Degradation of polychlorinated biphenyls (PCBs) by four bacterial isolates obtained from the PCB-contaminated soil and PCB-contaminated sediment

We investigated the PCB-degrading abilities of four bacterial strains isolated from long-term PCB-contaminated soil (Alcaligenes xylosoxidans and Pseudomonas stutzeri) and sediments (Ochrobactrum anthropi and Pseudomonas veronii) that were co-metabolically grown on glucose plus biphenyl which is an inducer of the PCB catabolic pathway. The aim of study was to determine the respective contribution of biomass increase and expression of degrading enzymes on the PCB degrading abilities of each isolate. Growth on 5 g l⁻¹ glucose alone resulted in the highest stimulation of the growth of bacterial strains, whereas grown on 10 mg l⁻¹, 100 mg l⁻¹, 1 g l⁻¹, or 5 g l⁻¹ biphenyl did not effected the bacterial growth. None of the strains used in this study was able to grow on PCBs as the sole carbon source. Cells grown on glucose exhibited enhanced degradation ability due to an increased biomass. Addition of biphenyl at concentrations of 1 or 5 g l⁻¹ did not increase total PCB degradation, but stimulated the degradation of highly chlorinated congeners for some of the strains. The degradation of di- and tri-chlorobiphenyls was significantly lower for cells grown on 5 g l⁻¹ biphenyl independently on glucose addition. The highest degradation of the PCBs was obtained for A. xylosoxidans grown in the presence of glucose. Thus A. xylosoxidans appears to be the most promising among the four bacterial isolates for the purpose of bioremediation.     

Application of soil slurry respirometry to optimise and subsequently monitor ex situ bioremediation of hydrocarbon-contaminated soils

A protocol to monitor respiration as O₂ consumption in soil slurries using the Strathtox^® respirometer was developed and tested on four soils from brownfield sites. Respiration rates (mg l⁻¹ h⁻¹) of soil slurries in the Strathtox^® were compared with rates (μl min⁻¹) of field moist soils analysed using the Columbus Oxymax^® ER10 respirometer. One of the soils (99612B), historically contaminated with diesel, was further studied by monitoring the effect of inorganic NH₄NO₃ liquid nutrient on enhancing respiration rate. Soil microcosms were monitored continuously on the Oxymax^® or sampled at 24, 48 and 72 h intervals, prepared as soil slurries, and analysed on the Strathtox^®. On the full-scale remediation project (∼6000 m³) soil 99612B was treated as a biopile with spent mushroom compost (SMC) amendment and respiration rates monitored in samples over an 8-week period. In the laboratory microcosm experiment and full-scale bioremediation treatment described, correlation was found for respiration rates between the two respirometry systems.     

Bioremediation of metal contamination in the Plankenburg River,Western Cape,South Africa

Three bioreactors (two laboratory-scale and one on-site) were evaluated for their efficiency to reduce metal concentrations in water collected from the Plankenburg River, South Africa. Water (bioreactors one, two and on-site) and bioballs (bioreactors two and on-site) collected throughout the study periods were digested and analysed using Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES). Aluminium (Al), nickel (Ni), and zinc (Zn) concentrations decreased from 0.41 mg l^?1 to 0.06 mg l^?1 (85%), 0.2 mg l^?1 to 0.07 mg l^?1 (65%) and 75 mg l^?1 to 0.02 mg l^?1 (97%), respectively (bioreactor one). Aluminium [(1.55–0.38 mg l^?1 (75%)], copper (Cu) [57% (from 0.33 mg l^?1 to 0.14 mg l^?1)], iron (Fe) [71.99–40.4 mg l^?1 (44%)] and manganese (Mn) [57% (0.07–0.03 mg l^?1)] concentrations also decreased in the water samples from bioreactor two. In the on-site, six-tank bioreactor system, concentrations for Fe, Cu, Mn and Ni decreased, while Zn and Al concentrations increased. The concentrations recorded in biofilm samples were higher than the corresponding water samples. The bioballs employed in the bioreactor were thus shown to be efficient attachment surfaces for biofilm development and subsequent metal accumulation. Potentially metal-tolerant organisms (Pseudomonas sp., Sphingomonas sp., and Bacillus sp.) were also identified using phylogeny.     

