Petroleum-degrading Microorganisms Research Articles

The influence of benzene on the composition, diversity and performance of the anodic bacterial community in glucose-fed microbial fuel cells.

Bioelectrochemical systems offer unique opportunities to remove recalcitrant environmental pollutants in a net positive energy process, although it remains challenging because of the toxic character of such compounds. In this study, microbial fuel cell (MFC) technology was applied to investigate the benzene degradation process for more than 160 days, where glucose was used as a co-metabolite and a control. We have applied an inoculation strategy that led to the development of 10 individual microbial communities. The electrochemical dynamics of MFC efficiency was observed, along with their 1H NMR metabolic fingerprints and analysis of the microbial community. The highest power density of 120 mW/m2 was recorded in the final period of the experiment when benzene/glucose was used as fuel. This is the highest value reported in a benzene/co-substrate system. Metabolite analysis confirmed the full removal of benzene, while the dominance of fermentation products indicated the strong occurrence of non-electrogenic reactions. Based on 16S rRNA gene amplicon sequencing, bacterial community analysis revealed several petroleum-degrading microorganisms, electroactive species and biosurfactant producers. The dominant species were recognised as Citrobacter freundii and Arcobacter faecis. Strong, positive impact of the presence of benzene on the alpha diversity was recorded, underlining the high complexity of the bioelectrochemically supported degradation of petroleum compounds. This study reveals the importance of supporting the bioelectrochemical degradation process with auxiliary substrates and inoculation strategies that allow the communities to reach sufficient diversity to improve the power output and degradation efficiency in MFCs beyond the previously known limits. This study, for the first time, provides an outlook on the syntrophic activity of biosurfactant producers and petroleum degraders towards the efficient removal and conversion of recalcitrant hydrophobic compounds into electricity in MFCs.

Pilot-scale field studies on activated microbial remediation of petroleum-contaminated soil.

Soil contamination by petroleum, including crude oil from various sources, is increasingly becoming a pressing global environmental concern, necessitating the exploration of innovative and sustainable remediation strategies. The present field-scale study developed a simple, cost-effective microbial remediation process for treating petroleum-contaminated soil. The soil treatment involves adding microbial activators to stimulate indigenous petroleum-degrading microorganisms, thereby enhancing the total petroleum hydrocarbons (TPH) degradation rate. The formulated microbial activator provided a growth-enhancing complex of nitrogen and phosphorus, trace elements, growth factors, biosurfactants, and soil pH regulators. The field trials, involving two 500 m3 soil samples with the initial TPH content of 5.01% and 2.15%, were reduced to 0.41% and 0.02% in 50 days, respectively, reaching the national standard for cultivated land category II. The treatment period was notably shorter than the commonly used composting and bioaugmentation methods (typically from 8 to 12 weeks). The results indicated that the activator could stimulate the functional microorganisms in the soil and reduce the phytotoxicity of the contaminated soil. After 40 days of treatment, the germination rate of rye seeds increased from 20 to 90%, indicating that the microbial activator could be effectively used for rapid on-site remediation of oil-contaminated soils.

Urea addition as an enhanced strategy for degradation of petroleum contaminants during co-composting of straw and pig manure: Evidences from microbial community and enzyme activity evaluation

Alterations in microbial community succession patterns and enzyme activities by petroleum pollutants during co-composting of straw and swine manure with the supplementary nitrogen source are unclear. In this study, urea was added into co-composting systems, and the removal performance of petroleum, microbial enzyme activity and community changes were investigated. Results showed that the polyphenol oxidase and catalase activities which were both related to the degradation of petroleum contaminants were accordingly increased from 20.65 to 30.31 U/g and from 171.87 to 231.86 U/g due to urea addition. The removal efficiency of petroleum contaminants in composting with urea increased from 45.06% to 82.29%. The addition of urea increased the diversity and abundance of petroleum-degrading microorganisms, and enhanced microbial linkages. This study provides a novel strategy for the degradation of petroleum hydrocarbon as well as a new insight into the effect of urea on both microbial processes and composting phases.

Legacy Effects of Phytoremediation on Plant-Associated Prokaryotic Communities in Remediated Subarctic Soil Historically Contaminated with Petroleum Hydrocarbons.

