Degradation of Phthalate and Di-(2-Ethylhexyl)phthalate by Indigenous and Inoculated Microorganisms in Sludge-Amended Soil

The metabolism of phthalic acid (PA) and di-(2-ethylhexyl)phthalate (DEHP) in sludge-amended agricultural soil was studied with radiotracer techniques. The initial rates of metabolism of PA and DEHP (4.1 nmol/g [dry weight]) were estimated to be 731.8 and 25.6 pmol/g (dry weight) per day, respectively. Indigenous microorganisms assimilated 28 and 17% of the carbon in [¹⁴C]PA and [¹⁴C]DEHP, respectively, into microbial biomass. The rates of DEHP metabolism were much greater in sludge assays without soil than in assays with sludge-amended soil. Mineralization of [¹⁴C]DEHP to ¹⁴CO₂ increased fourfold after inoculation of sludge and soil samples with DEHP-degrading strain SDE 2. The elevated mineralization potential was maintained for more than 27 days. Experiments performed with strain SDE 2 suggested that the bioavailability and mineralization of DEHP decreased substantially in the presence of soil and sludge components. The microorganisms metabolizing PA and DEHP in sludge and sludge-amended soil were characterized by substrate-specific radiolabelling, followed by analysis of ¹⁴C-labelled phospholipid ester-linked fatty acids (¹⁴C-PLFAs). This assay provided a radioactive fingerprint of the organisms actively metabolizing [¹⁴C]PA and [¹⁴C]DEHP. The ¹⁴C-PLFA fingerprints showed that organisms with different PLFA compositions metabolized PA and DEHP in sludge-amended soil. In contrast, microorganisms with comparable ¹⁴C-PLFA fingerprints were found to dominate DEHP metabolism in sludge and sludge-amended soil. Our results suggested that indigenous sludge microorganisms dominated DEHP degradation in sludge-amended soil. Mineralization of DEHP and PA followed complex kinetics that could not be described by simple first-order equations. The initial mineralization activity was described by an exponential function; this was followed by a second phase that was described best by a fractional power function. In the initial phase, the half times for PA and DEHP in sludge-amended soil were 2 and 58 days, respectively. In the late phase of incubation, the apparent half times for PA and DEHP increased to 15 and 147 days, respectively. In the second phase (after more than 28 days), the half time for DEHP was 2.9 times longer in sludge-amended soil assays than in sludge assays without soil. Experiments with radiolabelled DEHP degraders suggested that a significant fraction of the ¹⁴CO₂ produced in long-term degradation assays may have originated from turnover of labelled microbial biomass rather than mineralization of [¹⁴C]PA or [¹⁴C]DEHP. It was estimated that a significant amount of DEHP with poor biodegradability and extractability remains in sludge-amended soil for extended periods of time despite the presence of microorganisms capable of degrading the compound (e.g., more than 40% of the DEHP added is not mineralized after 1 year).     

Aerobic and Anaerobic Biodegradation of Aged Pentachlorophenol by Indigenous Microorganisms

Pentachlorophenol (PCP) use as a general biocide, particularly for treating wood, has led to widespread environmental contamination. Biodegradation has emerged as the main mechanism for PCP degradation in soil and groundwater and a key strategy for remediation. Examining the microbial biodegrading potential for PCP at a contaminated site is crucial in determining its fate. Hundreds of studies have been published on PCP microbial degradation, but few have described the biodegradation of PCP that has been in contact with soils for many years. The bioavailability of “aged” hydrophobic organics is a significant concern. PCP- and 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP)-contaminated soil samples from several depths at a former wood treatment site were placed under varying conditions in the laboratory to determine the anaerobic and aerobic potential for biodegradation of chlorophenols at the site. PCP biodegradation occurred in both anaerobic and aerobic soil samples. Rapid aerobic degradation occurred in samples spiked with 2- and 4-chlorophenol, but not with 3-chlorophenol. Reductive dechlorination of PCP in anaerobic samples resulted in the accumulation of 3-chlorophenol. In most anaerobic replicates, 3-chlorophenol was degraded with the appearance of detectable, but not quantifiable amounts of phenol. These results indicate excellent potential for remediation at the site using the indigenous microorganisms under both aerobic and anaerobic conditions. However, a fraction of the PCP was unavailable for degradation.     

