How can petroleum be degraded by bacterial action




















Screening and isolation of petroleum hydrocarbon degrading bacteria Separation of petroleum hydrocarbon degrading microorganisms. Strain culture and sequence. Crude oil degradation efficiency Petroleum degradation efficiency was determined by spectrophotometric method described as Bao et al.

Construction of bacterial consortium The oil degradation experiment was done with the five strains and combination among them. Immobilization of free microorganisms Method of preparing calcium alginate—Activated carbon microsphere. Then, 2. Experiments of environmental factors of biodegradation Influence of crude oil concentration. Influence of liquid temperature. Influence of initial pH. Influence of salinity. Influence of degradation time. Determination of n-alkanes. Determination of PAHs.

Download: PPT. Table 1. Morphology and biochemical characterization of the strains. Fig 1. Phylogenetic tree of the strains based on bootstrap test. Construction of optimal mixed flora The bacterial consortium constructing was done based on the five strains in order to get better degrading performance of crude oil. Table 2. Degradation efficiencies of bacterial consortia and single strains.

SEM of bio-carrier and immobilized bacterial consortium SEM photos of bio-carrier of calcium alginate, bio-carrier of calcium alginate—activated carbon and immobilized bacterial consortium are shown in Fig 2.

Fig 2. Photos of bio-carrier and immobilized bacterial consortium. Effect of environmental factors on crude oil biodegradation Crude oil concentration. Fig 3. Effects of crude oil concentration on crude oil degradation efficiency of free and immobilized bacterial consortium. Fig 4. Effects of temperature on crude oil degradation efficiency of free and immobilized bacterial consortium.

Initial pH. Fig 5. Effects of initial pH on crude oil degradation efficiency of free and immobilized bacterial consortium. Fig 6. Effects of salinity on crude oil degradation efficiency of free and immobilized bacterial consortium.

Degradation time. Fig 7. Effects of degradation time on crude oil degradation efficiency of free and immobilized bacterial consortium. Fig 8. Fig 9. Changes of the major normal alkanes content in crude oil samples before and after biodegradation. Fig Degradation efficiency of the main normal alkanes by the free bacteria group and the immobilized bacteria group.

PAHs gas chromatograms in crude oil samples before and after biodegradation. Degradation of the major PAHs by free bacteria and immobilized bacteria.

Discussion The microorganisms with efficient crude oil biodegradability, which were belonging to Exiguobacterium sp. Supporting information. S1 File. S2 File. S3 File. S4 File. S5 File. Acknowledgments We acknowledge and thank for the crude oil sample support by Xingzhong sinochem oil transport Zhoushan Co.

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Applied and Environmental Microbiology, 80 14 , Environmental Microbiology, 12 10 , Environmental Microbiology, 14 10 , Navigation menu Personal tools Log in. Biodegradation by natural populations of microorganisms represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants can be removed from the environment [ 6 ] and is cheaper than other remediation technologies [ 7 ].

Numerous scientific review articles have covered various factors that influence the rate of oil biodegradation [ 7 — 12 ]. One important requirement is the presence of microorganisms with the appropriate metabolic capabilities. If these microorganisms are present, then optimal rates of growth and hydrocarbon biodegradation can be sustained by ensuring that adequate concentrations of nutrients and oxygen are present and that the pH is between 6 and 9.

The physical and chemical characteristics of the oil and oil surface area are also important determinants of bioremediation success. There are the two main approaches to oil spill bioremediation: a bioaugmentation, in which known oil-degrading bacteria are added to supplement the existing microbial population, and b biostimulation, in which the growth of indigenous oil degraders is stimulated by the addition of nutrients or other growth-limiting cosubstrates.

The success of bioremediation efforts in the cleanup of the oil tanker Exxon Valdez oil spill of [ 13 ] in Prince William Sound and the Gulf of Alaska created tremendous interest in the potential of biodegradation and bioremediation technology. Most existing studies have concentrated on evaluating the factors affecting oil bioremediation or testing favored products and methods through laboratory studies [ 14 ]. Only limited numbers of pilot scale and field trials have provided the most convincing demonstrations of this technology which have been reported in the peer-reviewed literature [ 15 — 18 ].

The scope of current understanding of oil bioremediation is also limited because the emphasis of most of these field studies and reviews has been given on the evaluation of bioremediation technology for dealing with large-scale oil spills on marine shorelines. This paper provides an updated information on microbial degradation of petroleum hydrocarbon contaminants towards the better understanding in bioremediation challenges.

