Enhancing biodegradation of aged hydrocarbon-contaminated soils through toluene addition: assessing effects on solid and slurry phase treatments.

The main challenge in treating aged soils highly contaminated with total petroleum hydrocarbons (TPH) is to enhance their bioavailability for microbial degradation. Hydrocarbons in soils undergo chemical changes that make them more resistant to biodegradation. This study investigates toluene's efficacy in enhancing the biodegradation of aged hydrocarbon-contaminated soil containing 292,000 mg TPH kg-1 dry soil. Toluene's effect was compared between solid phase (SOP) and slurry phase (SLP) treatments using a microbial consortium isolated from Cyperus laxus rhizosphere. TPH biodegradation and microbial respiration were measured, the latter to estimate the respiratory quotient (RQ, the ratio between moles of carbon dioxide released and moles of oxygen absorbed during respiration). Toluene significantly accelerated TPH biodegradation in both treatments, achieving ~ 30% higher removal than in a non-solvent control, possibly through improved bioavailability of aromatic compounds and other low molecular weight compounds. According to the RQ analysis, toluene enhanced microbial respiratory processes and hydrocarbon catabolism with higher hydrocarbon mineralization (RQ = ~ 0.5) in both SOP and SLP assays. Our results reveal toluene's potential to increase hydrocarbon availability and microbial degradation efficiency in aged contaminated soils; its use in various bioremediation techniques could be of broad applicability across diverse soil types and pollutants.

Sorption and inhibitory effect of octylphenol ethoxylate Triton X-100 on methanogenic and denitrifying granular sludges.

The aims of this work were to characterize the sorption and evaluate the inhibitory effect of octylphenol ethoxylate Triton X-100 (OPEOTx) on methanogenic and denitrifying sludges. According to Langmuir isotherm, maximums OPEOTx sorption values on methanogenic and denitrifying sludges were 60.70 mg (gVSS)-1 and 87.47 mg (gVSS)-1 respectively. The specific removal rate of chemical oxygen demand (rCOD) and the accumulated volume biogas (VBG) were used to evaluate the OPEOTx inhibitory effect on sludges. Experimental inhibition data were fitted to the models of non-competitive inhibition and modified Gompertz. Methanogenic sludges reached higher levels inhibition in the rCOD and biogas production potential Pmax (84.0 and 88.5%) comparing with denitrifying sludges (24.3 and 21.9%). Furthermore, in all OPEOTx concentrations, carbohydrates-proteins quotient value of the extracellular polymeric substances for the denitrifying sludges remained below respect to the same quotient in methanogenic sludges. The above contributes in part to explain the greater sorption capacity of the denitrifying sludges by OPEOTx and their granules resistance to be damaged by OPEOTx amphiphilic nature. The study gives insights to understand OPEOs interactions and their effects on methanogenic and denitrifying granular sludges.

In vitro evaluation of germination and growth of five plant species on medium supplemented with hydrocarbons associated with contaminated soils.

The effect of a hydrocarbon mixture (HCM) of three polycyclic aromatic hydrocarbons (PAH) and Maya crude oil on germination, growth and survival of four grasses (Bouteloua curtipendula, Cenchrus ciliaris, Echinochloa crusgalli and Rhynchelytrum repens) was studied and compared to a control (Festuca arundinacea) under in vitro conditions. The species were cultured on MS medium with different HCM initial concentrations. Germination was not affected for any assayed concentration; however, the length of the stems and roots decreased when HCM increased and the survival of the four species also diminished. Except for F. arundinacea, a direct link between hydrocarbon concentration and plant survival was observed. In vitro studies are clean and easy to handle techniques allowing isolation of the plant activity from that derived from associations with microorganisms in non-sterile cultures. To our knowledge, this is the first work towards phytoremediation assisted by in vitro plant cultivation.

Improvement of the hydrocarbon phytoremediation rate by Cyperus laxus Lam. inoculated with a microbial consortium in a model system.