Removal of Phthalate Esters in Soil Column Inoculated with Microorganisms

The removal of phthalate esters, such as di-2-ethyl hexyl phthalate (DEHP) was efficiently effected by inoculating and retaining viable cells of Nocardia erythropolis, a bacterium known capable of rapidly degrading phthalate esters, in soil column. When an influent containing 3000 ppm of DEHP was passed through a column inoculated with Nocardia erythropolis, the eluent from the column was gas-chromatographically free of DEHP after 1 day. Residual DEHP on the support after 32 days in the column inoculated with Nocardia erythropolis was only 0.14% against the total amount of DEHP fed, whereas it was 5.2% in the uninoculated column. Microorganisms capable of utilizing DEHP were isolated from the inoculated and un- inoculated columns after 32 days operation and identified. The DEHP utilizing microorganisms in the inoculated column were found to belong to Nocardia erythropolis, Nocardia restricta and Pseudomonas putida (Biotype B), and those in the uninoculated column to Nocardia erythropolis, Pseudomonas putida (Biotype A and B) and Pseudomonas acidovorans. In particular, strain 1-1 of Nocardia erythropolis isolated from the inoculated column was morphologically and biochemically identical with the inoculated Nocardia erythropolis S-l. Ratio of all Nocardia erythropolis to total cells recovered increased from 10.8% immediately after inoculation to 27.2% after 32 days in inoculated column.     

Precipitation of Calcite by Indigenous Microorganisms to Strengthen Liquefiable Soils

Enrichments for indigenous microorganisms capable of hydrolyzing urea in the presence of CaCl₂ were performed on potentially liquefiable saturated soils in both the laboratory and in situ. Following enrichment, treatment of soils with nutrients, CaCl₂ and urea resulted in significant in situ precipitation of calcite, even at depth, by indigenous microorganisms. The biomineralized soils showed properties that indicate calcite precipitation increased their resistance to seismic-induced liquefaction.     

Crude Oil Biodegradation by a Soil Indigenous Bacillus sp. Isolated from Lavan Island

The application of microorganisms for removing crude oil pollution from contaminated sites as a type of bioremediation has long been a matter of study in scientific communities. In this study, 35 morphologically different spore-forming Bacilli were isolated from an oil-contaminated soil in Lavan Island. The objective of this study was to investigate the oil-biodegrading ability of these indigenous bacilli. Therefore, their biosurfactant production, using Du Neuy ring, and the crude oil aliphatic and aromatic content alteration after bacterial treatment, respectively, using gas chromatography and high-performance liquid chromatography, were studied. An isolate with high endurance of a wide range of temperature and pH and optimized growth at 30°C and pH 6.8 that could reduce the surface tension from 60 to 40 mN/m and cause the most alteration in aliphatic and aromatic content of crude oil was selected. Using biochemical and molecular analyses of 16SrRNA, this suitable bacterium for oil biodegradation was characterized as Bacillus cereus sp. 4.     

Stimulation of Diesel Fuel Biodegradation by Indigenous Nitrogen Fixing Bacterial Consortia