Characterization of polycyclic aromatic hydrocarbons degradation and arsenate reduction by a versatile Pseudomonas isolate

A Pseudomonas isolate, designated PAHAs-1, was found capable of reducing arsenate and degrading polycyclic aromatic hydrocarbons (PAHs) independently and simultaneously. This isolate completely reduced 1.5 mM arsenate within 48 h and removed approximately 100% and 50% of 60 mg l⁻¹ phenanthrene and 20 mg l⁻¹ pyrene within 60 h, respectively. Using PAHs as the sole carbon source, however, this isolate showed a slow arsenate reduction rate (4.62 μM h⁻¹). The presence of arsenic affected cell growth and concurrent PAHs removal, depending on PAH species and arsenic concentration. Adding sodium lactate to the medium greatly enhanced the arsenate reduction and pyrene metabolism. The presence of the alpha subunit of the aromatic ring-hydroxylating dioxygenase (ARHD) gene, arsenate reductase (arsC) and arsenite transporter (ACR3(2)) genes supported the dual function of the isolate. The finding of latter two genes indicated that PAHAs-1 possibly reduced arsenate via the known detoxification mechanism. Preliminary data from hydroponic experiment showed that PAHAs-1 degraded the majority of phenanthrene (>60%) and enhanced arsenic uptake by Pteris vittata L. (from 246.7 to 1187.4 mg kg⁻¹ As in the fronds). The versatile isolate PAHAs-1 may have potentials in improving the bioremediation of PAHs and arsenic co-contamination using the plant-microbe integrated strategy.     

Evaluation of pink-pigmented facultative methylotrophic bacteria for phosphate solubilization

Thirteen pink-pigmented facultative methylotrophic (PPFM) strains isolated from Adyar and Cooum rivers in Chennai and forest soil samples in Tamil Nadu, India, along with Methylobacterium extorquens, M. organophilum, M. gregans, and M. komagatae were screened for phosphate solubilization in plates. P-solubilization index of the PPFMs grown on NBRIP—BPB plates for 7 days ranged from 1.1 to 2.7. The growth of PPFMs in tricalcium phosphate amended media was found directly proportional to the glucose concentration. Higher phosphate solubilization was observed in four strains MSF 32 (415 mg l^−l), MDW 80 (301 mg l^−l), M. komagatae (279 mg l^−l), and MSF 34 (202 mg l^−l), after 7 days of incubation. A drop in the media pH from 6.6 to 3.4 was associated with an increase in titratable acidity. Acid phosphatase activity was more pronounced in the culture filtrate than alkaline phosphatase activity. Adherence of phosphate to densely grown bacterial surface was observed under scanning electron microscope after 7-day-old cultures. Biochemical characterization and screening for methanol dehydrogenase gene (mxaF) confirmed the strains as methylotrophs. The mxaF gene sequence from MSF 32 clustered towards M. lusitanum sp. with 99% similarity. This study forms the first detailed report on phosphate solubilization by the PPFMs.     

Analysis of phenanthrene degradation by Ascomycota fungi isolated from contaminated soil from Reynosa,Mexico

Polycyclic aromatic hydrocarbons (PAHs) are organic compounds generated mainly by anthropogenic sources. They are considered toxic to mammals, since they have carcinogenic, mutagenic and genotoxic properties, among others. Although mycoremediation is an efficient, economical and eco-friendly technique for degrading PAHs, the fungal degradation potential of the phylum Ascomycota has not been widely studied. In this work, we evaluated different fungal strains from the polluted soil of ‘La Escondida’ lagoon in Reynosa, Mexico to know their potential to degrade phenanthrene (PHE). Forty-three soil isolates with the capacity to grow in the presence of PHE (0·1% w/v) were obtained. The fungi Aspergillus oryzae MF13 and Aspergillus flavipes QCS12 had the best potential to degrade PHE. Both fungi germinated and grew at PHE concentrations of up to 5000 mg l⁻¹ and degraded 235 mg l⁻¹ of PHE in 28 days, with and without an additional carbon source. These characteristics indicate that A. oryzae MF13 and A. flavipes QCS12 could be promising organisms for the remediation of sites contaminated with PAHs and detoxification of recalcitrant xenobiotics.     

