Biodegradation of oily sludge in Kuwait soil

Soil microorganisms were not inhibited by mixing oily sludge in soil up to 8.7% (w/w) oil (15% sludge). Adding NH ₄ ⁺ and phosphate increased microbial activity. Microbial activity was also affected by seasonal variation. Thermotolerant microorganisms were more predominant during the summer. After 29 months, 72%, 84%, and 83% of the soil was degraded in fertilized soils dosed with 2.9, 5.8 and 8.7% oil, respectively.     

Enhancing the biodegradation of total petroleum hydrocarbons in oily sludge by a modified bioaugmentation strategy

The effect of successive inoculation with hydrocarbon-degrading bacteria on the dynamics of petroleum hydrocarbons degradation in soil was investigated in this study. Oily sludge was used as a source of mixed hydrocarbons pollutant. Two bacterial consortia composed of alkanes and polycyclic aromatic hydrocarbon degraders were constructed from bacteria isolated from soil and oily sludge. These consortia were applied to incubated microcosms either in one dose at the onset of the incubation or in two doses at the beginning and at day 62 of the incubation period, which lasted for 198 days. During this period, carbon mineralization was evaluated by respirometry while total petroleum hydrocarbons and its fractions were gravimetrically evaluated by extraction from soil and fractionation. Dosing the bacterial consortia resulted in more than 30% increase in the overall removal of total petroleum hydrocarbons from soil. While alkane removal was only slightly improved, aromatic and asphaltic hydrocarbon fraction removal was significantly enhanced by the addition of the second consortium. Polar compounds (resins) were enriched only as a result of aromatics and asphaltene utilization. Nonetheless, their concentration declined back to the original level by the end of the incubation period.     

Recovery of mitosporic fungi actively growing in soils after bacterial bioremediation of oily sludge and their potential for removing recalcitrant hydrocarbons

Bacterial bioremediation is a widely used technique to remove or neutralize contaminants. However, the enzymatic capabilities of bacteria are limited and, consequently, recalcitrant compounds remain in the soil. Fungi can help to overcome this drawback, since their enzymatic repertoire is extensive. In this study, the diversity of viable, actively growing, filamentous fungi was explored in soils previously subjected to bioremediation with bacterial consortia from three petroleum exploitation fields. Diversity was estimated using both morphological traits and ITS rDNA sequencing. We recovered a highly diverse group of morphotypes from each field, most of them previously reported genera of fungi associated with bioremediation (Aspergillus, Paecilomyces, and Penicillium), but a high proportion (40%) of the fungal species detected have never previously been reported as being involved in degradation of hydrocarbons. To build evidence of the isolates as potential bioremediation agents, their laccase and peroxidase activities were measured in vitro; peroxidase activity was a common trend in these fungi. The detection of peroxidase activity suggests adaptation of these fungi to the residual contaminants after bacterial action. Bioaugmentation of the fungal isolates in microcosms contaminated with oily sludge resulted in higher removal of the asphaltenic fraction compared to no bioaugmented microcosms. Our method allowed us to screen for and isolate viable mycelia within a contaminated environment, a strategy efficient for our environmental protection goals.     

Biodegradation of heavy oily sludge by a two-step inoculation composting process using synergistic effect of indigenous isolated bacteria

The impact of two-step inoculation of indigenous strains and their synergistic effect in the scaling-up of petroleum hydrocarbons biodegradation from a mineral-based medium (MBM) to a two-phase composting process were investigated. After isolating the strains KA3 and KA4 from heavy oily sludge (HOS), their emulsification index (E₂₄), bacterial adhesion to hydrocarbon (BATH), and oil degradation efficiency were evaluated in the MBM. Then, they were inoculated twice into the composting bioreactors lasted for the primary 8 weeks as the first phase (FP) and subsequent 8 weeks as the second phase (SP). The results indicated that the consortium of the two strains degraded 16-61% of crude oil (1-5% concentration) in the MBM. In the composting reactors, removals of 20 g kg⁻¹ initial concentration of total petroleum hydrocarbons (TPH) were found to be 63.95, 61.00, and 89.35% for the strains KA3, KA4, and their consortium, respectively. The computed biodegradation constants indicated the synergistic effect of the two strains and the effectiveness of the second-step inoculation. The study demonstrated the successful scaling-up of HOS biodegradation from MBM to the two-phase composting process through two-step inoculation of the isolated strains.     

