Recovery of microbial diversity and activity during bioremediation following chemical oxidation of diesel contaminated soils

To improve the coupling of in situ chemical oxidation and in situ bioremediation, a systematic analysis was performed of the effect of chemical oxidation with Fenton's reagent, modified Fenton's reagent, permanganate, or persulfate, on microbial diversity and activity during 8 weeks of incubation in two diesel-contaminated soils (peat and fill). Chemical oxidant and soil type affected the microbial community diversity and biodegradation activity; however, this was only observed following treatment with Fenton's reagent and modified Fenton's reagent, and in the biotic control without oxidation. Differences in the highest overall removal efficiencies of 69 % for peat (biotic control) and 59 % for fill (Fenton's reagent) were partially explained by changes in contaminant soil properties upon oxidation. Molecular analysis of 16S rRNA and alkane monooxygenase (alkB) gene abundances indicated that oxidation with Fenton's reagent and modified Fenton's reagent negatively affected microbial abundance. However, regeneration occurred, and final relative alkB abundances were 1–2 orders of magnitude higher in chemically treated microcosms than in the biotic control. 16S rRNA gene fragment fingerprinting with DGGE and prominent band sequencing illuminated microbial community composition and diversity differences between treatments and identified a variety of phylotypes within Alpha-, Beta-, and Gammaproteobacteria. Understanding microbial community dynamics during coupled chemical oxidation and bioremediation is integral to improved biphasic field application.     

Aryl hydrocarbon hydroxylase: Degradation of product due to contaminant in acetone

A contaminant present in reagent grade acetone causes degradation of fluorescent phenolic metabolites of benzo(a)pyrene as measured in the aryl hydrocarbon hydroxylase assay. Although the contaminant was not identified, its properties suggest that it is a relatively volatile organic material, possibly an oxidizing agent. The acetone may be readily purified by distillation.     

A role for arabinogalactan-proteins in root epidermal cell expansion

Arabinogalactan-proteins (AGPs) are abundant plant proteoglycans that react with (β-d-Glc)₃ but not (β-d-Man)₃ Yariv reagent. We report here that treatment with (β-d-Glc)₃ Yariv reagent caused inhibition of root growth of Arabidopsis thaliana (L.) Heynh. seedlings. Moreover, the treated roots exhibited numerous bulging epidermal cells. Treatment with (β-d-Man)₃ Yariv reagent did not have any such effects. These results indicate a role for AGPs in root growth and control of epidermal cell expansion. Because treatment with (β-d-Glc)₃ Yariv reagent phenocopies the reb1 (root epidermal cell bulging) mutant of Arabidopsis, AGPs were extracted from the reb1-1 mutant and compared with those of the wild type. The reb1-1 roots contained an approximately 30% lower level of AGPs than the wild type. More importantly, while the profile of AGPs from wild-type roots showed two major peaks upon crossed electrophoresis, the profile of AGPs from reb1-1 roots exhibited only one of the major peaks. Therefore, the reb1 phenotype appears to be a result of defective or missing root AGPs. Taken together, this pharmacological and genetic evidence strongly indicates a function of AGPs in the control of root epidermal cell expansion. Received: 13 February 1997 / Accepted: 1 April 1997     

DNA Breaking Reaction with Catecholamines

Breaking activity of catecholamines and their structural analogues on λ DNA were investigated by agarose slab gel electrophoresis. Since λ DNA has a homogenous molecular size, it is a favorable material to detect the activity of DNA breaking reagent. Among the compounds tested, those having enediol group were only active, though their activities remarkably differed owing to their side chains. The profile of the breaking reaction was studied in detail by the use of one of the catecholamines, epinephrine.     

Measurement of constitutive L-pyrrolidonyl peptidase activity from Streptococcus and Enterococcus using tetrazotized 0-dianisidine

The detection of L-pyrrolidonyl peptidase activity is extremely useful for the differentiation of Enterococcus species and Streptococcus pyogenes from other members of the family Streptococaceae. This analysis has generally been performed utilizing the hydrolyzable substrate L-pyrrolidonyl beta-naphthylamine. After the substrate was hydrolyzed, free beta-naphthylamine has been detected utilizing the reagent para-dimethylaminocinnamaldehyde. The cinnamaldehyde and naphthylamine reagents combine to form an orange color, much like the indole reaction. The use of the cinnamaldehyde reagent had several drawbacks however: color development was not sharp, and the reagent was difficult to produce, and it was not stable. A new indicator system employing tetrazotized 0-dianisidine was developed. An extremely deep burgundy colored complex resulted from the reaction between the new indicator and B-Naphthylamine. This diazo reagent showed excellent correlation with results obtained with para-dimethylaminocinnamaldehyde and yielded more objective, distinct endpoints. This inexpensive reagent can be utilized either in a liquid form or dired on paper discs.     

