Impact of bioremediation strategies on slurry phase treatment of aged oily sludge from a refinery.

Aged oily sludge was subjected to long term (90 day) slurry phase treatment (10% w/v oily sludge) using various biodegradation strategies involving intermittent spiking with nutrients (N), surfactant (S) and microorganisms (M), employed singly or in combination. The strategy involving simultaneous addition of N, S, and M (BNMS) resulted in the highest rate (0.0126 day-1) and extent of oil degradation (68.4%). However, oil degradation rate from aged sludge was almost half that observed for freshly procured sludge. In addition to removal of maltenes (85.7%), removal of asphaltenes (53.6%) was also achieved during BNMS treatment. Two-dimensional gas chromatograph equipped with time of flight mass spectrometer could resolve the unresolved complex mixture hump observed in both degraded and un-degraded samples and could provide greater insights on compositional changes in residual oil due to biodegradation. Although the BNMS strategy significantly enhanced oil degradation from aged sludge, treatment of fresh sludge would be faster and more cost effective.

Characterization, phylogenetic distribution and evolutionary trajectories of diverse hydrocarbon degrading microorganisms isolated from refinery sludge.

Phylogenic association between bacteria living under harsh conditions can provide important information on adaptive mechanism, survival strategy and their potential application. Indigenous microorganisms isolated from toxic refinery oily sludge with ability to degrade a diverse range of hydrocarbons were identified and characterized. The strains including Pseudomonas aeruginosa RS1, Microbacterium sp. RS2, Bacillus sp. RS3, Acinetobacter baumannii RS4 and Stenotrophomonas sp. RS5 could utilize n-alkanes, cycloalkanes, polynuclear aromatic hydrocarbons (PAHs) with 2-4 rings and also substituted PAHs as sole substrate. The phylogenetic position of Bacillus sp. RS3 and Pseudomonas sp. RS1 was tested by applying the maximum likelihood (ML) method to the aligned 16S rRNA nucleotide sequences of PAH and aliphatic hydrocarbon degrading strains belonging to the corresponding genus. The base substitution matrix created with each set of organisms capable of degrading aromatic and aliphatic hydrocarbons showed significant transitional event with high values of transition: transversion ratio (R) under all conditions. The guanine-cytosine (GC) content of the hydrocarbon degrading test strains was also found to be highest for the clade which harbored them. The test strains consistently occupied a distinct terminal end within the phylogenetic tree constructed by ML analysis. This study reveals that the refinery sludge imposed environmental stress on the bacterial strains which possibly caused significant genetic alteration and phenotypic adaptation. Due to the divergent evolution of the Pseudomonas and Bacillus strains in the sludge, they appeared distinctly different from other hydrocarbon degrading strains of the same genus.

Characterization of oily sludge from a refinery and biodegradability assessment using various hydrocarbon degrading strains and reconstituted consortia.

Oily sludge obtained from a refinery in India contained 10-11% oil associated with fine particulates. Along with Fe, Ca and Mg various toxic elements were associated with the sludge solids (Pb, Mn, Cu, Zn, As, Bi, Cd, Cr, Co, Ni and V). The oil contained 41-56% asphaltenes and the maltenes comprised of 49 ± 4%, 42 ± 2% and 4 ± 2%, aliphatic, aromatic and polar fractions, respectively. Biodegradation studies with the maltene fraction of oil provided as sole substrate revealed higher degradation by various 3-5 membered reconstituted consortia compared to pure bacterial strains and up to 42 ± 8% degradation could be achieved over 30 days. In contrast, over the same period up to 71.5 ± 2% oil degradation could be achieved using dried oily sludge (15% w/v) as sole substrate. Significant biodegradation observed in the un-inoculated controls indicated the presence of indigenous microorganisms in oily sludge. However, large variability in oil degradation was observed in the un-inoculated controls. Greater biodegradation of the maltene fraction led to significant enrichment of asphaltenes in residual oil associated with the sludge.

Evaluation of bioaugmentation and biostimulation effects on the treatment of refinery oily sludge using 2(n) full factorial design.

Bioremediation approaches for the treatment of oily sludge from a refinery were evaluated using a 2(3) factorial design. The three strategies tested were bioaugmentation with indigenous microbial consortia (MO) isolated from oily sludge, biostimulation with nutrients (NP) and biostimulation with the surfactant Triton X-100 (TX). Eight experimental runs were conducted in triplicate with factor settings ± (high/low) as per the 2(3) design. The main effects and the effects of various interactions of the factors on oil degradation and microbial growth in suspension were evaluated during a 30 day study. Multifactor ANOVA could reveal the significant effects while the normal order score approach failed in this scenario. The main effect of biostimulation with nutrients in the form of nitrate and phosphate, as well as biostimulation with Triton X-100, was positive and significant when both oil degradation and microbial growth in suspension were chosen as the response variables. However, the main effect of bioaugmentation was only significant for oil degradation but was insignificant for microbial growth at a 90% confidence level. The MO-NP binary interaction and the MO-NP-TX ternary interactions were positive and significant, indicating the synergistic effect of these strategies on oil degradation and microbial growth. All other binary interactions were found to be insignificant.

Biodegradation of pyrene by a Pseudomonas aeruginosa strain RS1 isolated from refinery sludge.

High molecular weight (HMW) polynuclear aromatic hydrocarbons (PAHs) with more than three rings are inherently difficult to degrade. Degradation of HMW PAHs is primarily reported for actinomycetes, such as, Rhodococcus and Mycobacterium. This study reports pyrene degradation by a Pseudomonas aeruginosa strain isolated from tank bottom sludge in a refinery. High cell surface hydrophobicity induced during growth on pyrene facilitated its utilization as sole carbon source. Specific growth rate (µ) in the range of 0.03-0.085 h(-1) could be achieved over the concentration range 25-500 mg/L. The specific growth rate and specific pyrene utilization rate increased linearly with increase in total pyrene concentration. Although various degradation intermediates were identified in the aqueous phase, accumulation of total organic carbon (TOC) in the aqueous phase was only a small fraction of TOC equivalents of pyrene lost from the cultures. The degradation pathway appears to be similar to that reported for Mycobacterium sp. PYR-I.

Practical considerations and challenges involved in surfactant enhanced bioremediation of oil.

Surfactant enhanced bioremediation (SEB) of oil is an approach adopted to overcome the bioavailability constraints encountered in biotransformation of nonaqueous phase liquid (NAPL) pollutants. Fuel oils contain n-alkanes and other aliphatic hydrocarbons, monoaromatics, and polynuclear aromatic hydrocarbons (PAHs). Although hydrocarbon degrading cultures are abundant in nature, complete biodegradation of oil is rarely achieved even under favorable environmental conditions due to the structural complexity of oil and culture specificities. Moreover, the interaction among cultures in a consortium, substrate interaction effects during the degradation and ability of specific cultures to alter the bioavailability of oil invariably affect the process. Although SEB has the potential to increase the degradation rate of oil and its constituents, there are numerous challenges in the successful application of this technology. Success is dependent on the choice of appropriate surfactant type and dose since the surfactant-hydrocarbon-microorganism interaction may be unique to each scenario. Surfactants not only enhance the uptake of constituents through micellar solubilization and emulsification but can also alter microbial cell surface characteristics. Moreover, hydrocarbons partitioned in micelles may not be readily bioavailable depending on the microorganism-surfactant interactions. Surfactant toxicity and inherent biodegradability of surfactants may pose additional challenges as discussed in this review.