Hydrocarbon phytoremediation by Cyperus laxus Lam. growing on perlite and inoculated with hydrocarbon-degrading microorganisms was evaluated. Total petroleum hydrocarbons (TPH) were extracted from weathered soil (60.7 g of TPH kg(-1) of dry soil) and spiked on perlite at initial concentration of 5 g of TPH kg(-1) of dry perlite. Phenological characteristics, total microbial viable counts, hydrocarbon degraders and residual hydrocarbons were determined through 180 days of culture. Phenological characteristics of inoculated plants were improved as compared with non-inoculated plants: root biomass was 1.6 times greater, flowering time was reduced (13%), and the number of inflorescences was 1.5 times higher. The rhizospheric bacterial and fungi counts were higher for planted treatments (inoculated and not inoculated) than for unplanted pots. The maximum phytoremediation rate (0.51 mg of TPH g(-1) of dry plant d(-1)) for inoculated plants was reached at 60 days of culture, and was two times higher than for non-inoculated plants (55% TPH removal). Similar hydrocarbon phytoremediation extent values for inoculated (90%) and non-inoculated (85%) plants were obtained at 180 days of culture. The present study demonstrated that mutual benefits between C. laxus and inoculated hydrocarbon-degrading microorganisms are improved during phytoremediation. It is pertinent to note that this is the first report of hydrocarbon phytoremediation by Cyperus laxus Lam., a native plant growing in highly contaminated swamps.

Mass transfer and hydrocarbon biodegradation of aged soil in slurry phase.

Addition of toluene into slurry phase laboratory microcosm is proposed in order to increase desorption rate of hydrocarbons and as an alternative to improve bioavailability of hydrocarbon in aged soils. Our studies showed that toluene has a positive effect on desorption of total petroleum hydrocarbons (TPH). Addition of 14,000 mg toluene/kg of soil, in highly polluted soil, increased the consumption rate of hydrocarbons three times in comparison to control without solvent. In 30 days the initial TPH concentration in soil, 292,000 mg/kg, diminished 45%. Although toluene was able to dissolve complex organic compounds such as asphaltene fraction, it probably yielded a highly toxic toluene-hydrocarbons phase. The inhibitory effect of toluene-TPH was also studied. A substrate inhibition model was used: the k(m) and k(i) constants were 57 and 490 mg TPH/L liquid phase, respectively. Experimental data were well described when the proposed model included sequential desorption and biodegradation phenomena. Damköhler number evaluation showed that rate of mass transfer was the limiting step in overall biodegradation in nonsolvent control. When high concentration of toluene was added, then bioreaction was the limiting step, but inhibitory effect should be considered. However, toluene addition at low concentrations facilitates the biodegradation of aromatic compounds.

Biodegradation of hexadecane in liquid and solid-state fermentations by Aspergillus niger.

The biodegradation and mineralisation of hexadecane (HXD) by Aspergillus niger were studied in SmF and Solid-state fermentation (SSF). HXD concentrations ranging from 45 to 180 g/l (SSF) and from 20 to 80 g/l (SmF) were tested. HXD consumption was three times higher and fungal growth was up to 30 times faster in SSF than in SmF. The maximum HXD consumption in SmF was 62% (18% mineralised) and in SSF 100% (52% mineralised) for initial HXD concentrations of 20 and 45 g/l, respectively. The respiratory quotient in SmF increased (from 0.85 to 1.08) with increase in HXD concentration, while it was independent (approximately 0.68) of the initial HXD concentration in SSF. These results showed that the consumption rate and biodegradation efficiency for HXD were higher in SSF than in SmF.

Anaerobic digestion of a nonionic surfactant: inhibition effect and biodegradation.

Nonionic surfactants are used worldwide in various industrial and household applications. Since these compounds are used in aqueous solutions, they primarily enter the environment through sewage and industrial wastewater treatment plants. The objective of this work was to evaluate the inhibitory effect of Triton X-100, a commercial nonionic surfactant, on the anaerobic digestion of lactose. Thus non-ionic surfactants acts as a non-competitive inhibitor with K1 = 250 mgL-1 and a inhibition order of 2.4. Nonetheless if give enough time the sludge was able to degrade 79% of Triton at 0.1 gL-1 d-1 in a UASB reactor. An activity test of this sludge showed that Triton inhibited the acetogenic (both propionic and butyric) and acetoclastic activities, while there were high fermentative and hydrogenotrophic activities (80% and 95%, respectively).

Phytoremediation and removal mechanisms in Bouteloua curtipendula growing in sterile hydrocarbon spiked cultures.

