Mercury-Resistant Bacteria and Petroleum Degradation

The concentration of mercury in water and sediment and in the oil extracted from water and sediment was determined for samples collected in Colgate Creek, located in Baltimore Harbor of the Chesapeake Bay. The concentration of mercury in the oil was 4,000 times higher than in sediment and 300,000 times higher than in water samples. The mercury-resistant bacterial populations of the samples studied have been shown to degrade oil, suggesting these bacteria to be a significant factor in the degradation of oil in Colgate Creek.     

Numerical taxonomy and ecology of petroleum-degrading bacteria.

A total of 99 strains of petroleum-degrading bacteria isolated from Chesapeake Bay water and sediment were identified by using numerical taxonomy procedures. The isolates, together with 33 reference cultures, were examined for 48 biochemical, cultural, morphological, and physiological characters. The data were analyzed by computer, using both the simple matching and the Jaccard coefficients. Clustering was achieved by the unweighted average linkage method. From the sorted similarity matrix and dendrogram, 14 phenetic groups, comprising 85 of the petroleum-degrading bacteria, were defined at the 80 to 85% similarity level. These groups were identified as actinomycetes (mycelial forms, four clusters), coryneforms, Enterobacteriaceae, Klebsiella aerogenes, Micrococcus spp. (two clusters), Nocardia species (two clusters), Pseudomonas spp. (two clusters), and Sphaerotilus natans. It is concluded that the degradation of petroleum is accomplished by a diverse range of bacterial taxa, some of which were isolated only at given sampling stations and, more specifically, from sediment collected at a given station.     

Enumeration of petroleum-degrading microorganisms.

A variety of factors, including concentration of oil, antibiotics, dyes, and inoculum washes, were examined to determine their effect on the total counts of microorganisms on oil-containing media. The media found to be best for enumerating petroleum-degrading microorganisms contained 0.5% (vol/vol) oil and 0.003% phenol red, with Fungizone added for isolating bacteria and streptomycin and tetracycline added for isolating yeasts and fungi. Washing the inoculum did not improve recovery of petroleum degraders. Specifically, silica gel-oil medium and a yeast medium are recommended for enumeration of petroleum-degrading bacteria and yeasts and fungi, respectively. It is suggested that counts of petroleum degraders be expressed as percentage of the total population rather than total numbers of petroleum degraders per se. Incubation temperature and presence of oil was found to influence the numbers of petroleum-degrading microorganisms at a given sampling site.     

Microorganisms form exocellular structures, trophosomes, to facilitate biodegradation of oil in aqueous media

Cytochemical staining and microscopy were used to study the trophic structures and cellular morphotypes that are produced during the colonization of oil-water interfaces by oil-degrading yeasts and bacteria. Among the microorganisms studied here, the yeasts (Schwanniomyces occidentalis, Torulopsis candida, Candida tropicalis, Candida lipolytica, Candida maltosa, Candida paralipolytica) and two representative bacteria (Rhodococcus sp. and Pseudomonas putida) produced exocellular structures composed of biopolymers during growth on petroleum hydrocarbons. Four of the yeasts including S. occidentalis, T. candida, C. tropicalis and C. maltosa excreted polymers through modified sites in their cell wall ('canals'), whereas C. lipolytica and C. paralipolytica and the two bacterial species secreted polymers over the entire cell surface. These polymers took the form of fibrils and films that clogged pores and cavities on the surfaces of the oil droplets. A three-dimensional reconstruction of the cavities using serial thin sections showed that the exopolymer films isolated the ambient aqueous medium together with microbial cells and oil to form both closed and open granules that contained pools of oxidative enzymes utilized for the degradation of the oil hydrocarbons. The formation of such granules, or 'trophosomes,' appears to be a fundamental process that facilitates the efficient degradation of oil in aqueous media.     