Contaminant-Adaptation and Community Tolerance in Ecological Risk Assessment: Introduction

Contaminant tolerance, either at the level of the community or an adaptation within populations, has important implications to the risk assessment field. Such tolerance has alternatively been described as a nuisance variable, complicating the extrapolation of toxicity data to field conditions, or as a ‘good weather indicator’, suggesting environmental resilience to a contaminant. These and other issues are explored in this set of invited papers, in which experienced workers from the field of contaminant tolerance have been invited to comment on the relationship between tolerance and the analysis of environmental risk. In addition, recent decades have seen the use of tolerance as a tool for assessing contaminant stress, particularly when establishing causality between specific contaminant exposure and significant ecological impact. The paradigm suggests that an increased tolerance to a contaminant is powerful causal evidence that this contaminant has exerted significant stress. Review, commentary and original data contributions within this Debate and Commentary section explore both the complicating and advantageous aspects of tolerance in risk assessment. The papers conclude that complications associated with tolerance demand careful consideration during risk assessments, and that while population adaptation does not appear to be a promising tool, community-level resistance might be a powerful instrument in ecological risk assessment.     

A Critical Review of Discrete Soil Sample Data Reliability: Part 1—Field Study Results

Part 1 of this study summarizes data for a field investigation of contaminant concentration variability within individual, discrete soil samples (intra-sample variability) and between closely spaced, “co-located” samples (inter-sample variability). Hundreds of discrete samples were collected from three sites known respectively to be contaminated with arsenic, lead, and polychlorinated biphenyls. Intra-sample variability was assessed by testing soil from ten points within a minimally disturbed sample collected at each of 24 grid points. Inter-sample variability was assessed by testing five co-located samples collected within a 0.5-m diameter of each grid point. Multi Increment soil samples (triplicates) were collected at each study site for comparison. The study data demonstrate that the concentration of a contaminant reported for a given discrete soil sample is largely random within a relatively narrow (max:min <2X) to a very wide (max:min >100X) range of possibilities at any given sample collection point. The magnitude of variability depends in part on the contaminant type and the nature of the release. The study highlights the unavoidable randomness of contaminant concentrations reported in discrete soil samples and the unavoidable error and inefficiency associated with the use of discrete soil sample data for decision making in environmental investigations.     

Ecological Effects of Metals in Streams on a Defense Materials Processing Site in South Carolina,USA

The Savannah River Site (SRS) is a 780 km² U.S. Department of Energy facility near Aiken, South Carolina, established in 1950 to produce nuclear materials. SRS streams are “integrators” that potentially receive water transportable contaminants from all sources within their drainage basins, necessitating a watershed approach to organize contaminant distribution data and characterize the effects of multiple contaminants on aquatic organisms. This study used several lines-of-evidence to assess the ecological effects of metals in SRS streams, including contaminant exposure models for apex predators and bioassessments of fish and macroinvertebrate assemblages. Concentrations of metals in sediments, fish, and water were elevated in streams affected by SRS operations, but contaminant exposure models for the river otter Lontra Canadensis and belted kingfisher Ceryle alcyon indicated that toxicological reference values were exceeded only by Hg and Al. Macroinvertebrate assemblage structure was unrelated to sediment metal concentrations. Fish assemblage data were inconclusive. This study indicated that (1) modeling studies and field bioassessments provide a complementary basis for addressing the individual and cumulative effects of contaminants, (2) habitat effects must be controlled when assessing contaminant impacts, (3) sensitivity analyses of contaminant exposure models can help to apportion sampling effort, and (4) most individual metals in SRS streams are unlikely to have significant ecological effects.     

