Fatima Menezes Bento

Microbial contamination of stored hydrocarbon fuels and its control

The major microbial problem in the petroleum refining industry is contamination of stored products, which can lead to loss of product quality, formation of sludge and deterioration of pipework and storage tanks, both in the refinery and at the end-user. Three major classes of fuel are discussed in this article - gasoline, aviation kerosene and diesel, corresponding to increasingly heavy petroleum fractions. The fuel that presents the most serious microbiological problems is diesel. The many microorganisms that have been isolated from hydrocarbon fuel systems are listed. The conditions required for microbial growth and the methods used to monitor and to control this activity are discussed. The effects of various fuel additives, including biocides, are considered.

Biodegradation potential of oily sludge by pure and mixed bacterial cultures.

The biodegradation capacity of aliphatic and aromatic hydrocarbons of petrochemical oily sludge in liquid medium by a bacterial consortium and five pure bacterial cultures was analyzed. Three bacteria isolated from petrochemical oily sludge, identified as Stenotrophomonas acidaminiphila, Bacillus megaterium and Bacillus cibi, and two bacteria isolated from a soil contaminated by petrochemical waste, identified as Pseudomonas aeruginosa and Bacillus cereus demonstrated efficiency in oily sludge degradation when cultivated during 40 days. The bacterial consortium demonstrated an excellent oily sludge degradation capacity, reducing 90.7% of the aliphatic fraction and 51.8% of the aromatic fraction, as well as biosurfactant production capacity, achieving 39.4% reduction of surface tension of the culture medium and an emulsifying activity of 55.1%. The results indicated that the bacterial consortium has potential to be applied in bioremediation of petrochemical oily sludge contaminated environments, favoring the reduction of environmental passives and increasing industrial productivity.

Biodeterioration of stored diesel oil: studies in Brazil

Abstract The problems of hydrocarbon fuel storage in Brazil are particularly acute for diesel fuel. Visits to bus depots showed that many foremen did not understand the importance of draining water bottoms regularly and most systems were microbially contaminated. Common fungal isolates from refineries and distribution systems, Hormoconis resinae , Aspergillus niger , Aspergillus fumigatus , Paecilomyces variotii , and Candida silvicola , grew equally well in laboratory diesel/water systems with or without a chemical additive mixture, showing that this package of compounds neither promoted nor retarded fungal growth. Non-sterilised diesel was stored for 450 days over a water bottom, with or without an isothiazolone biocide, in the laboratory. The fungi most frequently detected in the non-biocide treated systems were H. resinae , A fumigatus , P. variotii , a Penicillium sp., and the yeasts, Rhodotorula glutinis and Candida silvicola . Bacterial isolates included oxidative Gram negative rods, sulphate-reducing bacteria and a Micrococcus sp. Biocide at 0.1 ppm maintained the systems clean for up to 30 days, and at 1 or 10 ppm for 400 days. After 400 days, the biomass (dry weight) from non-additive-containing diesel in control, 1 and 10 ppm biocide-containing systems was 24.6, 4.6 and 3.3 mg, respectively. The system treated with 0.1 ppm biocide yielded 38.2 mg biomass, indicating that sub-effective doses may lead to increased microbial growth. Within 24 h of addition of 10 ppm biocide to a highly contaminated control flask (145 days storage) there was a 2-log reduction in total aerobic bacterial and yeast population and the filamentous fungal count was >5 ml −1 .

Biodiversity of diazotrophic bacteria within the soil, root and stem of field-grown maize

