Hydrocarbon bioremediation of a mineral-base contaminated waste from crude oil extraction by indigenous bacteria

The susceptibility to bioremediation of the hydrocarbons contained in a waste from crude oil extraction was examined. Laboratory scale batch reactors were inoculated with indigenous bacteria and biodegradation was followed for 45 days. The total hydrocarbon content was reduced to 70% of its initial value at the end of the experiments. Saturated and aromatic hydrocarbons were the most readily degraded fractions with, respectively, 70% and 60% of the fraction remaining at the end of the experiment. A minor degradation was observed in the resins fraction (20%), whereas the asphaltenes fraction remained almost constant.The substrate preferences of the natural population towards various fractions of the crude oil were determined by both the length of the lag phase and the slope of the exponential growth in a mineral salt-base medium containing either of the different hydrocarbon fraction as the sole source of carbon. The highest consumption rate for every fraction during the time course experiments was in agreement with the shortest lag phase and the greatest exponential growth slope in the corresponding selective media, indicating changes in the population composition.     

Short-Term Effects of South Louisiana and Kuwait Crude Oils on Glucose Utilization by Marine Bacterial Populations

Two crude oils, South Louisiana and Kuwait, were examined for their impact on glucose utilization by bacterial populations from the Gulf of Mexico. The uptake and mineralization of [U-¹⁴C]glucose was assayed after a 4- to 23-h exposure to various concentrations of added crude oil (0, 0.001, 0.01, and 0.1% [vol/vol]). The effects of oil were determined in a total of 15 sediment and 13 water samples collected from offshore, open-bay, and salt marsh environments. The utilization of glucose by bacterial populations usually was not affected by added oil; in 10 sediment and 11 water samples, oil had no significant effect on either glucose uptake or mineralization. Stimulation by oil was recorded in four sediment samples. Oil inhibition occurred in one sediment and two water samples, but only in the presence of the highest concentration of added oil, i.e., 0.1%. Our data suggest that short-term exposure to either South Louisiana or Kuwait crude oil, even at 0.1%, usually has no toxic effect on glucose utilization by marine bacterial populations.     

Biodegradation of a crude oil by three microbial consortia of different origins and metabolic capabilities

Microbial consortia were obtained three by sequential enrichment using different oil products. Consortium F1AA was obtained on a heavily saturated fraction of a degraded crude oil; consortium TD, by enrichment on diesel and consortium AM, on a mixture of five polycyclic aromatic hydrocarbons [PAHs]. The three consortia were incubated with a crude oil in order to elucidate their metabolic capabilities and to investigate possible differences in the biodegradation of these complex hydrocarbon mixtures in relation to their origin. The efficiency of the three consortia in removing the saturated fraction was 60% (F1AA), 48% (TD) and 34% (AM), depending on the carbon sources used in the enrichment procedures. Consortia F1AA and TD removed 100% of n-alkanes and branched alkanes, whereas with consortium AM, 91% of branched alkanes remained. Efficiency on the polyaromatic fraction was 19% (AM), 11% (TD) and 7% (F1AA). The increase in aromaticity of the polyaromatic fraction during degradation of the crude oil by consortium F1AA suggested that this consortium metabolized the aromatic compounds primarily by oxidation of the alkylic chains. The 500-fold amplification of the inocula from the consortia by subculturing in rich media, necessary for use of the consortia in bioremediation experiments, showed no significant decrease in their degradation capability. Journal of Industrial Microbiology & Biotechnology (2002) 28, 252–260 DOI: 10.1038/sj/jim/7000236 Received 12 July 2001/ Accepted in revised form 11 November 2001     

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.     

