Surfactant aided biodegradation of pyrene using immobilized cells of Mycobacterium frederiksbergense

Low aqueous phase solubility is the major limiting factor in successful biodegradation of pyrene and other polycyclic aromatic hydrocarbons (PAH), which can, however, be overcome by using a suitable surfactant. Biodegradation of pyrene by immobilized cells of Mycobacterium frederiksbergense in presence of non-ionic surfactant Tween 80 was evaluated. For cell immobilization, beads were prepared using calcium alginate as the immobilizing material based on immobilized cell viability and mechanical stability of the beads. Complete degradation of pyrene was achieved employing the immobilized cells in batch shake flask experiments for all four different initial concentrations of the PAH at 100 mg l⁻¹, 200 mg l⁻¹, 400 mg l⁻¹ and 1000 mg l⁻¹. The experimental results of biodegradation of pyrene at very high initial concentration of 1000 mg l⁻¹ using the cell immobilized beads was further investigated in a 3 l fermentor operated at controlled conditions of 150 rpm, 28 °C, pH 7 and 1.5 l min⁻¹ aeration. The results confirmed complete degradation of the PAH with a very higher degradation rate of 250 mg l⁻¹ d⁻¹, which is so far the highest value reported for pyrene biodegradation.     

2-Mercaptobenzothiazole degradation in laboratory fed-batch systems

Rubber additives manufacture yields waste-waters with recalcitrant and/or toxic benzothiazole compounds. Biodegradation of such compounds was investigated in fed-batch systems. 2-Mercaptobenzothiazole (MBT) was best degraded by a mixture of MBT-history and non-MBT-history sludge. Concentrations up to 200 mg·l^–1 were removed. From 100 mg·l^–1 onwards, high percentages of the recalcitrant disulphide were accumulated in the sludge. MBT slowed down the biodegradation of benzothiazole-2-sulphonate. MBT and benzothiazole did not mutually influence their degradation. Under some experimental conditions high levels of unidentified so-called polar compounds were formed.     

Microcosm assays and Taguchi experimental design for treatment of oil sludge containing high concentration of hydrocarbons

Microcosm assays and Taguchi experimental design was used to assess the biodegradation of an oil sludge produced by a gas processing unit. The study showed that the biodegradation of the sludge sample is feasible despite the high level of pollutants and complexity involved in the sludge. The physicochemical and microbiological characterization of the sludge revealed a high concentration of hydrocarbons (334,766 ± 7001 mg kg⁻¹ dry matter, d.m.) containing a variety of compounds between 6 and 73 carbon atoms in their structure, whereas the concentration of Fe was 60,000 mg kg⁻¹ d.m. and 26,800 mg kg⁻¹ d.m. of sulfide. A Taguchi L₉ experimental design comprising 4 variables and 3 levels moisture, nitrogen source, surfactant concentration and oxidant agent was performed, proving that moisture and nitrogen source are the major variables that affect CO₂ production and total petroleum hydrocarbons (TPH) degradation. The best experimental treatment yielded a TPH removal of 56,092 mg kg⁻¹ d.m. The treatment was carried out under the following conditions: 70% moisture, no oxidant agent, 0.5% of surfactant and NH₄Cl as nitrogen source.     

Influence of environmental factors on reductive bioprecipitation of uranium by sulfate reducing bacteria

Microbial reduction of soluble uranyl [U (VI)] to insoluble uraninite by sulfate reducing bacteria (SRB) is a promising remediation strategy for uranium-contaminated groundwater. Effects of environmental factors, including pH and coexisting ions, on U (VI) bioreduction processes (UBP) remain unknown. Anaerobic batch experiments were performed to evaluate impact on UBP. Kinetic investigations with varied pH demonstrated that U (VI) was reduced mostly within 48 h. The bioprecipitation yields depended strongly on pH, increasing from 12.9% to 99.4% at pH 2.0 and 6.0, respectively. Sulfate concentration 4000 mg l⁻¹ did not affect UBP; however, sulfate concentration 5000 mg l⁻¹ significantly slowed UBP. Biogenic H₂S produced during sulfate reduction was not directly involved in UBP. At 20 mg l⁻¹ Zn or 10 mg l⁻¹ Cu, no UBP inhibition was observed and uraninite was detected in metal sulfide precipitate. However, 25 mg l⁻¹ Zn or 15 mg l⁻¹ Cu stopped UBP completely. Cu toxicity mechanism probably differed from Zn. The ability to reduce U (VI) was lost permanently with exposure to 15 mg l⁻¹ Cu, but not for Zn 25 mg l⁻¹. No uraninite could be detected before nitrate removal, suggesting nitrate strongly inhibited UBP, which may possibly be related to denitrification intermediates controlling the solution redox potential.     

