Experimental Vibriosis Induction with Vibrio alginolyticus of Larvae of the Catarina Scallop (Argopecten ventricosus =circularis) (Sowerby II, 1842)

Abstract Six-day-old larvae of the catarina scallop, Argopecten ventricosus (=circularis), were infected with different concentrations of Vibrio alginolyticus to determine virulence and to describe vibriosis in this species. The development of vibriosis was compared to the effect of the supernatant of a 24-h V. alginolyticus culture. An experimental larvae culture system (ELCS) yielded a maximum survival of 80% from the 6th to the 19th day (control and low concentrations of V. alginolyticus). No effect was shown with concentrations of V. alginolyticus below 0.5 × 10⁵ CFU ml⁻¹. At concentrations higher than 5.0 × 10⁵ CFU ml⁻¹, swimming depletion, empty stomachs, lipidic granules in the digestive system, velum degradation, and massive mortality were observed. The supernatant of V. alginolyticus culture showed similar effects to the highest concentrations of V. alginolyticus cells. Received: 13 November 1996; Accepted 28 March 1997     

Potential of Trametes trogii culture fluids and its purified laccase for the decolorization of different types of recalcitrant dyes without the addition of redox mediators

Trametes trogii BAFC 463 culture fluids (containing 110 U ml⁻¹ laccase; 0.94 U ml⁻¹ manganese peroxidase), as well as its purified laccase were capable of decolorizing azoic, indigoid, triphenylmethane, anthraquinonic and heterocyclic dyes, in the absence of redox mediators. Six dyes: RBBR, Indigo Carmine, Xylidine, Malachite Green, Gentian Violet and Bromophenol Blue were almost completely degraded (more than 85% decolorization after 1 d) by either laccase or T. trogii itself in culture, proving the role of the enzyme in dye decolorization. The purified laccase also decolorized 65% of Fast Blue RR and 30% of Azure B and Methylene Blue after 24 h. The use of redox mediators significantly increased the decolorization rates (90% decolorization of Azure B after 1 h). 1-hydroxybenzotriazole resulted the best redox mediator, but the natural mediator p-hydroxybenzoic acid also demonstrated its efficiency for dye decolorization. Due to their ability to decolorize recalcitrant dyes without addition of redox mediators, high laccase activities, high thermostability and efficient decolorization at 70 °C and pH 7.0, even in the presence of high concentrations of heavy metals (100 mM Cu⁺², Pb⁺² or Cd⁺²) or in a synthetic dyebath, T. trogii culture fluids could be effectively used to decolorize synthetic dyes from effluents.     

Decolorization of dye and molasses by continuous and semi-continuous jar-fermentor cultures ofGeotrichum candidum Dec 1

Two culture modes, continuous and semi-continuous, of the decolorization fungus,Geotrichum candidum Dec 1, were compared to obtain a high treatment efficiency of molasses decolorization and a large productivity of peroxidase (DyP) to simultaneously decolorize dyes and molasses. The continuous culture ofG. candidum Dec 1 using a 5-l jar-fermentor showed high DyP activity at a low dilution ratio of 0.005h⁻¹, and decolorization ratio of molasses of 80% was obtained concomitantly. Therefore, a semi-continuous culture was performed by repeated refill and draw. In this mode, approximately 1.5 liters of the culture broth was replaced per cycle when the decolorization ratio of molasses was near 80%. The molasses medium (1.0 liter per day) was treated and the peroxidase productivity in the drawn culture broth was 26.6 U/day, whereas the peroxidase productivity was 17.9 U/day in the continuous culture with a dilution rate of 0.005 h⁻¹. The semi-continuous treatment system was an efficient decolorization method for the strain,G. candidum Dec 1.     

Rapid biodegradation and decolorization of Direct Orange 39 (Orange TGLL) by an isolated bacterium <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain BCH

A newly isolated novel bacterium from sediments contaminated with dyestuff was identified as Pseudomonas aeruginosa strain BCH by 16S rRNA gene sequence analysis. The bacterium was extraordinarily active and operative over a wide rage of temperature (10–60°C) and salinity (5–6%), for decolorization of Direct Orange 39 (Orange TGLL) at optimum pH 7. This strain was capable of decolorizing Direct Orange 39; 50 mg l⁻¹ within 45 ± 5 min, with 93.06% decolorization, while maximally it could decolorize 1.5 g l⁻¹ of dye within 48 h with 60% decolorization. Analytical studies as, UV–Vis spectroscopy, FTIR, HPLC were employed to confirm the biodegradation of dye and formation of new metabolites. Induction in the activities of lignin peroxidases, DCIP reductase as well as tyrosinase was observed, indicating the significant role of these enzymes in biodegradation of Direct Orange 39. Toxicity studies with Phaseolus mungo and Triticum aestivum revealed the non-toxic nature of degraded metabolites.     