Biodegradation of petroleum hydrocarbons is a complex process that depends on the nature and on the amount of the hydrocarbons present. Petroleum hydrocarbons can be divided into four classes: the saturates, the aromatics, the asphaltenes phenols, fatty acids, ketones, esters, and porphyrins , and the resins pyridines, quinolines, carbazoles, sulfoxides, and amides [ 19 ]. Different factors influencing hydrocarbon degradation have been reported by Cooney et al.

One of the important factors that limit biodegradation of oil pollutants in the environment is their limited availability to microorganisms. Petroleum hydrocarbon compounds bind to soil components, and they are difficult to be removed or degraded [ 21 ].

Hydrocarbons differ in their susceptibility to microbial attack. The susceptibility of hydrocarbons to microbial degradation can be generally ranked as follows: linear alkanes branched alkanes small aromatics cyclic alkanes [ 6 , 22 ].

Some compounds, such as the high molecular weight polycyclic aromatic hydrocarbons PAHs , may not be degraded at all [ 23 ]. Microbial degradation is the major and ultimate natural mechanism by which one can cleanup the petroleum hydrocarbon pollutants from the environment [ 24 — 26 ]. The recognition of biodegraded petroleum-derived aromatic hydrocarbons in marine sediments was reported by Jones et al. They studied the extensive biodegradation of alkyl aromatics in marine sediments which occurred prior to detectable biodegradation of n-alkane profile of the crude oil and the microorganisms, namely, Arthrobacter, Burkholderia, Mycobacterium, Pseudomonas, Sphingomonas, and Rhodococcus were found to be involved for alkylaromatic degradation.

Microbial degradation of petroleum hydrocarbons in a polluted tropical stream in Lagos, Nigeria was reported by Adebusoye et al. Nine bacterial strains, namely, Pseudomonas fluorescens, P.

Hydrocarbons in the environment are biodegraded primarily by bacteria, yeast, and fungi. Many scientists reported that mixed populations with overall broad enzymatic capacities are required to degrade complex mixtures of hydrocarbons such as crude oil in soil [ 33 ], fresh water [ 34 ], and marine environments [ 35 , 36 ].

Bacteria are the most active agents in petroleum degradation, and they work as primary degraders of spilled oil in environment [ 37 , 38 ]. Several bacteria are even known to feed exclusively on hydrocarbons [ 39 ]. Floodgate [ 36 ] listed 25 genera of hydrocarbon degrading bacteria and 25 genera of hydrocarbon degrading fungi which were isolated from marine environment. A similar compilation by Bartha and Bossert [ 33 ] included 22 genera of bacteria and 31 genera of fungi.

In earlier days, the extent to which bacteria, yeast, and filamentous fungi participate in the biodegradation of petroleum hydrocarbons was the subject of limited study, but appeared to be a function of the ecosystem and local environmental conditions [ 7 ]. Crude petroleum oil from petroleum contaminated soil from North East India was reported by Das and Mukherjee [ 40 ]. Acinetobacter sp.

Bacterial genera, namely, Gordonia , Brevibacterium , Aeromicrobium, Dietzia , Burkholderia, and Mycobacterium isolated from petroleum contaminated soil proved to be the potential organisms for hydrocarbon degradation [ 42 ]. The degradation of poly-aromatic hydrocarbons by Sphingomonas was reported by Daugulis and McCracken [ 43 ].

Fungal genera, namely, Amorphoteca , Neosartorya , Talaromyces, and Graphium and yeast genera, namely, Candida, Yarrowia, and Pichia were isolated from petroleum-contaminated soil and proved to be the potential organisms for hydrocarbon degradation [ 42 ]. Singh [ 44 ] also reported a group of terrestrial fungi, namely, Aspergillus , Cephalosporium, and Pencillium which were also found to be the potential degrader of crude oil hydrocarbons.

The yeast species, namely, Candida lipolytica, Rhodotorula mucilaginosa, Geotrichum sp , and Trichosporon mucoides isolated from contaminated water were noted to degrade petroleum compounds [ 45 ]. Though algae and protozoa are the important members of the microbial community in both aquatic and terrestrial ecosystems, reports are scanty regarding their involvement in hydrocarbon biodegradation.

Walker et al. Cerniglia et al. Protozoa, by contrast, had not been shown to utilize hydrocarbons. A number of limiting factors have been recognized to affect the biodegradation of petroleum hydrocarbons, many of which have been discussed by Brusseau [ 53 ]. The composition and inherent biodegradability of the petroleum hydrocarbon pollutant is the first and foremost important consideration when the suitability of a remediation approach is to be assessed.