Interaction Among Copper Toxicity,Temperature and Salinity on the Population Dynamics of <Emphasis Type="Italic">Brachionus Rotundiformis</Emphasis> (Rotifera)

Heavy metals may interact with ecological factors such as temperature, food level and salinity, causing both mortality and reduced reproduction in organisms. Among different heavy metals, copper compounds are commonly used for eliminating algal blooms in aquaculture tanks. At certain concentrations, copper is toxic to rotifers. In the present work, we evaluated the combined effects of salt concentrations (2.5 and 5.0 g l⁻¹ NaCl), copper levels (0, 0.03125, 0.0625, 0.125 and 0.25 mg l⁻¹ as CuCl₂) and two temperatures (20 and 25 °C) on the population growth of B. rotundiformis using Chlorella as the algal food (at 0.5 × 10⁶ cells ml⁻¹ for every 24 h). Regardless of salinity and temperature, copper at concentrations as low as 0.03 mg l⁻¹ had an adverse effect on the population growth of rotifers and above 0.125 mg l⁻¹, the populations did not grow. The effect of the toxicant on B. rotundiformis was more severe at 25° than at 20 °C at lower salinity. In general, we observed peak densities of rotifers around day 12 at 20 °C but 6–8 days earlier at 25 °C. Peak population densities of B. rotundiformis in the controls at the salinity of 2.5 g l⁻¹ ranged from 90 to 180 ind. ml⁻¹, depending on temperature; at a salinity of 5.0 g l⁻¹, these were lower. The population growth rates, r, in our study varied from +0.31 to –0.12 depending on the test conditions. There was a significant impact of temperature, salinity and toxicity level on the population growth rate of B. rotundiformis. Our results suggested that even narrow changes in salinity could negatively influence the toxicity of heavy metal on the population growth rates of B. rotundiformis.     

Adhesion to the hydrocarbon phase increases phenanthrene degradation by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> LP6a

Microbial adhesion is an important factor that can influence biodegradation of poorly water soluble hydrocarbons such as phenanthrene. This study examined how adhesion to an oil–water interface, as mediated by 1-dodecanol, enhanced phenanthrene biodegradation by Pseudomonas fluorescens LP6a. Phenanthrene was dissolved in heptamethylnonane and added to the aerobic aqueous growth medium to form a two phase mixture. 1-Dodecanol was non-toxic and furthermore could be biodegraded slowly by this strain. The alcohol promoted adhesion of the bacterial cells to the oil–water interface without significantly changing the interfacial or surface tension. Introducing 1-dodecanol at concentrations from 217 to 4,100 mg l⁻¹ increased phenanthrene biodegradation by about 30% after 120 h incubation. After 100 h incubation, cultures initially containing 120 or 160 mg l⁻¹ 1-dodecanol had mineralized >10% of the phenanthrene whereas those incubated without 1-dodecanol had mineralized only 4.5%. The production and accumulation of putative phenanthrene metabolites in the aqueous phase of cultures likewise increased in response to the addition of 1-dodecanol. The results suggest that enhanced adhesion of bacterial cells to the oil–water interface was the main factor responsible for enhanced biodegradation of phenanthrene to presumed polar metabolites and to CO₂.     

Significant enhancement of oleanolic acid accumulation by biotic elicitors in cell suspension cultures of Calendula officinalis L.