A transcriptome-wide assessment of differentially expressed genes among two highly divergent,yet sympatric,lineages of the freshwater Atyid shrimp, <Emphasis Type="Italic">Paratya australiensis</Emphasis>

The current study evaluated the possible toxic effects of the water-soluble fraction of crude oil on the general cellular stress-response mechanisms of two dominant representatives of Lake Baikal’s littoral community, the endemic amphipod species Eulimnogammarus verrucosus and E. cyaneus. The acute toxicity effects on the cellular stress-response mechanisms of amphipods were studied in the laboratory by exposing amphipods in water from Lake Baikal to addition of a water-soluble fraction of crude oil at concentrations considered safe for the aquatic environment. The present study found that even short-term exposure to a water-soluble fraction of crude oil at concentration of 50 µg/L, established as the threshold limit for fishery and aquaculture water reservoirs in the Russian Federation, directly affected the general stress-response markers HSP70 and lipid peroxidation and significantly changed the activity of antioxidant enzymes in both studied species. This result confirms the high sensitivity of Baikal endemics to crude oil. Thus, it also indicates that established standards and threshold limit values of oil concentrations estimated for ecological monitoring of general water reservoirs cannot be applied directly to the unique Lake Baikal ecosystem.     

Changes in cardiac activity, oxygen uptake and perfusion indices in Carcinus maenas (L.) exposed to crude oil and dispersant

Cardiac activity and oxygen consumption increased when C. maenas were exposed to a 20% solution of the water-soluble fraction of Fortes crude oil, a 10% solution of the dispersant BP1100WD or a combination of both. Normal feeding behaviour was disrupted. Perfusion indices (Q/VO2) decreased as locomotor activity increased following exposure to crude oil. However, exposure to dispersant or dispersant + crude oil resulted in elevation of perfusion index despite crabs becoming active. All test animals survived for at least 6 weeks following exposure to the pollutants. The acute, sublethal effects of dispersant and dispersant + crude oil were more severe than the effects of crude oil alone.     

Effect of Four Dispersants on Biodegradation and Growth of Bacteria on Crude Oil

Four chemical dispersants, Corexit 8666, Gamlen Sea Clean, G. H. Woods Degreaser-Formula 11470, and Sugee 2 were examined singly and in individual combinations with Arabian Crude Oil (1:1 ratio) at 10 and 25 C for their effects on the growth of bacteria indigenous to local marine waters, the bacterial population composition, and biodegradation of crude oil; in addition, their emulsifying capacities, at approximately 24 C, were determined. None of the dispersants used alone were toxic even at relatively high concentrations (1.25%), although Gamlen Sea Clean and G. H. Woods Degreaser-Formula 11470 did cause an increase in the lag phase which was more pronounced at 10 than at 25 C; addition of the crude oil reduced the lag phase increase. All of the dispersants used alone supported good growth of microorganisms, but qualitative population shifts were caused by the dispersant-oil combinations. The degrees of degradation of the n-alkane fraction of the crude oil varied depending upon the dispersant used. Under these test conditions, only Sugee 2, which had the poorest emulsifying capacity, promoted n-alkane degradation compared with the values obtained by using the crude oil alone.     

Degradation of crude oil by Acinetobacter calcoaceticus and Alcaligenes odorans

Two types of Indian crude oil (Bombay High and Gujarat) were tested for their biodegradability by Acinetobacter calcoaceticus and Alcaligenes odorans. Acinetobacter calcoaceticus S30 and Alc. odorans P20 degraded Bombay High crude oil by 50% and 45%, while only 29% and 37% of Gujarat crude oil (heavy crude oil) was degraded by these isolates, respectively. Acinetobacter calcoaceticus and Alc. odorans in combination deraded 58% and 40% of Bombay High and Gujarat crude oils, respectively, which were significantly higher than that of by individual cultures. Acinetobacter calcoaceticus S30 degraded more of the alkanes fraction than the aromatics fraction of both crude oils. GC fingerprinting of alkane fraction showed major degradation of heptadecane (C₁₇), octadecane (C₁₈), nonadecane (C₁₉), eicosane (C₂₀), docosane (C₂₂), tricosane (C₂₃) and tetracosane (C₂₄) of crude oil, while the Alc. odorans P20 degraded alkanes and aromatics equally. The asphaltenic component increased in both types of crude oil after biodegradation. The two strains grew very well on n -alkane up to C₃₃ as well as on pristane (branched-chain alkane) but could not grow on cycloalkanes. Acinetobacter calcoaceticus S30 could not grow on pure polycyclic aromatic hydrocarbon (PAH) compounds except naphthalene but Alc. odorans P20 could grow on anthracene, phenanthrene, dibenzothiophene, fluorene, fluoranthene, pyrene and chrysene.     