Phytoremediation of Soils Contaminated with Wood Preservatives: Greenhouse and Field Evaluations

Phytoremediation was evaluated as a potential treatment for the creosote-contaminated surface soil at the McCormick and Baxter (M&B) Superfund Site in Portland, Oregon. Soil at the M&B site is contaminated with pentachlorophenol (PCP) and polyaromatic hydrocarbons (PAHs). Eight individual PAH compounds (containing four to six aromatic rings) were included in the investigation. Greenhouse and field studies were carried out using perennial ryegrass (Lolium perenne). The following three treatments were compared in both studies: T1 = planted-amended soil; T2 = unplanted-amended soil; and T3 = unplanted-unamended soil. The amendments were mineral nutrients and dolomite, which was used to raise the acidic pH of the soil. Contaminant concentrations in the soil were measured initially and at regular intervals for several months. In the greenhouse study, the concentrations of certain contaminants decreased as a function of time. Thus, PCP, fluoranthene, pyrene, chrysene, and benzo(k)fluoranthene appeared to undergo biodegradation in all of the treatments. On the other hand, certain larger molecular weight PAHs were relatively recalcitrant. These “recalcitrant PAHs” included benzo(b)fluoranthene, benzo(a)pyrene, benzo(g,h,i)perylene and indeno(1,2,3-cd)pyrene. Statistical methods were used to compare the concentrations of the more easily biodegraded contaminants in treatments T1, T2, and T3. The statistical analysis was facilitated by normalizing the contaminant concentrations relative to the sum of the recalcitrant PAHs in the same sample. Thus, ratios were created that could be compared directly to benchmark values indicative of the contaminant at the beginning of the study. In the greenhouse study, statistically significant differences between T1 and T2, across all treatment times, were obtained for fluoranthene, pyrene, and chrysene (p ≤0.05), suggesting enhanced rhizosphere biodegradation for these compounds. Significant differences between T2 and T3 were obtained for pyrene (p ≤0.03), indicating that nutrients stimulated the biodegradation of this contaminant. Although the greenhouse study was carried out with a well-mixed soil sample from the M&B site, an extremely uneven distribution of contaminants was encountered in the field study. The resulting scatter in the field data made comparisons difficult, and treatment-specific effects observed in the greenhouse study were not statistically significant in the field study. However, analysis of the normalized data from the field revealed the same time-dependent decreases in contaminant concentration as observed in the greenhouse study.     

Cytochemical localization of catalase activity in glyoxysomes from castor bean endosperm

Glyoxysomes isolated from castor bean (Ricinus communis L. var. zanzibariensis) endosperm have been stained by the cytochemical diaminobenzidine reaction. The reaction product obtained by preincubation with 3,3′-diaminobenzidine and incubation with the reagent and H₂O₂ is distributed uniformly throughout the matrix of the organelles. Ricinosomes or dilated cisternae may be completely absent from the organelle preparation or are, at the most, a minor contaminant.     

New anamorphic yeast species: Candida infanticola sp. nov., Candida polysorbophila sp. nov., Candida transvaalensis sp. nov. and Trigonopsis californica sp. nov

Three new species of Candida and a new species of Trigonopsis are described based on their recognition from phylogenetic analysis of gene sequences from large subunit ribosomal RNA, ITS1/ITS2 rRNA, mitochondrial small subunit rRNA and cytochrome oxidase II. Candida infanticola sp. nov. (type strain NRRL Y-17858, CBS 7922) was isolated from the ear of an infant in Germany and is closely related to Candida sorbophila. Candida polysorbophila sp. nov. (type strain NRRL Y-27161, CBS 7317) is a member of the Zygoascus clade and was isolated in South Africa as a contaminant from an emulsion of white oil and polysorbate. Candida transvaalensis sp. nov. (type strain NRRL Y-27140, CBS 6663) was obtained from forest litter, the Transvaal, South Africa, and forms an isolated clade with Candida santjacobensis. Trigonopsis californica sp. nov. (type strain NRRL Y-27307, CBS 10351) represents a contaminant from wine in California, and forms a well-supported clade with Trigonopsis cantarellii, Trigonopsis variabilis and Trigonopsis vinaria.     

A New N-Acyl Derivative of (S)-Cysteine for Quantitative Determination of Enantiomers of Amino Compounds by HPLC with a Precolumn Modification with o-Phthalaldehyde

N-Phenylacetyl-(R)-phenylglycyl-(S)-cysteine (NPPC) was used for the determination of enantiomers of primary amines by rpHPLC with a precolumn modification with o-phthalaldehyde. NPPC was compared with the classic SH reagent N-acetyl-(S)-cysteine (NAC) in the analysis of stereomers of nonfunctionalized amines and amino alcohols. After the NAC-modification, the resulting diastereomeric isoindoles were difficult to separate by HPLC, and satisfactory resolution was achieved only for some aliphatic amino alcohols. The use of NPPC improved the chromatographic analysis of stereomeric amino alcohols and, in addition, allowed the enantiomeric analysis of the nonfunctionalized amines. Similarity between the side radicals of the amino component and the thiol reagent favored the diastereomer separation. This method was used for determination of the absolute concentration of individual enantiomers of amines in the course of stereoselective enzymatic reactions. The optically active NPPC was prepared with a high yield by a chemoenzymatic synthesis based on a regioselective acylation of the (S)-cysteine amino group in aqueous medium by the action of penicillin acylase.     

