Bioremediation of sago industry effluent and its impact on seed germination (green gram and maize)

In this study, we attempted two investigational systems: one is treatment of sago industry effluent by aerobic bacterial consortium and the other is impact of treated and untreated effluent on seed germination. For the treatment system, the starch degrading bacteria were isolated from sago industry effluent and effluent contaminated soil. The genera, Alcaligenes, Bacillus and Corynebacterium were found efficient in starch degradation. The selected isolates were tested for their efficiency on the degradation of starch both in Mineral Salts Medium (MSM) and in sago industry effluent. About 85% of the starch was degraded in MSM by a bacterial consortium composed of Alcaligenes, Bacillus and Corynebacterium, whereas in effluent the degradation of starch was only 63%. The physico-chemical properties such as electrical conductivity, total solids, suspended solids, dissolved solids, BOD, COD, nitrogen and phosphate were found decreased in effluent after 72 h. The pH of the effluent was relatively increased from 3 to 6.7. The study of seed germination (maize and green gram) was carried out at 25, 50, 75 and 100% concentrations of treated and untreated effluent using soil sowing method. Shoot length, root length, fresh weight, dry weight and chlorophyll content showed an increase when treated effluent was tested whereas a decrease of growth was noticed in untreated effluent tested seedlings. The results revealed that effluent treated by aerobic microorganisms has no negative impact on the seed germination and can be effectively used for irrigation.     

Decolorization and detoxification of Congo red and textile industry effluent by an isolated bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. SU-EBT

The 16S rRNA sequence and biochemical characteristics revealed the isolated organism as Pseudomonas sp. SU-EBT. This strain showed 97 and 90% decolorization of a recalcitrant dye, Congo red (100 mg l⁻¹) and textile industry effluent with 50% reduction in COD within 12 and 60 h, respectively. The optimum pH and temperature for the decolorization was 8.0 and 40°C, respectively. Pseudomonas sp. SU-EBT was found to tolerate the dye concentration up to 1.0 g l⁻¹. Significant induction in the activity of intracellular laccase suggested its involvement in the decolorization of Congo red. The metabolites formed after decolorization of Congo red, such as p-dihydroxy biphenyl, 8-amino naphthol 3-sulfonic acid and 3-hydroperoxy 8-nitrosonaphthol were characterized using FTIR and GC–MS. Phytotoxicity study revealed nontoxic nature of the degradation metabolites to Sorghum bicolor, Vigna radiata, Lens culinaris and Oryza sativa plants as compared to Congo red and textile industry effluent. Pseudomonas sp. SU-EBT decolorized several individual textile dyes, dye mixtures and textile industry effluent, thus it is a useful strain for the development of effluent treatment methods in textile processing industries.     

Biotreatment of Simulated Textile Dye Effluent Containing malachite green by an Up-Flow Immobilized Cell Bioreactor

An up-flow immobilized cell bioreactor was developed using a microbial consortium, consisting of Bacillus sp., Alcaligenes sp. and Aeromonas sp., immobilized on refractory brick pieces as immobilization support. malachite green, a model triphenylmethane dye was decolourized by more than 93% within 48 h (operating conditions: initial dye concentration 30 mg l⁻¹; flow rate 6 ml h⁻¹). The analytical studies based on TLC and ¹H NMR showed degradation of the aromatic rings of the malachite green into simpler metabolic intermediates.     