Abstract Successful stimulation of N₂ fixation and petroleum hydrocarbon degradation in indigenous microbial consortia may decrease exogenous N requirements and reduce environmental impacts of bioremediation following petroleum pollution. This study explored the biodegradation of petroleum pollution by indigenous N₂ fixing marine microbial consortia. Particulate organic carbon (POC) in the form of ground, sterile corn-slash (post-harvest leaves and stems) was added to diesel fuel amended coastal water samples to stimulate biodegradation of petroleum hydrocarbons by native microorganisms capable of supplying a portion of their own N. It was hypothesized that addition of POC to petroleum amended water samples from N-limited coastal waters would promote the growth of N₂ fixing consortia and enhance biodegradation of petroleum. Manipulative experiments were conducted using samples from coastal waters (marinas and less polluted control site) to determine the effects of POC amendment on biodegradation of petroleum pollution by native microbial consortia. Structure and function of the microbial consortia were determined by measurement of N₂ fixation (acetylene reduction), hydrocarbon biodegradation (¹⁴C hexadecane mineralization), bacterial biomass (AODC), number of hydrocarbon degrading bacteria (MPN), and bacterial productivity (³H-thymidine incorporation). Throughout this study there was a consistent enhancement of petroleum hydrocarbon degradation in response to the addition of POC. Stimulation of diesel fuel biodegradation following the addition of POC was likely attributable to increases in bacterial N₂ fixation, diesel fuel bioavailability, bacterial biomass, and metabolic activity. Toxicity of the bulk phase water did not appear to be a factor affecting biodegradation of diesel fuel following POC addition. These results indicate that the addition of POC to diesel-fuel-polluted systems stimulated indigenous N₂ fixing microbial consortia to degrade petroleum hydrocarbons. Received: 29 December 1998; Accepted: 6 April 1999     

Aerobic Reduction of Hexavalent Chromium in Soil by Indigenous Microorganisms

Surface soil containing 25,100 mg/kg total Cr [12,400 mg/kg Cr(VI)] obtained from a Superfund site was used in laboratory microcosm studies to evaluate the potential for aerobic reduction of Cr(VI) by the indigenous soil microbial community. Hexavalent chromium in soil was reduced by as much as 33% (from 1840 to 1240 mg/L) within 21 days under enrichment conditions. Reduction of Cr(VI) in this system was biologically mediated and depended on the availability of usable energy sources. Mass balance studies suggested that the microbial populations removed Cr(VI) from the soil solutions by reduction. Indigenous microbial soil communities even with no history of Cr(VI) contamination were capable of mediating this process. However, Cr(VI) removal was not observed when microbial populations from a sewage sludge sample were added to the soil microcosms. The results suggest that Cr(VI)-reducing microbial populations are widespread in soil, and thus the potential exists for in situ remediation of environmentally significant levels of Cr( VI) contamination.     

Enhanced Biodegradation of Diesel Oil in Seawater Supplemented with Nutrients

The imbalance of C, N, and P caused by the spilled oil could be regulated by the addition of nitrogen and phosphorous. Moreover, different kinds of N and P sources were used in order to stimulate oil biodegradation under laboratory and field conditions, but the results were conflicting. To evaluate the effectiveness of nutrient supplementation, N sources (NO₃‐N and NH₄‐N) and P sources (PO₄‐P) were applied to the simulated diesel‐polluted seawater in the N/P ratio of 10:1 and 20:1, respectively. The results showed that the addition of nutrients increased the oil biodegradation rate and the counts of petroleum degrading bacteria (PDB) and heterotrophic bacteria (HB). A strongly positive correlation existed (the interrelated coefficient was nearly 0.9) between the percentage ratio of PDB/HB and the oil biodegradation rates, and therefore the percentage ratio of PDB/HB could be used as a good indicator to predict oil biodegradation. Among the four samples treated with nutrients, the biodegradation efficiency of the group amended with NO₃‐N and PO₄‐P in the ratio of 10:1 (10NO₃‐P group) was as much as 75.8 %, while in the 10NH₄‐P, 20NO₃‐P and 20NH₄‐P groups this value was 61.3 %, 52.4 % and 40.5, respectively. It would take natural degradation without nutrient supplementation about 78 days to achieve the result obtained within 14 days with 10NO₃‐P amendment . Chemical and microbiological analyses confirmed that the addition of nutrients in the same N/P ratio remarkably enhanced the biodegradation rate and the counts of microorganisms in the NO₃‐N treated groups, indicating that the microorganisms tend to utilize NO₃‐N rather than NH₄‐N as their growth N source. When the same kind of N source was added to the system, the promoted efficiency in the 10:1 (N/P ratio) groups was notable compared to the 20:1 groups, i.e., adding nutrients in the ratio of 10:1 is superior in the stimulation of oil biodegradation to the ratio of 20:1.     