Microbial communities and their ability to oxidize n-alkanes in the area of release of gas- and oil-containing fluids in Mid-Baikal (Cape Gorevoi Utes)

The microbial community in the area of oil seep in Mid-Baikal (Cape Gorevoi Utes) was studied. The number of microorganisms that oxidize normal hydrocarbons, petroleum, and easily accessible organic matter in the water mass of the lake, bottom sediments, and bitumen structures was studied in 2005?C2009. The high heterogeneity of the distribution of microorganisms associated with the deparaffination of oil in the areas of oil seeps was noted. The maximum concentrations of hydrocarbon-oxidizing microorganisms in the samples of bottom water above bitumen structures (up to 2200 ± 175 CFU/mL) and in bitumen structures themselves (up to 170 000 ± 13 000 CFU/g) were determined. A model experiment showed that in the conditions of low temperatures (4°C) the degradation of the fraction of oil n-alkanes by the natural microbial community reaches 90% over a period of 60 days.     

Effect of Estuarine Sediment pH and Oxidation-Reduction Potential on Microbial Hydrocarbon Degradation

Microbial mineralization rates of two petroleum hydrocarbons, as affected by pH and oxidation-reduction potential, were determined in a Barataria Bay, Louisiana, sediment using ¹⁴C-labeled hydrocarbons. Hydrocarbon mineralization rates were inferred from the activity of respired ¹⁴CO₂. Sediment pH and oxidation-reduction potential were important factors in governing the population of hydrocarbon-degrading microorganisms in the sediment and subsequent mineralization rates. Highest mineralization rates occurred at pH 8.0, and the lowest occurred at pH 5.0. At all pH levels mineralization decreased with decreasing oxidation-reduction potential (i.e., increasing sediment anaerobiosis). Generally, mineralization rates for octadecane were greater than those for naphthalene. Aerobic microorganisms in the oxidized sediment were more capable of degrading hydrocarbons than anaerobic microorganisms in reduced sediment of the same pH.     

海洋石油降解微生物及其降解机理

微生物修复作为一种新型环保的生物修复技术,已成为海洋石油污染生物修复的核心技术。对海洋石油降解微生物的种类即细菌、蓝藻、真菌以及藻类进行了总结,对微生物对石油烃的降解途径与降解机理进行了综述。微生物降解烷烃的过程包括末端氧化、烷基氢过氧化物以及环己烷降解3种形式。微生物对芳香烃的降解是通过芳香烃被氧化酶氧化导致苯环开环来实现的。微生物对多环芳烃的降解是在单加氧酶或双加氧酶的催化作用下被最终降解为二氧化碳和水而被分解。并对影响石油烃降解微生物的因素包括温度、营养物质等因素进行了分析。     

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.     

胜利油田滩涂区石油降解菌的筛选、鉴定及其多样性分析

基于传统的实验方法,对胜利油田滩涂区土壤中石油降解菌进行了筛选和鉴定,并利用PCR-DGGE (变性梯度凝胶电泳)技术分析了胜利油田滩涂区的菌群多样性.结果表明：由研究区土壤中筛选出13株石油降解菌,其中,6株石油降解菌的石油降解率>90%,能降解大部分C₁₂～C₂₆的石油烷烃,系统发育学鉴定为Alcanivorax、Halomonas和Marinobacter,均属于γ-proteobacteria分支;胜利油田滩涂区未培养优势菌有Sulfurovum、Gillisia和Arcobacter;该区优势菌群中γ-proteobacteria所占比重较大,其次为α-proteobactiria、ε-proteobactiria、Actinobacteria 和Flavobacteria.     

