Alicja Szulc

Biodegradation of diesel/biodiesel blends by a consortium of hydrocarbon degraders: Effect of the type of blend and the addition of biosurfactants

Biodegradation experiments for diesel/biodiesel blends in liquid cultures by-petroleum degrading microbial consortium showed that for low amendments of biodiesel (10%) the overall biodegradation efficiency of the mixture after seven days was lower than for petroleum diesel fuel. Preferential usage of methyl esters in the broad biodiesel concentration range and diminished biodegradation of petroleum hydrocarbons for 10% biodiesel blend was confirmed. Rhamnolipids improved biodegradation efficiency only for blends with low content of biodiesel. Emulsion formation experiments showed that biodiesel amendments significantly affected dispersion of fuel mixtures in water. The presence of rhamnolipids biosurfactant affected stability of such emulsions and altered cell surface properties of tested consortium.

Biodegradation and surfactant-mediated biodegradation of diesel fuel by 218 microbial consortia are not correlated to cell surface hydrophobicity

In this study, we elucidated the role of cell surface hydrophobicity (microbial adhesion to hydrocarbons method, MATH) and the effect of anionic rhamnolipids and nonionic Triton X-100 surfactants on biodegradation of diesel fuel employing 218 microbial consortia isolated from petroleum-contaminated soils. Applied enrichment procedure with floating diesel fuel as a sole carbon source in liquid cultures resulted in consortia of varying biodegradation potential and diametrically different cell surface properties, suggesting that cell surface hydrophobicity is a conserved parameter. Surprisingly, no correlations between cell surface hydrophobicity and biodegradation of diesel fuel were found. Nevertheless, both surfactants altered cell surface hydrophobicity of the consortia in similar manner: increased for the hydrophilic and decreased for the hydrophobic cultures. In addition to this, the surfactants exhibited similar influence on diesel fuel biodegradation: Increase was observed for initially slow-degrading cultures and the opposite for fast degraders. This indicates that in the surfactant-mediated biodegradation, effectiveness of surfactants depends on the specification of microorganisms and not on the type of surfactant. In contrary to what was previously reported for pure strains, cell surface hydrophobicity, as determined by MATH, is not a good descriptor of biodegrading potential for mixed cultures.

The influence of bioaugmentation and biosurfactant addition on bioremediation efficiency of diesel-oil contaminated soil: Feasibility during field studies

The study focused on assessing the influence of bioaugmentation and addition of rhamnolipids on diesel oil biodegradation efficiency during field studies. Initial laboratory studies (measurement of emitted CO2 and dehydrogenase activity) were carried out in order to select the consortium for bioaugmentation as well as to evaluate the most appropriate concentration of rhamnolipids. The selected consortium consisted of following bacterial taxa: Aeromonas hydrophila, Alcaligenes xylosoxidans, Gordonia sp., Pseudomonas fluorescens, Pseudomonas putida, Rhodococcus equi, Stenotrophomonas maltophilia, Xanthomonas sp. It was established that the application of rhamnolipids at 150xa0mg/kg of soil was most appropriate in terms of dehydrogenase activity. Based on the obtained results, four treatment methods were designed and tested during 365 days of field studies: I) natural attenuation; II) addition of rhamnolipids; III) bioaugmentation; IV) bioaugmentation and addition of rhamnolipids. It was observed that bioaugmentation contributed to the highest diesel oil biodegradation efficiency, whereas the addition of rhamnolipids did not notably influence the treatment process.

Relative quantitative PCR to assess bacterial community dynamics during biodegradation of diesel and biodiesel fuels under various aeration conditions

The degradation of diesel fuel, B20 blend and biodiesel in liquid cultures by a seven-member bacterial consortium was compared under conditions with full aeration or with limited aeration with nitrate added as main electron acceptor. Community dynamics was assessed employing real-time PCR and the ddCt method for relative quantification. Biodegradation rates increased with increasing biodiesel content, but were significantly reduced under conditions with nitrate. Despite large variations in biodegradation rates, magnitude changes in population numbers were typically observed only from zero to one order, regardless the type of fuel and electron acceptor. Only Comamonadaceae and Variovorax sp. distinctly preferred aerobic conditions, and during aerobic growth showed suppression as fuel contained more biodiesel. Thus, the consortium is relatively stable and most of the degraders can shift their metabolism from hydrocarbons to biodiesel. The stability of the consortium is of interest in the context of biodiesel-mediated biodegradation of petroleum hydrocarbons.