Degradation of Petroleum by an Alga, Prototheca Zopfii

Prototheca zopfii is an achlorophyllous alga which degrades oil. It has been found to degrade 10 and 40% of a motor oil and crude oil, respectively, when tested under appropriate conditions. Degradation of the crude oil observed in this study compares well with the amount of degradation accomplished by bacteria. P. zopfii was found to degrade a greater percentage of the aromatic hydrocarbons in motor oil than of the saturated hydrocarbons and a greater percentage of saturated hydrocarbons in crude oil than of aromatic hydrocarbons. Resins and asphaltens were produced during degradation of motor oil, whereas these fractions in crude oil were degraded. P. zopfii did not demonstrate preferential utilization of lower homologues of cycloalkanes and aromatics as has been observed with bacteria.     

The effect of several crude oils and some petroleum distillation fractions on intestinal absorption in ducklings (Anas platyhynchos).

Ducklings given hypertonic saline drinking water show significant increases in the rates of Na+ and water transfer across the intestinal mucosa. These increased rates of transfer are maintained as long as the birds are fed dypertonic saline. Oral administration of a single small dose of crude oil had no effect on the basal rate of mucosal transfer in freshwater-maintained ducklings but the adaptive response of the mucosa is suppressed in birds given hypertonic saline. When crude oils from eight different geographical locations were tested, the degree of inhibition varied between them; the greatest and smallest degrees of inhibition being observed following administration of Kuwait and North Slope, Alaska, crude oils respectively. The effects of distallation fractions derived from two chemically different crude oils were also examined. The volume of each distallation fraction administered corresponded to its relative abundance in the crude oil from which it was derived. The inhibitory effect was not associated exclusively with the same distallation fractions from each oil. A highly naphthenic crude oil from the San Joaquin Valley, California, showed the greatest inhibitory activity in the least abundant (2%), low boiling point (smaller than 245 degrees C) fraction and the least inhibitory activity in the highest boiling point (greater than 482 degrees C) most abundant (47%) fraction. In contrast, a highly paraffinic crude oil from Paradox Basin, Utah, showed the greatest inhibitory effect with the highest boiling point fraction and a minimal effect with the lowest boiling point fraction; the relative abundances of these two fractions in the crude oil represented 27 and 28% respectively. Water-soluble extracts of both crude oils also had inhibitory effects on mucosal transfer rates and these roughly proportionate to the inhibitory potency of the low boiling point fraction of each oil. Weathered samples of San Joaquin Valley, California, and the Paradox Basin, Utah, oils showed greater effects than corresponding samples of unweathered oils even though most of the low molecular weight material from both oils was either evaporated or solubilized in the underlying water during the 36-h weathering period.     

Phosphatidylcholine removal from brain lipid extracts expands lipid detection and enhances phosphoinositide quantification by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