Phytoremediation of petroleum hydrocarbons in subarctic regions relies on the successful establishment of plants that stimulate petroleum-degrading microorganisms, which can be challenging due to the extreme climate, limited nutrients, and difficulties in maintaining sites in remote locations. A long-term phytoremediation experiment was initiated in Alaska in 1995 with the introduction of grasses and/or fertilizer to petroleum hydrocarbon (PHC)-contaminated soils that were subsequently left unmanaged. In 2011, the PHC concentrations were below detection limits in all soils tested and the originally planted grasses had been replaced by volunteer plant species that had colonized the site. Here, we sought to understand how the original treatments influenced the structure of prokaryotic communities associated with plant species that colonized the soils and to assess the interactions between the rhizospheric and endophytic communities of the colonizing vegetation 20 years after the experiment was established. Metataxonomic analysis performed using 16S rRNA gene sequencing revealed that the original type of contaminated soil and phytoremediation strategy influenced the structure of both rhizospheric and endophytic communities of colonizing plants, even 20 years after the treatments were applied and following the disappearance of the originally planted grasses. Our findings demonstrate that the choice of initial phytoremediation strategy drove the succession of microorganisms associated with the colonizing vegetation. The outcome of this study provides new insight into the establishment of plant-associated microbial communities during secondary succession of subarctic areas previously contaminated by PHCs and indicates that the strategies for restoring these ecosystems influence the plant-associated microbiota in the long term. IMPORTANCE Subarctic ecosystems provide key services to local communities, yet they are threatened by pollution caused by spills and disposal of petroleum waste. Finding solutions for the remediation and restoration of subarctic soils is valuable for reasons related to human and ecosystem health, as well as environmental justice. This study provides novel insight into the long-term succession of soil and plant-associated microbiota in subarctic soils that had been historically contaminated with different sources of PHCs and subjected to distinct phytoremediation strategies. We provide evidence that even after the successful removal of PHCs and the occurrence of secondary succession, the fingerprint of the original source of contamination and the initial choice of remediation strategy can be detected as a microbial legacy in the rhizosphere, roots, and shoots of volunteer vegetation even 2 decades after the contamination had occurred. Such information needs to be borne in mind when designing and applying restoration approaches for PHC-contaminated soils in subarctic ecosystems.

Advances in the bioaugmentation-assisted remediation of petroleum contaminated soil

Bioremediation is considered as a cost-effective, efficient and free-of-secondary-pollution technology for petroleum pollution remediation. Due to the limitation of soil environmental conditions and the nature of petroleum pollutants, the insufficient number and the low growth rate of indigenous petroleum-degrading microorganisms in soil lead to long remediation cycle and poor remediation efficiency. Bioaugmentation can effectively improve the biodegradation efficiency. By supplying functional microbes or microbial consortia, immobilized microbes, surfactants and growth substrates, the remediation effect of indigenous microorganisms on petroleum pollutants in soil can be boosted. This article summarizes the reported petroleum-degrading microbes and the main factors influencing microbial remediation of petroleum contaminated soil. Moreover, this article discusses a variety of effective strategies to enhance the bioremediation efficiency, as well as future directions of bioaugmentation strategies.

Adsorptive bioremediation of soil highly contaminated with crude oil

Due to the extended oil extraction and transportation in Russia and other oil-producing countries, many lands remain contaminated because of accidental spills. This situation requires the cost-effective and efficient remediation of petroleum-contaminated soils. Bioremediation of soils contaminated with high concentrations of crude oil is usually hampered by high toxicity thresholds for microbial degraders. We have performed a two-year microfield experiment on the influence of a mixed adsorbent (ACD) composed of granular activated carbon and diatomite on bioremediation of a grey forest soil contaminated with crude oil at concentrations (5–15 % w/w) that would theoretically not result in a successful pollutant removal due to toxicity. Remediation of these soils was evaluated after treating with the ACD adsorbent (from 4 to 12% w/w) and a biopreparation (BP) containing hydrocarbon-degrading bacteria, separately or in combination. Reduction of total petroleum hydrocarbons content was significantly greater in highly contaminated soils with the combined amendments than in the respective controls (through the activation of indigenous degrading microorganisms by fertilizing and mixing) by 9-10% and 5-8% at the end of the first and second years, respectively, depending on the contamination level. Significantly higher counts of petroleum-degrading microorganisms (as indigenous and introduced by the BP), as well as much less phytotoxicity was detected in the ACD-amended soils, as compared with the samples without adsorbent. In addition, the ACD mixture drastically reduced the wash-out of polar petroleum metabolites (evidently oxidized hydrocarbons) and the phytotoxicity of the lysimetric waters, especially in highly contaminated soils. The results indicate that the mixture of activated carbon and diatomite is a prospective adsorbent for the in situ bioremediation of soils highly contaminated with crude oil.