The response of maize (Zea mays L.) plant assisted with bacterial consortium and fertilizer under oily sludge

The objective of this study was to evaluate the role of PGPR consortium and fertilizer alone and in combination on the physiology of maize grown under oily sludge stress environment as well on the soil nutrient status. Consortium was prepared from Bacillus cereus (Acc KR232400), Bacillus altitudinis (Acc KF859970), Comamonas (Delftia) belonging to family Comamonadacea (Acc KF859971) and Stenotrophomonasmaltophilia (Acc KF859973). The experiment was conducted in pots with complete randomized design with four replicates and kept in field. Oily sludge was mixed in ml and Ammonium nitrate and Diammonium phosphate (DAP) were added at 70 ug/g and 7ug/g at sowing. The plant was harvested at 21 d for estimation of protein, proline and antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD). To study the degradation, total petroleum hydrocarbon was extracted by soxhelt extraction and extract was analyzed by GC-FID at different period after incubation. Combined application of consortium and fertilizer enhanced the germination %, protein and, proline content by 90,130 and 99% higher than untreated maize plants. Bioavailability of macro and micro nutrient was also enhanced with consortium and fertilizer in oily sludge. The consortium and fertilizer in combined treatment decreased the superoxide dismutase (SOD), peroxidase dismutase (POD) of the maize leaves grown in oily sludge. Degradation of total petroleum hydrocarbon (TPHs) was 59% higher in combined application of consortium and fertilizer than untreated maize at 3 d. The bacterial consortium can enhanced the maize tolerance to oily sludge and enhanced degradation of total petroleum hydrocarbon (TPHs). The maize can be considered as tolerant plant species to remediate oily sludge contaminated soils.     

Biodegradation and detoxification of phenolic compounds by pure and mixed indigenous cultures in aerobic reactors

Degradation and detoxification of a mixture of persistent compounds (2-chlorophenol, phenol and m-cresol) were studied by using pure and mixed indigenous cultures in aerobic reactors. Biodegradation assays were performed in batch and continuous flow reactors. Biodegradation was evaluated by determining total phenols, ultraviolet spectrophotometry and chemical oxygen demand (COD). Microbial growth was measured by the plate count method. Scanning electronic microscopy was employed to observe the microbial community in the reactor. Detoxification was evaluated by using Daphnia magna toxicity tests. Individual compounds were degraded by pure bacteria cultures within 27 h. The mixture of 2-clorophenol (100 mgl⁻¹), phenol (50 mgl⁻¹) and m-cresol (50 mgl⁻¹) was degraded by mixed bacteria cultures under batch conditions within 36 h: 99.8% of total phenols and 92.5% of COD were removed; under continuous flow conditions 99.8% of total phenols and 94.9% of COD were removed. Mineralization of phenolic compounds was assessed by gas chromatography performed at the end of the batch assays and in the effluent of the continuous-flow reactor. Toxicity was not detected in the effluent of the continuous-flow reactor.     

Comparison of bioremediation strategies for soil impacted with petrochemical oily sludge

Different bioremediation techniques (natural attenuation, biostimulation and bioaugmentation) in contaminated soils with two oily sludge concentrations (1.5% and 6.0%) in open and closed microcosms systems were assessed during 90 days. The results showed that the highest biodegradation rates were obtained in contaminated soils with 6% in closed microcosms. Addition of microbial consortium and nutrients in different concentrations demonstrated higher biodegradation rate of total petroleum hydrocarbons (TPH) than those of the natural attenuation treatment. Soils treated in closed microcosms showed highest removal rate (84.1 ± 0.9%) when contaminated at 6% and bacterial consortium and nutrients in low amounts were added. In open microcosms, the soil contaminated at 6% using biostimulation with the highest amounts of nutrients (C:N:P of 100:10:1) presented the highest degradation rate (78.7 ± 1.3%). These results demonstrate that the application of microbial consortium and nutrients favored biodegradation of TPH present in oily sludge, indicating their potential applications for treatment of the soils impacted with this important hazardous waste.     