The effects of elicitors on cell growth and oleanolic acid (OA) accumulation in shaken cell suspension cultures of Calendula officinalis were investigated. Elicitors were added individually at various concentrations to 5-day-old cell cultures and their effects monitored at 24 h intervals for 4 days. Different effects on OA accumulation were observed depending on the day of treatment. Jasmonic acid was the most efficient elicitor. After 72 h of treatment with 100 μM JA, the intracellular content of OA reached its maximum value (0.84 mg g⁻¹ DW), which was 9.4-fold greater than that recorded in an untreated control cultures. The addition of chitosan at 50 mg l⁻¹ produced a 5-fold enhancement of OA accumulation (0.37 mg g⁻¹ DW) after 48 h of treatment. Treatment with yeast extract at 200 mg l⁻¹ for 96 h or with pectin at 2 mg l⁻¹ for 48 h produced identical cellular levels of OA (0.22 mg g⁻¹ DW). Lastly, 48 h elicitation with homogenate of the fungus Trichoderma viride produced a 1.8-fold increase in oleanolic acid content (0.12 mg g⁻¹ DW). In addition to significantly stimulating OA accumulation and its secretion into the culture medium, the elicitors also caused slight inhibition of cell growth.     

Biodegradation kinetics of 1,4-benzoquinone in batch and continuous systems

Combining chemical and biological treatments is a potentially economic approach to remove high concentration of recalcitrant compounds from wastewaters. In the present study, the biodegradation of 1,4-benzoquinone, an intermediate compound formed during phenol oxidation by chlorine dioxide, was investigated using Pseudomonas putida (ATCC 17484) in batch and continuous bioreactors. Batch experiments were conducted to determine the effects of 1,4-benzoquinone concentration and temperature on the microbial activity and biodegradation kinetics. Using the generated data, the maximum specific growth rate and biodegradation rate were determined as 0.94 h⁻¹ and 6.71 mg of 1,4-benzoquinone l⁻¹ h⁻¹. Biodegradation in a continuous bioreactor indicated a linear relationship between substrate loading and biodegradation rates prior to wash out of the cells, with a maximum biodegradation rate of 246 mg l⁻¹ h⁻¹ observed at a loading rate of 275 mg l⁻¹ h⁻¹ (residence time: 1.82 h). Biokinetic parameters were also determined using the steady state substrate and biomass concentrations at various dilution rates and compared to those obtained in batch cultures.     

Treatment of an artificial sulphide containing wastewater in subsurface horizontal flow laboratory-scale constructed wetlands

In general, treatment wetlands seem to be a potential method of tackling the sulphide problem of post-treatment of anaerobic digester effluents.Because of insufficient practical experience and lack of knowledge of sulphide removal, sulphur transformation was investigated, particularly in horizontal subsurface flow constructed wetlands (depth of 35 cm) under laboratory-scale conditions with artificial wastewater.The plants affected a clear stimulation of the sulphide and ammonia removal rates. Sulphide concentration in the range of 1.5–2.0 mg l⁻¹ was tolerated by the plants and completely removed in the planted model wetlands; sulphide concentration of >2.0 mg l⁻¹ caused instabilities in sulphide and nitrogen removal. Area-specific sulphide removal rates of up to 94 mg sulphide m⁻² d⁻¹ were achieved in the planted beds at hydraulic retention times of 2.5 d. Sulphate affected the sulphide removal. While in the unplanted control bed an almost stable removal in the range of 150–300 mg N m⁻² d⁻¹ was observed variations of hydraulic retention time, sulphide and sulphate concentrations influenced the ammonia removal rate within the planted beds in a broader range (600–1400 mg N m⁻² d⁻¹).These results showed that nitrification, sulphide oxidation, denitrification and sulphate reduction can occur simultaneously in the rhizosphere of treatment wetlands caused by dynamic redox gradients (aerobic–anaerobic) conditions.     

Replicative and integrative plasmids for production of human interferon gamma in Bacillus subtilis

Integrative and replicative plasmids for the expression driven by the P₄₃ promoter and secretion of recombinant proteins in Bacillus subtilis were constructed. The plasmids named pInt and pRep respectively were tested for the production of recombinant human interferon gamma (rhIFN-γ). A synthetic hIFN-γ gene employing the optimized B. subtilis codon usage was fused with the Bacillus licheniformis α-amylase signal peptide (sp-amyL) encoding sequence. The integrative construct produced 2.5 ± 0.2 mg l⁻¹ and the replicative system produced 20.3 ± 0.8 mg l⁻¹ of total recombinant rhIFN-γ. The results showed that secretion of hIFN-γ was the bottleneck for the overexpression of mature rhIFN-γ by B. subtilis.     