Study on Extreme Microbial Degradation of Petroleum Hydrocarbons

Some oil-contaminated areas are often accompanied by extreme environments including high salinity, low temperature, and high temperature. The petroleum-degrading microorganisms isolated from these extreme environments can quickly adapt to extreme environments, grow and metabolize in extreme environments, and show great potential in the field of oil pollution control. This article reviews species, adaptation mechanism and research progress of oil-degrading bacteria in three extreme environments of hypersaline, low temperature and high temperature, so as to provide theoretical basis for further research and application.

Effects of bioaugmentation on sorption and desorption of benzene, 1,3,5-trimethylbenzene and naphthalene in freshly-spiked and historically-contaminated sediments

This work investigated the frequently observed “rebounds” of contaminants of concern in groundwater systems. Specifically, influences of bioaugmented microorganisms on the sorption and desorption of representative petroleum constituents [benzene, 1,3,5-trimethylbenzene and naphthalene (BTN)] were studied in freshly-spiked and historically–contaminated sediments. Capable microorganisms were enriched and supplemented to contaminated sediments to enhance biodegradation of petroleum hydrocarbons. In freshly-spiked sediments, when petroleum-degrading microorganisms were added, concentrations of dissolved petroleum constituents appeared to increase initially, and 12.4, 14.0 and 20.0 mg/kg of benzene, 1,3,5-trimethylbenzene and naphthalene, respectively desorbed from the sediments into the water phase. In the historically-contaminated sediments, the augmentation of petroleum-degrading microorganisms led to the desorption of 0.023–0.059, 0009–0.016, and 1.731–2.763 mg/L of previously sequestrated BTN into the water phase, and also triggered the desorption of 0.051–0.223, −0.133–2.630, and 2.324–1.200 mg/kg of previously sequestrated BTN as the methanol extraction quantity. The mechanisms of the enhanced desorption at the presence of microbes remain to be determined; however, we presumed that microbially produced constituents such as biosurfactants and cell mass could have attributed to the partition of petroleum compounds from the sediments. Findings from this study may partially explain “rebounds” of certain petroleum constituents into the groundwater during in situ bioremediation practice, although such immediate rebounds sometimes are weak, and the desorbed constituents can be eventually biodegraded under proper biogeochemical conditions.

A Correlation to Estimate the Bioventing Degradation Rate Constant

ABSTRACT Estimating the biodegradation rate is essential when designing a bioremediation strategy for petroleum-contaminated sites, and when evaluating assessment guidelines. However, estimating the biodegradation rate is difficult as the rate constant varies from site to site due to changing site conditions, which include soil type, biological activity, and type of contaminant. Accordingly, bench-scale biodegradation studies were completed using respirometers to measure first-order biodegradation rate constants for gasoline in several soils over 30 days of incubation. A total of seven soils were tested at various gasoline concentrations with constant nutrient ratios and water content. No microbial inhibition was observed for the range of gasoline concentrations studied. Analysis showed that the statistically significant parameters were the initial population of petroleum-degrading microorganisms and the organic matter content. The developed empirical correlation is a simple tool that practioners can use to estimate the biodegradation rate without conducting lengthy and expensive experiments.

Bioremediation of oil-contaminated soil using Candida catenulata and food waste

Even though petroleum-degrading microorganisms are widely distributed in soil and water, they may not be present in sufficient numbers to achieve contaminant remediation. In such cases, it may be useful to inoculate the polluted area with highly effective petroleum-degrading microbial strains to augment the exiting ones. In order to identify a microbial strain for bioaugmentation of oil-contaminated soil, we isolated a microbial strain with high emulsification and petroleum hydrocarbon degradation efficiency of diesel fuel in culture. The efficacy of the isolated microbial strain, identified as Candida catenulata CM1, was further evaluated during composting of a mixture containing 23% food waste and 77% diesel-contaminated soil including 2% (w/w) diesel. After 13 days of composting, 84% of the initial petroleum hydrocarbon was degraded in composting mixes containing a powdered form of CM1 (CM1-solid), compared with 48% of removal ratio in control reactor without inoculum. This finding suggests that CM1 is a viable microbial strain for bioremediation of oil-contaminated soil with food waste through composting processes.