Biodegradation of Petroleum Hydrocarbons in Seawater at Low Temperatures (0–5 °C) and Bacterial Communities Associated with Degradation

In this study biodegradation of hydrocarbons in thin oil films was investigated in seawater at low temperatures, 0 and 5 °C. Heterotrophic (HM) or oil-degrading (ODM) microorganisms enriched at the two temperatures showed 16S rRNA sequence similarities to several bacteria of Arctic or Antarctic origin. Biodegradation experiments were conducted with a crude mineral oil immobilized as thin films on hydrophobic Fluortex adsorbents in nutrient-enriched or sterile seawater. Chemical and respirometric analysis of hydrocarbon depletion showed that naphthalene and other small aromatic hydrocarbons (HCs) were primarily biodegraded after dissolution to the water phase, while biodegradation of larger polyaromatic hydrocarbons (PAH) and C₁₀–C₃₆ n-alkanes, including n-hexadecane, was associated primarily with the oil films. Biodegradation of PAH and n-alkanes was significant at both 0 and 5°C, but was decreased for several compounds at the lower temperature. n-Hexadecane biodegradation at the two temperatures was comparable at the end of the experiments, but was delayed at 0°C. Investigations of bacterial communities in seawater and on adsorbents by PCR amplification of 16S rRNA gene fragments and DGGE analysis indicated that predominant bacteria in the seawater gradually adhered to the oil-coated adsorbents during biodegradation at both temperatures. Sequence analysis of most DGGE bands aligned to members of the phyla Proteobacteria (Gammaproteobacteria) or Bacteroidetes. Most sequences from experiments at 0°C revealed affiliations to members of Arctic or Antarctic consortia, while no such homology was detected for sequences from degradation experiment run at 5°C. In conclusion, marine microbial communities from cold seawater have potentials for oil film HC degradation at temperatures ≤5°C, and psychrotrophic or psychrophilic bacteria may play an important role during oil HC biodegradation in seawater close to freezing point.     

Effects of nutrient and temperature on degradation of petroleum hydrocarbons in sub-Antarctic coastal seawater

In an attempt to evaluate the potential of petroleum bioremediation at high latitudes environments, microcosm studies using Antarctic coastal seawater contaminated with diesel or crude oil were conducted in Kerguelen Archipelago (49°22′S, 70°12′E). Microcosms were incubated at three different temperatures (4, 10 and 20°C). During experiments, changes observed in microbial assemblages (total direct count, heterotrophic cultivable microorganisms and hydrocarbon-degrading microorganisms) were generally similar for all incubation temperatures, but chemical data showed only some slight changes in biodegradation indices [Σ(C12–C20)/Σ(C21–C32) and C17/pristane]. The complete data set provided strong evidence of the presence of indigenous hydrocarbon-degrading bacteria in Antarctic seawater and their high potential for hydrocarbon bioremediation. The rate of oil degradation could be increased by the addition of a commercial fertilizer, but water temperature had little effects on biodegradation efficiency which is in conflict with the typical temperature-related assumption predicting 50% rate reduction when temperature is reduced by 10°C. Global warming of Antarctic seawater should not increase significantly the rate of oil biodegradation in these remote regions.     

Isolation and assessment of phytate-hydrolysing bacteria from the DelMarVa Peninsula

The Delaware-Maryland-Virginia (DelMarVa) Peninsula, flanking one side of the Chesapeake Bay, is home to a substantial broiler chicken industry. As such, it produces a significant amount of manure that is typically composted and spread onto local croplands as a fertilizer. Phytate (myo inositol hexakisphosphate), the major form of organic phosphorus in the manure, can be hydrolysed by microorganisms to produce orthophosphate. Orthophosphate is a eutrophication agent which can lead to algal blooms, hypoxia and fish kills in the Chesapeake Bay and its tributaries. This transect study reveals a subpopulation of heterotrophic, thiosulfate-utilizing bacteria that can degrade phytate within the watershed as well as its receiving water sediment. Aerobic isolates were typical soil bacteria, e.g. Pseudomonad, Bacillus and Arthrobacter species, as well as a less common Staphylococcus inhabitant. Bacillus pumilus, Staphyloccocus equorum, Arthrobacter bergei and Pseudomonas marginalis strains have not been previously described as phytate-degrading. Each site along the transect - from manure pile to receiving sediment - was host to a population of bacteria that can degrade phytate and hence, each is a possible non-point source of orthophosphate pollution. Each new isolate could provide an enzyme additive for monogastric feed, thus reducing the impact of excessive phytate load on the environment.     

