Enhanced Degradation of Hexachlorocyclohexane Isomers by Sphingomonas paucimobilis

Hexachlorocyclohexane (HCH) has been banned for use in technologically advanced countries; however, it is still in use in tropical countries like India. Earlier we reported the degradation of HCH isomers by Sphingomonas paucimobilis within 12 days of incubation. Here we report the role of different factors that could enhance the degradation rate of HCH isomers. We found that an increase in the cell number from 10² to 10⁸ cells/ml resulted in an increased degradation rate of HCH isomers viz. α, β, γ, and δ-HCH. While α-HCH and γ-HCH disappeared completely from the medium within 3 days of incubation, a maximum of only 90% and 85% degradation was observed for β and δ-HCH, respectively. We have also observed that adapted cultures degraded HCH isomers more efficiently than did the normal cultures. Received: 16 February 2000 / Accepted: 23 May 2000     

Rhizosphere microbial community and hexachlorocyclohexane degradative potential in contrasting plant species

The organochlorine 1,2,3,4,5,6 hexachlorocyclohexane (HCH) is a broad-spectrum insecticide that was used on a large-scale worldwide. The soil–plant–microbe system and its influence on HCH biodegradation are evaluated. A greenhouse experiment was designed to evaluate HCH dissipation and several microbial parameters among rhizosphere and bulk soil of two contrasting plants, Cytisus striatus (Hill) Rothm and Holcus lanatus L. Plants were grown for 180 days in three treatments: uncontaminated soil (control), uncontaminated soil inoculated with soil (3% w/w) from a HCH-contaminated site (INOC), and uncontaminated soil inoculated with soil (3% w/w) from the HCH-contaminated site and artificially contaminated to obtain 100 mg HCH kg⁻¹ dry soil (100HCH-INOC). At harvest, plant biomass, soil water-extractable organic C, pH and Cl concentration, rhizosphere microbial densities (total heterotrophs, ammonifiers, amylolytics) and C substrate utilization patterns, and degradation of α-, β-, δ- and γ-HCH isomers were determined in bulk and rhizosphere soils. Soil solution Cl concentration was determined every 30 days throughout the entire growth period. Results demonstrate that both Cytisus striatus and Holcus lanatus can grow in soils with up to 100 mg HCH kg⁻¹. An enhanced degradation of α-HCH, but not β- or δ-HCH, was observed in the rhizosphere. Significant changes in the microbial densities were observed between bulk and rhizosphere soils of Cytisus, and an increase in C source utilization indicated changes in community level physiological profiles (CLPP) in the rhizosphere of this species when grown in contaminated soils. HCH dissipation was also greater in soils planted with this species. In accordance, increases in soil extractable C, Cl concentration and acidity were greater at the rhizosphere of Cytisus. Concentration of Cl in soil solutions also indicates greater HCH dechlorination in soils planted with Cytisus than Holcus. Results suggest that phytostimulation of bacteria present or added to soil is a promising approach to cleaning HCH-contaminated sites, and especially for biodegradation of α-HCH.     

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.     

Fungal degradation of chlorpyrifos by Verticillium sp. DSP in pure cultures and its use in bioremediation of contaminated soil and pakchoi

Pesticides residues in soils and on vegetables are a public safety concern. Pretreatment with microorganisms degrading pesticides has the potential to alleviate the conditions. For this purpose, the degradation characteristics of chlorpyrifos by an isolated fungal strain Verticillium sp. DSP in pure cultures, soil, and on pakchoi (Brassica chinensis L.) were investigated. Degradation rate of chlorpyrifos in the mineral salts medium was proportional to the concentrations of chlorpyrifos ranging from 1 to 100 mg l⁻¹. The rate of degradation for chlorpyrifos (1 mg l⁻¹) in the mineral salts medium was 1.12 and 1.04 times faster at pH 7.0 than those at pHs 5.0 and 9.0, and the degradation at 35 °C was 1.15 and 1.12 times faster, respectively, than those at 15 and 20 °C. The addition of the fungal strain DSP into the contaminated soils was found to significantly increase the degradation of chlorpyrifos. Degradation rates of chlorpyrifos in inoculated soils were 3.61, 1.50 and 1.10 times faster in comparison with the sterilized soil, previously chlorpyrifos-untreated soil, and previously chlorpyrifos-treated soil under laboratory conditions. In contrast to the controls, the half-lives of chlorpyrifos were significantly shortened by 10.9% and 17.6% on treated pakchoi, 12.0% and 37.1% in inoculated soils, respectively, in the greenhouse and open field. The results indicate that the fungal strain DSP can be used successfully for the removal or detoxification of chlorpyrifos residues in/on contaminated soil and vegetable.     