Enhanced lindane removal from soil slurry by immobilized Streptomyces consortium

The aim of this work was to assess lindane removal from soil slurry by a Streptomyces consortium immobilized in cloth sachets, at different inoculum, lindane and slurry concentrations. In concentrated slurry (soil/water ratio of 2:3), the higher lindane removal (35.3 mg Kg⁻¹) was obtained with the medium inoculum (10⁷ CFU g⁻¹) and the highest lindane concentration tested, at 7 days of incubation. Although, lindane removal was also detected in abiotic controls, probably caused by pesticide adsorption to soil particles. Thus, these parameters were selected for evaluating the pesticide removal in diluted slurry (soil/water ratio of 1:4). After 14 days of incubation, 28.7 mg Kg⁻¹ of lindane were removed. Also, a phytotoxicity assay demonstrated that seeds growing on diluted slurries bioremediated during 7 and 14 days, showed an improvement in biological parameters, compared to those growing on non-bioremediated slurries. Thus, bioremediated slurries would not have toxic effects on lettuce seeds.     

Phosphorus release from sediments in a river-valley reservoir in the northern Great Plains of North America

Aside from a companion investigation to this study, there are currently no peer-reviewed phosphorus (P) release rate data for northern North American (i.e., Canadian) reservoirs. Using Lake Diefenbaker, Saskatchewan, Canada as a case study, we tested the effect of variation in overlying water DO conditions on the P release rates from sediment cores. Sediment cores from four down-reservoir locations in Lake Diefenbaker were incubated under high (>8 mg l⁻¹), low (2–3 mg l⁻¹), or anoxic (<1 mg l⁻¹) DO concentrations. Sediment cores were then analyzed for total P (TP) and three geochemical P fractions to assess how the DO regime influenced sediment P inventory. Maximum P release rates were highest under anoxic conditions and similar among sites (15.0–20.3 mg m⁻² day⁻¹), with the low-DO rates intermediate to the high-DO and anoxic P fluxes. Predictive internal P loading models considering only hypolimnetic anoxia may therefore oversimplify and thus underestimate P mobilization in situ. Non-apatite inorganic P (54 ± 10% across sites) from the top 1 cm of the sediment profile was the main source of P released during incubations, indicating that sampling on a coarser scale of resolution could obscure the relationship between sediment geochemistry and short-term P flux.

     

Application of soil slurry respirometry to optimise and subsequently monitor ex situ bioremediation of hydrocarbon-contaminated soils

A protocol to monitor respiration as O₂ consumption in soil slurries using the Strathtox^® respirometer was developed and tested on four soils from brownfield sites. Respiration rates (mg l⁻¹ h⁻¹) of soil slurries in the Strathtox^® were compared with rates (μl min⁻¹) of field moist soils analysed using the Columbus Oxymax^® ER10 respirometer. One of the soils (99612B), historically contaminated with diesel, was further studied by monitoring the effect of inorganic NH₄NO₃ liquid nutrient on enhancing respiration rate. Soil microcosms were monitored continuously on the Oxymax^® or sampled at 24, 48 and 72 h intervals, prepared as soil slurries, and analysed on the Strathtox^®. On the full-scale remediation project (6000 m³) soil 99612B was treated as a biopile with spent mushroom compost (SMC) amendment and respiration rates monitored in samples over an 8-week period. In the laboratory microcosm experiment and full-scale bioremediation treatment described, correlation was found for respiration rates between the two respirometry systems.     

