更高。     

Stimulating in-soil rhamnolipid production in a bioslurry reactor by limiting nitrogen

A soil with aged contamination from lubricating oil (LO) and polychlorinated biphenyls (PCBs) was treated in a bioslurry reactor to investigate in-soil biosurfactant production by Pseudomonas aeruginosa, the most abundant indigenous, culturable, hydrocarbon-degrading microorganism. After 2 days of growth on LO, a depletion of nitrogen stimulated the production and accumulation of rhamnolipids to levels roughly 20 times the critical micelle concentration. Surface tensions and concentrations of monorhamnolipid and dirhamnolipid, PCBs, and total petroleum hydrocarbons (TPH) were measured in a slurry filtrate. Soil-bound PCBs and TPH were also quantified. Rhamnolipid production was observed within 1 to 2 days after nitrogen depletion in each of the 10 batches tested. By day 6, total rhamnolipid concentrations increased from below detection to average values over 1,000 mg/L, which caused over 98% of soil-bound PCBs and over 99% of TPH to be emulsified and recovered in the filtrate. After 70 days, rhamnolipid concentrations were only reduced by 15%, because of nitrogen-limited rates of rhamnolipid biodegradation. The results show that in-soil biosurfactant production can be stimulated in a controlled way with nutrient limitation and can be used to achieve soil washing.     

Effect of addition ofPseudomonas aeruginosa UG2 inocula or biosurfactants on biodegradation of selected hydrocarbons in soil

Summary A laboratory study was undertaken to assess the effect of adding eitherPseudomonas aeruginosa UG2 cells or the biosurfactants produced by this m microorganism on the biodegradation of a hydrocarbon mixture in soil at 20°C over a 2-month incubation period. The addition of 100 g of UG2 biosurfactants per g soil significantly enhanced the degradation of tetradecane, hexadecene and pristane but not 2-methylnaphthalene, the most water-soluble of the hydrocarbons. Addition of UG2 cells at densities of 10⁶, 10⁷, and 10⁸ per g soil did not have a significant effect on biodegradation of the hydrocarbon mixture.     

Comparison of mono-rhamnolipids and di-rhamnolipids on microbial enhanced oil recovery (MEOR) applications

Rhamnolipids (RMLs) have more effectiveness for specific uses according to their homologue proportions. Thus, the novelty of this work was to compare mono-RMLs and di-RMLs physicochemical properties on microbial enhanced oil recovery (MEOR) applications. For this, RML produced by three strains of Pseudomonas aeruginosa containing different homologues proportion were used: a mainly mono-RMLs producer (mono-RMLs); a mainly di-RMLs producer (di-RMLs), and the other one that produces relatively balanced amounts of mono-RML and di-RML homologues (mono/di-RML). For mono-RML, the most abundant molecules were Rha-C₁₀C₁₀ (m/z 503.3), for di-RML were RhaRha-C₁₀C₁₀ (m/z 649.4) and for Mono/di-RML were Rha-C₁₀C₁₀ (m/z 503.3) and RhaRha-C₁₀C₁₀ (m/z 649.4). All RMLs types presented robustness under high temperature and variation of salinity and pH, and high ability for oil displacement, foam stability, wettability reversal and were classified as safe for environment according to the European Union Directive No. 67/548/EEC. For all these properties, it was observed a highlight for mono-RML. Mono-RML presented the lowest surface tension (26.40 mN/m), interfacial tension (1.14 mN/m), and critical micellar concentration (CMC 27.04 mg/L), the highest emulsification index (EI₂₄ 100%) and the best wettability reversal (100% with 25 ppm). In addition, mono-RML showed the best acute toxicity value (454 mg/L), making its application potential even more attractive. Based on the results, it was concluded that all RMLs homologues studied have potential for MEOR applications. However, results showed that mono-RML stood out and have the best mechanism of oil incorporation in micelles due their most effective surface-active physicochemical features.     

Effect of rhamnolipid on the aerobic removal of polyaromatic hydrocarbons (PAHs) and COD components from petrochemical wastewater

The removal efficiencies of 15 PAHs and some COD components (inert, readily degradable, slowly degradable and metabolic products) from a wastewater taken from a petrochemical industry treatment plant (İzmir, Turkey) have been determined using an aerobic completely stirred tank reactor (CSTR). Addition of rhamnolipid surfactant (15 mg l⁻¹) increased the removal efficiencies of PAHs and soluble COD from 72% and 90% to 80% and 99%, respectively. The rhamnolipid treatment caused a significant increase of 5- and 6-ring PAH degradation. The soluble COD removal efficiency was 93%, in CSTR reactors with rhamnolipid added. The inert COD removal efficiency was 60% in a CSTR reactor containing rhamnolipid. Batch tests showed that removal arising from the adsorption of the PAHs was low (between 1.88% and 4.84%) while the removal of PAHs from the petrochemical industry wastewater via volatilization varied between 0.69% and 5.92%. Low sorption capacity (K_p) values for refinery activated sludge (approximately 2.98 l g⁻¹) confirmed that bio-sorption was not an important mechanism controlling the fate of PAHs in aerobic CSTR reactors. Models proposed to simulate the PAH removal indicated that 94% of the PAHs were removed via biodegradation.     