Among physical factors, temperature plays an important role in biodegradation of hydrocarbons by directly affecting the chemistry of the pollutants as well as affecting the physiology and diversity of the microbial flora. Atlas [ 54 ] found that at low temperatures, the viscosity of the oil increased, while the volatility of the toxic low molecular weight hydrocarbons were reduced, delaying the onset of biodegradation.

Temperature also affects the solubility of hydrocarbons [ 62 ]. Although hydrocarbon biodegradation can occur over a wide range of temperatures, the rate of biodegradation generally decreases with the decreasing temperature.

Figure 1 shows that highest degradation rates that generally occur in the range 30—40 C in soil environments, 20—30 C in some freshwater environments and 15—20 C in marine environments [ 33 , 34 ]. Venosa and Zhu [ 63 ] reported that ambient temperature of the environment affected both the properties of spilled oil and the activity of the microorganisms. Significant biodegradation of hydrocarbons have been reported in psychrophilic environments in temperate regions [ 64 , 65 ].

Nutrients are very important ingredients for successful biodegradation of hydrocarbon pollutants especially nitrogen, phosphorus, and in some cases iron [ 34 ]. Some of these nutrients could become limiting factor thus affecting the biodegradation processes.

Atlas [ 35 ] reported that when a major oil spill occurred in marine and freshwater environments, the supply of carbon was significantly increased and the availability of nitrogen and phosphorus generally became the limiting factor for oil degradation.

In marine environments, it was found to be more pronounced due to low levels of nitrogen and phosphorous in seawater [ 36 ]. Freshwater wetlands are typically considered to be nutrient deficient due to heavy demands of nutrients by the plants [ 66 ]. Therefore, additions of nutrients were necessary to enhance the biodegradation of oil pollutant [ 67 , 68 ]. On the other hand, excessive nutrient concentrations can also inhibit the biodegradation activity [ 69 ].

Several authors have reported the negative effects of high NPK levels on the biodegradation of hydrocarbons [ 70 , 71 ] especially on aromatics [ 72 ]. The effectiveness of fertilizers for the crude oil bioremediation in subarctic intertidal sediments was studied by Pelletier et al. Use of poultry manure as organic fertilizer in contaminated soil was also reported [ 73 ], and biodegradation was found to be enhanced in the presence of poultry manure alone.

Maki et al. The most rapid and complete degradation of the majority of organic pollutants is brought about under aerobic conditions. Figure 2 shows the main principle of aerobic degradation of hydrocarbons [ 75 ]. The initial intracellular attack of organic pollutants is an oxidative process and the activation as well as incorporation of oxygen is the enzymatic key reaction catalyzed by oxygenases and peroxidases.

Peripheral degradation pathways convert organic pollutants step by step into intermediates of the central intermediary metabolism, for example, the tricarboxylic acid cycle. Biosynthesis of cell biomass occurs from the central precursor metabolites, for example, acetyl-CoA, succinate, pyruvate. Sugars required for various biosyntheses and growth are synthesized by gluconeogenesis. The degradation of petroleum hydrocarbons can be mediated by specific enzyme system.

Figure 3 shows the initial attack on xenobiotics by oxygenases [ 75 ]. Other mechanisms involved are 1 attachment of microbial cells to the substrates and 2 production of biosurfactants [ 76 ]. The uptake mechanism linked to the attachment of cell to oil droplet is still unknown but production of biosurfactants has been well studied. Cytochrome P alkane hydroxylases constitute a super family of ubiquitous Heme-thiolate Monooxygenases which play an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds [ 77 ].

Depending on the chain length, enzyme systems are required to introduce oxygen in the substrate to initiate biodegradation Table 1. Higher eukaryotes generally contain several different P families that consist of large number of individual P forms that may contribute as an ensemble of isoforms to the metabolic conversion of given substrate. In microorganisms such P multiplicity can only be found in few species [ 78 ]. Cytochrome P enzyme systems was found to be involved in biodegradation of petroleum hydrocarbons Table 1.

The capability of several yeast species to use n-alkanes and other aliphatic hydrocarbons as a sole source of carbon and energy is mediated by the existence of multiple microsomal Cytochrome P forms.

These cytochrome P enzymes had been isolated from yeast species such as Candida maltosa , Candida tropicalis , and Candida apicola [ 79 ]. The diversity of alkaneoxygenase systems in prokaryotes and eukaryotes that are actively participating in the degradation of alkanes under aerobic conditions like Cytochrome P enzymes, integral membrane di-iron alkane hydroxylases e. Biosurfactants are heterogeneous group of surface active chemical compounds produced by a wide variety of microorganisms [ 57 , 58 , 60 , 81 — 83 ].

