Soil Chemical Analysis

Recent advances in molecular biology have extended our understanding of the metabolic processes related to microbial transformation of petroleum hydrocarbons. The physiological responses of microorganisms to the presence of hydrocarbons, including cell surface alterations and adaptive mechanisms for uptake and efflux of these substrates, have been characterized. New molecular techniques have enhanced our ability to investigate the dynamics of microbial communities in petroleum-impacted ecosystems. By establishing conditions which maximize rates and extents of microbial growth, hydrocarbon access, and transformation, highly accelerated and bioreactor-based petroleum waste degradation processes have been implemented. Biofilters capable of removing and biodegrading volatile petroleum contaminants in air streams with short substrate-microbe contact times ( 2 S and sulfoxides from petrochemical waste streams. Microbes also have potential for use in removal of nitrogen from crude oil leading to reduced nitric oxide emissions provided that technical problems similar to those experienced in biodesulfurization can be solved. Enzymes are being exploited to produce added-value products from petroleum substrates, and bacterial biosensors are being used to analyze petroleum-contaminated environments.

Recent Advances in Petroleum Microbiology

Polycyclic aromatic hydrocarbons: environmental pollution and bioremediation

The recent explosion of newly described species of Nocardia results from the impact in the last decade of newer molecular technology, including PCR restriction enzyme analysis and 16S rRNA sequencing. These molecular techniques have revolutionized the identification of the nocardiae by providing rapid and accurate identification of recognized nocardiae and, at the same time, revealing new species and a number of yet-to-be-described species. There are currently more than 30 species of nocardiae of human clinical significance, with the majority of isolates being N. nova complex, N. abscessus, N. transvalensis complex, N. farcinica, N. asteroides type VI (N. cyriacigeorgica), and N. brasiliensis. These species cause a wide variety of diseases and have variable drug susceptibilities. Accurate identification often requires referral to a reference laboratory with molecular capabilities, as many newer species are genetically distinct from established species yet have few or no distinguishing phenotypic characteristics. Correct identification is important in deciding the clinical relevance of a species and in the clinical management and treatment of patients with nocardial disease. This review characterizes the currently known pathogenic species of Nocardia, including clinical disease, drug susceptibility, and methods of identification.

/pdf/clinical-and-laboratory-features-of-the-nocardia-spp-based-40fuz52wrw.pdf

Clinical and Laboratory Features of the Nocardia spp. Based on Current Molecular Taxonomy

Recent development in the treatment of oily sludge from petroleum industry: A review

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 (103 to 104 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.

/pdf/evaluation-of-inoculum-addition-to-stimulate-in-situ-21t9i9cbo6.pdf

Evaluation of Inoculum Addition To Stimulate In Situ Bioremediation of Oily-Sludge-Contaminated Soil

A field-scale study was conducted in a 4000 m2 plot of land contaminated with an oily sludge by use of a carrier-based hydrocarbon-degrading bacterial consortium for bioremediation. The land belonged to an oil refinery. Prior to this study, a feasibility study was conducted to assess the capacity of the bacterial consortium to degrade oily sludge. The site selected for bioremediation contained approximately 300 tons of oily sludge. The plot was divided into four blocks, based on the extent of contamination. Blocks A, B, and C were treated with the bacterial consortium, whereas Block D was maintained as an untreated control. In Block A, at time zero, i.e., at the beginning of the experiment, the soil contained as much as 99.2 g/kg of total petroleum hydrocarbon (TPH). The application of a bacterial consortium (1 kg carrier-based bacterial consortium/10 m2 area) and nutrients degraded 90.2% of the TPH in 120 days, whereas in block D only 16.8% of the TPH was degraded. This study validates the large-scale use of a carrier-based bacterial consortium and nutrients for the treatment of land contaminated with oily sludge, a hazardous hydrocarbon waste generated by petroleum industry.

In Situ Bioremediation Potential of an Oily Sludge-Degrading Bacterial Consortium

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.

Evaluation of Genetic Diversity among Pseudomonas citronellolis Strains Isolated from Oily Sludge-Contaminated Sites

A hydrocarbon degrading Acinetobacter baumannii S30 strain, isolated from crude oil-contaminated soil, was inserted with the lux gene from the luciferase gene cassette luxCDABE. Soil microcosms were designed to study the degradation efficacy for total petroleum hydrocarbon (TPH) of crude oil by lux-tagged A. baumannii S30 pJES. Bioaugmentation of a TPH-contaminated microcosm with A baumannii S30 pJES showed that TPH levels were reduced from 89.3 to 53.9 g/kg soil in 90 days. Biodegradation of TPH by A baumannii S30 pJES was also monitored in shake flask conditions, which showed a reduction of initial TPH levels by over 50% at the end of 120 h. A lux-PCR-based approach along with the standard dilution plating with selective antibiotics was successfully utilized to monitor the survivability of the lux-tagged strain A. baumannii S30 pJES in soil microcosms and stability of the lux insert in the host strain A. baumannii S30. The selective plating technique indicated the population of A. baumannii S30 pJES to be 6.5 ± 0.13 × 108 CFU/g at day zero (just after bioaugmentation) and 2.09 ± 0.08 × 108 CFU/g of soil after 90 days of incubation. lux-PCR confirmed the stability of the insert in all the randomly selected colonies of A. baumannii strains from the antibiotic plates. The lux insert was stable after 50 generations in Luria Bertini broth and storage at −70 °C as glycerol stocks for over a year. These results revealed that the lux insert was stable and lux-tagged A. baumannii S30 strain could survive in a TPH-contaminated soil microcosm and could degrade TPH in the soil microcosm conditions. It can be used as an effective marker to monitor the survival of augmented strains at a bioremediation site.

Crude oil degradation efficiency of a recombinant Acinetobacter baumannii strain and its survival in crude oil-contaminated soil microcosm.

The principal findings of a study for further evaluating paraffin baiting as a routine laboratory diagnostic procedure in the isolation of Nocardia asteroides, the etiological agent of nocardiosis, are reported.

https://aem.asm.org/content/aem/18/4/686.full.pdf

Sanjeet Mishra

Papers

Evaluation of Inoculum Addition To Stimulate In Situ Bioremediation of Oily-Sludge-Contaminated Soil

In Situ Bioremediation Potential of an Oily Sludge-Degrading Bacterial Consortium

Evaluation of Genetic Diversity among Pseudomonas citronellolis Strains Isolated from Oily Sludge-Contaminated Sites

Crude oil degradation efficiency of a recombinant Acinetobacter baumannii strain and its survival in crude oil-contaminated soil microcosm.

Application of paraffin bait technique to the isolation of Nocardia asteroides from clinical specimens.