微生物降解多环芳烃的研究进展

多环芳烃是一类长久存在于环境中,具有毒性、致突变与致癌等特性的环境优先污染物。本文对降解多环芳烃的微生物类群进行了阐述,介绍了在土壤与厌氧条件下细菌降解多环芳烃的研究情况,最后介绍了降解多环芳烃的相关酶类以及分子生物学的研究,并对消除环境中多环芳烃的相关生物技术提出展望。     

Effect of salt on aerobic biodegradation of petroleum hydrocarbons in contaminated groundwater

Hydrocarbon-contaminated soil and groundwater at oil and gas production sites may be additionally impacted by salts due to release of produced waters. However, little is known about the effect of salt on the in-situ biodegradation of hydrocarbons by terrestrial microbes, especially at low temperatures. To study this effect, we prepared a groundwater-soil slurry from two sites in Canada: a former flare pit site contaminated with flare pit residue (Site A), and a natural gas processing facility contaminated with natural gas condensate (Site B). The slurry with its indigenous microbes was amended with radiolabeled hydrocarbons dissolved in free product plus nutrients and/or NaCl, and incubated in aerobic biometer flasks with gyrotory shaking at either 25 or 10°C for up to 5 weeks. Cumulative production of ¹⁴CO₂ was measured and the lag time, rate and extent of mineralization were calculated. For Site A, concentrations of NaCl ≥1% (w/v) delayed the onset of mineralization of both ¹⁴C-hexadecane and ¹⁴C-phenanthrene under nutrient-amended conditions, but once biodegradation began the degradation rates were similar over the range of salt concentrations tested (0–5% NaCl). For Site B, increasing concentrations of NaCl ≥1% (w/v) increased the lag time and decreased the rate and extent of mineralization of aliphatic and aromatic substrates. Of particular interest is the observation that low concentrations of salt (≤1% NaCl) slightly stimulated mineralization in some cases.     

Swarming of Pseudomonas aeruginosa PAO1 without differentiation into elongated hyperflagellates on hard agar minimal medium

Polar flagellated Pseudomonas aeruginosa PAO1 demonstrated extensive spreading growth in 2 days on 1.5% agar medium. Such spreading growth of P. aeruginosa PAO1 strains was absent on Luria-Bertani 1.5% agar medium, but remarkable on Davis minimal synthetic agar medium (especially that containing 0.8% sodium citrate and 1.5% Eiken agar) under aerobic 37 degrees C conditions. Analyses using isogenic mutants and complementation transformants showed that bacterial flagella and rhamnolipid contributed to the surface-spreading behavior. On the other hand, a type IV pilus-deficient pilA mutant did not lose the spreading growth activity. Flagella staining of PAO1 T cells from the frontal edge of a spreading colony showed unipolar and normal-sized rods with one or two flagella. Thus, the polar flagellate P. aeruginosa PAO1 T appears to swarm on high-agar medium by producing biosurfactant rhamnolipid and without differentiation into an elongated peritrichous hyperflagellate.     

Effect of Rhamnolipids on Chromium-Contaminated Kaolinite

Hexavalent chromium Cr(VI) is a common environmental pollutant that is treated by its reduction to the trivalent form Cr(III). The latter can be re-oxidized to the toxic form, Cr(VI), under specific conditions. A study was conducted on the removal of Cr(III) to eliminate the hazard imposed by its presence in soil as there has been some evidence that organic compounds can decrease its sorption. The effect of addition of negatively-charged biosurfactants (rhamnolipids) on chromium contaminated kaolinite was studied. Results showed that the rhamnolipids have the capability of extracting 25% portion of the stable form of chromium, Cr(III), from the kaolinite, under optimal conditions. The removal of hexavalent chromium was also enhanced compared to water by a factor of 2 using a solution of rhamnolipids. Results from the sequential extraction procedure showed that rhamnolipids remove Cr(III) mainly from the carbonate and oxide/hydroxide portions of the kaolinite. The rhamnolipids had also the capability of reducing close to 100% of the extracted Cr(VI) to Cr(III) over a period of 24 days. This study indicated that rhamnolipids could be beneficial for the removal or long–term conversion of chromium Cr(VI) to Cr(III).     