Biodegradation kinetics of trans-4-methyl-1-cyclohexane carboxylic acid

Naphthenic acids are a complex mixture of organic compounds which naturally occur in crude oil. Low molecular weight components of the naphthenic acids are known to be toxic in aquatic environments and there is a need to better understand the factors controlling the kinetics of their biodegradation. In this study, a relatively low molecular weight naphthenic acid compound (trans-isomer of 4-methyl-1-cyclohexane carboxylic acid) and a microbial culture developed in our laboratory were used to study the biodegradation of this naphthenic acid and to evaluate the kinetics of the process in batch cultures. The initial concentration of trans-4-methyl-1-cyclohexane carboxylic acid (50–750 mg l⁻¹) did not affect the maximum specific growth rate of the bacteria at 23°C (0.52 day⁻¹) to the maximum biodegradable concentration (750 mg l⁻¹). The maximum yield observed at this temperature and at a neutral pH was 0.21 mg of biomass per milligram of substrate. Batch experiments indicated that biodegradation can be achieved at low temperatures; however, the biodegradation rate at room temperature (23°C) and neutral pH was 5 times faster than that observed at 4°C. Biodegradation at various pH conditions indicated a maximum specific growth rate of 1.69 day⁻¹ and yield (0.41 mg mg⁻¹) at a pH of 10.     

Linoleic and α-linolenic fatty acids affect biomass and secondary metabolite production and nutritive properties of Panax ginseng adventitious roots cultured in bioreactors

The effect of elicitation with linoleic (C18:2) and α-linolenic (C18:3) fatty acids (LLA and α-LNA) was investigated in Panax ginseng C.A. Meyer adventitious roots cultured in 5 l balloon-type bioreactors. Fatty acids were added in culture medium at 0.0, 1.0, 2.5, 5.0, 10.0, and 20.0 μmol l⁻¹ at day 40, at the end of exponential growth phase. Roots were harvested and assayed at day 47. Elicitation with both LLA and α-LNA enhanced accumulation of total polyphenolics and flavonoids in roots compared with control without elicitation. The highest accumulation of flavonoids was observed at 5.0 μmol l⁻¹ for both elicitors. Total phenolics production reached its highest value of about 4.0 mg g⁻¹ DW under the elicitation with 5.0 μmol l⁻¹ LLA and 5.0–20.0 μmol l⁻¹ α-LNA. Meanwhile, α-LNA was more effective than LLA for increasing biomass and ginsenoside production. The biomass of 11.1 g DW l⁻¹ and maximal total ginsenoside content of 7.9 mg g⁻¹ DW were achieved at 5 μmol l⁻¹ α-linolenic acid. The essential polyunsaturated linoleic (C18:2) and α-linolenic (C18:3) fatty acids were accumulated in roots in response to elicitation while content of palmitic (C16:0) and oleic (C18:1) acids declined. The activities of SOD, G-POD and CAT were enhanced by two elicitors to similar extent while APX activity was preferably stimulated by α-LNA. Our results demonstrate that elicitation with α-linolenic acid stimulates production of biomass and secondary metabolites in bioreactor-cultured P. ginseng adventitious roots.     

Effect of keratinous waste addition on improvement of crude oil hydrocarbon removal by a hydrocarbon-degrading and keratinolytic mixed culture

The aim of this work was to evaluate the effect of keratinous waste addition on oil-hydrocarbon removal, through a mixed culture of oil-degrading bacteria, with the ability to secrete keratinases. The mixed culture was grown in the media with oil, or oil supplemented with chicken-feathers as the keratinous waste. Residual oil-hydrocarbons were determined as total petroleum hydrocarbons (TPHs) and oil fractions and then quantified by GC–FID and GC–MS.Results showed that in presence of the keratinous waste, the removal of oil-hydrocarbons was 57,400 mg l^?1, meanwhile the treatment without waste presented an oil-hydrocarbons removal of 35,600 mg l^?1. The aliphatic fraction was the most removed in both treatments. In addition, chromatographic profiles indicated that the aliphatic fraction showed different degradation pattern; in the presence of keratinous wastes, the C₁₈ to C₂₈ compounds were preferably removed over the C₁₀ to C₁₇. The addition of keratinous waste not only improved the oil-hydrocarbons removal but, it changed the removal pattern of the target hydrocarbons.     