Biodegradation of chrysene by the bacterial strains isolated from oily sludge

The biodegradation studies were conducted to test the ability of the bacterial strains (Chry2 and Chry3) isolated from the oily sludge obtained from Gujarat refinery, India, for utilization of chrysene in the liquid medium. Biodegradation of the compound was confirmed using gas chromatography and the percent degradation was calculated to be 15.0 and 17% by Chry2 and Chry3, respectively. The biodegradation results were supported by increase in viable cell count and dry biomass, in the presence of chrysene as the sole carbon source. Both the cultures produced biosurfactant which was indicated by the reduction in surface tension of the growth medium. Presence of catechol 2, 3-dioxygenase gene in Chry3 indicated its potential for degradation of PAHs through meta cleavage degradation pathway. Both the strains were found to possess catechol 1,2-dioxygenase and catechol 2,3-dioxygenase enzyme activities. Based on morphological and biochemical tests, the cultures were tentatively identified as Bacillus sp. (Chry2) and Pseudomonas sp. (Chry3).     

Design of bacterial defined mixed cultures for biodegradation of specific crude oil fractions, using population dynamics analysis by DGGE

Hydrocarbon-degrading bacteria isolated from oil-polluted soils, were used to design three defined mixed cultures (DMC) for biodegradation of Maya crude oil fractions. The first degrading culture, DMC A was made up with 10 strains. Design of DMC B (six strains) and DMC C (three strains) was based on DGGE profiles obtained throughout biodegradation assays of different petroleum fractions. Biodegradation of the aliphatic fraction (10 000 mg l⁻¹) and an aromatic–polar mixture (5000 mg l⁻¹) was evaluated for the DMC B. Biodegradation of total hydrocarbons (10 000 mg l⁻¹) and its fractions was evaluated for DMC B and DMC C. During biodegradation assays, O₂ consumption and CO₂ production were assessed by respirometry, while population dynamics of predominant strains was based on PCR-DGGE profiles of partial 16S rDNA. Aliphatic fraction was completely biodegraded by DMC B, while degradation of the aromatic–polar mixture was 12.5% and for total hydrocarbons 40.5%. DMC B was able to degrade the aromatic fraction (31%) and even the polar fraction (19.6%) present in total hydrocarbons. DMC C degraded the aromatic and polar fractions (5.6% and 2%, respectively) present in total hydrocarbons. DGGE profiles of the DMCs indicated that Pseudomonas sp., Gordonia rubripertincta and a non-identified strain were predominant and probably responsible of the hydrocarbons biodegradation. The use of DGGE-fingerprinting to track microbial populations, allowed selecting strains to design efficient oil-degrading defined mixed cultures.     

Biodegradation of naphthalene in the oil refinery wastewater by enriched activated sludge

The main purpose of this paper is to study naphthalene (NAP) biodegradation by acclimated activated sludge, employing the culture-enrichment method in a continuous flow bioreactor of the wastewater treatment process. The effects of various COD loadings and influent flow rates of an artificial wastewater containing 15 mg l⁻¹ NAP on the biodegradation rates of the activated sludge will be investigated, in order to determine the biodegradation kinetics and minimum mean cell residence time of the activated sludge. From the experimental results, it was found that the resulting enriched activated sludge follows the growth rate of the Monod type and can biodegrade those COD and NAP loadings in the influents efficiently, and its bio-treatment efficiency on NAPs increases with the decrease of influent flow rate. The sludge volume index (SVI) of the resulting enriched activated sludge meets the design value required by the convectional activated sludge process for the treatment of wastewater.     