Factors influencing MOW deinking: Laboratory scale studies

The deinking of MOW is examined at laboratorial scale. The effect of deinking aids, pre-washing and mixing are studied. The operating conditions during pulp treatment affect the pulp and paper properties, interfering with the mechanism of ink removal and modifying the ink particle characteristics. Pre-washing the pulp facilitates the deinking process. Cellulolytic enzymes and deinking chemicals are comparable in terms of ink removal ability.     

Isolation and Purification of Staphylococcal Lipase

An extracellular lipase was isolated from the cell-free supernatant fluid of a 24-hr culture of Staphylococcus aureus grown in Trypticase Soy Broth at 37 C with continuous agitation. The purification was achieved by precipitation with alcohol followed by differential precipitation at pH 8.6 and 4.3. Subsequent purification with Sephadex G 200 and BioGel 300 yielded a preparation which showed a 350- to 450-fold increase in specific activity over the original cell-free supernatant fluid. The purified lipase was homogeneous over a BioGel 300 column and showed a single peak on electrophoresis in a Veronal buffer (pH 8.6, Γ/2 = 0.1). The electrophoretic mobility was -7.78 × 10^-5 cm² per v per sec.     

Hexavalent chromium reduction by a dichromate-resistant gram-positive bacterium isolated from effluents of tanneries

A gram-positive, chromium (Cr)-resistant bacterial strain (ATCC 700729) was isolated from effluent of tanneries. It was grown in media containing potassium dichromate concentration up to 80 mg ml⁻¹ of the medium. The dichromate reducing capability of the bacterium was checked by estimating the amount of Cr VI in the medium before and after introduction of bacterial culture. The influence of factors like pH of the medium, concentration of Cr, and the amount of the inoculum was studied to determine the ability of the bacterium to reduce Cr VI in the medium under various conditions. In a medium containing dichromate 20 mg ml⁻¹ more than 87% reduction of dichromate ions was achieved within 72 h. The feasibility of the use of this bacterial strain for detoxification of dichromate in the industrial wastewater has been assessed. The isolated strain can be exploited for specific environmental clean-up operations. Received: 7 April 1999 / Accepted: 12 September 1999     

Decolorization of anthraquinone dye by Shewanella decolorationis S12

A new species of genus Shewanella, Shewanella decolorationis S12, from activated sludge of a textile-printing wastewater treatment plant, can decolorize Reactive Brilliant Blue K-GR, one kind of anthraquinone dye, with flocculation first. Although S. decolorationis displayed good growth in an aerobic condition, color removal was the best in an anaerobic condition. For color removal, the most suitable pH values and temperatures were pH 6.0–8.0 and 30–37°C under anaerobic culture. More than 99% of Reactive Brilliant Blue K-GR was removed in color within 15 h at a dye concentration of 50 mg/l. Lactate was the suitable carbon source for the dye decolorization. A metal compound, HgCl₂, had the inhibitory effect on decolorization of Reactive Brilliant Blue K-GR, but a nearly complete decolorization also could be observed at a HgCl₂ concentration of 10 mg/l. The enzyme activities, which mediate the tested dye decolorization, were not significantly affected by preadaptation of the bacterium to the dye.     

Bacterial Decolorization of Azo Dyes by Rhodopseudomonas palustris

Summary The ability of Rhodopseudomonas palustris AS1.2352 possessing azoreductase activity to decolorize azo dyes was investigated. It was demonstrated that anaerobic conditions were necessary for bacterial decolorization, and the optimal pH and temperature were pH 8 and 30–35 °C, respectively. Decolorization of dyes with different molecular structures was performed to compare their degradability. The strain could decolorize azo dye up to 1250 mg l⁻¹, and the correlation between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Long-term repeated operations showed that the strain was stable and efficient during five runs. Cell extracts from the strain demonstrated oxygen-insensitive azoreductase activity in vitro.     