POTENTIAL OF BRASSICA JUNCEA IN ORDER TO TREAT TEXTILE—EFFLUENT—CONTAMINATED SITES

Three plant species (Brassica juncea, Sorghum vulgare, and Phaseolus mungo) of different agronomic consequence were evaluated for the decolorization of the dyes from textile effluent. B. juncea, S. vulgare, and P. mungo showed textile effluent decolorization up to 79, 57, and 53%, respectively. A significant decrease in shoot and root height, but no significant injury, was observed in the case of P. mungo and S. vulgare. B. juncea (Indian mustard), the most tolerant and more effective metals accumulator than other tested agricultural plant species, showed enhanced growth with respect to the height of the shoot and root, 129 and 178%, respectively, when grown using original textile effluent. Textile effluent induced intracellular nicotinamide adenine dinucleotide reduced (NADH)–dichlorophenol indophenol reductase significantly in the case of S. vulgare and B. juncea with 209 and 194%, respectively. The extracellular riboflavin reductase activity was induced by 223% in the case of P. mungo as compared to control plants. Significant induction of intracellular laccase (266%) was observed in the case of B. juncea, indicating their crucial role for a potential metabolism and further degradation of the textile effluent. The metabolites were identified as napthalenesufamide (m/z 372) and 2-amino-4, 6-dichlorotriazine (m/z 167), when B. juncea was used to degrade a model dye, Reactive red 2.     

Biodegradation of malachite green by Ochrobactrum sp.

This study presents the biodegradation of malachite green (MG), a triphenylmethane dye, using a novel microorganism isolated from textile effluent contaminated environment. The organism responsible for degradation was identified as Ochrobactrum sp JN214485 by 16S rRNA analysis. The effect of operating parameters such as temperature, pH, immobilized bead loading, and initial dye concentration on % degradation was studied, and their optimal values were found to be 30 °C, 6, 20 g/L and 100 mg/L, respectively. The analysis showed that the extracellular enzymes were responsible for the degradation. The biodegradation of MG was confirmed by UV–visible spectroscopic and FTIR analysis. The phytotoxicity test concluded that the degradation products were less toxic compared to MG. The kinetics of biodegradation was studied and the activation energy was found to be 10.65 kcal/mol.     

Malachite green treatment of industrial Penicillium chrysogenum protoplasts results in increased penicillin-V formation

We attempted protoplast fusion in order to generate gene transfer between an industrial strain of Penicillium chrysogenum and a fission yeast, Schizosaccharomyces pombe. The Penicillium strain was treated with malachite green. The S. pombe strain was auxotrophic for lysine. The regenerated colonies showed Penicillium morphology. The number of Penicillium colonies was significantly higher when the inactivated Penicillium protoplasts were fused to S. pombe protoplasts than in the self-fusion control experiments. We randomly isolated colonies from the regeneration plates and measured beta-lactam formation in cultures from shaken flasks. Antibiotic production was increased in colonies originated from the malachite green-treated protoplasts. Received 2 June 1998/ Accepted in revised form 30 November 1998     

Plant growth promoting bacteria enhance water stress resistance in green gram plants

Plant growth promoting bacterial (PGPB) strains Pseudomonas fluorescens Pf1 and endophytic Bacillus subtilis EPB5, EPB22, EPB 31 were tested for their capacity to induce water stress related proteins and enzymes in green gram (Vigna radiata) plants. Among the different bacteria used, P. fluorescens Pf1 increased the vigour index, fresh weight and dry weight of green gram seedlings in vitro. Quantitative and qualitative analyses of stress-related enzymes indicated the greater activity of catalase and peroxidase in green gram plants bacterized with P. fluorescens Pf1 against water stress when compared to untreated plants. The greater accumulation of proline was recorded in Pf1 treated plants compared to untreated plants. The pot culture study revealed the greater resistance to water stress by green gram plants treated with P. fluorescens Pf1 compared to untreated plants. The greater activity of stress-related enzymes in green gram plants mediated by PGPB could pave the way for developing drought management strategies.     