Biodegradation of bisphenol A by bacterial consortia

The effect of light on BPA degradation by an adapted bacterial consortium was investigated. BPA was completely degraded up to 50 mg l⁻¹, and the degradation followed first-order reaction kinetics both in the light and in the dark. The degradation half-life of BPA when the consortium was grown in presence of light was 21.9, 17.2, and 12.6 h for concentrations of 10, 20, and 50 mg l⁻¹, respectively; the degradation half-life of BPA in the dark was 13.1, 10.8, and 10.2 h for concentrations of 10, 20, and 50 mg l⁻¹, respectively. Therefore, light inhibited BPA biodegradation. However, under both conditions, BPA was completely depleted. The bacterial consortium effectively utilised BPA as a growth substrate to sustain a cell yield of 0.95 g g⁻¹ and 0.97 g g⁻¹ in the light and dark, respectively. A total of ten and nine biodegradation intermediates were detected in the light and dark, respectively. Three bacterial metabolic pathways and one photodegradation pathway were proposed to explain their occurrence. This study demonstrated that bacterial consortia may assemble a wide range of catabolic pathways to allow for efficient degradation of BPA, converting BPA to principally bacterial biomass and metabolites exhibiting low or no oestrogenic activity.     

Decolourization of Amaranth dye by bacterial biofilm in batch and continuous packed bed bioreactor

The aim of this study was to exploit the bacterial biofilms to remove dyes from industrial effluents. Biofilms of strains AK1, AK2, VKY1 and a consortium on sheep bone chips were examined in batch, repeated batch and continuous packed bed bioreactor. Biofilms are more efficient for decolourization of Amaranth dye at three different dye concentrations (200, 400, and 600 mg l⁻¹). 100% decolourization of Amaranth dye was observed even at higher concentrations (400 and 600 mg l⁻¹) by all the tested biofilms in 24 h than that of corresponding free cells. The biofilms were superior over those of free cells and could be reused for more than 18 repeated cycles. In a packed bed bioreactor, biofilms could be operated with much higher dilution rates and at lower hydraulic retention time. Further, the decolourization of dye was confirmed by UV–visible spectrophotometer, TLC and HPLC analysis of Amaranth dye degradation products from packed bed bioreactor effluent.     

Effective biodegradation of 2,4,6-trinitrotoluene using a novel bacterial strain isolated from TNT-contaminated soil

In this environmental-sample based study, rapid microbial-mediated degradation of 2,4,6-trinitrotoluene (TNT) contaminated soils is demonstrated by a novel strain, Achromobacter spanius STE 11. Complete removal of 100 mg L⁻¹ TNT is achieved within only 20 h under aerobic conditions by the isolate. In this bio-conversion process, TNT is transformed to 2,4-dinitrotoluene (7 mg L⁻¹), 2,6-dinitrotoluene (3 mg L⁻¹), 4-aminodinitrotoluene (49 mg L⁻¹) and 2-aminodinitrotoluene (16 mg L⁻¹) as the key metabolites. A. spanius STE 11 has the ability to denitrate TNT in aerobic conditions as suggested by the dinitrotoluene and NO₃ productions during the growth period. Elemental analysis results indicate that 24.77 mg L⁻¹ nitrogen from TNT was accumulated in the cell biomass, showing that STE 11 can use TNT as its sole nitrogen source. TNT degradation was observed between pH 4.0–8.0 and 4–43 °C; however, the most efficient degradation was at pH 6.0–7.0 and 30 °C.     

Phenanthrene degradation and strategies to improve its bioavailability to microorganisms isolated from brackish sediments

Samborombón Bay, Argentina, is a highly productive area exposed to chronic contamination, including polycyclic aromatic hydrocarbons. Four phenanthrene-degrading strains were isolated from sediments collected in this area. Analysis of partial 16S rRNA sequencing and a BLAST search indicated that three of the strains belong to genus Pseudomonas and one to Sphingomonas. All the strains were able to grow in 150 mg l⁻¹ phenanthrene as the sole carbon and energy source, with high degradation efficiency (75–100% in 72–168 h). Growth in sodium salicylate indicated that the Pseudomonas strains used this pathway to degrade phenanthrene.Strategies that may enhance substrate bioavailability, such as surfactant production and chemotactic responses, were tested. Two Pseudomonas strains showed significant production of surface-active compounds, and a strong chemotactic response toward phenanthrene. Together with the ability to consume the supplied phenanthrene to completion, these characteristics make the mentioned strains good candidates for bioremediation strategies intended to clean up polluted areas.     