Arbuscular Mycorrhiza and Petroleum-Degrading Microorganisms Enhance Phytoremediation of Petroleum-Contaminated Soil

While plants can phytoremediate soils that are contaminated with petroleum hydrocarbons, adding microbes to remediate contaminated sites with petroleum-degrading microorganisms and arbuscular mycorrhizal fungi (AMF) is not well understood. The phytoremediation of Arabian medium crude oil (ACO) was done with a Lolium multiflorum system inoculated with an AMF (Glomus intraradices) and a mixture of petroleum-degrading microorganisms—the bacterium, Sphingomonas paucimobilis (Sp) and the filamentous fungus, Cunninghamella echinulata (Ce, SpCe)—or with a combination of microorganisms (AMF + SpCe). Based on an earlier study on screening plants for phytoremediation of ACO, L. multiflorum (Italian ryegrass) was selected for its tolerance and rapid growth response (Alarcón, 2006). The plants were exposed to ACO-contaminated soil (6000 mg kg−1) for 80 d under greenhouse conditions. A modified Long Ashton Nutrient Solution (LANS) was supplied to all treatments at 30 μg P mL−1, except for a second, higher P, control treatment at 44 μg P mL−1. Inoculation with AMF, SpCe, or AMF + SpCe resulted in significantly increased leaf area as well as leaf and pseudostem dry mass as compared to controls at 30 μg P mL−1. Populations of bacteria grown on a nitrogen-free medium and filamentous fungi increased with AMF + SpCe and SpCe treatments. The average total colonization and arbuscule formation of AMF-inoculated plants in ACO-contaminated soil were 25% and 8%, respectively. No adverse effects were caused by SpCe on AMFcolonization. Most importantly, ACOdegradation was significantly enhanced by the addition of petroleum-degrading microorganisms and higher fertility controls, as compared to plants at 30 μg P mL−1. The highest ACOdegradation (59%) was observed with AMF + SpCe. The phytoremediation of ACO was also enhanced by single inoculation of AMF or SpCe. The effect of AMF and petroleum-degrading microorganisms on plant growth and ACOdegradation was not attributable to differences in proline, total phenolics, nitrate reductase levels, or variation in plant–gas exchange.

Phytoremediation of aged petroleum sludge: effect of irrigation techniques and scheduling.

The use of higher plants to accelerate the remediation of petroleum contaminants in soil is limited by, among other factors, rooting depth and the delivery of nutrients to the microsites at which remediation occurs. The objective of this study was to test methods of enhancing root growth and remediation in the subsurface of a contaminated petroleum sludge. The phytoremediation of highly contaminated petroleum sludge (total petroleum hydrocarbons >35 g kg(-1) was tested in the greenhouse as a function of the frequency and the depth of irrigation and fertilization. Water and dissolved plant nutrients were added to the soil surface or at a depth of 30 cm, either daily or weekly. Equivalent quantities of water and nutrients were added in all cases. Daily irrigation at a depth of 30 cm invoked greater root growth and enhanced contaminant degradation relative to all other treatments. In the absence of plants, residual concentrations of petroleum hydrocarbons after 7 mo were higher than with plants. The presence of plant roots clearly improved the physical structure of the soil and increased microbial populations. Thus, the plant roots in conjunction with daily additions of soluble N and P appeared to enhance oxygen transport to greater depths in the soil, stimulate petroleum-degrading microorganisms, and provide microbial access to soil micropores. Subsurface irrigation with frequent, small amounts of water and nutrients could significantly accelerate phytoremediation of field soils contaminated with petroleum hydrocarbons.