Recent studies suggest a high diversity of diazotrophic bacteria in maize. However, none of these works have been based on a sufficient number of samples to provide reasonable quantitative estimates of diazotrophic bacterial diversity. Here we present the use of molecular tools and statistical inference to assess diazotrophic bacterial diversity within rhizosphere soils, roots and stems of field grown maize. DNA was isolated from the latter collected from six maize growing regions within the southern most state in Brazil, Rio Grande do Sul. Using conserved primers, nifH Cluster I gene fragments were amplified from each of the three zones, and the products cloned and sequenced. The majority of the sequences were classified within the Proteobacteria with the α-proteobacteria and β-proteobacteria being the most abundant in the rhizosphere soil and stem samples. The γ-proteobacteria were most abundant in rhizosphere soils, less so in roots, and least in the stem samples. According to three different diversity measures, the rhizosphere soil samples possessed greater diazotrophic bacterial diversity than the roots and stems of the maize plants. Only two genera, Azospirillum and Azotobacter, were found in virtually all samples at an abundance of over 1% of the total nifH sequences obtained. Other genera were largely restricted to soil (Methylocystis, Beijerinckia, Geobacter, Rhodovulum, Methylobacterium, Gluconacetobacter, Methylocella, and Delftia), roots (Dechloromonas), or stems (Methylosinus, Raoultella, and Rhizobium). Three genera, Herbaspirillum, Ideonella, and Klebsiella, appeared to dominate in the interior of the plant but were much rarer in soil.

Bioremediation of soil contaminated by diesel oil

Were evaluated natural attenuation, biostimulation and bioaugmentation on the degradation of total petroleum hydrocarbons (TPH) in soils contaminated with diesel oil. Bioaugmentation showed the greatest degradation in the light (C12 - C23) fractions (72.7%) and heavy (C23 - C40) fractions of TPH (75.2%) and natural attenuation was more effective than biostimulation. The greatest dehydrogenase activity was observed upon bioaugmentation of the Long Beach soil (3.3-fold) and the natural attenuation of the Hong Kong soil sample (4.0-fold). The number of diesel oil degrading microorganisms and heterotrophic population was not influenced by the bioremediation treatments. The best approach for bioremediation of soil contaminated with diesel oil is the inoculum of microorganisms pre-selected from their own environment.

Characterization of a Polycyclic Aromatic Hydrocarbon–Degrading Microbial Consortium from a Petrochemical Sludge Landfarming Site

ABSTRACT Anthracene, phenanthrene, and pyrene are polycyclic aromatic hydrocarbon (PAHs) that display both mutagenic and carcinogenic properties. They are recalcitrant to microbial degradation in soil and water due to their complex molecular structure and low solubility in water. This study presents the characterization of an efficient PAH (anthracene, phenanthrene, and pyrene)-degrading microbial consortium, isolated from a petrochemical sludge landfarming site. Soil samples collected at the landfarming area were used as inoculum in Warburg flasks containing soil spiked with 250 mg kg−1 of anthracene. The soil sample with the highest production of CO2-C in 176 days was used in liquid mineral medium for further enrichment of anthracene degraders. The microbial consortium degraded 48%, 67%, and 22% of the anthracene, phenanthrene, and pyrene in the mineral medium, respectively, after 30 days of incubation. Six bacteria, identified by 16S rRNA sequencing as Mycobacterium fortuitum, Bacillus cereus, Microbacterium sp., Gordonia polyisoprenivorans, two Microbacteriaceae bacteria, and a fungus identified as Fusarium oxysporum were isolated from the enrichment culture. The consortium and its monoculture isolates utilized a variety of hydrocarbons including PAHs (pyrene, anthracene, phenanthrene, and naftalene), monoaromatics hydrocarbons (benzene, ethylbenzene, toluene, and xylene), aliphatic hydrocarbons (1-decene, 1-octene, and hexane), hydrocarbon mixtures (gasoline and diesel oil), intermediary metabolites of PAHs degradation (catechol, gentisic acid, salicylic acid, and dihydroxybenzoic acid) and ethanol for growth. Biosurfactant production by the isolates was assessed by an emulsification index and reduction of the surface tension in the mineral medium. Significant emulsification was observed with the isolates, indicating production of high-molecular-weigh surfactants. The high PAH degradation rates, the wide spectrum of hydrocarbons utilization, and emulsification capacities of the microbial consortium and its member microbes indicate that they can be used for biotreatment and bioaugumentation of soils contaminated with PAHs.