Isolation of a malachite green-degrading Pseudomonas sp. MDB-1 strain and cloning of the tmr2 gene

The release of malachite green, a commonly used triphenylmethane dye, into the environment is causing increasing concern due to its toxicity, mutagenicity, and carcinogenicity. A bacterial strain that could degrade malachite green was isolated from the water of an aquatic hatchery. It was identified as a Pseudomonas sp. based on the morphological, physiological, and biochemical characteristics, as well as the analysis of 16S rRNA gene sequence and designated as MDB-1. This strain was capable of degrading both malachite green and leucomalachite green, as well as other triphenylmethane dyes including Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase from MDB-1, was cloned, sequenced and effectively expressed in E. coli. These results highlight the potential of this bacterium for the bioremediation of aquatic environments contaminated by malachite green.     

Inhibition of chloroplastic fructose 1,6-bisphosphatase in tomato fruits leads to decreased fruit size,but only small changes in carbohydrate metabolism

A potato (Solanum tuberosum L. ) cDNA coding for the chloroplastic isoform of fructose 1,6-bisphosphatase (cp-FBPase) was utilized to repress its activity in tomatoes (Lycopersicon esculentum Mill.) using antisense techniques. The patatin B33 promoter was used to ensure fruit specificity of the antisense effect. Transgenic plants were isolated in which fructose 1,6-bisphosphatase activity was reduced by more than 50% of the control in green fruits. Immunoblots indicated that the plastidial isoform was almost completely eliminated in the most strongly inhibited lines. Fruits of the transgenic plants were analyzed for levels of metabolites during fruit development. Glucose and fructose concentrations were increased in green fruits in the transgenic lines, but unchanged at later stages of development. The sucrose concentration was low, and was not significantly altered in the transgenic lines. There was net degradation of starch over the developmental period, but the starch content was not decreased. In green fruit the levels of hexose phosphates were unchanged, whilst the level of 3-phosphoglyceric acid was significantly increased in one line. Most importantly the deduced ratio of hexose phosphate to 3-phosphoglyceric acid decreased, consistent with an in vivo inhibition of fructose 1,6-bisphosphatase activity. One consequence of this reduction of in vivo activity of cp-FBPase was that the average weight of fully ripe fruits was significantly decreased by up to 20% in all transgenic lines in comparison with the control.Abbreviations AGPase ADP-glucose pyrophosphorylase - cp-FBPase Chloroplastidic fructose 1,6-bisphosphatase - cy-FBPase Cytoplasmic fructose 1,6-bisphosphatase - DAF Days after flowering     

Gas chromatographic–mass spectrometric investigation of metabolites from the needles and roots of pine seedlings at early stages of pathogenic fungi <Emphasis Type="Italic">Armillaria ostoyae</Emphasis> attack

An investigation was carried out of the composition of metabolites in pine seedlings tissues at the initial stages of the infectious process caused by pathogenic fungi Armillaria ostoyae, which causes a root rot of trees and degradation of forest resources. With the help of successive extraction with organic solvents of different polarity, more than 190 metabolites were extracted from the needles and roots of the seedlings and then identified by GC–MS method. The composition of the extracts from control plants and those inoculated with Armillaria ostoyae were compared. It was established that part of secondary metabolites (glucosamines and free amino acids, carbohydrates raffinose and trehalose) were present only in the tissues of inoculated plants. Possible roles of some of these compounds appearing in the roots of seedlings infected with the fungus are also discussed in the paper.     

Significant reduction in toxicity,BOD, and COD of textile dyes and textile industry effluent by a novel bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. LBC1

The 16S rRNA sequence analysis and biochemical characteristics were confirmed that the isolated bacterium is Pseudomonas sp. LBC1. The commonly used textile dye, Direct Brown MR has been used to study the fate of biodegradation. Pseudomonas sp. LBC1 showed 90% decolorization of Direct Brown MR (100 mg/L) and textile industry effluent with significant reduction in COD and BOD. The optimum condition for decolorization was 7.0 pH and 40°C. Significant increase in a activity of extracellular laccase suggested their possible involvement in decolorization of Direct Brown MR. Biodegradation metabolites viz. 3,6-dihydroxy benzoic acid, 2-hydroxy-7-aminonaphthol-3-sulfonic acid, and p-dihydroperoxybenzene were identified on the basis of mass spectra and using the 1.10 beta Shimadzu NIST GC–MS library. The Direct Brown MR and textile industry effluent were toxic to Sorghum bicolor and Vigna radiata plants as compared to metabolites obtained after decolorization. The Pseudomonas sp. LBC1 could be useful strain for decolorization and detoxification of textile dyes as well as textile industry effluent.     