Purification of xylanase from Trichoderma viride by precipitation with an anionic polymer Eudragit S 100

Summary Endo-xylanase from T. viride has been purified 4.2 fold by precipitation with a commercially available enteric polymer Eudragit S-100. Electrophoretic analysis also indicated removal of contaminant proteins. The enzyme could be recovered in more than 89% yield. The binding of the enzyme to the polymer was predominantly by electrostatic interaction.     

The influence of local plant growth conditions on non-fastidious bacterial contamination of meristem-tips ofHydrangea culturedin vitro

A high incidence ofin vitro bacterial contamination (69%) has been detected in meristem-tip explants ofHydrangea from widely differing locations in Ireland and the UK. The bacteria were characterised by API 20E biochemical test kits and by fatty acid profile analysis. The results obtained from the different methods were compatible and anomalies were explicable in terms of the limitations of the respective methods. The majority of the isolates were environmental or animal-associated bacteria with clusters ofEnterobacter isolates in Dublin, and ofEscherichia coli in the main Cork location. A cluster of Pseudomonads was detected in the Derby (UK) plants. The main association was between the location and the contaminant clusters. The main finding was that the nature of organic soil amendments may influence inoculum for the contamination of plants and the conclusion was that fertilisation with organic materials should be avoided in the preparation of plants for micropropagation.     

Inheritance Profile of Weathered Chlordane and P,P′-DDTs Accumulation by cucurbita pepo hybrids

Cucurbita pepo ssp pepo (zucchini) accumulates significant levels of persistent organic pollutants in its roots, followed by unexpectedly high contaminant translocation to the stems. Most other plant species, including the closely related C. pepo ssp ovifera (squash), do not have this ability. To investigate the mechanism of contaminant accumulation, two cultivars each of parental zucchini and squash, as well as previously created first filial (F1) hybrids and F1 backcrosses (BC) of those parental cultivars, were grown under field conditions in a soil contaminated with weathered chlordane (2.29 μg/g) and DDX residues (0.30 μg/g; sum of DDT, DDE, DDD). The parental zucchini had stem-to-soil bioconcentration factors (BCF, contaminant ratio of stem to soil) for chlordane and DDX of 6.23 and 3.10; these values were 2.2 and 3.7 times greater than the squash, respectively. Chlordane and DDX translocation factors, the ratio of contaminant content in the stems to that in the root, were 2.1 and 3.2 times greater for zucchini than for squash. The parental zucchini and squash also differed significantly in chlordane component ratios (relative amounts of trans-nonachlor [TN], cis-chlordane [CC], trans-chlordane [TC]) and enantiomer fractions for the chiral CC and TC. Hybridization of the parental squash and zucchini resulted in significant differences in contaminant uptake. For both the three separate component ratios (CR) and two sets of enantiomer fraction (EF) values, subspecies specific differences in the parental generation became statistically equivalent in the F1 hybrid zucchini and squash. When backcrossed (BC) with the original parental plants, the zucchini and squash F1 BC cultivars reverted to the statistically distinct CR and EF patterns. This pattern of trait segregation upon hybridization suggests either single gene or single locus control for persistent organic pollutant (POP) uptake ability by C. pepo ssp pepo.     

Use of mycelia as paths for the isolation of contaminant‐degrading bacteria from soil

Mycelia of fungi and soil oomycetes have recently been found to act as effective paths boosting bacterial mobility and bioaccessibility of contaminants in vadose environments. In this study, we demonstrate that mycelia can be used for targeted separation and isolation of contaminant‐degrading bacteria from soil. In a ‘proof of concept’ study we developed a novel approach to isolate bacteria from contaminated soil using mycelia of the soil oomycete Pythium ultimum as translocation networks for bacteria and the polycyclic aromatic hydrocarbon naphthalene (NAPH) as selective carbon source. NAPH‐degrading bacterial isolates were affiliated with the genera Xanthomonas, Rhodococcus and Pseudomonas. Except for Rhodococcus the NAPH‐degrading isolates exhibited significant motility as observed in standard swarming and swimming motility assays. All steps of the isolation procedures were followed by cultivation‐independent terminal 16S rRNA gene terminal fragment length polymorphism (T‐RFLP) analysis. Interestingly, a high similarity (63%) between both the cultivable NAPH‐degrading migrant and the cultivable parent soil bacterial community profiles was observed. This suggests that mycelial networks generally confer mobility to native, contaminant‐degrading soil bacteria. Targeted, mycelia‐based dispersal hence may have high potential for the isolation of bacteria with biotechnologically useful properties.     