The use of poultry litter as co-substrate and source of inorganic nutrients and microorganisms for the ex situ biodegradation of petroleum compounds

The purpose of this investigation was to determine the feasibility of utilizing poultry litter as a source of microorganisms, C co-substrate, N, and P to enhance the biodegradation of petroleum compounds in contaminated soil. An initial laboratory-scale study utilized soil contaminated with approximately 3,000 mg/kg (ppm) total petroleum hydrocarbons (TPHC) as diesel fuel. Biotreatment units, each containing 10 L of contaminated soil, were supplemented (0, 1, 10, and 20%, total weight basis) with broiler litter containing 3.65% N and 1.89% P. Petroleum-degrading microorganisms previously enriched from broiler and turkey litter were also inoculated into the litter-treated units. A significant first order rate of TPHC biodegradation was measured for all treatment units containing broiler litter (P < 0.05). Based on these results, a subsequent study was conducted at the site of a commercial facility permitted to treat soil (ex situ) contaminated with hazardous compounds. Soil treatment plots, each containing approximately 1 ton of soil contaminated with approximately 1,200 to 1,600 mg/kg diesel fuel were established. Each plot was replicated four times and the experiment was conducted for 35 d. Treatments were as follows: control, soil only; soil + commercial blend of bioremediation nutrients; soil + commercial fertilizer; soil + poultry litter (1% by volume); soil + poultry litter (10% by volume); soil + pelleted poultry litter (10% by volume). The results showed that the remediation of soil contaminated with petroleum compounds is significantly (P < 0.05) enhanced when supplemented with poultry litter (pelleted or nonpelleted) in concentrations of 10% soil volume. These results demonstrate the potential for a specialized market for the use of poultry litter.

Immobilization of Prototheca zopfü in calcium-alginate beads for the degradation of hydrocarbons

A petroleum-degrading achlorophyllous micro-alga, Prototheca zopfü, was immobilized in calcium-alginate beads and the rate of degradation for n-alkanes (C 14, C 15 and C 16) measured. The rate of biodegradation for the hydrocarbon mixture could be described by a second-order rate expression which is a function of both population of algal cells and substrate concentration. The volumetric biodegradation rates observed in free cells were comparable to those reported in other systems of marine petroleum-degrading micro-organisms, such as Bacillus sp. and Pseudomonas sp. A modified bacterial adherence to hydrocarbon assay revealed the strong hydrophobicity of the outermost surface of the algal cells. Repeated cycles of subculture were carried out and the stability of the immobilized algal cells for biodegradation confirmed.

Ecological distribution of the petroleum-degrading microorganisms in the Bohai Sea : Wang, Wenxing, 1984. In: Fate and fluxes of oil pollutants in the KAP region. Symposium, Marine Science Center, Basrah, Iraq. DouAbul, A.A.Z., organizer; in press

Ecological distribution of the petroleum-degrading microorganisms in the Bohai Sea : Wang, Wenxing, 1984. In: Fate and fluxes of oil pollutants in the KAP region. Symposium, Marine Science Center, Basrah, Iraq. DouAbul, A.A.Z., organizer; in press

Effect of Crude Oil and Petroleum-degrading Micro-organisms on the Growth of Freshwater and Soil Protozoa

SUMMARY: Growth responses of six species of freshwater and soil protozoa were determined for cells grown monoxenically on various petroleum-utilizing microflora. Protozoa were cultured either in the presence or absence of partially degraded crude oil. The regression coefficient characterizing the slope of the growth curve was used as a comparison index throughout. The nature of the prey species determined the magnitude of the protozoan growth response. The presence of crude oil did not consistently raise or lower an individual species' rate of division. Crude oil had no overall influence on protozoan size although there was a slight tendency towards larger cells in the presence of oil.

Enumeration of petroleum-degrading marine and estuarine microorganisms by the most probable number method

Several media designed for use in a most probable number (MPN) determination of petroleum-degrading microorganisms were compared. The best results, i.e., largest numbers, were obtained using a buffered (32 mM PO4=) liquid medium containing 1% hydrocarbon substrate. Of 104 presumptive oil degraders tested, 20 grew on oil agar medium but did not utilize oil or a mixture of pure paraffinic hydrocarbons (C10 to C16 n-alkanes) in liquid (MPN) medium. Visible turbidity in the liquid medium was correlated with hydrocarbon utilization. Counts of petroleum degraders obtained using liquid medium (MPN) were in most cases higher than those obtained on an oil-amended silica gel medium. Both procedures yield an estimation of oil degraders, and the oil-amended agar permits growth of organisms which do not degrade crude oil. All strains of oil-degrading microorganisms examined in this study were lipolytic, but the converse was not always true.