Phytoremediation potential of Portulaca grandiflora Hook. (Moss-Rose) in degrading a sulfonated diazo reactive dye Navy Blue HE2R (Reactive Blue 172)

Wild and tissue cultured plants of Portulaca grandiflora Hook. have shown to be able to decolorize a sulfonated diazo dye Navy Blue HE2R (NBHE2R) up to 98% in 40 h. A significant induction in the activities of lignin peroxidase, tyrosinase and DCIP reductase was observed in the roots during dye decolorization. The wild plants and tissue cultures could independently decolorize and degrade NBHE2R into metabolites viz. N-benzylacetamide and 6-diazenyl-4-hydroxynaphthalene-2-sulfonic acid. A dye mixture and a textile effluent were also decolorized efficiently by P. grandiflora. The phytotoxicity study revealed reduction in the toxicity due to metabolites formed after dye degradation.     

Effect of detoxified Aflatoxin B1 contaminated products on some in vivo and in vitro plant physiological systems

Aflatoxin B₁ was detoxified (60 %) enzymatically by horseradish peroxidase. In another set of experiments the toxin was detoxified (97 %) by a combination of enzyme and microwave treatments. Chloroform extracts of these reaction products were used to study their effects on some important physiological processes of higher plants. Cell mass growth in suspension culture of Catharanthus roseus showed 34 % and 77 % respective increase in dry weight after 4 to 8 weeks of incubation at room temperature (22 EC) without toxin but in presence of toxin only 21 % and 43 % dry weight increase occurred in the same time interval. Aflatoxin B₁ showed a profound effect on pollen germination and pollen tube morphology leading to only 40 % germination and several morphological anomalies in Catharanthus roseus and Haemanthus Katherinae. Embryogenic callus of Santalum album could give rise to only 60 % somatic embryo in presence of 1mM toxin with several abnormalities which is much less compared to 94 % conversion to distinct bipolar embryos in case of control set without toxin. Only 49 % seeds of Arachis hypogaea germinated in presence of 1mM aflatoxin in the germinating medium compared to 71 % germination in control media.     

Role of microalgal ligninolytic enzymes in industrial dye decolorization

Abstract

In this study, the decolorization efficiency of seven microalgae isolates; Nostoc muscorum, Nostoc humifusum, Spirulina platensis, Anabaena oryzae, Wollea saccata, Oscillatoria sp. and Chlorella vulgaris was investigated for dye decolorization. The highest decolorization percentages of Brazilwood, Orange G, and Naphthol Green B dyes (99.5%, 99.5%, and 98.5%, respectively) were achieved by Chlorella vulgaris. However, the maximum efficiency for dye decolorization percentages of CV and malachite green dyes were exhibited by A. oryzae (97.4%) and W. saccata (93.3%). Ligninolytic enzymes activity assay was carried out for laccase and lignin peroxidase enzymes, which revealed a high efficiency of the C. vulgaris, A. oryzae and W. saccata to lignin containing compound degradation. The highest laccase production recorded by C. vulgaris with Brazilwood, Orange G, and Naphthol Green B dyes (665.0, 678.6, and 659.5?U/ml, respectively). Similarly, C. vulgaris gave a high lignin peroxidase enzyme production with the above three dyes respectively (306.00, 298.34, and 311.45?U/ml). In addition, A. oryzae and W. saccata showed the highest production of the laccase enzyme (634.6 and 577.45?U/ml, respectively) with CV and malachite green dyes. The degradation products have been characterized after decolorization and verified using FTIR analysis. The high decolorization percentages achieved by C. vulgaris, A. oryzae and W. saccata make them potential candidates for bioremediation and pre-processing to remove dyes from textile effluents.     