Biodegradation of Diesel Fuel in Soil Under Various Nitrogen Addition Regimes

Bioremediation is a growing technology for treating fuel-contaminated soils. Many biological, physical, and chemical parameters control the rate and efficiency of this process, including type and concentration of contaminants, temperature, oxygen content, and nutrient status. This study investigated the effect that nitrogen sources and concentrations had on the degradation rate of diesel fuel in nutrient limited soil at two carbon-to-nitrogen ratios. The different sources of nitrogen studied were ammonium nitrate, ammonium sulfate, potassium nitrate, urea, and urea oligomers (control release fertilizer). Laboratory experiments were conducted on field-contaminated soil using sealed bioreactors at a controlled temperature of 25°C. For both carbon-to-nitrogen ratios tested, hydrocarbon degradation rates were the highest for the ammonium sulfate (20:1 at 0.032?d^?1; 40:1 at 0.019d^?1) and urea treatments (20:1 at 0.025?d^?1; 40:1 at0.011?d^?1). A degradation rate correlation as a function of nitrate and ammonia concentrations was developed. The correlation suggests the occurrence of nitrate inhibition at elevated nitrate concentrations.     

Potential of Bioaugmentation and Biostimulation for Enhancing Intrinsic Biodegradation in Oil Hydrocarbon-Contaminated Soil

Studies were conducted using a 10-chamber Micro-Oxymax (Columbus, OH, USA) respirometer to determine the effect of bioaugmentation and biostimulation (by diverse ways of O₂ supply) on enhancing biodegradation of oil hydrocarbons to reduce risk at a former military airport in Kluczewo, Poland. Indigenous or exogenous bacteria bioaugmentation was used to degrade hydrocarbons. Aerated water and/or aqueous solutions of H₂O₂ or KMnO₄ were used to supply O₂. The intrinsic and enhanced biodegradation was evaluated by the O₂ uptake and CO₂ production rates obtained using a linear regression of the cumulative O₂ uptake and CO₂ production curves. Generally, in all cases biodegradation rates enhanced by bioaugmentation were two to four times higher than the rates of intrinsic biodegradation. Moreover, application of indigenous bacteria was more efficient in comparison to the exogenous consortia. The highest CO₂ production rates were achieved when aqueous solution of KMnO₄ was applied, as the increase of CO₂ production rates were about 71% to 97% higher compared to a control. The aqueous solution of H₂O₂ did not cause any significant improvement of the biodegradation rates. Compared to a control, the addition of aerated water resulted in a decrease of CO₂ production rates. Most probably the excessive soil moisture could reduce the air-filled porosity and, consequently, the oxygen contents in soil.     

Surface Percolation for Soil Improvement by Biocementation Utilizing In Situ Enriched Indigenous Aerobic and Anaerobic Ureolytic Soil Microorganisms

The use of biocementation via microbially induced carbonate precipitation (MICP) for improving the mechanical properties of weak soils in the laboratory has gained increased attention in recent years. This study proposes an approach for applying biocementation in situ, by combining the surface percolation of nutrients and cementation solution (urea/CaCl₂) with in situ cultivation of indigenous soil urease positive microorganisms under non-sterile conditions. The enrichment of indigenous ureolytic soil bacteria was firstly tested in batch reactors. Using selective conditions (i.e., pH of 10 and urea concentrations of 0.17 M), highly active ureolytic microorganisms were enriched from four diverse soil samples under both oxygen-limited (anoxic) and oxygen-free (strictly anaerobic) conditions, providing final urease activities of more than 10 and 5 U/mL, respectively. The enrichment of indigenous ureolytic soil microorganisms was secondly tested in pure silica sand columns (300 and 1000 mm) for biocementation applications using the surface percolation approach. By applying the same selective conditions, the indigenous ureolytic soil microorganisms with high urease activity were also successfully enriched for both the fine and coarse sand columns. However, the in situ enriched urease activity was highly related to the dissolved oxygen of the percolated growth medium. The results showed that the in situ cultivated urease activity may produce non-clogging cementation over the entire 1000-mm columns, with unconfined compressive strength varying between 850–1560 kPa (for coarse sand) and 150–700 kPa (for fine sand), after 10 subsequent applications of cementation solution. The typically observed loss of ureolytic activity during the repeated application of the cementation solution was recovered by providing more growth medium under selective enrichment conditions, enabling the in situ enriched ureolytic microorganisms to increase in numbers and urease activity in such a way that continued cementation was possible.     