Novel LinA Type 3 δ-Hexachlorocyclohexane Dehydrochlorinase

LinA is the first enzyme of the microbial degradation pathway of a chlorinated insecticide, hexachlorocyclohexane (HCH), and mediates the dehydrochlorination of α-, γ-, and δ-HCH. Its two variants, LinA type 1 and LinA type 2, which differ at 10 out of 156 amino acid residues, have been described. Their activities for the metabolism of different HCH isomers differ considerably but overall are high for γ-HCH, moderate for α-HCH, low for δ-HCH, and lacking for β-HCH. Here, we describe the characterization of a new variant of this enzyme, LinA type 3, whose gene was identified from the metagenome of an HCH-contaminated soil sample. Its deduced primary structure in the region spanning amino acid residues 1 to 147 of the protein exhibits 17 and 12 differences from LinA type 1 and LinA type 2, respectively. In addition, the residues GIHFAPS, present at the region spanning residues 148 to 154 in both LinA type 1 and LinA type 2, are deleted in LinA type 3.The activity of LinA type 3 for the metabolism of δ-HCH is several orders of magnitude higher than that of LinA type 1 or LinA type 2 and can be useful for improvement of the metabolism of δ-HCH.     

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.     

Biodegradation of lindane pesticide by non white- rots soil fungus <Emphasis Type="Italic">Fusarium</Emphasis> sp.

Lindane or γ- hexachlorocyclohexane (γ-HCH) is a chlorinated pesticide and its toxic effects on biota necessitate its removal. Microbial degradation is an important process for pesticide bioremediation and the role of soil fungi in recycling of organic matter prompted us to study the biodegradation of lindane using fungi. This study aims at enrichment, isolation and screening of soil fungi capable of metabolizing lindane. Two Fusarium species (F. poae and F. solani) isolated from the pesticide contaminated soil showed better growth on the plates supplemented with lindane as a sole carbon source, when compared with the growth performance of other fungal isolates from the same contaminated soil. However, ANOVA revealed a significant difference in fungal biomass production in both F. poae (F = 22.02; N = 15; P < 0.001) and F. solani (F = 268.75; N = 15; P < 0.001) across different lindane concentrations (0–600 μg ml⁻¹). Growth of both Fusarium sp. was maximum at a lindane concentration of 100 μg ml⁻¹, while minimum at 600 μg ml⁻¹ concentrations. Results on the time dependent release of chlorine by the Fusarium strains in the presence of various concentration of lindane showed the highest mineralization of the pesticide on 10th day of incubation. Time dependent variations in the release of chlorine from 1st to 10th day by both the selected fungal strains were found to be statistically significant. A significant positive relationship exists between fungal biomass increase and chlorine release existed for both F. solani (R2 = 0.960) and F. poae (R2 = 0.628). The results of gas chromatograph analysis of γ- HCH confirmed the biodegradation and utilization of γ- HCH by F. poae and F. solani. The data on lindane degradation by the two fungal strains demonstrated that the biodegradation of lindane by F. solani (59.4%) was slightly higher than that by the F. poae (56.7%).     

Enhanced biodegradation of hexachlorocyclohexane (HCH) in contaminated soils via inoculation with Sphingobium indicum B90A