ALLELOPATHIC GROWTH INHIBITION OF NODULATED AND UNNODULATED ALNUS CRISPA SEEDLINGS BY POPULUS BALSAMIFERA

Water extracts of fresh leaves, buds, and leaf litter of Populus balsamifera (balsam poplar) were tested at different dilutions for allelopathic effects on nodulation, nitrogenase activity and growth of nodulated green alder (Alnus crispa) seedlings, and on growth of unnodulated green alder seedlings. All extracts inhibited height growth, root elongation and dry weight increment of nodulated and unnodulated green alder seedlings to some degree during a 2-month experiment. Foliar nitrogen content of both nodulated and unnodulated seedlings was significantly lower in extract treated plants than in controls. Growth inhibition was about 25% less in nodulated than in unnodulated seedlings. The number of nodules per plant in seedlings treated with any balsam poplar extract was only 51% that of control plants. Acetylene reduction by seedlings treated with bud and leaf litter extracts indicated a decrease of 62% compared to controls. Growth inhibition was not mediated by pH or osmotic effects of the extracts. The possible ecological significance is discussed.     

Studies on phytoremediation potentiality of Typhonium flagelliforme for the degradation of Brilliant Blue R

In vitro culture plants of Typhonium flagelliforme were found to decolorize a variety of dyes, including Malachite Green, Red HE 8B, Methyl Orange, Reactive Red 2, Direct Red 5B (DR5B), Red HE 7B, Golden Yellow HER, Patent Blue, and Brilliant Blue R (BBR), to varying extents within 4 days. The enzymatic analysis of plant roots of aseptically raised plantlets performed before and after degradation of the dye BBR by these plantlets showed a significant induction in the activities of peroxidase, laccase, tyrosinase, and 2,6-dichlorophenol-indophenol reductase, which indicated the involvement of these enzymes in the metabolism of the dye. Comparative study of the enzyme status of the plants Typhonium flagelliforme and Blumea malcolmii during the degradation of DR5B and BBR showed marked variations in the enzyme profile with respect to the use of different sources of the enzyme. Phytoremediation of BBR using Typhonium flagelliforme was confirmed with high performance liquid chromatography and Fourier transform infrared spectroscopy analysis performed before and after the degradation of the dye. One of the products of the metabolism of the dye was identified as 4-(4-ethylimino-cyclohexa-2,5-dienylidinemethyl)-phenylamine with the aid of gas chromatography–mass spectroscopy (GC–MS) analysis. Significant decrease in the American Dye Manufacturer’s Institute, biological oxygen demand, and chemical oxygen demand values of synthetic mixture of textile dyes and industrial effluent confirmed the decolorization and detoxification. Phytotoxicity studies also revealed the nontoxic nature of the metabolites of BBR.     

13C NMR cross polarization and magic angle spinning (CPMAS) and gas chromatography/mass spectrometry analysis of the products from a soda pulp mill effluent decolourised by two Streptomyces strains