Biodegradation of methyl t-butyl ether by pure bacterial cultures

Three pure bacterial cultures degrading methyl t-butyl ether (MTBE) were isolated from activated sludge and fruit of the Gingko tree. They have been classified as belonging to the genuses Methylobacterium, Rhodococcus, and Arthrobacter. These cultures degraded 60 ppm MTBE in 1–2 weeks of incubation at 23–25 °C. The growth of the isolates on MTBE as sole carbon source is very slow compared with growth on nutrient-rich medium. Uniformly-labeled [¹⁴C]MTBE was used to determine ¹⁴CO₂ evolution. Within 7 days of incubation, about 8% of the initial radioactivity was evolved as ¹⁴CO₂. These strains also grow on t-butanol, butyl formate, isopropanol, acetone and pyruvate as carbon sources. The presence of these compounds in combination with MTBE decreased the degradation of MTBE. The cultures pregrown on pyruvate resulted in a reduction in ¹⁴CO₂ evolution from [¹⁴C]MTBE. The availability of pure cultures will allow the determination of the pathway intermediates and the rate-limiting steps in the degradation of MTBE. Received: 8 December 1995 / Received last revision: 5 August 1996 / Accepted: 12 August 1996     

Biodegradation by activated sludge and toxicity of tetracycline into a semi-industrial membrane bioreactor

Much attention has been devoted recently to the fate of pharmaceutically active compounds such as tetracycline antibiotics in soil and water. Tetracycline (TC) biodegradability by activated sludge derived from membrane bioreactor (MBR) treating swine wastewater via CO₂-evolution was evaluated by means of modified Sturm test, which was also used to evaluate its toxicity on carbon degradation. The impact of tetracycline on a semi-industrial MBR process was also examined and confronted to lab-scale experiments. After tetracycline injection in the pilot, no disturbance was detected on the elimination of organic matters and ammonium (nitrification), reaching after injection 88% and 99% respectively; only denitrification was slightly affected. Confirming the ruggedness and the superiority of membrane bioreactors over conventional bioreactors, no toxicity was observed at the considered level of TC in the pilot (20 mg TOC L⁻¹), while at lab-scale sodium benzoate biodegradation was completely inhibited from 10 mg TOC L⁻¹ TC. The origin of the activated sludge showed a significant impact on the performances, since the ultimate biodegradation was in the range −50% to −53% for TC concentrations in the range 10–20 mg TOC L⁻¹ with conventional bioreactor sludge and increased to 18% for 40 mg TOC L⁻¹ of TC with activated sludge derived from the MBR pilot. This confirmed the higher resistance of activated sludge arising from membrane bioreactor.     

Biodegradation of ammonium sulphite spent liquor by pure bacterial cultures

Summary Several bacterial strains able to grow on ammonium sulphite spent liquor (ASSL) were isolated by an enrichment culture technique and identified. The capacities of these bacteria to degrade ASSL in pure culture was compared with the modification of the methoxyl groups of the lignosulphonates. A rapid demethylation followed by remethylation, observed in some species, showed a complex biodegradation mechanism.Attempts to correlate the ability to degrade the substrate studied with that of the micro-organism selected to grow upon aromatic carbon sources were made. The isolate might have numerous activities regarding ASSL.     

Comparison of bio-augmentation and composting for remediation of oily sludge: A field-scale study in China

Two bioremediation technologies were performed in order to explore a better treatment process for an oily sludge restoration in China during 2004. The bioremediation by augmentation of biopreparation was compared with a conventional composting. The oily sludge and oil-polluted soil were received from an oil production plant. The total hydrocarbon content (THC) varied from 327.7 to 371.2 g kg⁻¹ of dry sludge and the THC in contaminated soil was 151.0 g kg⁻¹. Before application of preparation, straw, sawdust, top sand and pure soil were added in different proportions to the sludge and soil and mixed thoroughly. Such sludge and soil composites were used for negative controls and for activation of indigenous oil degrading microorganisms with addition of fertilizer (positive controls). For composting, crude manure and straw were added to the oily sludge and the THC was 101.4 g kg⁻¹. The biopreparation was applied every 2 weeks and experiment lasted 56 days under the ambient temperature. The sludge was mixed and watered every 3 days. After three times of biopreparation application, the THC decreased by 46–53% in the oily sludge and soil, while in the positive controls (activation of indigenous microorganisms) the THC decreased by 13–23%, and there was no oil degradation in negative controls After composting, the THC decreased by 31% in the oily sludge. The planting of Tall Fescue (Festuca arundinace) revealed a decrease of sludge toxicity after application of both bioremediation technologies and additionally decreased the THC by 5–7%.     