Ex-Situ Studies on Biodegradation of Artificially Enriched Kerosene and Diesel Soils by Fungal Isolates

To demonstrate the potential of biodegradation of soils enriched with kerosene and diesel, an ex-situ study with the objective of evaluating and comparing the effects of three different fungal isolates P. janthinellum, P. decumbens, and A. terreus was performed. The study dealt with the biodegradation of artificially enriched kerosene and diesel soils by 5%, 10%, and 15% (w/w). The experiment was performed by ex-situ large-scale tray method using 24 plastic trays 6′′ X 3′′ X 1′′ in each containing 60 kg enriched soil. After eight weeks of inoculation of the fungal isolates, P. janthinellum was found to have potential compared to the other two and displayed the highest kerosene and diesel degradative capacity, resulting in 98.29%, 97%, 96%, 82%, 70%, and 62% degradation at 5, 10, and 15% kerosene- and diesel-enriched soils after 45 and 60 days, respectively. Moreover, the total fungal population was found to increase as a function of time. A first-order kinetic model equation showed that the specific biodegradation rate constant “k” value were 0.1023 and 0.0285 day^?1 for 5% kerosene and diesel enrichment by P. janthinellum treatment strategy, which was comparatively higher than the values for the other two organisms tested. Thus, the degree of effectiveness of these bioremediation strategies in the soils enriched with kerosene and diesel is in the following order: P. janthinellum>P. decumbens>A. terreus.     

Bioremediation Potential of Cr(VI)-Contaminated Soil Using Indigenous Microorganisms

Amendment of Cr(VI)-contaminated soil (approx. 200 mg/kg) with various treatments resulted in greater CO₂ evolution and Cr(VI) reduction with organic amendments relative to controls receiving no organics, indicating bacterial reduction of Cr(VI) under anaerobic conditions. Isolation of Cr(VI)-reducing, indigenous bacteria, representative of the dominant soil population, further indicated Cr(VI) reduction by indigenous bacteria. Although desorption of Cr(VI) was evident with some treatments, its reduction was not affected.     

Heterotrophic Potentials and Hydrocarbon Biodegradation Potentials of Sediment Microorganisms Within the Athabasca Oil Sands Deposit

Techniques for the enumeration and the determination of the potential activity of disturbed sediment mixed populations at control sites and sites within the Athabasca oil sands formation were applied to August and December samples. These techniques included the determination of general heterotrophic potential for the assimilation and respiration of glutamate, which indicated no oil sand-related changes in the sediments but which indicated a significant seasonal change. Enumeration by epifluorescence direct counts, oil sand hydrocarbon plate counts, and most-probable-number determinations of [¹⁴C]hexadecane and [¹⁴C]-naphthalene degraders indicated that only the plate count was sensitive to increased numbers of oil sand-related hydrocarbon-oxidizing microorganisms within the oil sands deposit. Unlike the most probable number determinations of [¹⁴C]hexadecane and [¹⁴C]naphthalene degraders, however, the biodegradation potential results of these substrates indicated a significant increase in activity at oil sands sites. These biodegradation potentials also showed a marked seasonal fluctuation. Although the biodegradation potentials and the endogenous hydrocarbon plate counts indicated an oil sand-adapted mixed sediment population, the results of these techniques did not correlate well with the concentrations of bituminous hydrocarbons in the sediments. The results suggest that a general capability for hydrocarbon oxidation exists in the Athabasca River system and that this capability is enhanced within the natural bounds of the Athabasca oil sands.     