Two Streptomyces strains, UAH 30 and UAH 51, have been shown to decolourise a paper-mill effluent obtained after semichemical alkaline pulping of wheat straw. Fractionation of the effluent decolourised by strains UAH 30 and UAH 51 showed that 60% and 80% respectively of the alkali-lignin fraction have been removed from the effluent after 7 days of growth. ¹³C NMR cross polarization and magic angle spinning (CPMAS) spectra of the alkali-lignin remaining in the effluent after decolourisation revealed a decrease in the relative amount of aromatic lignin units compared to that obtained from the untreated effluent along with a reduction in the ratio of syringyl:guaiacyl units. Gas chromatography/mass spectrometry analysis of the low-molecular-mass compounds extracted from the decolourised effluent revealed the presence of new aromatic lignin-related compounds that were not present in the untreated control effluent. This was linked to a general depolymerization of larger lignin molecules occurring during decolourisation by the two Streptomyces strains. Identification of low-molecular-mass aromatic compounds extracted from the decolourised effluent revealed only the presence of p-hydroxyphenyl units in effluents decolourised by the strain UAH 30 while p-hydroxyphenyl, guaiacyl and syringyl units were detected in effluents decolourised by Streptomyces strain UAH 51. The study indicates that, while decolourisation is a common feature of the two Streptomyces strains, the mechanisms involved in the degradation of the lignin fractions may be different and strain-specific. Received: 8 July 1996 / Received revision: 9 October 1996 / Accepted: 14 October 1996     

Effects of Crude and Bioremediated Thermal Power Plant Effluents in Brassica Juncea

Crude phenol and cyanides are the major components of the effluents discharged by industries involved in the manufacture of many synthetic inorganic and organic compounds, pharmaceuticals, electroplating units, and thermal power plants.

In this study, an effort was made to use effluents as manure. The effect of various amendments (10, 40, 70, and 100%) of bioremediated and crude effluents was checked on plants of Brassica juncea. Roots, shoot lengths, and the mean dry weights were maximum in the plants irrigated with 10% treated and 10% crude effluent as compared to the control plants. Total plant phenolics increased with an increase in the amendment of the effluents in the plants irrigated with bioremediated as well as crude effluents. Applications of lower amendments of treated and untreated effluents in Brassica juncea improved the chlorophyll levels in the experimental plants and the values obtained were similar to the control plants. Effect of bioremediated and crude effluents on antioxidant enzymes of Brassica juncea showed a differential effect. The activity of antioxidant enzymes peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) was observed to increase with an increase in the effluent concentration.

Here we demonstrate that the industrial effluents, when bioremediated of their harmful components, can also serve as a nourishment for plants, which can further help in the rehabilitation of wastelands.     

Toxicity and metabolism of malachite green and leucomalachite green during short-term feeding to Fischer 344 rats and B6C3F1 mice

Malachite green, an N-methylated diaminotriphenylmethane dye, has been widely used as an antifungal agent in commercial fish hatcheries. Malachite green is reduced to and persists as leucomalachite green in the tissues of fish. Female and male B6C3F1 mice and Fischer 344 rats were fed up to 1200 ppm malachite green or 1160 ppm leucomalachite green for 28 days to determine the toxicity and metabolism of the dyes. Apoptosis in the transitional epithelium of the urinary bladder occurred in all mice fed the highest dose of leucomalachite green. This was not observed with malachite green. Hepatocyte vacuolization was present in rats administered malachite green or leucomalachite green. Rats given leucomalachite green also had apoptotic thyroid follicular epithelial cells. Decreased T4 and increased TSH levels were observed in male rats given leucomalachite green. A comparison of adverse effects suggests that exposure of rats or mice to leucomalachite green causes a greater number of and more severe changes than exposure to malachite green. N-Demethylated and N-oxidized malachite green and leucomalachite green metabolites, including primary arylamines, were detected by high performance liquid chromatography/mass spectrometry in the livers of treated rats. 32P-Postlabeling analyses indicated a single adduct or co-eluting adducts in the liver DNA. These data suggest that malachite green and leucomalachite green are metabolized to primary and secondary arylamines in the tissues of rodents and that these derivatives, following subsequent activation, may be responsible for the adverse effects associated with exposure to malachite green.     

用复合孔雀绿分析无机磷的改良方法

在研究酸度、钼酸钠浓度及孔雀绿浓度对显色影响的基础上,用复合孔雀绿试剂分析无机磷,该方法平行程度很好．测定10个标准管光吸收的标准差=0.0065,变异系数=1.26%,可以用于测定Na⁺,K⁺-ATP酶活力等．