Biorremediação de solos contaminados com hidrocarbonetos aromáticos policíclicos

Polycyclic aromatic hydrocarbons (PAHs) are mutagenic and carcinogenic compounds to the humans and animals, released through the environment by anthropogenic activities related to the extraction, transport, refine, transformation and use of the petroleum and its derivatives. Most of the soils microorganisms do not possess the capacity to degrade them, which results in its accumulation in the atmosphere and contamination of the ecosystems. A strategy for PAHs elimination from the soil is through the bioremediation, where microorganisms having capacity to metabolize these compounds will transform them in inert substances, CO2 and water. However, this biotechnology can be limited by the lack of specific HAP microbial-degraders in soil, by unfavorable environmental conditions to these microorganisms or by the low bioavailability of those contaminants to the microorganisms. To overcome these limitations and to promote an efficient removal of the pollutants to the atmosphere, several bioremediation techniques were developed as passive bioremediation, bioaugmentation, biostimulation, phytoremediation, landfarming, composting and bioreactors. This revision aims at discussing microbial metabolism of PAHs, present the main chemical and physical factors that influence the survival and the activity of these microorganisms and to show the bioremediation techniques that are being used now for the PAHs removal in soil.

Improved Enrichment and Isolation of Polycyclic Aromatic Hydrocarbons (PAH)-Degrading Microorganisms in Soil Using Anthracene as a Model PAH

Lack of attention to soil and microbial characteristics that influence PAHs degradation has been a leading cause of failures in isolation of efficient PAH degraders and bioaugumentation processes with microbial consortia. This study compared the classic method of isolation of PAHs-degraders with a modified method employing a pre-enrichment respirometric analysis. The modified enrichment of PAH degrading microorganisms using in vitro microcosm resulted to reduced enrichment period and more efficient PAH-degrading microbial consortia. Results indicate that natural soils with strong heterotrophic microbial activity determined through pre-enrichment analysis, are better suited for the isolation of efficient PAH degrading microorganisms with significant reduction of the enrichment period.

Diversidade de bactérias diazotróficas endofíticas associadas a plantas de milho

Endophytic diazotrophic bacteria are capable of promoting maize growth through biological nitrogen fixation (BNF) or by the production of plant hormones. The aim of this study was to characterize diversity of endophytic bacteria in maize at sites with different climate and soil conditions in Rio Grande do Sul, Brazil. A PCR-RFLP approach and sequence analysis of nifH Cluster I clone libraries were used to assess diversity in maize plants. The Shannon-Weaver and Equitability indices were calculated to estimate the diazotroph diversity as well as the nucleotide diversity and the average sequence divergence to estimate genetic diversity. To evaluate the variability in populations we performed the FST test. A greater variation in bacterial communities was detected between rather than within regions, particularly among communities of different soil types and varying water regimes and geographical regions. The Shannon-Weaver index indicated a high difference in terms of diversity of taxonomic units among the communities. The diversity of the communities in the northern region, with higher precipitation and clay content, tended to be higher than that in the south. The Equitability index indicated that there was a dominant taxonomic unit within each community. All clones grouped into nifH gene cluster I. The nifH sequence types from Alpha, Beta and Gammaproteobacteria were recovered. These results demonstrate that there is a large diversity of endophytic nitrogen-fixing bacteria able to colonize maize tissue and that nifH diversity is correlated with the different environmental conditions.

Enzymatic activity of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase produced by Gordonia polyisoprenivorans

This study aimed to evaluate the environmental conditions for enzyme activity of catechol 1,2-dioxygenase (C1,2O) and catechol 2,3-dioxygenase (C2,3O) produced by Gordonia polyisoprenivorans in cell-free and immobilized extracts. The optimum conditions of pH, temperature, time course and effect of ions for enzyme activity were determined. Peak activity of C1,2O occurred at pH 8.0. The isolate exhibited the highest activity of C2,3O at pH 7.0 and 8.0 for the cell-free extract and immobilized extract, respectively. This isolate exhibited important characteristics such as broad range of pH, temperature and time course for enzyme activity.