Biodegradation of rhamnolipids in liquid cultures: Effect of biosurfactant dissipation on diesel fuel/B20 blend biodegradation efficiency and bacterial community composition

Bacterial utilization of rhamnolipids during biosurfactant-supplemented biodegradation of diesel and B20 (20% biodiesel and 80% diesel v/v) fuels was evaluated under conditions with full aeration or with nitrate and nitrite as electron acceptors. Rhamnolipid-induced changes in community dynamics were assessed by employing real-time PCR and the ddCt method for relative quantification. The experiments with rhamnolipids at 150 mg/l, approx. double critical micelle concentration (CMC) and diesel oil confirmed that rhamnolipids were readily degraded by a soil-isolated consortium of hydrocarbon degraders in all samples, under both aerobic and nitrate-reducing conditions. The presence of rhamnolipids increased the dissipation rates for B20 constituents under aerobic conditions, but did not influence the biodegradation rate of pure diesel. No effect was observed under nitrate-reducing conditions. The biodegradation of rhamnolipids did not favor the growth of any specific consortium member, which proved that the employed biosurfactant did not interfere with the microbial equilibrium during diesel/biodiesel biodegradation.

Utilization of Triton X-100 and polyethylene glycols during surfactant-mediated biodegradation of diesel fuel

The hypothesis regarding preferential biodegradation of surfactants applied for enhancement of microbial hydrocarbons degradation was studied. At first the microbial degradation of sole Triton X-100 by soil isolated hydrocarbon degrading bacterial consortium was confirmed under both full and limited aeration with nitrate as an electron acceptor. Triton X-100 (600 mg/l) was utilized twice as fast for aerobic conditions (t(1/2)=10.3h), compared to anaerobic conditions (t(1/2)=21.8h). HPLC/ESI-MS analysis revealed the preferential biodegradation trends in both components classes of commercial Triton X-100 (alkylphenol ethoxylates) as well as polyethylene glycols. The obtained results suggest that the observed changes in the degree of ethoxylation for polyethylene glycol homologues occurred as a consequence of the central fission mechanism during Triton X-100 biodegradation. Subsequent experiments with Triton X-100 at approx. CMC concentration (150 mg/l) and diesel oil supported our initial hypothesis that the surfactant would become the preferred carbon source even for hydrocarbon degrading bacteria. Regardless of aeration regimes Triton X-100 was utilized within 48-72 h. Efficiency of diesel oil degradation was decreased in the presence of surfactant for aerobic conditions by approx. 25% reaching 60 instead of 80% noted for experiments without surfactant. No surfactant influence was observed for anaerobic conditions.

Bioaugmentation with Petroleum-Degrading Consortia Has a Selective Growth-Promoting Impact on Crop Plants Germinated in Diesel Oil-Contaminated Soil

Rhizoremediation is a complex type of green clean-up technology that involves both plants and the rhizosphere-associated microorganisms to decompose hazardous compounds. The success of the strategy strongly depends on plant tolerance towards the pollutant, as well as plants interactions with the rhizospheric microbes. The microorganisms may be stimulated by the secreted root exudates, which results in an increased breakdown of contaminants in the rhizosphere. The main goal of this study was to establish a potential rhizoremediation combination for a diesel-polluted site. Inoculation of plant roots or seeds with indigenous rhizospheric populations is a common approach in the rhizoremediation. However, we introduced hydrocarbon-degrading consortia (M10, R3, and K52) that were previously isolated from crude oil-contaminated soil instead of indigenous microbes. Bioaugmentation with these petroleum degraders was applied to screen four high biomass crop species (Indian mustard, alfalfa, high erucic acid rapeseed, HEAR, and low erucic acid rapeseed, LEAR) for their tolerance towards diesel oil. At no pollution, a promoting effect of M10 bacteria could be observed on germination and root elongation of all plant species. Moreover, M10 consortiums increased the germination index at 6,000xa0mg diesel oil per kilogram dry soil in the case of Indian mustard, alfalfa, and HEAR. The latter species was found to increment its dry weight upon bioaugmentation with M10 bacteria and all diesel oil treatments (6,000 and 24,000xa0mg diesel oil per kilogram dry soil). The initial results indicate HEAR and the M10 bacterial consortium as a promising plant–microbe tandem for a long-term rhizoremediation process.