The utilization of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the analytical detection and quantification of phosphoinositides and other lipids in lipid extracts from biological samples was explored. Since phosphatidylcholine species in crude extracts have been shown to cause ion suppression of the MS signals for other lipids, a minicolumn of a silica gel cation exchanger was used to adsorb the cationic lipids including the phosphatidylcholine species from the chloroform phase of fetal and adult murine brain extracts. In positive ion mode, lipid peaks that had been completely suppressed in the crude extract became readily detectable and quantifiable in the flow-through fraction from the column. In negative ion mode, improved sensitivity made it possible to readily detect and measure phosphatidylinositol-4,5-bisphosphate (PIP(2)) which had been only marginally detectable before the fractionation. By incorporating an internal standard into the samples, the relative MALDI-TOF MS signals obtained for increasing concentrations of mammalian phosphatidylinositol (PtdIns) increased linearly with correlation coefficients >0.95. Using strong cation exchange minicolumn treated extracts, the levels of PtdIns and PIP(2) in adult and fetal murine brains were measured and compared. The removal of cationic lipids from the chloroform-methanol murine brain extracts resulted in improved overall detection of neutral and anionic lipids and quantification of phosphoinositides by MALDI-TOF MS.     

Evidence for in situ crude oil biodegradation after the Prestige oil spill

In November 2002, the oil tanker Prestige sank off the Spanish coast after releasing approximately 17,000 tones of heavy fuel, coating several hundred kilometers of coastline in oil sludge. In December 2002 and February 2003, samples were collected from the shore of the Galician coast to analyse the indigenous population ability to carry out crude oil degradation in situ. Carbon isotopic ratio of the dissolved inorganic carbon (DIC) in seawater samples was used as a rapid method to directly assess activity of microbes on the oil components. 12CO2/13CO2 ratio in samples from certain locations along the coast revealed degradation of a very delta13C-negative source such as the Prestige crude oil (-30.6 per thousand). Putative biodegradation processes taking place at areas with high income of fresh seawater could not be detected with this technique. Laboratory-scale biostimulation processes carried out in samples with the highest oil biodegradation activity showed that N/P deficiency in seawater is a limiting factor for crude oil degradation. The most probable number (MPN) of crude oil component degraders was estimated for several aromatic compounds (naphthalene, anthracene, phenanthrene, pyrene) and for undecane. Our results clearly show that bacteria present in the contaminated water are readily able to transform components of the crude oil into inorganic carbon.     

Consolidating biofuel platforms through the fermentative bioconversion of crude glycerol to butanol

Economic realities for the rising industrial biofuel production have changed substantially during the low oil price period starting in the mid 2010’s. Increased competition requires the sector to increase productivity through the reduction of low-value by-products and full utilization of all value and energy stored in their respective feedstock. Biodiesel is produced commercially from substrates such as animal fat and vegetable oil, generating approximately 10 wt% crude glycerol as its main, currently underutilized, by-product. This crude glycerol is contaminated with catalyst, soap, free fatty acids, glycerides and methyl esters; hence only a small fraction enters the existing glycerol markets, while the purification costs for the majority of crude glycerol are simply too high. However, this presents a unique opportunity to generate additional value. One technical possibility is to use crude glycerol as a carbon source for butanol production, a compound of higher value and energy, a potential additive for gasoline and diesel fuels and bulk chemical commodity. Conversion facilities could be co-located with biodiesel plants, utilizing established infrastructure and adding significant value and productivity to the existing biodiesel industry. This review focuses on the current activities geared towards the bioconversion of crude glycerol to butanol.     

高效石油降解菌群的构建及对芳香烃化合物萘的协同降解性能

【背景】石油作为一类混杂有机化合物,一旦产生污染就会对人类和环境造成严重的危害。【目的】从新疆石油污染土壤中分离筛选石油降解菌,为石油污染土壤的生物修复提供数据支持及技术参考。【方法】以石油为唯一碳源,通过富集培养、筛选分离得到123株单菌,根据菌落形态挑选出30个不同形态菌株,通过16S rRNA基因序列确定其种属,构建系统发育树;通过原油降解实验筛选出高效石油降解菌,以芳香烃的标志化合物萘为唯一碳源筛选出高效降解菌株,并分别筛选可降解水杨酸、邻苯二酚的菌株。【结果】分离筛选出5株高效石油降解菌,降解率高于85%;萘、水杨酸和邻苯二酚降解菌株各获得一株,将3种菌株按照1:1:1的接种比例对萘进行降解,萘的降解率从单菌60.74%提升到89.40%,菌株间的分工协作可以提高有机物的降解效率。【结论】筛选得到的菌株丰富了石油降解微生物菌种库,不同微生物菌株之间的分工协作为石油污染物的降解提供了新思路,为进一步研究石油污染治理提供参考。     