Enzymatic deinking of secondary fibers: cellulases/hemicellulases versus laccase-mediator system

The use of enzymes has been suggested as an environmentally friendly alternative to complement conventional chemical deinking in the recycling of recovered paper. This study compares the use of cellulases/hemicellulases versus the laccase-mediator system for deinking printed fibers from newspapers and magazines. For this purpose, two commercial enzyme preparations with endoglucanase and endoxylanase activities (Viscozyme Wheat from Aspergillus oryzae and Ultraflo L from Humicola insolens, Novozymes) and a commercial laccase (NS51002 from Trametes villosa, Novozymes), the latter in the presence of synthetic or natural (lignin-related) mediators, were evaluated. The enzymatic treatments were studied at the laboratory scale using a standard chemical deinking sequence consisting of a pulping stage; an alkaline stage using NaOH, sodium silicate and fatty acid soap; and a bleaching stage using hydrogen peroxide. The handsheets were then prepared and their brightness, residual ink concentration, and strength properties were measured. Among the different enzymatic treatments assayed, both carbohydrate hydrolases were found to deink the secondary fibers more efficiently. Brightness increased up to 3–4% ISO on newspaper fibers, being Ultraflo 20% more efficient in the ink removal. Up to 2.5% ISO brightness increase was obtained when magazine fibers were used, being Viscozyme 9% more efficient in the ink removal. Regarding the laccase-mediator system, alone or in combination with carbohydrate hydrolases, it was ineffective in deinking both newspaper and magazine fibers, resulting in pulps with worse brightness and residual ink concentration values. However, pulp deinking by the laccase-mediator system was displayed when secondary fibers from printed cardboard were used, obtaining up to 3% ISO brightness increase and lower residual ink concentrations.     

Simultaneous production and induction of cellulolytic and xylanolytic enzymes in aStreptomyces sp.

Extracellular cellulolytic and xylanolytic enzymes ofStreptomyces sp. EC22 were produced during submerged fermentation. The cell-free culture supernatant of the streptomycete grown on microcrystalline cellulose contained enzymes able to depolymerize both crystalline and soluble celluloses and xylans. Higher cellulase and xylanase activities were found in the cell-free culture supernatant of the strain when grown on microcrystalline cellulose than when grown on xylan. Total cellulase and endoglucanase [carboxymethyl-cellulase (CMCase)] activities reached maxima after 72 h and xylanase activity was maximal after 60h. Temperature and pH optima were 55°C and 5.0 for CMCase activity and 60°C and 5.5 for total crystalline cellulase and xylanase activities. At 80°C, approximate half-lives of the enzymes were 37, 81 and 51 min for CMCase, crystalline cellulose depolymerization and xylanase, respectively.     

Decolorization of an industrial effluent by free and immobilized cells of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> AAP56<Emphasis Type="Italic">.</Emphasis> Implementation of efficient down flow column reactor

A bacterial strain AAP56, isolated from a polluted soil (from Kelibia city) and identified as Stentrophomonas maltophilia, was particularly interesting for its ability to decolorize recalcitrant dyes of an industrial effluent: SITEX Black. The final percentage decolorization 60% was shown by bacterial culture after incubation in LB medium at 30°C under shaking conditions. The decolorization was closely correlated with the metabolic bacterial growth. The replacement of yeast extract in LB medium composition by soya flour was clearly efficient to enhance the percentage decolorization by 20% and also to reduce the growth medium cost 60-fold. The bacteria were able to reduce 23% from the initial COD and 28% from the initial BOD₅ of the effluent. The immobilization of bacterial cells in calcium alginate beads improved by 25% the effectiveness of the biotransformation within 24 h in batch conditions. The potential of a downflow fixed column reactor (DFCR) to decolorize SITEX Black was evaluated under dilution rate. The best decolorization percentage (82%) was recorded at 0.3 h⁻¹. This bioprocess seems to be a potentially useful method to remediate the colored textile wastewater.     

Extracellular α-amylase from Thermus filiformis Ork A2: purification and biochemical characterization