Rhamnolipids Increase the Phytotoxicity of Diesel Oil Towards Four Common Plant Species in a Terrestrial Environment

The study focused on assessing the influence of rhamnolipids on the phytotoxicity of diesel oil-contaminated soil samples. Tests evaluating the seed germination and growth inhibition of four terrestrial plant species (alfalfa, sorghum, mustard and cuckooflower) were carried out at different rhamnolipid concentrations (ranging from 0 to 1.200xa0mg/kg of wet soil). The experiments were performed in soil samples with a different diesel oil content (ranging from 0 to 25xa0ml/kg of wet soil). It was observed that the sole presence of rhamnolipids may be phytotoxic at various levels, which is especially notable for sorghum (the germination index decreased to 41xa0%). The addition of rhamnolipids to diesel oil-contaminated soil samples contributed to a significant increase of their phytotoxicity. The most toxic effect was observed after a rhamnolipid-supplemented diesel oil biodegradation, carried out with the use of a hydrocarbon-degrading bacteria consortium. The supplemention of rhamnolipids (600xa0mg/kg of wet soil) resulted in a decrease of seed germination of all studied plant species and an inhibition of microbial activity, which was measured by the 2,3,5-triphenyltetrazolium chloride tests. These findings indicate that the presence of rhamnolipids may considerably increase the phytotoxicity of diesel oil. Therefore, their use at high concentrations, during in situ bioremediation processes, should be avoided in a terrestrial environment.

Comparative study on the biodegradability of morpholinium herbicidal ionic liquids.

This study focused on evaluating the toxicity as well as primary and ultimate biodegradability of morpholinium herbicidal ionic liquids (HILs), which incorporated MCPA, MCPP, 2,4-D or Dicamba anions. The studied HILs were also subjected to determination of surface active properties in order to assess their influence on toxicity and biodegradability. The study was carried out with microbiota isolated from different environmental niches: sediments from river channel, garden soil, drainage trench collecting agricultural runoff stream, agricultural soil and municipal waste repository. The obtained results revealed that resistance to toxicity and biodegradation efficiency of the microbiota increased in the following order: microbiota from the waste repositoryxa0>xa0microbiota from agricultural soilxa0≈xa0microbiota from an agricultural runoff streamxa0>xa0microbiota from garden soilxa0>xa0microbiota from the river sludge. It was observed that the toxicity of HILs increased with the hydrophobicity of the cation, however the influence of the anion was more notable. The highest toxicity was observed when MCPA was used as the anion (EC50 values ranging from 60 to 190xa0mgxa0L−1). The results of ultimate biodegradation tests indicated that only HILs with 2,4-D as the anion were mineralized to some extent, with slightly higher values for HILs with the 4-decyl-4-ethylmorpholinium cation (10–31xa0%) compared to HILs with the 4,4-didecylmorpholinium cation (9–20xa0%). Overall, the cations were more susceptible (41–94xa0%) to primary biodegradation compared to anions (0–61xa0%). The obtained results suggested that the surface active properties of the studied HILs may influence their toxicity and biodegradability by bacteria in different environmental niches.

Biodegradation of diesel fuel by a microbial consortium in the presence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues

Fast development of ionic liquids as gaining more and more attention valuable chemicals will undoubtedly lead to environmental pollution. New formulations and application of ionic liquids may result in contamination in the presence of hydrophobic compounds, such as petroleum mixtures. We hypothesize that in the presence of diesel fuel low-water-soluble ionic liquids may become more toxic to hydrocarbon-degrading microorganisms. In this study the influence of 1-alkoxymethyl-2-methyl-5-hydroxypyridinium chloride homologues (side-chain length from C3 to C18) on biodegradation of diesel fuel by a bacterial consortium was investigated. Whereas test performed for the consortium cultivated on disodium succinate showed that toxicity of the investigated ionic liquids decreased with increase in side-chain length, only higher homologues (C8–C18) caused a decrease in diesel fuel biodegradation. As a result of exposure to toxic compounds also modification in cell surface hydrophobicity was observed (MATH). Disulphine blue active substances method was employed to determine partitioning index of ionic liquids between water and diesel fuel phase, which varied from 1.1 to 51% for C3 and C18 homologues, respectively. We conclude that in the presence of hydrocarbons acting as a solvent, the increased bioavailability of hydrophobic homologues is responsible for the decrease in biodegradation efficiency of diesel fuel.