Use of bacteria-immobilized cotton fibers to absorb and degrade crude oil

Using enrichment culture technique, two isolates that brought a significant degradation and dispersion of crude oil were obtained from contaminated sediments of the Bohai Bay, China. 16S rRNA gene sequencing and phylogenetic analysis indicated that the two bacterial strains affiliated with the genera Vibrio and Acinetobacter. Subsequently, the bacterial cells were immobilized on the surface of cotton fibers. Cotton fibers were used as crude oil sorbent as well as a biocarrier for bacteria immobilization. Among the two isolates, the marine bacteria Acinetobacter sp. HC8-3S showed a strong binding to the cotton fibers, possibly enhanced through extracellular dispersant excreted by Acinetobacter sp. HC8-3S. Both planktonic and immobilized bacteria showed relatively high biodegradation (>60%) of saturated hydrocarbons fraction of crude oil, in the pH range of 5.6–8.6. The degradation activities of planktonic and immobilized bacteria were not affected significantly when the NaCl concentration reached 70 g/L. The immobilized bacterial cells exhibited an enhanced biodegradation of crude oil. The efficiency of saturated hydrocarbons degradation by the immobilized bacterial cells increased about 30% compared to the planktonic bacterial cells.     

GROWTH OF MAIZE SEEDLINGS AS AFFECTED BY GLUCOKININ AND INSULIN

1. Solutions of glucokinin and insulin, particularly those from which the easily dialyzable substances had been removed, increased the growth of roots and tops of young maize seedlings, as shown by comparisons with untreated seedlings grown in distilled water. 2. Strong solutions of crude glucokinin or of crude insulin repressed growth. 3. Seedlings from which the tips of the primary roots had been removed just before placing the plants in the test solutions made greater gains in both top growth and root growth than seedlings with uncut roots treated with solutions of the same strength. Control experiments showed that this difference in growth was not the result of cutting the roots, and that crude glucokinin and crude insulin contained several substances some of which were more readily absorbed by the plant than others. 4. Purification of crude glucokinin and crude insulin by dialysis showed that the residue of relatively non-dialyzable substance was the growth-promoting fraction. 5. The dialysate of crude glucokinin contained at least three types of material, one of which repressed growth. 6. Ammonium sulfate, one of the possible impurities of glucokinin, repressed the growth of seedlings but did not produce the other changes in metabolism shown by seedlings treated with dialysate of onion glucokinin. 7. The endosperm of plants treated with growth-promoting solutions of purified insulin did not lose weight as rapidly as the endosperms of untreated plants, indicating that the treated plants made their greater gains in growth by more efficient utilization of the endosperm, or as a result of greater photosynthetic activity, or by a combination of these. 8. Experiments with albino seedlings suggested that the greater gain in weight made by plants treated with insulin was the result in part of increased photosynthetic activity.     

Laboratory evaluation of crude oil biodegradation with commercial or natural microbial inocula.