An extracellular α-amylase produced by the thermophilic bacterium Thermus filiformis Ork A2 was purified from cell-free culture supernatant by ion exchange chromatography. The molecular mass was estimated to be 60 000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was rich in both basic and hydrophobic amino acids, presenting the following NH₂-terminal amino acid sequence: Thr-Ala-Asp-Leu-Ile-Val-Lys-Ile-Asn-Phe. Amylolytic activity on soluble starch was optimal at pH 5.5–6.0 and 95°C, and the enzyme was stable in the pH range of 4.0–8.0. Calcium enhanced thermostability at temperatures above 80°C, increasing the half-life of activity to more than 8 h at 85°C, 80 min at 90°C, and 19 min at 95°C. Ethylenediaminetetraacetic acid (EDTA) inhibited amylase activity, the inhibition being reversed by the addition of calcium or strontium ions. The α-amylase was also inhibited by copper and mercuric ions, and p-chloromercuribenzoic acid, the latter being reversed in the presence of dithiothreitol. Dithiothreitol and β-mercaptoethanol activated the enzyme. The α-amylase exhibited Michaelis-Menten kinetics for starch, with a K _m of 5.0 mg·ml⁻¹ and k _cat/K _m of 5.2 × 10⁵ ml·mg⁻¹ s⁻¹. Similar values were obtained for amylose, amylopectin, and glycogen. The hydrolysis pattern was similar for maltooligosaccharides and polysaccharides, with maltose being the major hydrolysis product. Glucose and maltotriose were generated as secondary products, although glucose was produced in high levels after a 6-h digestion. To our knowledge this is the first report of the characterization of an α-amylase from a strain of the genus Thermus. Received: June 2, 1997 / Accepted: September 16, 1997     

Autoinduction of a genetic locus encoding putative acyltransferase in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

A genetic locus, encoding putative acyltransferase, was induced by autoinducers in Corynebacterium glutamicum. The autoinducers were maximally produced by the bacterium after 24 h culture. Those molecules are resistant to proteinase K treatment (300 μg ml⁻¹) for 30 min at 37°C or at 121°C for 15 min, and remained stable after extensive storage at 4°C. Autoinducers in the cell-free culture fluids from Corynebacterium ammoniagenes and Pseudomonas aeruginosa also induced the expression of acyltransferase in C. glutamicum, suggesting possible cross-recognition of the autoinducers by C. glutamicum. C. glutamicum thus possesses an autoinduction system which secretes autoinducers during growth, triggering the expression of downstream genes, exemplified by the putative acyltransferase gene.     

Indigestible dextrin is an excellent inducer for α-amylase, α-glucosidase and glucoamylase production in a submerged culture of Aspergillus oryzae

α-Amylase activities of Aspergillus oryzae grown on dextrin or indigestible dextrin were 7·8 and 27·7 U ml⁻¹, respectively. Glucoamylase activities of the cultures grown on dextrin or indigestible dextrin were 5·4 and 301 mU ml⁻¹, respectively. The specific glucoamylase production rate in indigestible dextrin batch culture reached 1·35 U g DW⁻¹ h⁻¹. In contrast, biomass concentration of A. oryzae in indigestible dextrin culture was 35% of that in dextrin culture. Thus, the culture method using indigestible dextrin has the potential to improve amylolytic enzyme production and fungal fermentation broth rheology.     

Evaluation Antimicrobial and Antiadhesive Properties of the Biosurfactant Lunasan Produced by <Emphasis Type="Italic">Candida sphaerica</Emphasis> UCP 0995

Different groups of biosurfactants exhibit diverse properties and display a variety of physiological functions in producer microorganisms; these include enhancing the solubility of hydrophobic/water-insoluble compound, heave metal binding, bacterial pathogenesis, cell adhesion and aggregation, quorum sensing and biofilm formation. Candida sphaerica was grown in a low cost medium, consisting of distilled water supplemented with 9% refinery residue of soybean oil and 9% corn steep liquor, for 144 h at 28°C and 150 rpm. The cell-free supernatant obtained at the end of the experiments was submitted to extraction, and afterward the biosurfactant was isolated using methanol with a yield of 9 g l⁻¹. The critical micelle concentration of the biosurfactant was found to be 0.25 mg ml⁻¹ with a surface tension of 25 mN m⁻¹. Several concentrations of the biosurfactant (0.625–10 mg ml⁻¹) were used to evaluate its antimicrobial and antiadhesive activities against a variety of microorganisms. The biosurfactant showed antimicrobial activity against Streptococcus oralis (68%), Candida albicans (57%), and Staphylococcus epidermidis(57.6%) for the highest concentration tested. Furthermore, the biosurfactant at a concentration of 10 mg ml⁻¹ inhibited the adhesion between 80 and 92% of Pseudomonas aeruginosa, Streptococcus agalactiae, Streptococcus sanguis12. Inhibition of adhesion with percentages near 100% occurred for the higher concentrations of biosurfactant used. Results gathered in this study point to a potential use of the biosurfactant in biomedical applications.     