Surfactants enhance solubilization and removal of contaminants [ 84 , 85 ]. Biodegradation is also enhanced by surfactants due to increased bioavailability of pollutants [ 86 ].

Bioremediation of oil sludge using biosurfactants has been reported by Cameotra and Singh [ 87 ]. Microbial consortium consisting of two isolates of Pseudomonas aeruginosa and one isolate Rhodococcus erythropolis from soil contaminated with oily sludge was used in this study. The ability of the consortium to degrade sludge hydrocarbons was tested in two separate field trials.

In addition, the effect of two additives a nutrient mixture and a crude biosurfactant preparation on the efficiency of the process was also assessed. The biosurfactant used was produced by a consortium member and was identified as being a mixture of 11 rhamnolipid congeners. The data substantiated the use of a crude biosurfactant for hydrocarbon remediation. Pseudomonads are the best known bacteria capable of utilizing hydrocarbons as carbon and energy sources and producing biosurfactants [ 37 , 87 — 89 ].

Among Pseudomonads , P. However, glycolipid type biosurfactants are also reported from some other species like P. Biosurfactants increase the oil surface area and that amount of oil is actually available for bacteria to utilize it [ 90 ]. Table 2 summarizes the recent reports on biosurfactant production by different microorganisms. Biosurfactants can act as emulsifying agents by decreasing the surface tension and forming micelles.

The microdroplets encapsulated in the hydrophobic microbial cell surface are taken inside and degraded. Figure 4 demonstrates the involvement of biosurfactant rhamnolipids produced by Pseudomonas sp. Immobilized cells have been used and studied for the bioremediation of numerous toxic chemicals.

Immobilization not only simplifies separation and recovery of immobilized cells but also makes the application reusable which reduces the overall cost. Wilsey and Bradely [ 91 ] used free suspension and immobilized Pseudomonas sp. The study indicated that immobilization resulted in a combination of increased contact between cell and hydrocarbon droplets and enhanced level of rhamnolipids production. Rhamnolipids caused greater dispersion of water-insoluble n-alkanes in the aqueous phase due to their amphipathic properties and the molecules consist of hydrophilic and hydrophobic moieties reduced the interfacial tension of oil-water systems.

This resulted in higher interaction of cells with solubilized hydrocarbon droplets much smaller than the cells and rapid uptake of hydrocarbon in to the cells. Diaz et al. Immobilization can be done in batch mode as well as continuous mode. Packed bed reactors are commonly used in continuous mode to degrade hydrocarbons.

Cunningham et al. They constructed laboratory biopiles to compare immobilised bioaugmentation with liquid culture bioaugmentation and biostimulation.

Immobilised systems were found to be the most successful in terms of percentage removal of diesel after 32 days. Rahman et al. The results showed that there was no decline in the biodegradation activity of the microbial consortium on the repeated use. It was concluded that immobilization of cells are a promising application in the bioremediation of hydrocarbon contaminated site. Microbiological cultures, enzyme additives, or nutrient additives that significantly increase the rate of biodegradation to mitigate the effects of the discharge were defied as bioremediation agents by U.

EPA [ 95 ]. Bioremediation agents are classified as bioaugmentation agents and biostimulation agents based on the two main approaches to oil spill bioremediation. The U. But the list was modified, and the number of bioremediation agents was reduced to nine. Studies showed that bioremediation products may be effective in the laboratory but significantly less so in the field [ 14 , 17 , 18 , 98 ].

This is because laboratory studies cannot always simulate complicated real world conditions such as spatial heterogeneity, biological interactions, climatic effects, and nutrient mass transport limitations. Therefore, field studies and applications are the ultimate tests or the most convincing demonstration of the effectiveness of bioremediation products.

Compared to microbial products, very few nutrient additives have been developed and marketed specifically as commercial bioremediation agents for oil spill cleanup. It is probably because common fertilizers are inexpensive, readily available, and have been shown effective if used properly.

However, due to the limitations of common fertilizers e. This nutrient product is a microemulsion-containing urea as a nitrogen source, sodium laureth phosphate as a phosphorus source, 2-butoxyethanol as a surfactant, and oleic acid to give the material its hydrophobicity. The claimed advantages of Inipol EAP22 include 1 preventing the formation of water-in-oil emulsions by reducing the oil viscosity and interfacial tension; 2 providing controlled release of nitrogen and phosphorus for oil biodegradation; 3 exhibiting no toxicity to flora and fauna and good biodegradability [ 99 ].