Simultaneous and sequential fermentations with yeast and lactic acid bacteria in apple juice

A complex substrate, reconstituted concentrated apple juice, was used for testing the principal processes during yeast and malolactic bacteria fermentations. Interactions between microorganisms were studied based on two controlled inoculation procedures, and at different fermentation temperatures. Temperature had a more important effect on yeast growth than the presence of malolactic bacteria in the medium. Acceleration of the death phase of the bacterial population was detected at increased temperatures. In all cases, malic acid degradation was affected by the fermentation temperature. When experiments were carried out with simultaneous inoculation, acidification of the medium took place at both temperatures tested (15°C and 22°C), that was not observed when the malolactic bacteria were inoculated after completion of alcoholic fermentation by yeasts. Received 4 August 1998/ Accepted in revised form 9 December 1998     

Persistence and Biodegradation of Spilled Residual Fuel on an Estuarine Beach

The enrichment of hydrocarbon-degrading bacteria and the persistence of petroleum hydrocarbons on an estuarine beach after a spill of residual fuel oil on 11 April 1973 in Upper Narragansett Bay, R.I. was investigated. A rapid enrichment occurred during days 4 to 16 after the oil spill and a significant population of hydrocarbon-degrading bacteria was maintained in the beach sand for at least a year. The concentration of petroleum hydrocarbons in the mid-tide area declined rapidly during the bacterial enrichment period, remained fairly constant throughout the summer, and then declined to a low concentration after 1 year. An increased concentration of branched and cyclic aliphatic hydrocarbons in the low-tide sediment 128 days after the spill suggested a migration of hydrocarbons during the summer. Hydrocarbon biodegradation was apparent during the winter months at a rate of less than 1 mug of hydrocarbon per g of dry sediment per day.     

Isolation and Identification of hydrocarbon degrading bacteria from Ennore creek

The widespread problem caused due to petroleum products, is their discharge and accidental spillage in marine environment proving to be hazardous to the surroundings as well as life forms. Thus remediation of these hydrocarbons by natural decontamination process is of utmost importance. Bioremediation is a non-invasive and cost effective technique for the clean-up of these petroleum hydrocarbons. In this study we have investigated the ability of microorganisms present in the sediment sample to degrade these hydrocarbons, crude oil in particular, so that contaminated soils and water can be treated using microbes. Sediments samples were collected once in a month for a period of twelve months from area surrounding Ennore creek and screened for hydrocarbon degrading bacteria. Of the 113 crude oil degrading isolates 15 isolates were selected and cultivated in BH media with 1% crude oil as a sole carbon and energy source. 3 efficient crude oil bacterial isolates Bacillus subtilis I1, Pseudomonas aeruginosa I5 and Pseudomonas putida I8 were identified both biochemically and phylogenetically. The quantitative analysis of biodegradation is carried out gravimetrically and highest degradation rate, 55% was recorded by Pseudomonas aeruginosa I5 isolate.     

Degradation of Crude Oil and PAHs in Iron–Manganese Concretions and Sediment from the Northern Baltic Sea

The ability of different sea bottom types to recover from oil contamination under oxic and anoxic conditions is poorly known in the brackish Baltic Sea. We carried out microcosm experiments to examine the capacity of iron–manganese concretions and sediment to remove petroleum compounds at 10°C. The biological degradation potential of polycyclic aromatic hydrocarbons (PAHs) was indicated by the 1000-fold increase in the copy numbers of PAH-degradation genes resulting in 2.2 × 10⁶ gene copies g^?1 DW. In the experiments 35?81% of the crude oil and 96% of PAHs disappeared through abiotic and biotic pathways, and there was little difference between the concretions and sediment. Bacterial community analysis revealed that the bacterial sequences obtained from the oil-treated concretions were affiliated to groups typical for concretions and metal-rich environments, whereas oil-treated sediment sequences were similar to those originating from sediments and oil-contaminated environments. The high frequency of concretion clones affiliated with specific taxonomic groups of α-, β-, γ-, and δ-proteobacteria may indicate the existence of new clades of bacteria within the Rhizobiales and Rhodospirillales of the α-proteobacteria, Burkholderiales—incertae sedis of the β-proteobacteria, Chromatiales of the γ-proteobacteria and the Syntrophobacteriales of the δ-proteobacteria. Our study suggests that concretions and bottom sediment maintain rich, distinct bacterial communities under oil exposure that have the potential to remove petroleum compounds in oxic and anoxic conditions.     