Soil pollution with hexachlorocyclohexane (HCH) has caused serious environmental problems. Here we describe the targeted degradation of all HCH isomers by applying the aerobic bacterium Sphingobium indicum B90A. In particular, we examined possibilities for large-scale cultivation of strain B90A, tested immobilization, storage and inoculation procedures, and determined the survival and HCH-degradation activity of inoculated cells in soil. Optimal growth of strain B90A was achieved in glucose-containing mineral medium and up to 65% culturability could be maintained after 60 days storage at 30°C by mixing cells with sterile dry corncob powder. B90A biomass produced in water supplemented with sugarcane molasses and immobilized on corncob powder retained 15–20% culturability after 30 days storage at 30°C, whereas full culturability was maintained when cells were stored frozen at −20°C. On the contrary, cells stored on corncob degraded γ-HCH faster than those that had been stored frozen, with between 15 and 85% of γ-HCH disappearance in microcosms within 20 h at 30°C. Soil microcosm tests at 25°C confirmed complete mineralization of [¹⁴C]-γ-HCH by corncob-immobilized strain B90A. Experiments conducted in small pits and at an HCH-contaminated agricultural site resulted in between 85 and 95% HCH degradation by strain B90A applied via corncob, depending on the type of HCH isomer and even at residual HCH concentrations. Up to 20% of the inoculated B90A cells survived under field conditions after 8 days and could be traced among other soil microorganisms by a combination of natural antibiotic resistance properties, unique pigmentation and PCR amplification of the linA genes. Neither the addition of corncob nor of corncob immobilized B90A did measurably change the microbial community structure as determined by T-RFLP analysis. Overall, these results indicate that on-site aerobic bioremediation of HCH exploiting the biodegradation activity of S. indicum B90A cells stored on corncob powder is a promising technology.     

Biodegradation of hexachlorocyclohexane (HCH) by microorganisms

The organochlorine pesticide Lindane is the -isomer of hexachlorocyclohexane (HCH). Technical grade Lindane contains a mixture of HCH isomers which include not only -HCH, but also large amounts of predominantly -, - and -HCH. The physical properties and persistence of each isomer differ because of the different chlorine atom orientations on each molecule (axial or equatorial). However, all four isomers are considered toxic and recalcitrant worldwide pollutants. Biodegradation of HCH has been studied in soil, slurry and culture media but very little information exists on in situ bioremediation of the different isomers including Lindane itself, at full scale. Several soil microorganisms capable of degrading, and utilizing HCH as a carbon source, have been reported. In selected bacterial strains, the genes encoding the enzymes involved in the initial degradation of Lindane have been cloned, sequenced, expressed and the gene products characterized. HCH is biodegradable under both oxic and anoxic conditions, although mineralization is generally observed only in oxic systems. As is found for most organic compounds, HCH degradation in soil occurs at moderate temperatures and at near neutral pH. HCH biodegradation in soil has been reported at both low and high (saturated) moisture contents. Soil texture and organic matter appear to influence degradation presumably by sorption mechanisms and impact on moisture retention, bacterial growth and pH. Most studies report on the biodegradation of relatively low ( 500 mg/kg) concentrations of HCH in soil. Information on the effects of inorganic nutrients, organic carbon sources or other soil amendments is scattered and inconclusive. More in-depth assessments of amendment effects and evaluation of bioremediation protocols, on a large scale, using soil with high HCH concentrations, are needed.     

Ergosterol contents of some wood-rotting basidiomycete fungi grown in liquid and solid culture conditions

Ergosterol contents of six wood-rotting basidiomycetes were analyzed under different cultivation conditions. Four white-rot and two brown-rot fungi were cultivated in liquid synthetic medium with low nutrient nitrogen (2 mM) and 0.1% glucose, and ergosterol in mycelial biomasses were measured weekly for 35 days. The highest ergosterol content per fungal dry mass in the white-rot fungi was found in Phanerochaete chrysosporium being 2100 μg g⁻¹, while in Ceriporiopsis subvermispora it was 1700 μg g⁻¹, Phlebia radiata 700 μg g⁻¹, and Physisporinus rivulosus 560 μg g⁻¹. In brown-rot fungi the ergosterol content was in Poria placenta 2868 μg g⁻¹ and in Gloeophyllum trabeum 3915 μg g⁻¹. On agar media, P. chrysosporium and P. radiata reached the highest ergosterol value in 7 days, while in wood block cultures the ergosterol contents were quite stable. The conversion factors for ergosterol-to-fungal biomass varied from 48 and 243, which were lower than values for ascomycetous soil fungi reported in the literature.     

Degradation of β-Hexachlorocyclohexane by Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

β-Hexachlorocyclohexane (β-HCH) is the most recalcitrant among the α-, β-, γ-, and δ-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of γ-HCH in Sphingomonas paucimobilis UT26, metabolizes β-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.     