Tolerance index and phytoremediation factors of side oats grama (Bouteloua curtipendula) with recalcitrant polycyclic aromatic hydrocarbons (PAH) phenanthrene (PHE), pyrene (PYR), and benzo[a]pyrene (BaP) and the resulting impact on phenotypic response, were evaluated in sterile conditions with whole plant growing in test-tube cultures with MS medium with PAH and compared with Tall fescue (Festuca arundinacea), control for this study. PAH mixture of PHE, PYR and BaP (1:1:1 w/w/w) blended with Maya crude oil (1:1 w/w), final concentration of 1500 mg kg(-1) was used. After 40 days, BaP removal, in the presence of Maya crude was superior compared with PHE and PYR removal Although the presence of PAH negatively affects the phenotypic response of the plants; sterile conditions experiments were helpful to evaluate phytoremediation factors to elucidate some important questions regarding phytoremediation mechanisms; in this study, B. curtipendula was able to phytostabilizate BaP associated to a significant hydrocarbon removal (57.4%) with high root accumulation but attenuated transport to stems, here reported as translocation factor. To our knowledge, this is the first time that quantifiable phytoremediation factors were used to evaluate the tolerance and removal capacity of a native semi-arid climate plant which is probably able to phytoremediate hydrocarbon contaminated soils.

Contaminated soil phytoremediation by Cyperus laxus Lam. cytochrome p450 EROD-activity induced by hydrocarbons in roots.

Laboratory and greenhouse experiments with Cyperus laxus Lam were conducted to determine the rate and extent of phytoremediation and the effect of hydrocarbons on the cytochrome P450 EROD (7-ethoxyresorufin-O-deethylase) enzymatic activity in roots. Plants were cultivated on hydrocarbon-contaminated soil (HCS) and spiked perlite. Phytoremediation was evaluated using 6.5 kg HCS (173 +/- 15 mg total petroleum hydrocarbons [TPH] g(-1) of dry soil) pots at different moisture contents; the average removal rate was 3.46-0.25 mg TPH g(-1) dry soil month(-1) and 48% was removed when moisture was kept at 60%. The aromatic hydrocarbon fraction was the mostly removed, 60%; aliphatic, 51%; and polar 24% after 24-month experiments. In unplanted pots, TPH concentration did not exhibit significant differences with respect to the initial concentration. We confirmed that the presence of hydrocarbons induced ERODactivity up to 6.5-fold. Moreover, short-term experiments (up to 13 d) with spiked perlite demonstrated that two EROD activities in roots contributed to the total detected; 60% was found in the cytosolic and 40% in the microsomal fraction. To our knowledge, this is the first work that tries to build links between the hydrocarbon-inducible character of ERODactivity in roots and the phytoremediation ability of C. laxus in highly contaminated soils.

Hydrocarbon biodegradation in oxygen-limited sequential batch reactors by consortium from weathered, oil-contaminated soil.

We studied the use of sequential batch reactors under oxygen limitation to improve and maintain consortium ability to biodegrade hydrocarbons. Air-agitated tubular reactors (2.5 L) were operated for 20 sequential 21-day cycles. Maya crude oil-paraffin mixture (13,000 mg/L) was used as the sole carbon source. The reactors were inoculated with a consortium from the rhizosphere of Cyperus laxus, a native plant that grows naturally in weathered, contaminated soil. Oxygen limitation was induced in the tubular reactor by maintaining low oxygen transfer coefficients (k(L)a < 20.6 h(-1)). The extent and biodegradation rates increased significantly up to the fourth cycle, maintaining values of about 66.33% and 460 mg x L(-1) x d(-1), respectively. Thereafter, sequential batch reactor operation exhibited a pattern with a constant general trend of biodegradation. The effect of oxygen limitation on consortium activity led to a low biomass yield and non-soluble metabolite (0.45 g SS/g hydrocarbons consumed). The average number of hydrocarbon-degrading microorganisms increased from 6.5 x 10(7) (cycles 1-3) to 2.2 x 10(8) (cycles 4-20). Five bacterial strains were identified: Achromobacter (Alcaligenes) xylosoxidans, Bacillus cereus, Bacillus subtilis, Brevibacterium luteum, and Pseudomonas pseudoalcaligenes. Asphaltene-free total petroleum hydrocarbons, extracted from a weathered, contaminated soil, were also biodegraded (97.1 mg x L(-1) x d(-1)) and mineralized (210.48 mg CO2 x L(-1) x d(-1)) by the enriched consortium without inhibition. Our results indicate that sequential batch reactors under oxygen limitation can be used to produce consortia with high and constant biodegradation ability for industrial applications of bioremediation.

Production and properties of three pectinolytic activities produced by Aspergillus niger in submerged and solid-state fermentation.