Efficacy of carboxin and heat treatment for controlling the growth of Sclerotium rolfsii during culture of the mushroom Pleurotus flabellatus

Soaking paddy straw for 18 h in a carboxin suspension in water (5 mg a.i./l) effectively controlled the growth of Sclerotium rolfsii occurring as a natural contaminant in paddy straw, during culture of the mushroom Pleurotus flabellatus. Carboxin at this concentration delayed sporophore formation by 2–3 days, but did not affect total yield. No detectable carboxin residues were found in the mushrooms grown on the treated paddy straw. Treatment of the paddy straw with hot water at 60°C for 10 min completely controlled the contaminant without affecting mushroom growth and yield; but 20 mm treatments with hot air at 60°C or heating with the steam to 75–80°C did not control S. rolfsii.     

Combined Use of Life Cycle Assessment and Groundwater Transport Modeling to Support Contaminated Site Management

The subject of this study is a spent pot lining (SPL) landfill. The aim of this study was to identify the site remediation option, among four alternatives, that minimizes overall environmental impacts based on: 1) a comparative life cycle assessment (LCA); and 2) modeling of contaminant transport in groundwater. The four options were: leaving the landfill in place (Option 1); excavation of the landfill, with on-site disposal of the excavated materials in a secure cell (Option 2); excavation of the landfill, with treatment of the SPL fraction (Option 3); and excavation of the landfill, with incineration of the SPL fraction in a cement kiln (Option 4). The LCA was performed following the guidelines provided by the International Standard Organization (ISO). Furthermore, to improve the relevance of LCA to site remediation sector, impacts caused by residual in-situ contamination were assessed by applying a simulation of contaminant transport in groundwater, using site-specific data. The LCA identified Option 1 as having the least environmental impacts. However, the transport modeling concluded that contaminant concentrations 50 years from the present could be approximately 30 to 40 times the regulatory criteria if this option is retained. In addition, this study demonstrated that LCA can be used as a screening tool to help identify significant environmental issues; the LCA identified acute and chronic water ecotoxicity categories as being the dominant impact categories of the environmental profile and consequently, it is recommended that a complete environmental risk assessment (ERA) be performed for Option 1.     

Bolton-Hunter reagent as a vital stain for developing systems

The Bolton-Hunter reagent, N-succinimidyl 3-(4-hydroxy, 5-[¹²⁵I]iodophenyl)propionate, was used as a vital stain for developing amphibian and tunicate embryos and for isolated cells (human erythrocytes and cultured chick limb mesenchymal cells). We found that the Bolton-Hunter reagent can be used on living cells at room temperature with techniques that are quite similar to the techniques routinely used to label isolated macromolecules in vitro. At concentrations of vital stain that were sufficient to label intracellular proteins in intact-cells, labeled cells underwent normal developmental sequences. Under these conditions, vital staining with the Bolton-Hunter reagent disproportionately labeled exterior proteins, and it seems likely that the Bolton-Hunter reagent is an especially good vital stain for cell surface and cell membrane proteins. The Bolton-Hunter stain is covalently bound, is not reutilizable, and appears not to disrupt natural physiological and developmental processes. Thus, we used the Bolton-Hunter reagent to follow the natural life spans of proteins in vivo and we were able to distinguish particularly long-lived proteins in Xenopus embryos.     

An Improvement in the Sensitivity of the Salkowski Reagent for Tryptamine, Tryptophan and Indoleacetic Acid

The reaction of indoles with the Salkowski reagent has been examined. It was found that the concentration of acid as well as the concentration and anionic component of the iron salt employed are critical factors in the choice of a reagent that will fail to react—or will react maximally with a given indole. Tryptamine can be reproducibly assayed with a reagent containing 0.01 M Fe(NO₃)₃ in 7.0 M HCIO₄. Two ml of this reagent are added to two ml of the sample. The absorbancy is read at 450 nm after 90 minutes under uniform light conditions. Versions of this reagent can also be used for the quantitative colorimetric determination of tryptophan or indoleacetic acid.