The two nutrient products were derived from fish meals in a granular form with urea and super phosphate as nitrogen and phosphorus sources and proteinaceous material as the carbon source. The results showed that the presence of biosurfactant in BIOREN 1 was the most active ingredient which contributed to the increase in oil degradation rates whereas BIOREN 2 without biosurfactant was not effective in that respect.

The biosurfactant could have contributed to greater bioavailability of hydrocarbons to microbial attack. Phytoremediation is an emerging technology that uses plants to manage a wide variety of environmental pollution problems, including the cleanup of soils and groundwater contaminated with hydrocarbons and other hazardous substances.

The different mechanisms, namely, hydraulic control, phytovolatilization, rhizoremediation, and phytotransformation. Advantages of using phytoremediation include cost-effectiveness, aesthetic advantages, and long-term applicability Table 4.

Furthermore, the use of phytoremediation as a secondary or polishing in situ treatment step minimizes land disturbance and eliminates transportation and liability costs associated with offsite treatment and disposal. Research and application of phytoremediation for the treatment of petroleum hydrocarbon contamination over the past fifteen years have provided much useful information that can be used to design effective remediation systems and drive further improvement and innovation. Phytoremediation could be applied for the remediation of numerous contaminated sites.

However, not much is known about contaminant fate and transformation pathways, including the identity of metabolites Table 4. Little data exists on contaminant removal rates and efficiencies directly attributable to plants under field conditions. The potential use of phytoremediation at a site contaminated with hydrocarbons was investigated. Plant growth was found to vary depending upon the species.

Presence of some species led to greater TPH disappearance than with other species or in unvegetated soil. Among tropical plants tested for use in Pacific Islands, three coastal trees, kou Cordia subcordata , milo Thespesia populnea , and kiawe Prosopis pallida and the native shrub beach naupaka tolerated field conditions and facilitated cleanup of soils contaminated with diesel fuel [ ].

Grasses were often planted with trees at sites with organic contaminants as the primary remediation method. Grasses were often planted between rows of trees to provide soil stabilization and protection against wind-blown dust that could move contaminants offsite. Legumes such as alfalfa Medicago sativa , alsike clover Trifolium hybridum , and peas Pisum sp. Fescue Vulpia myuros , rye Elymus sp. Once harvested, the grasses could be disposed off as compost or burned.

Microbial degradation in the rhizosphere might be the most significant mechanism for removal of diesel range organics in vegetated contaminated soils [ ]. This occurs because contaminants such as PAHs are highly hydrophobic, and their sorption to soil decreases their bioavailability for plant uptake and phytotransformation. Applications for genetically engineered microorganisms GEMs in bioremediation have received a great deal of attention to improve the degradation of hazardous wastes under laboratory conditions.

There are reports on the degradation of environmental pollutants by different bacteria. Table 5 shows some examples of the relevant use of genetic engineering technology to improve bioremediation of hydrocarbon contaminants using bacteria.

The genetically engineered bacteria showed higher degradative capacity. However, ecological and environmental concerns and regulatory constraints are major obstacles for testing GEM in the field. These problems must be solved before GEM can provide an effective clean-up process at lower cost.

The use of genetically engineered bacteria was applied to bioremediation process monitoring, strain monitoring, stress response, end-point analysis, and toxicity assessment. Examples of these applications are listed in Table 6. The range of tested contaminants included chlorinated compounds, aromatic hydrocarbons, and nonpolar toxicants. The combination of microbiological and ecological knowledge, biochemical mechanisms, and field engineering designs are essential elements for successful in situ bioremediation using genetically modified bacteria.

Cleaning up of petroleum hydrocarbons in the subsurface environment is a real world problem. A better understanding of the mechanism of biodegradation has a high ecological significance that depends on the indigenous microorganisms to transform or mineralize the organic contaminants.

Microbial degradation process aids the elimination of spilled oil from the environment after critical removal of large amounts of the oil by various physical and chemical methods.

This is possible because microorganisms have enzyme systems to degrade and utilize different hydrocarbons as a source of carbon and energy. The use of genetically modified GM bacteria represents a research frontier with broad implications. The potential benefits of using genetically modified bacteria are significant.

But the need for GM bacteria may be questionable for many cases, considering that indigenous species often perform adequately but we do not tap the full potential of wild species due to our limited understanding of various phytoremediation mechanisms, including the regulation of enzyme systems that degrade pollutants.

Therefore, based on the present review, it may be concluded that microbial degradation can be considered as a key component in the cleanup strategy for petroleum hydrocarbon remediation. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



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