The degradation of biphenyl and chlorobiphenyls by mixed bacterial cultures

Abstract Pseudomonas sp. HV3 grows on naphthalene but not on biphenyl, as the sole source of carbon. When the cells of Pseudomonas sp. HV3 grown on naphthalene were shaken with biphenyl as the carbon source in a mineral salt solution, a yellow metabolite identified as the meta -cleavage product of biphenyl was excreted. The degradation of biphenyl stopped here, but was completed if either 2-methyl-4-chlorophenoxy acetic acid (MCPA)-degrading mixed culture or a Nocardia strain was added to the growth solution. Neither of these uses naphthalene or biphenyl as growth substrate. The mixed culture of Pseudomonas sp. HV3 and Nocardia sp. also degrades the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1221. A yellow metabolite was likewise produced in the degradation, and sometimes two different peaks of the yellow metabolite were observed. The gas chromatography-mass spectrometry (GC-MS) analyses showed that 40–87% of Aroclor 1221 was degraded during an incubation time of 6–21 days. Chlorobenzoic acids were found as metabolites.     

Biodegradation of chlorophenols by mixed and pure cultures from a fluidized-bed reactor

An aerobic, continuous-flow fluidized-bed reactor was established with inoculum from activated sludge, and fed a mixture of 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP) and pentachlorophenol (PCP) as the sole sources of carbon and energy for 2 years. Experiments with the enrichment were performed with material from the reactor. Later, degradation experiments were completed using pure cultures of bacteria that were isolated from suspended samples of the carrier biofilm. In batch-bottle bioassays, the reactor enrichment degraded PCP, TeCP and TCP both in mineral salts (MS) and tryptone-yeast extract-glucose (TGY) media. ortho-Methoxylated chlorophenols including 4,5-dichloroguaiacol (4,5-DCG), tetrachloroguaiacol (TeCG) and trichlorosyringol (TCS) resisted biodegradation by the enrichment both in MS and TGY media, whereas 5,6-dichlorovanillin (5,6-DCV) was readily transformed to an unidentified metabolite. Experiments with ¹⁴C labeled chlorophenols showed mineralization of 2,4-dichlorophenol (DCP) and 2,3,5-TCP to ¹⁴CO₂ by the enrichment. Material from the suspended biofilm after continuous chlorophenol feeding for 2 years was inoculated onto TGY-agar plates, and showed predominantly two colony, types accounting for over 99% of the total colony counts. The two colony types, were equal in abundance. Six Gram-negative, oxidase- and catalase-positive, non-fermentative small rods were isolated in TGY agar media supplemented with 10 mg/l of TeCP or PCP. All isolates formed colonies in TGY plus 150 mg/l of PCP. The isolates degraded TCP and TeCP but not PCP. In mixtures of isolated bacteria the rates of chlorophenol degradation were similar to those observed with individual isolates. Three isolates were identified as Pseudomonas saccharophila and three were an unidentified species of Pseudomonas.     

Biodegradation of Phenanthrene by the Green Alga Scenedesmus obliquus ES‐55

While the degradation of polycyclic aromatic hydrocarbons by bacteria and fungi has been broadly investigated, less is known about the metabolism of these compounds by algae. The goal of the experiments was to test the degradability of phenanthrene by the green alga Scenedesmus obliquus ES‐55 (Chlorophyceae) and to identify the metabolites. It was shown that S. obliquus ES‐55 metabolized phenanthrene. Under light conditions, phenanthrene (14 mg/L) inhibits cell division by more than twice. However, the metabolic processes in the cells affected by phenanthrene continued because the content of chlorophyll increased. In the exponential phase under phototrophic conditions the alga degraded phenanthrene. Phenanthrene was removed by algae up to 42 % in BBM medium and up to 24 % in Kuhl medium. Dihydroxy‐dihydro‐phenanthrene, a degradation metabolite in fungi, bacteria and cyanobacteria, could also be detected as a transformation product of S. obliquus ES‐55. Further detected common metabolites foster the assumption that both phototrophic and non‐photothrophic organisms metabolize phenanthrene via a similar pathway. The present study is the first evidence of the ability of an axenic culture of the green alga S. obliquus to biotransform phenanthrene into other metabolites.     