On‐site bioremediation treatment system design and on‐site analytical screening of hydrocarbon‐contaminated dredge sediments

In early 1991, a petroleum refining facility located on the Blair Waterway in Commencement Bay near Tacoma, Washington, wished to deepen its berthing facility. Sediments had accumulated in the berthing area adjacent to the facility's petroleum handling dock to the extent that tanker ships could go aground during low tides. A preliminary sediment characterization program had indicated that elevated polynuclear aromatic hydrocarbons such as anthracene and phenanthrene, and total petroleum hydrocarbons exceeded Puget Sound Dredge Disposal Analysis maximum level guidelines for unconfined, open‐water disposal. Enviros designated an on‐shore sediment treatment facility to receive the dredged sediments. Design criteria and construction details of a treatment area capable of accommodating 8000 yd³ of sediments are presented. Onsite, real‐time sediment analysis for total petroleum hydrocarbon (TPH) concentrations was conducted by an onsite mobile laboratory. Laboratory techniques to expedite sediment analysis for TPH using infrared spectrophotometry are described.     

Experimentation on Degradation of Petroleum in Contaminated Soils in the Root Zone of Maize (Zea Mays L.) Inoculated with Piriformospora Indica

Plant-based methods such as rhizodegradation are very promising for the remediation of petroleum-contaminated soils. Associations of plants with endophytes can further enhance their phytoremediation potential. In this study, a rhizobox experiment was conducted to investigate whether inoculation with the root-colonizing fungus Piriformospora indica could further enhance the degradation of petroleum hydrocarbons in the root zone of maize (Zea mays L.). The rhizoboxes were subdivided into compartments in accordance with distance from the plants. After filling the boxes with soil from a petroleum-contaminated site, seedlings that had either been inoculated with P. indica or not were grown in the middle compartments of the rhizoboxes and grown for 64 days. A plant-free treatment was included for control. The presence of roots strongly increased the counts of total and petroleum-degrading soil bacteria, respiration, dehydrogenase activity, water-soluble phenols and petroleum degradation. All these effects were also found in the soil adjacent to the middle compartments of the rhizoboxes, but strongly decreased further away from it. Inoculation with P. indica further enhanced all the recorded parameters without changing the spatial pattern of the effects. Inoculated plants also produced around 40% more root and shoot biomass than noninoculated plants and had greener leaves. Together, the results indicate that the treatment effects on the recorded soil microbial and biochemical parameters including petroleum hydrocarbon degradation were primarily due to increased root exudation. Irrespectively of this, they show that maize can be used to accelerate the rhizodegradation of petroleum hydrocarbons in soil and that inoculation with P. indica can substantially enhance the phytoremediation performance of maize.     