Enhanced yield of medium-chain-length polyhydroxyalkanoates from nonanoic acid by co-feeding glucose in carbon-limited, fed-batch culture

Medium-chain-length polyhydroxyalkanoates (MCL-PHAs) were produced in carbon-limited, single-stage, fed-batch fermentations of Pseudomonas putida KT2440 by co-feeding nonanoic acid (NA) and glucose (G) to enhance the yield of PHA from NA. An exponential (μ = 0.25 h⁻¹) followed by a linear feeding strategy at a NA:G ratio of 1:1 (w/w) achieved 71 g l⁻¹ biomass containing 56% PHA. Although the same overall PHA productivity (1.44 g l⁻¹ h⁻¹) was obtained when NA alone was fed at the same specific growth rate, the overall yield of PHA from NA increased by 25% (0.66 g PHA g NA⁻¹ versus 0.53 g g⁻¹) with glucose co-feeding. Further increasing glucose in the feed (NA:G = 1:1.5) resulted in a slightly higher yield (0.69 g PHA g NA⁻¹) but lower PHA content (48%) and productivity (1.16 g l⁻¹ h⁻¹). There was very little change in the PHA composition.     

Can soil Chytridiomycota survive and grow in different osmotic potentials?

Twenty isolates from soil in the orders Spizellomycetales, Blastocladiales and Chytridiales (Chytridiomycota) grew on complex solid media supplemented with 10 g l⁻¹ sodium chloride. In a synthetic liquid medium, 4.4 g l⁻¹ sodium chloride strongly inhibited growth in three of the five isolates, possibly because of the effect of the ions or osmolarity of the solution. The maximum concentration for growth in synthetic liquid medium with different osmotic potentials using polyethylene glycol (PEG) varied considerably amongst the isolates. Three patterns of growth with increasing concentrations of PEG were evident among isolates within the genus Rhizophydium. Up to the concentration where growth ceased, the dry weight of each isolate either decreased, remained constant, or in one case, increased. Most of the fungi survived when incubated at room temperature for 7 d in complex liquid media supplemented with 35 g l⁻¹ sodium chloride or 300 g l⁻¹ PEG. These data indicate that soil Chytridiomycota can survive various osmotic potentials that may occur during the wetting and drying phases in soils.     

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.     

Isolation and characterization of a white rot fungus <Emphasis Type="Italic">Bjerkandera</Emphasis> sp. strain capable of oxidizing phenanthrene

Strain BOL13 was selected from 18 fungal strains isolated from an oil-spill contaminated site in Oruro, Bolivia. It was identified as a basidiomycete with high homology to Bjerkandera. The fungus degraded 100 mg phenanthrene l⁻¹ at 0.17 mg l⁻¹ d⁻¹ at 30 °C at pH 7. During phenanthrene degradation, a maximum manganese peroxidase activity of 100–120 U l⁻¹ was measured after 10 days of incubation. The ability of Bjerkandera sp. to produce lignin-modifying enzymes and to oxidize phenanthrene under various pH and temperature conditions was confirmed.     

Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii. The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1 μM of l-phenylalanine (6.2 g L⁻¹). Addition of 1 μM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7 μg L⁻¹), which was not detected in the untreated control culture. Supplementation with 100 μM of l-phenylalanine together with 100 μM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3 μg L⁻¹ (control untreated culture) to 568.2 μg L⁻¹, the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8 μg L⁻¹) was observed when the medium was supplemented with 100 μM of the precursor in combination with methyl jasmonate.Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium.     

Nitrogenous preference of toxigenic Pseudo-nitzschia australis (Bacillariophyceae) from field and laboratory experiments