Anaerobic digestion of cellulose by pure and mixed bacterial cultures

Summary By enrichment technique, nine anaerobic mixed bacterial cultures were isolated, five of which showed stable cellulolysis. All cultures fermented cellulose and produced different fermentative products. Mixed culture BOC 25 yielded major levels of acetate and ethanol (39.6 and 12.0 mmol/l, respectively) and minor levels of propionate (2.5 mmol/l) and digested filter paper cellulose to the extent of 32.5% w/v. BOC 25 digested cellulosic and lignocellulosic substrates and produced filter paper cellulase, carboxymethyl cellulase, Avicelase and -glucosidase. Strain DC 25, a cellulolyticClostridium was purified from one of the mixed cultures. The fermentation products of DC 25 from cellulose, cellobiose or glucose were ethanol, acetate, formate, H₂ and CO₂.     

Inhibition of bacterial alpha-glucosidases by castanospermine in pure cultures and activated sludge

Castanospermine (CAST) is a known and potent inhibitor of various alpha-glucosidases in eukaryotes. In this work, we elucidated whether CAST could also be used for determining bacterial alpha-glucosidase activity, when measured with 4-methylumbelliferyl-alpha- D-glucoside as a substrate, both in a complex bacterial community, in activated sludge and in pure cultures of bacterial isolates. We found that 140 microM CAST inhibited alpha-glucosidase activity by 30% in a pure culture of Pseudomonas stutzeri. The alpha-glucosidase activity in Chryseobacterium gleum was inhibited by 90% at a concentration of 150 microM CAST, whereas the alpha-glucosidase in Paracoccus denitrificans was resistant to the inhibitor. CAST (140 microM) reduced alpha-glucosidase activity in activated sludge by 40%, the respiration rate being reduced by only 12%. No significant inhibition of the respiration rate was observed in Ps. stutzeri or Pa. denitrificans, whereas the respiration rate in C. gleum grown in a medium containing starch was inhibited by 50% with 140 microM CAST. No effect of CAST was observed in C. gleum grown in a complex medium. This indicated that CAST, at the concentration used, did not cause a general negative effect on bacterial activity. The results suggest that the CAST assay may potentially be useful in determining whether alpha-glucosidase activity, starch, poly- and disaccharides contribute appreciably to the overall activity of a bacterial community. However, the assay should not be used for quantitative measurements of such activity.     

Biodegradation of wastewater pollutants by activated sludge encapsulated inside calcium-alginate beads in a tubular packed bed reactor

The wastewater treatment plants produce large quantities of biomass (sludge) that require about one-third of the total inversion and operation plant costs for their treatment. By the microorganisms immobilization it is possible to handle high cell concentration in the reactor, increasing its efficiency, reducing the loss of biomass and the wash out is avoided. Moreover, there is no cell growth then the sludge production is reduced. In this study, the COD removal and VSS variation were modeled in a tubular reactor with activated sludge immobilized in Ca-alginate. Moreover, two aspects that are commonly not considered in the performance of the actual reactors of this kind were introduced; the performance in non-steady state and the dispersion effect. The model was calibrated with an actual wastewater taken out from a Mexican wastewater treatment plant. The results of the performance of the tubular bioreactor at different scenarios (i.e., different residence time and VSS in the reactor) are presented. With longer residence times and higher VSS concentration in the Ca-alginate beads in the tubular bioreactor it is possible to increase the time operation of the bioreactor and to treat higher volumes of wastewater. During the process, the sludge generation was drastically reduced and it is possible to remove nitrogen form the wastewater making this process more attractive.