Biodegradation of petroleum hydrocarbons in estuarine sediments: metal influence

In this work, the potential effect of metals, such as Cd, Cu and Pb, on the biodegradation of petroleum hydrocarbons in estuarine sediments was investigated under laboratory conditions. Sandy and muddy non-vegetated sediments were collected in the Lima River estuary (NW Portugal) and spiked with crude oil and each of the metals. Spiked sediments were left in the dark under constant shaking for 15 days, after which crude oil biodegradation was evaluated. To estimate microbial abundance, total cell counts were obtained by DAPI staining and microbial community structure was characterized by ARISA. Culturable hydrocarbon degraders were determined using a modified most probable number protocol. Total petroleum hydrocarbons concentrations were analysed by Fourier Transform Infrared Spectroscopy after their extraction by sonication, and metal contents were determined by atomic absorption spectrometry. The results obtained showed that microbial communities had the potential to degrade petroleum hydrocarbons, with a maximum of 32 % degradation obtained for sandy sediments. Both crude oil and metals changed the microbial community structure, being the higher effect observed for Cu. Also, among the studied metals, only Cu displayed measurable deleterious effect on the hydrocarbons degradation process, as shown by a decrease in the hydrocarbon degrading microorganisms abundance and in the hydrocarbon degradation rates. Both degradation potential and metal influence varied with sediment characteristics probably due to differences in contaminant bioavailability, a feature that should be taken into account in developing bioremediation strategies for co-contaminated estuarine sites.     

A Probabilistic Ecological Risk Assessment of Tributyltin in Surface Waters of the Chesapeake Bay Watershed

The goal of this study was to conduct a probabilistic ecological risk assessment for tributyltin (TBT) in surface waters of the Chesapeake Bay watershed. Ecological risk was characterized by comparing the probability distributions of environmental exposure concentrations with the probability distributions of species response data determined from laboratory studies. The overlap of these distributions was a measure of risk to aquatic life. Tributyltin exposure data from the Chesapeake Bay watershed were available from over 3600 water column samples from 41 stations in nine basins from 1985 through 1996. Most of the stations were located in the Virginia waters of Chesapeake Bay, primarily the James, Elizabeth and York Rivers. In Maryland waters of the Bay, various marina, harbor and river systems were also sampled. As expected, the highest environmental concentrations of tributyltin (based on 90th percentiles) were reported in and near marina areas. The sources of TBT causing these high concentrations were primarily boat hulls and painting/depainting operations. Lower concentrations of TBT were reported in open water areas, such as the Potomac River, Choptank River and C and D Canal, where the density of boats was minimal. Temporal data from a ten year data base (1986-1996) from two areas in Virginia showed that TBT water column concentrations have declined since 1987 legislation prohibited the use of TBT paints on recreation boats (<25?m). Acute saltwater and freshwater TBT toxicity data were available for 43 and 23 species, respectively. Acute effects for saltwater species were reported for concentrations exceeding 420?ng/L; the lowest acute value for a freshwater species was 1110?ng/L. The acute 10th percentiles for all saltwater and freshwater species were 320 and 103?ng/L, respectively. The order of sensitivity from most to least sensitive for saltwater trophic groups and corresponding acute 10th percentiles were as follows: zooplankton (5?ng/L), phytoplankton (124?ng/L), benthos (312?ng/L) and fish (1009?ng/L). For freshwater species, the order of sensitivity from most to least sensitive trophic groups and corresponding acute 10th percentiles were: benthos (44?ng/L), zooplankton (400?ng/L), and fish (849?ng/L). Chronic data for both saltwater and freshwater species were limited to a few species in each water type. Based on these limited data, the saltwater and freshwater chronic 10th percentiles were 5 and 102?ng/L, respectively. Limited mesocosm and microcosm studies in saltwater suggested that TBT concentrations less than 50?ng/L did not impact the structure and function of biological communities. The saltwater acute (320?ng/L) and chronic (5?ng/L) 10th percentiles were used to determine ecological risk because all exposure data were from saltwater areas of the Chesapeake Bay watershed. Highest ecological risk was reported for marina areas in Maryland waters of Chesapeake Bay and for areas in Virginia such as the Elizabeth River, Hampton Creek and Sarah Creek. Low ecological risk was reported for areas such as the Potomac River, Choptank River, C and D Canal and Norfolk Harbor. Regulation of TBT on recreational watercraft in 1987 has successfully reduced water column concentrations of this organometallic compound. However, various studies have showed that TBT may remain in the sediment for years and continue to be source for water column exposures.