Assessment of the biodegradability of a monosulfonated azo dye and aromatic amines

Biodecolourisation of an azo dye by anaerobic cultures using a liposomal textile levelling agent as primary substrate was assessed. Liposomes seem to facilitate the uptake of the dye (Acid Orange 7) by anaerobic biomass, leading to a fast decolourisation (colour removal of 96% was achieved in the first sample port of the reactor profiles). On the other hand, the presence of dye (60–300 mg l⁻¹) caused a decrease in the chemical oxygen demand (COD) degradation rate (4.1–2.5 g COD removed l⁻¹ d⁻¹ for 60 and 300 mg l⁻¹ of dye, respectively), suggesting inhibitory effects.Aerobic degradation of aromatic amines was investigated in aerobic respirometric assays with different types of inocula. Sulfanilic acid and aniline were mineralised by inocula with a significant microbiological diversity, even with domestic effluent. These results were confirmed by a significant reduction of COD, total organic carbon (TOC) and a high oxygen consumption (biochemical oxygen demand/theoretical oxygen demand), 92±4%. Kinetic analysis showed that a sigmoid function describes quite well the experimental data, even better than the exponential model. Orthanilic and metanilic acids and 1-amino-2-naphtol were persistent under the tested conditions.     

Effects of carbon concentration and carbon to nitrogen ratio on the growth and sporulation of several biocontrol fungi

Effects of carbon concentration and carbon to nitrogen (C:N) ratio on six biocontrol fungal strains are reported in this paper. All fungal strains had extensive growth on the media supplemented with 6–12 g l⁻¹ carbon and C:N ratios from 10:1 to 80:1, and differed in nutrient requirements for sporulation. Except for the two strains of Paecilomyces lilacinus, all selected fungi attained the highest spore yields at a C:N ratio of 160:1 when the carbon concentration was 12 g l⁻¹ for Metarhizium anisopliae SQZ-1-21, 6 g l⁻¹ for M. anisopliae RS-4-1 and Trichoderma viride TV-1, and 8 g l⁻¹ for Lecanicillium lecanii CA-1-G. The optimal conditions for P. lilacinus sporulation were 8 g l⁻¹ carbon with a C:N ratio of 10:1 for M-14 and 12 g l⁻¹ carbon with a C:N ratio of 20:1 for IPC-P, respectively. The results indicated that the influence of carbon concentration and C:N ratio on fungal growth and sporulation is strain dependent; therefore, consideration for the complexity of nutrient requirements is essential for improving yields of fungal biocontrol agents.     

Sorption of Co, Cu, Ni and Zn from industrial effluents by the submerged aquatic macrophyte Myriophyllum spicatum L.

The submerged aquatic plant Myriophyllum spicatum L. (Eurasian water milfoil) has been suggested as an efficient plant species for the treatment of metal-contaminated industrial wastewater. The process of metal removal by plants involves a combination of rapid sorption on the surface and slow accumulation and translocation in the biomass. This study focussed on the sorption/desorption characteristics of the surface of M. spicatum for Co, Cu, Ni and Zn. Batch sorption tests with mixed metal solutions covering a range of 0, 1, 5, 10, 50 and 100 mg l⁻¹ of each metal, were performed. For Co, Ni and Zn, the sorption process was well described by the Langmuir model, whereas sorption of Cu was better described by the Freundlich model. The biomass showed the highest affinity for Cu and Zn. Langmuir sorption maxima of Co, Ni and Zn were 2.3, 3.0 and 6.8 mg g⁻¹ DM, respectively. At the highest initial concentration of 100 mg l⁻¹, a maximum of 29 mg g⁻¹ DM of Cu was sorbed onto the surface of the biomass. Desorption by 0.1 M HCl did not fully recover the metals sorbed onto the surface and there was evidence of leaching from within the biomass. Recovery of heavy metals and regeneration of the biomass by washing with 0.1 M HCl was therefore not suggested as a viable strategy.     