Somatic embryogenesis and in vitro plant regeneration in moth bean [Vigna aconitifolia (Jacq.) Marechal]: a recalcitrant grain legume

An efficient in vitro regeneration protocol for moth bean [Vigna aconitifolia (Jacq.) Marechal] via somatic embryogenesis has been developed. Embryogenic callus cultures were established from the cotyledonary node as explant on semi-solid Murashige and Skoog (MS) medium supplemented with 0.75 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ 6-benzylaminopurine (BA) and with various additives (50 mg l⁻¹ ascorbic acid and 25 mg l⁻¹ each of adenine sulphate, citric acid and l-arginine). Numerous somatic embryos differentiated on MS basal nutrient medium supplemented with 0.25 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ of kinetin (Kin). Sustained cell division resulted in the formation of cell aggregates, which progressed to the globular- and heart-shaped somatic embryos and then, if they differentiated properly, to the torpedo shape and cotyledonary stages. The transfer of embryos onto fresh MS basal medium containing 0.2 mg l⁻¹ BA and 2.0 mg l⁻¹ gibberellic acid enabled the embryos to achieve complete maturation and germination. More than 80% of somatic embryos were converted into true-to-type fertile plants. In vitro-regenerated plantlets with well-developed roots were successfully hardened in a greenhouse and established in soil.     

Aerobic cometabolic degradation of trichloroethene by methane and ammonia oxidizing microorganisms naturally associated with <Emphasis Type="Italic">Carex comosa</Emphasis> roots

The degradation potential of trichloroethene by the aerobic methane- and ammonia-oxidizing microorganisms naturally associated with wetland plant (Carex comosa) roots was examined in this study. In bench-scale microcosm experiments with washed (soil free) Carex comosa roots, the activity of root-associated methane- and ammonia-oxidizing microorganisms, which were naturally present on the root surface and/or embedded within the roots, was investigated. Significant methane and ammonia oxidation were observed reproducibly in batch reactors with washed roots incubated in growth media, where methane oxidation developed faster (2 weeks) compared to ammonia oxidation (4 weeks) in live microcosms. After enrichment, the methane oxidizers demonstrated their ability to degrade 150 μg l⁻¹ TCE effectively at 1.9 mg l⁻¹ of aqueous CH₄. In contrast, ammonia oxidizers showed a rapid and complete inhibition of ammonia oxidation with 150 μg l⁻¹ TCE at 20 mg l⁻¹ of NH₄ ⁺-N, which may be attributed to greater sensitivity of ammonia oxidizers to TCE or its degradation product. No such inhibitory effect of TCE degradation was detected on methane oxidation at the above experimental conditions. The results presented here suggest that microorganisms associated with wetland plant roots can assist in the natural attenuation of TCE in contaminated aquatic environments.     

Naphthalene and biphenyl oxidation by two marine Pseudomonas strains isolated from Venice Lagoon sediment

A sediment sample from Venice Lagoon was found to be contaminated with 475 mg Kg⁻¹ polycyclic aromatic hydrocarbons (PAHs). Naphthalene was the principal pollutant at 26% of total PAHs. Two strains of Pseudomonas SN1 and SB1 were isolated from sediment amended with 2% naphthalene. 16S rRNA gene sequence analysis indicated that the two strains have about 99% nucleotide identity with strains of the genus Pseudomonas, and are very close to Pseudomonas stutzeri. Their metabolic profiles showed significant nutritional differences, the most significant of which was that SN1 grows in marine mineral medium spiked with naphthalene and SB1 grows with biphenyl as sole carbon and energy sources. Pseudomonas sp. SN1 had a doubling time of 3.1 h with 2% naphthalene and SB1 had a doubling time of 19.5 h with 2% biphenyl. Strain SN1 oxidised naphthalene at 564±32 mg O₂ l⁻¹ d⁻¹ and SB1 oxidised biphenyl at 426±25 mg O₂ l⁻¹ d⁻¹ in respirometry reaction vessels under controlled conditions. Screening of the two strains for dioxygenase genes involved in the first step of the two hydrocarbon degradation pathways, by polymerase chain reaction, showed naphthalene dioxygenase in SN1 and biphenyl dioxygenase in SB1. The strains each have a different catechol 2,3-dioxygenase responsible for cleavage of the aromatic ring.     