Experiments have been performed to screen eight microbial commercial products that, according to the manufacturers, are able to degrade crude oil. This study compared the crude oil biodegradation activity of commercial inocula with that of natural inocula (activated sludge and tropical aquarium water). Some of the latter were previously adapted to the crude oil as the only carbon source. Nutrients and sorbents in the commercial formulations were eliminated, and each inoculum was precultured on marine yeast extract medium. Crude oil biodegradability tests were conducted with close initial substrate concentration to initial bacterial concentration ratios (S0/X0) of 0.94 g of crude oil/10(9) CFU, which allowed a comparison of biodegradation activity. The inocula oxidized the crude oil after a short lag time of less than 3-18 days. After that time, the rate of oxidation varied between 45 and 244 mg O2/(L.day). Crude oil biodegradation after a 28-day test was effective only for 10 out of 12 inocula (from 0.1 to 25% in weight). Biodegradation mainly corresponded to the saturated fraction of the crude oil; the asphaltene fraction was never significantly biodegraded. Our results led to the conclusion that natural inocula, either adapted or not adapted to crude oil, were the most active (from 16 to 25% of loss in crude oil weight) and only one commercial inoculum was able to degrade 18% of the crude oil. Other inocula had a biodegradation activity ranging from 0.1 to 14%.     

Microbial Degradation of Alkyl Carbazoles in Norman Wells Crude Oil

Norman Wells crude oil was fractionated by sequential alumina and silicic acid column chromatography methods. The resulting nitrogen-rich fraction was analyzed by gas chromatography-mass spectrometry and showed 26 alkyl (C₁ to C₅) carbazoles to be the predominant compounds. An oil-degrading mixed bacterial culture was enriched on carbazole to enhance its ability to degrade nitrogen heterocycles. This culture was used to inoculate a series of flasks of mineral medium and Norman Wells crude oil. Residual oil was recovered from these cultures after incubation at 25°C for various times. The nitrogen-rich fraction was analyzed by capillary gas chromatography, using a nitrogen-specific detector. Most of the C₁-, C₂-, and C₃- carbazoles and one of the C₄-isomers were degraded within 8 days. No further degradation occurred when incubation was extended to 28 days. The general order of susceptibility of the isomers to biodegradation was C₁ > C₂ > C₃ > C₄. The carbazole-enriched culture was still able to degrade n-alkanes, isoprenoids, aromatic hydrocarbons, and sulfur heterocycles in the crude soil.     

Bioreactor operated production of lipase: castor oil hydrolysis using partially-purified lipase.

A highly stable lipase from Pseudomonas aeruginosa KKA-5 was produced by batch cultivation technique employing shake flask and 5 L-bioreactor. The bioreactor was run at different airflow rates. Low airflow rates (1 and 3 L/min), did not lead to effective growth and lipase production. Growth increased by about one order and lipase production increased by about 6 times, at an airflow rate of 5 L/min. Lipase production occurred during decelerated cell growth. A highly stable lipase was produced which retained its activity in the running bioreactor, even after a period of one month. This stable lipase was partially-purified using ammonium sulphate precipitation technique. Castor oil was hydrolyzed using 300U crude and partially-purified lipase, each. Approximately 21-fold, partially-purified lipase could hydrolyze 81% castor oil within a period of 96 hr, where as only 63% hydrolysis was obtained, in 216 hour, when crude lipase was used.     

Hydrocarbon emulsification and enhanced crude oil degradation by lauroyl glucose ester

Enzymatically synthesized lauroyl glucose emulsified different hydrophobic substrates when assayed spectrophotometrically. Stable emulsions were formed with triglycerides as well as with hydrocarbons. There was a linear relation between the concentration of lauroyl glucose (50-450 microg) and emulsification activity under the assay conditions when tested with aromatic and aliphatic hydrocarbons. This sugar ester was able to emulsify the aromatic hydrocarbons benzene, toluene and xylene. Long chain alkanes (n-decane and n-hexadecane) as well as brominated long chain alkanes (1-bromodecane and 1-bromohexadecane) were efficiently emulsified. The effect of lauroyl glucose ester on degradation of crude oil by a known oil-degrading Rhodococcus species was also investigated. The culture showed enhanced degradation of crude oil when lauroyl glucose ester was used as an emulsifier. It degraded 70% of the aliphatic fraction of Bombay High crude oil in the presence of the sugar ester at a concentration of 200mg l(-1) as compared to 50% without the emulsifier.