High-performance liquid chromatographic method for determination of DDT and its degradation products in rat plasma, liver and brain: validation and application to a pharmacokinetic study

A sensitive and reliable high-performance liquid chromatographic (HPLC) method, using a solid-phase extraction (SPE), was established and validated for determination of p,p′-DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] and its metabolite p,p′-DDE [1,1-(2,2-dichloroethanylidene)-bis(4-chlorobenzene)] in rat plasma, liver and brain. After being diluted with water, plasma, liver and brain samples were applied to a solid-phase extraction C₁₈ cartridge. The extraction containing p,p′-DDT and p,p′-DDE from the cartridge were cleaned-up using a Florisil Sep-Pak cartridge. The samples were analyzed by HPLC using UV detection at 238 nm. The limit of detection for p,p′-DDT and p,p′-DDE was 0.1 mg kg⁻¹ liver or brain and 0.1 mg l⁻¹ plasma. For six replicate samples at 40, 4 and 0.2 mg kg⁻¹, intra-day precision values were within 4.9% for plasma, 6.4% for liver, and 9.7% for brain. Inter-day precision values at 4 mg kg⁻¹ were within 8.2% for plasma and tissues. The method performances were shown to be selective for p,p′-DDT and p,p′-DDE, and linear over the range 0.04–12 mg kg⁻¹ (mg l⁻¹ for plasma). The absolute recoveries of p,p′-DDT and p,p′-DDE in rat plasma and tissues were over 92%. The method was proved to be applicable to the pharmacokinetic study of DDT in rats after a single oral administration.     

Aerobic degradation of 3-chlorobenzoic acid by an indigenous strain isolated from a polluted river

An indigenous strain of Pseudomonas putida capable of degrading 3-chlorobenzoic acid as the sole carbon source was isolated from the Riachuelo, a polluted river in Buenos Aires. Aerobic biodegradation assays were performed using a 2-l microfermentor. Biodegradation was evaluated by spectrophotometry, chloride release, gas chromatography and microbial growth. Detoxification was evaluated by using Vibrio fischeri, Pseudokirchneriella subcapitata and Lactuca sativa as test organisms. The indigenous bacterial strain degrades 100 mg l⁻¹ 3-chlorobenzoic acid in 14 h with a removal efficiency of 92.0 and 86.1% expressed as compound and chemical oxygen demand removal, respectively. The strain was capable of degrading up to 1,000 mg of the compound l⁻¹. Toxicity was not detected at the end of the biodegradation process. Besides initial concentration, the effect of different factors, such as initial pH, initial inoculum, adaptation to the compound and presence of other substrates and toxic related compounds, was studied.     

Enzymatic pre-hydrolysis applied to the anaerobic treatment of effluents from poultry slaughterhouses

A pool of hydrolases with 21.4 U g⁻¹ lipase activity was produced through solid-state fermentation of the fungus Penicillium restrictum in waste from the Orbignya oleifera (babassu) oil processing industry. Enzymatic hydrolysis and anaerobic biodegradability tests were conducted on poultry slaughterhouse effluents with varying oil and grease contents (150–1200 mg l⁻¹) and solid enzymatic pool concentrations (0.1–1.0% w/v). Enhanced anaerobic treatment efficiency relative to raw effluent was achieved when a 0.1% concentration of enzymatic pool was used in the pre-hydrolysis stage with 1200 mg oil and grease l⁻¹ (chemical oxygen demand (COD) removal efficiency of 85% vs. 53% and biogas production of 175 ml vs. 37 ml after 4 d).     

Concentration dependent growth/non-growth linked kinetics of endosulfan biodegradation by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

The rates of biodegradation of endosulfan by P. aeruginosa were determined with different initial endosulfan concentrations (10, 50, 100, 150, 200 and 250 mg l⁻¹) and different growth linked kinetic models were fitted at these concentrations. At 10 mg endosulfan l⁻¹, Monod no growth model was well fitted. Monod with growth model described the biodegradation pattern at an initial concentration of 50, 100 and 150 mg endosulfan l⁻¹. Significant increases of P. aeruginosa MN2B14 density in broth culture during incubation further support this result. Conversely, zero order kinetic model was well fitted into the biodegradation data if initial endosulfan concentration was ≥200 mg endosulfan l⁻¹. The kinetics of endosulfan biodegradation by P. aeruginosa MN2B14 in liquid broth was highly dependent upon its initial concentration. The results of this study could be employed for predicting the persistence of endosulfan in water environment containing P. aeruginosa as an endosulfan degrading bacterium.