Fast versatile regeneration of <Emphasis Type="Italic">Trifolium alexandrinum</Emphasis> L.

Trifolium alexandrinum L. (Egyptian clover) is one of the most important forage crops in the world. Its regeneration in tissue culture has been described in a few reports but the efficiency, accurate time scales and applicability to various genotypes of the described procedures are uncertain. Therefore their suitability for genetic transformation is unclear. In this study, were report new fast procedures for regeneration of Egyptian clover that are applicable to the regeneration of various genotypes (Mescawi-ahaly, Sakha3 and Sakha4). Shoots were regenerated from intact and wounded cotyledons as well as hypocotyls of Mescawi-ahaly on naphthaleneacetic acid/benzyladenine (NAA/BA) and naphthaleneacetic acid/thidiazuron (NAA/TDZ) media. The highest shoot regeneration frequencies were obtained from intact cotyledons on NAA/BA (0.05 mg l⁻¹ NAA combined with 2.0 mg l⁻¹ BA) and NAA/TDZ (0.05 mg l⁻¹ NAA combined with 1.0 mg l⁻¹ TDZ) media (66.2 and 43.1% respectively) compared to 18.4 and 10.1% for wounded cotyledons on NAA/BA and NAA/TDZ respectively. 21.0% shoot regeneration frequency was observed for hypocotyls on NAA/BA (2.0 mg l⁻¹ NAA combined with 0.5 mg l⁻¹ BA) medium but no regeneration was obtained on NAA/TDZ medium. Rooting of the regenerated shoots was induced on indole butyric acid (IBA: 0.24 mg l⁻¹) or NAA (2.0 mg l⁻¹) media where IBA medium supported significantly higher frequencies of rooting as well as survival of the whole plantlets after transfer to soil. However, the rooting and survival frequencies also depended on the type of explant and the medium used for shoot regeneration. The two cultivars Sakha3 and Sakha4 were regenerated using the culture conditions optimized for Mescawi-ahaly with comparable efficiencies, indicating that the described procedure is not genotype dependent. The time scale of whole plantlet regeneration ranged from 7.5 weeks for intact and wounded cotyledons to 10 weeks for hypocotyl explants.     

Somatic embryogenesis and shoot organogenesis in the medicinal plant <Emphasis Type="Italic">Pulsatilla koreana</Emphasis> Nakai

We have developed a system for the in vitro regeneration of pasqueflowers (Pulsatilla koreana Nakai). The system was based on somatic embryogenesis and shoot organogenesis. Over a growth period of 6 weeks, multiple shoots were initiated from leaf, petiole, and pedicel explants on Murashige and Skoog (MS) medium containing 0.5 mg l⁻¹ indole-3-acetic acid (IAA) and zeatin (Zn), kinetin (Kin), or 6-benzyladenine (BA). We achieved 100% of adventitious shoot induced when petiole and pedicel explants were cultured on MS, 0.5–2.0 mg l⁻¹ Zn, and 0.5 mg l⁻¹ IAA. Somatic embryos developed from the explants and generated shoots on MS medium containing 0.25 mg l⁻¹ Zn and 0.5 mg l⁻¹ IAA. Globular and heart-shaped stages of somatic embryos were observed. Histological studies have revealed the stages of development of somatic embryos. For propagation and growth, the regenerated shoots from organogenic or embryogenic calluses were transferred to MS medium containing either (1) 1.5 mg l⁻¹ Zn and 0.05 mg l⁻¹ IAA or (2) 1.0 mg l⁻¹ BA and 0.05 mg l⁻¹ IAA. After the length of the shoots reached 3 cm, the shoots initiated by organogenesis as well as those initiated by somatic embryogenesis were transferred to the root induction medium. After 2 months of culture in half-strength MS with 1.5 mg l⁻¹ α-naphthalene acetic acid (NAA), the rooting ratio was 93%. Finally, the rooted plantlets were acclimatized in a mixture of mountain soil and perlite.