Three extracellular pectinases were produced by Aspergillus niger CH4 by submerged and solid-state fermentation, and their physicochemical and kinetic properties were studied. The highest productivities of endo- and exo-pectinase and pectin lyase were obtained with solid-state fermentation. The kinetic and physicochemical properties of these enzymes were influenced by the type of culture method used. All activities were very different in terms of pH and temperature optima, stability at different pH and temperature values and affinity for the substrate (Km values). In solid-state fermentation, all pectinase activities were more stable at extreme pH and temperature values but the Km values of endo-pectinase and pectin lyase were higher with respect to those activities obtained by the submerged-culture technique. The pectin lyase activity obtained by the submerged-culture technique showed substrate inhibition but the enzyme obtained by solid-state fermentation did not. Electrophoresis, using sodium dodecyl sulphate/polyacrylamide gel with enzymatic extracts obtained for both culture methods, showed the same number of protein bands but some differences were found in their electrophoretic position. The results obtained in this work suggest that the culture method (submerged or solid-state) may be responsible for inducing changes in some of the pectinolytic enzymes produced by A. niger.

Heat transfer simulation in solid substrate fermentation.

A mathematical model was developed and tested to simulate the generation and transfer of heat in solid substrate fermentation (SSF). The experimental studies were realized in a 1-L static bioreactor packed with cassava wet meal and inoculated with Aspergillus niger. A simplified pseudohomogeneous monodimensional dynamic model was used for the energy balance. Kinetic equations taking into account biomass formation (logistic), sugar consumption (with maintenance), and carbon dioxide formation were used. Model verification was achieved by comparison of calculated and experimental temperatures. Heat transfer was evaluated by the estimation of Biot and Peclet heat dimensionless numbers 5-10 and 2550-2750, respectively. It was shown that conduction through the fermentation fixed bed was the main heat transfer resistance. This model intends to reach a better understanding of transport phenomena in SSF, a fact which could be used to evaluate various alternatives for temperature control of SSF, i.e., changing air flow rates and increasing water content. Dimensionless numbers could be used as scale-up criteria of large fermentors, since in those ratios are described the operating conditions, geometry, and size of the bioreactor. It could lead to improved solid reactor systems. The model can be used as a basis for automatic control of SSF for the production of valuable metabolites in static fermentors.

Biodegradation of Maya crude oil fractions by bacterial strains and a defined mixed culture isolated from Cyperus laxus rhizosphere soil in a contaminated site.

Ten bacterial strains were isolated by enrichment culture, using as carbon sources either aliphatics or an aromatic-polar mixture. Oxygen uptake rate was used as a criterion to determine culture transfer timing at each enrichment stage. Biodegradation of aliphatics (10,000 mg L(-1)) and an aromatic-polar mixture (5000 mg L(-1), 2:1) was evaluated for each of the bacterial strains and for a defined culture made up with a standardized mixture of the isolated strains. Degradation of total hydrocarbons (10,000 mg L(-1)) was also determined for the defined mixed culture. Five bacterial strains were able to degrade more than 50% of the aliphatic fraction. The most extensive biodegradation (74%) was obtained with strain Bs 9A, while strains Ps 2AP and UAM 10AP were able to degrade up to 15% of the aromatic-polar mixture. The defined mixed culture degraded 47% of the aliphatics and 6% of the aromatic-polar mixture. The defined mixed culture was able to degrade about 40% of the aliphatic fraction and 26% of the aromatic fraction when grown in the presence of total hydrocarbons, while these microorganisms did not consume the polar hydrocarbons fraction. The proposed strategy that combines enrichment culture together with oxygen uptake rate allowed the isolation of bacterial strains that are able to degrade specific hydrocarbons fractions at high consumption rates.

Production of pectinases by Aspergillus niger in solid state fermentation at high initial glucose concentrations.

Exopectinase (exo-p) and endopectinase (endo-p) production by Aspergillus niger CH4 in solid state culture was studied at initial glucose concentrations of 100, 250, 350 and 450 g/l. The highest activity of exo-p (35 U/g) was produced at 72 and 120 h in the medium containing 100 and 250 g glucose/l, respectively. The maximum endo-p activity (9 U/g) was produced at 72 h in the medium with 250 g glucose/l. The reduction in pectinase production at 350 and 450 g/l initial glucose concentration was due neither to repression of the synthesis of the enzyme nor to the glucose consumption rate of the strain but due to a drastic drop in pH of the medium.