Field and laboratory experiments were designed to determine the differential growth and toxin response to inorganic and organic nitrogen additions in Pseudo-nitzschia spp. Nitrogen enrichments of 50 μM nitrate (KNO₃), 10 μM ammonium (NH₄Cl), 20 μM urea and a control (no addition) were carried out in separate carboys with seawater collected from the mouth of the San Francisco Bay (Bolinas Bay), an area characterized by high concentrations of macronutrients and iron. All treatments showed significant increases in biomass, with chlorophyll a peaking on days 4–5 for all treatments except urea, which maintained exponential growth through the termination of the experiment. Pseudo-nitzschia australis Frenguelli abundance was 10³ cells l⁻¹ at the start of the experiment and increased by an order of magnitude by day 2. Particulate domoic acid (pDA) was initially low but detectable (0.15 μg l⁻¹), and increased throughout exponential and stationary phases across all treatments. At the termination of the experiment, the urea treatment produced more than double the amount of pDA (9.39 μg l⁻¹) than that produced by the nitrate treatment (4.26 μg l⁻¹) and triple that of the control and ammonium treatments (1.36 μg l⁻¹ and 2.64 μg l⁻¹, respectively). The mean specific growth rates, calculated from increases in chlorophyll a and from cellular abundance of P. australis, were statistically similar across all treatments.These field results confirmed laboratory experiments conducted with a P. australis strain isolated from Monterey Bay, CA (isolate AU221-a) grown in artificial seawater enriched with 50 μM nitrate, 50 μM ammonium or 25 μM of urea as the sole nitrogen source. The exponential growth rate of P. australis was significantly slower for cells grown on urea (ca. 0.5 day⁻¹) compared to the cells grown on either nitrate or ammonium (ca. 0.9 day⁻¹). However the urea-grown cells produced more particulate and dissolved domoic acid (DA) than the ammonium- or nitrate-grown cells. The field and laboratory experiments demonstrate that P. australis is able to grow effectively on urea as the primary source of nitrogen and produced more pDA when grown on urea in both natural assemblages and unialgal cultures. These results suggest that the influence of urea from coastal runoff may prove to be more important in the development or maintenance of toxic blooms than previously thought, and that the source of nitrogen may be a determining factor in the relative toxicity of west coast blooms of P. australis.     

Bioremediation of hexachlorocyclohexane (HCH) in soil using spent mushroom compost of Pleurotus ostreatus

Abstract

Hexachlorocyclohexane (HCH), a highly chlorinated pesticide, was used worldwide in the 1950s and 1960s. HCH toxic residues are still detected in environmental compartments. Thus, effective, viable and eco-friendly strategy is required for its remediation. In this study, degradation of four HCH isomers was evaluated by amending contaminated soil using four treatments of spent mushroom compost (SMC) of Pleurotus ostraetus. The soil was incubated for 5 weeks and was sampled every seven days. Quantitative attenuation in HCH was calculated using gas chromatography–electron capture detector (GC-ECD) and metabolite was identified using gas chromatography–mass selective detector (GC-MSD). Maximum reduction 58%, 26%, 45%, and 64% for α-, β-, γ- and δ-HCH isomers, respectively, using SMC and soil (both unsterilized) showed that this treatment was the best for bioremediation of HCH in soil. However, when one of the factors, either soil or SMC, was sterilized, a significant reduction in HCH degradation was noticed. The second most reduction of isomers was seen during treatment where unsterilized SMC was added in sterilized soil followed by treatment where SMC was sterilized but soil was not. Abiotic control did not remove any significant quantities of HCH. Simple first-order (SFO) kinetic confirmed that SMC reduced the half-live manifolds as compared to biotic control. Only one metabolite δ-PCCH was identified during the course of study.     

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.     

Purification and characterization of a novel protease-resistant α-galactosidase from Rhizopus sp. F78 ACCC 30795

A novel extracellular α-galactosidase, named Aga-F78, from Rhizopus sp. F78 ACCC 30795 was induced, purified and characterized in this study. This soybean-inducible α-galactosidase was purified to homogeneity by ammonium sulfate precipitation and fast protein liquid chromatography (FPLC), with a yield of 14.6% and a final specific activity of 74.6 U mg⁻¹. Aga-F78 has an estimated relative molecular mass of 78 kDa from SDS-PAGE while native mass of 210 kDa and 480 kDa from non-denaturing gradient PAGE. This α-galactosidase had no N- or O-glycosylated. Amino acid sequences of three internal fragments were determined, and fragment 1, NQLVLDLTR, shared high homology with bacterial and fungal GH-36 α-galactosidases. The optimum pH and temperature on activity of Aga-F78 were 4.8 and 50 °C, respectively. The properties of pH and temperature stability, effect of ions and chemicals were also studied. Furthermore, the resistant to neutral and alkaline proteases and substrate specificity of natural substrates (melibiose, raffinose, stachyose and guar gum) were also studied to enlarged the application of Aga-F78 in more fields. Kinetic studies revealed a K_m and V_max of 2.9 mmol l⁻¹ and 246.1 μmol (mg min)⁻¹, respectively, using pNPG as substrate. To our knowledge, this is the first report of purification and characterization of α-galactosidase from Rhizopus with some special properties, which may aid its utilization in the food and feed industries.