Decolorization of Some Reactive Textile Dyes by White Rot Fungi Isolated in Pakistan

Summary Four white-rot fungi isolated in Pakistan were used for decolorization of widely used reactive textile dyestuffs. Phanerochaete chrysosporium, Coriolus versicolor, Ganoderma lucidum and Pleurotus ostreatus were grown in defined nutrient media for decolorization of Drimarene Orange K-GL, Remazol Brilliant Yellow 3GL, Procion BluePX-5R and Cibacron Blue P-3RGR for 10 days in shake flasks. Samples were removed every day, centrifuged and the absorbances of the supernatants were read to determine percentage decolorization. It was observed that P. chrysosporium and C. versicolor could effectively decolorize Remazol Brilliant Yellow 3GL, Procion BluePX-5R and Cibacron Blue P-3RGR. Drimarene Orange K-GL was completely decolorized (0.2 g/l after 8 days) only by P.chrysosporium, followed by P. ostreatus (0.17 g/l after 10 days). P. ostreatus also showed good decolorization efficiencies (0.19–0.2 g/l) on all dyes except Remazol Brilliant Yellow (0.07 g/l after 10 days). G. lucidum did not decolorize any of the dyestuffs to an appreciable extent except Remazol Brilliant Yellow (0.2 g/l after 8 days).     

Control of the rootknot nematode meloidogyne javanica by Bacillus cereus

Exposure of Meloidogyne javanica second‐stage juveniles to the bacterium Bacillus cereus in soil inhibited the penetration of the juvenile nematodes into tomato roots. Culture filtrate of the bacterium grown on nutrient broth and tryptic soy broth revealed nematocidal activity on M. javanica juveniles and eggs. Loss of the nematocidal activity of the media by lowering pH, boiling or dialysis raised the possibility that the active ingredient in the culture filtrate was ammonia, released during the breakdown process of peptides in the media by bacterial activity. Free ammonia (NH₃) concentrations in the nutrient broth and tryptic soy broth culture filtrates measured after 48 h were 140 and 190 µg ml^?1 respectively. Exposure of second‐stage juveniles to 9.3 µg ml^?1 ammonia for 40 h in vitro was lethal to 95% of the nematode population. In a nitrate medium, nitrite accumulated up to 250 µg ml^?1 during the growth of the bacterium, and its culture filtrate revealed nematocidal activity. The nematocidal activity of the bacterium increased when the bacterium was applied with various proteinaceous supplements to soil. Soil treated with the bacteria and peptone showed an earlier nematocidal activity than either the bacteria or peptone applied alone, and also had a higher level of ammonia than the individual treatments. However, the level of ammonia was lower than the lethal level for second‐stage juveniles recorded in vitro. The nematocidal activity exhibited by the bacterium‐proteinaceous amendment combination is not fully understood; the ammonia released during protein degradation by the bacterium may contribute significantly to the recorded nematocidal activity.     

Decolorization of Direct Red 28 by mixed bacterial culture in an up-flow immobilized bioreactor

Aerobic mixed bacterial culture comprised of five isolates (Bacillus vallismortis, B. pumilus, B. cereus, B. subtilis and B. megaterium) identified by 16srDNA analysis was developed from wastewater samples from the aeration tank of an effluent treatment plant of a textile and dyeing industry and evaluated for its ability to decolorize azo dye Direct Red 28 in an up-flow immobilized packed bed bioreactor using marble chips as support matrix. The bioreactor was operated under two parameters: an aeration rate of 0.4 and 0.6 mmol/min at a flow rate of 60, 90 and 120 ml/h, respectively. At a constant aeration rate of 0.4 mmol/min and with flow rates of 60, 90 and 120 ml/h, optimum decolorization of 91, 75 and 72% was observed, while at an aeration rate of 0.6 mmol/min and flow rates of 60, 90 and 120 ml/h, optimum decolorization of 93, 78 and 72% was observed over 10 days. The study concluded that across the two aeration rates and the respective flow rates, the higher aeration rate of 0.6 mmol/min along with a flow rate of 60 ml/h was best suited to decolorize Direct Red 28 in the packed bed bioreactor. Spectral changes of the input and output of the bioreactor by UV–visible spectroscopy indicated decolorization of the dye solution by degradation in addition to the visual observation of the biosorption process.     

Studies on Amylolytic Enzymes Produced by Endomyces sp.

Purification of amylase produced by Endomyces sp. IFO 0111 was carried out. A highly purified amyloglucosidase preparation was obtained from culture broth by means of precipitation with ammonium sulfate, decolorization with rivanol, precipitation with acetone and zone electrophoresis. The homogeneity of the preparation was proved by ultracentrifugation and electrophoresis. General properties of the preparation were investigated.