Utilization of monocyclic aromatic hydrocarbons individually and in mixture by bacteria isolated from petroleum-contaminated soil

The fate of benzene, ethylbenzene, toluene, xylenes (BTEX) compounds through biodegradation was investigated using two different bacteria, Ralstonia picketti (BP-20) and Alcaligenes piechaudii (CZOR L-1B). These bacteria were isolated from extremely polluted soils contaminated with petroleum hydrocarbons. PCR and Fatty Acid Methyl Ester (FAME) were used to identify the isolates. In this study, BTEX biodegradation, applied as a mixture or as individual compounds by the bacteria was evaluated. Both bacteria were shown to degrade each of the BTEX compounds individually and in mixture. However, Alcaligenes piechaudii was a better degrader of BTEXs both in the mixture and individually. Differences between BTEX biodegradation in the mixture and individually were observed, especially in the case of benzene. The degradation of all BTEXs in the mixture was lower than the degradation of individual compounds for both bacteria tested. In the all experiments, toluene and m + p- xylenes were better removed than the other BTEXs. No intermediates of biodegradation were detected. Biosurfactant production was observed by culture techniques. In addition, 3-hydroxy fatty acids, important in biosurfactant production, were observed by FAME analysis. The test results indicate that the bacteria could contribute to bioremediation of aromatic hydrocarbon pollution.     

Changes in the circulating hemocyte population of Manduca sexta larvae following injection of bacteria

A technique for the collection of stable hemolymph from larvae of Manduca sexta has been developed. The method avoids the cell clumping and melanization reactions commonly encountered with insect hemolymph by minimizing contact between hemocytes and surfaces which provoke defensive or repair responses. The circulating hemocyte population of second-day, fifth-instar larvae (2dL5) of M. sexta consisted of 4.5 ± 2.5 × 10⁶ cells/ml (n = 15, range 2–7 × 10⁶ cells/ml) and contained five cell types: prohemocytes, plasmatocytes, granulocytes, spherulocytes, and oenocytoids. Two strains of Pseudomonas aeruginosa which differ in pathogenicity (P11-1 and 9027) and Escherichia coli D31 grew well at 26°C in cell-free hemolymph prepared from naive (nonimmunized) 2dL5 M. sexta. When viable cells of any of the three bacteria were injected into M. sexta larvae, changes in both the total hemocyte count (THC) and differential hemocyte count were observed. Viable bacteria were not required to produce these changes since formalin-killed cells of P. aeruginosa 9027 produced a qualitatively and quantitatively similar response. Following injection of bacteria, the THC increased, reaching a maximal level at 1 hr postinjection, and remained elevated for at least 4 hr after injection. While prohemocytes, plasmatocytes, granulocytes, and spherulocytes all increased in number, 80% of the increased cell population at 1 hr postinjection of bacteria were the latter two cell types. Granulocytes and spherulocytes are cells with recognized defensive capabilities. The increased numbers of these cells in circulation soon after injection of bacteria may confer an advantage on M. sexta larvae in dealing with bacterial infections. This could explain in part the unusual resistance of M. sexta to certain bacterial pathogens.     

污染土壤中多环芳烃生物降解的调控研究

选用温度、湿度、表面活性剂ＴＷ８０和ＣＮＰ比４个因素为调控因子,采用正交法进行周期为１５０天的实验研究．结果表明,３０天后,土壤中ＰＡＨｓ的降解率可达４４．５～７４．６％,６０天后,达７０．４～９３．７％,降解率的不同与调控条件显著相关．在此期间,降解最佳条件为４０℃,湿度２５％,ＣＮＰ比为１２０１０１,ＴＷ８０分别为２００～５００ｍｇ·ｋｇ－１．实验结束时,土壤中ＰＡＨｓ的降解率达９１．２～９９．８％．降解的最佳条件是４０℃,湿度１５％．经Ｒ值判别表明,不同时期各因子对ＰＡＨｓ降解影响有所不同．温度对ＰＡＨｓ降解影响较大,表面活性剂对土壤中ＰＡＨｓ的生物降解有调控作用．     

填埋场氯代烃生物降解过程的机制转化与调控研究及展望

明晰氯代烃在复杂污染体系中的生物转化机制对强化污染物原位生物修复有重要意义。填埋场属典型复合污染场地,本文对不同地区填埋场填埋气中氯代烃种类、含量和其在覆盖层中的降解情况进行统计分析,发现填埋气中主要包括氯代烷烃和氯代烯烃两大类污染物,其浓度分别为0.20–32.45μg/m~3和0.50–32.45μg/m~3;覆盖土对氯代烃降解速率随着氯原子取代的增多而降低。基于覆盖层中微生物种类多、生长底物复杂多样和不同梯度氧气含量差异等特点,总结得出氯代烃在覆盖土中的降解途径主要是好氧共代谢、直接氧化和厌氧还原脱氯;并基于不同工况特点构建了氯代烃在填埋场覆盖层底部扩散至大气界面过程的生物转化机制模型。最后就复杂环境体系中氯代烃类污染物的去除进行了展望。