Prepared Bed Land Treatment of Soils Containing Diesel and Crude Oil Hydrocarbons

An ex situ, field-scale, prepared bed land treatment unit (LTU) was used to bio-remediate soils containing petroleum hydrocarbons. Two soils were treated in side-by-side units to compare performance: (1) a clayey silt containing crude oil hydrocarbons from releases 30 to 40 years ago and (2) a silty sand containing diesel fuel hydrocarbons from a leak about three years prior to the bioremediation. The effectiveness of the bioremediation in the LTU was evaluated over a period of 18 months. The results indicated that: (1) prepared bed bioremediation reduced the hydrocarbon concentration, mobility, and relative toxicity in the soil with the diesel fuel, and (2) chemical bioavailability appeared to limit bioremediation of the soil containing the crude oil hydrocarbons. Although the soils containing the crude oil hydrocarbons contained an average of 10,000?mg TPH/kg dry soil, these soils had limited hydrocarbon availability, nontoxic conditions, and low potential for chemical migration. For the soils containing the diesel fuel, active prepared bed bioremediation of about 15 weeks was adequate to reach an environmentally acceptable endpoint. At that time, there was little further TPH loss, no Microtox^TM toxicity, and limited hydrocarbon mobility.     

基因工程菌在生物降解中的应用及其发展

在简要阐述基因工程菌构建方法的基础上,系统综述和评价了基因工程构建方法的研究进展及其在生物降解中存在的问题。     

Effect of Shale Kerogen Oxidation Products on Biodegradation of Oil and Oil Products in Soil and Water

The effect of oxidation products of shale kerogen (high-molecular-weight acids with 6–22 carbon atoms) on biodegradation of oil and oil products in soil and water was studied. High-molecular-weight acids (HMWA) not only affected the layer of oil and/or oil products and dispersed it into small particles, but also stimulated growth of Rhodococcus erythropolis VKM AS-1339D, degraders of oil and oil products. Addition of 0.001–0.003% HMWA to a medium to be purified from oil products increased the extent of bacterial biodegradation by a factor of 1.1–5.0.     

The Influence of Rhamnolipids on Aliphatic Fractions of Diesel Oil Biodegradation by Microorganism Combinations

Twelve different bacteria–yeast combinations were tested for determination of their ability to biodegrade diesel oil. The cell surface properties of the bacterial and yeast strains were correlated with the type of carbon source used in the experiments. The highest biodegradation of diesel oil after 7 days was obtained for the following combinations: Aeromonas hydrophila MR4–Yarrowia lipolytica EH 56 (87 %) and Xantomonas maltophila MRP7–Candida maltosa EH15 (90 %). Degradation performances of 10 of 12 combinations were enhanced by the presence of rhamnolipids. The highest increases were observed for A. hydrophila MR4–C. maltosa EH15 (from 34 to 67 %), A. hydrophila MR4–C. maltosa EH60 (from 47 to 76 %) and for Pseudomonas stutzeri MR7–C. maltosa EH60 (from 29 to 79 %). The addition of rhamnolipids to the system reduces the removal time of diesel oil from the contaminated water and changes the microbial adhesion to hydrocarbons. Modification of the cell surface of the tested strain during biodegradation is a very important factor determining the removal of hydrophobic compounds.     

Enzymatic Evaluation During Biodegradation of Kerosene and Diesel by Locally Isolated Fungi from Petroleum-Contaminated Soils of Western India

The current study suggests that the fungal isolates P. decumbens PDX7, P. janthinellum SDX7, and A. terreus PKX4 degraded kerosene by 95%, 96%, and 75% and diesel by 79%, 75%, and 70% after 16 days based on the ability of utilizing these compounds as sole carbon sources. GC-MS chromatograms revealed that n-alkane fractions are easily degraded; however, the rate is lower for branched alkanes, n-alkyl aromatics, cyclic alkanes, and polynuclear aromatics displaying delayed and lower degradation. The ratio of aromatic/aliphatic hydrocarbons >0.8 indicates the efficiency of these fungi in removing the aromatic hydrocarbons of the petroleum products. All of the treated fungal strains exhibited higher MnP, laccase, and dehydrogenase activities on the twelfth and sixteenth days as compared to the initial fourth and eighth days. In addition, P. decumbens PDX7 and P. janthinellum SDX7 displayed higher enzymatic activities as compared to A. terreus PKX4. Fungal isolates were also tested for their growth on various xenobiotic compounds as sole carbon sources.