Petroleum-degrading achlorophyllous alga Prototheca zopfii

ONE of the objectives of this research programme is to enumerate and identify petroleum-degrading microorganisms occurring in Chesapeake Bay, to assess the potential of these microorganisms to degrade petroleum in vitro and in situ and to determine the types of petroleum degraded1. We are sampling two locations in Chesapeake Bay at monthly intervals. One is Colgate Creek, an oil-contaminated site in Baltimore Harbour, the other is an area south of Parson's Island in Eastern Bay, a non-polluted shellfish harvesting region of Chesapeake Bay. The presence of petroleum in Colgate Creek and its absence in Eastern Bay has been confirmed by computerised low-resolution mass spectrometry analysis of benzene extracts of sediments collected at both sites2,3. We have routinely isolated petroleum-degrading bacteria, yeasts and filamentous fungi from Colgate Creek and Eastern Bay, but during April and May 1973, and again in 1974, an unusual organism, believed to be an algal species, was isolated from sediment of Colgate Creek. Corresponding isolates have not been isolated from Eastern Bay.

Microbial degradation of model petroleum at low temperatures

Two areas of Chesapeake Bay, Colgate Creek in Baltimore Harbor and Eastern Bay, are presently under study, with routine sampling of water and sediment for petroleum-degrading microorganisms (bacteria, yeasts, and fungi) by direct plating and enrichment culture. Selected physical and chemical parameters are recorded for each sampling site, and water and sediment samples are extracted for hydrocarbons. Numbers of petroleum-degrading microorganisms enumerated by direct plating were found to correlate with the concentration of benzene-extractable material and were higher for the Colgate Creek than for the Eastern Bay site. Petroleum-degrading microorganisms were isolated from water and sediment samples at environmental temperatures of 0°, 5°, and 10°C.A salts medium supplemented with nitrate and phosphate was used to provide optimum conditions for petroleum degradation, whereas Chesapeake Bay water was used to simulate natural environmental conditions. Use of a model petroleum permitted quantitative measurement of utilization of individual hydrocarbons ranging in complexity from simple alkanes to polynuclear aromatic hydrocarbons. Higher growth yields and maximum hydrocarbon degradation was observed for microorganisms in the salts medium at 0°, 5°, and 10°C, although significant quantities of hydrocarbons were utilized in some samples grown in a medium for which Chesapeake Bay water was the diluent. Bacterial hydrocarbon degradation accounted for most of the model petroleum utilization at 0° and 5°C. However, oscillations of bacterial populations, with significant growth of yeasts, was observed at 10°C. Photomicroscopy and scanning electron microscopy revealed aggregates of bacteria, yeasts, and fungi associated with oil globules. From preliminary identification and classification of the hydrocarbon-utilizing bacteria, members of the generaVibrio, Aeromonas, Pseudomonas, andAcinetobacter were present in the enrichment cultures. From results of this study, it is concluded that utilization of model petroleum at low temperatures is a function of the types and numbers of microorganisms present in an original inoculum taken from the natural environment.

Microbial Ecology of Petroleum Utilization in Chesapeake Bay

ABSTRACT Analysis of water and sediments collected at two stations in Chesapeake Bay demonstrated four to five times the concentration of petroleum in an oil polluted site in Baltimore Harbor compared with the station in Eastern Bay which served as a control. The numbers of petroleum-degrading microorganisms, measured by direct and replica plating, in the water and sediment samples were related to the concentration of oil in each sample. Total yields of petroleum-degrading microorganisms grown on an oil substrate were greater for those organisms exposed to oil in the natural environment. Microorganisms isolated from an oil-contaminated environment produced cell yields under “natural” conditions, i.e., laboratory simulation of growth conditions in the natural environment, which equaled the yields of microorganisms which had not been previously exposed to oil and were grown under optimum conditions. Microorganisms isolated from water and sediment samples collected in Baltimore Harbor grew on substrates representative of the aliphatic, aromatic and refractory hydrocarbons. The hydrocarbon-utilizing fungus, Cladosporium resinae and actinomycetes were predominant among the hydrocarbon-utilizing isolates. Microbial degradation of petroleum in Chesapeake Bay appears to be mediated by the autochthonous microbial flora, the seasonal incidence of which is presently under study.