Azo dye decolorization with a mutant Escherichia coli strain

A recombinant Escherichia coli strain (E. coli NO3) containing genomic DNA fragments from azo-reducing wild-type Pseudomonas luteola strain decolorized a reactive azo dye (C.I. Reactive Red 22) at approx. 17 mg dye h^–1 g cell. The ability to decolorize the azo dye probably did not originate from the plasmid DNA. Acclimation in azo-dye-containing media gave a nearly 10% increase in the decolorization rate of E. coli NO3. Growth with 1.25 g glucose l^–1 completely stopped the decolorization activity. When the decolorization metabolites from E. coli NO3 were analyzed by HPLC and MS, the results suggested that decolorization of the azo dye may be due to cleavage of the azo bond.     

Degradation of dye-containing textile effluent by the agaric white-rot fungus Clitocybula dusenii

Raw mixed-dye wastewater from a textile dye-producing plant was partly decolorized by the agaric white-rot fungus, Clitocybula dusenii. The fungus had higher Mn peroxidase (MnP) and laccase activities when grown with dye effluent than in control cultures. The activity of MnP increased commensurately with the proportion of the raw dye wastewater in the medium (control: 20 U l^–1; 10% v/v effluent: 67 U l^–1; 25% v/v effluent: 130 U l^–1; and 33% v/v effluent: 180 U l^–1). Maximal decolorization rates were achieved over 20 d at 28 °C using four-fold diluted dye-containing effluent on a 5 d pre-grown mycelium.     

Formation of phenylethanoid glycosides by Cistanche deserticola callus grown on solid media

The optimal growth of Cistanche deserticola callus and formation of phenylethanoid glycosides (PeG) was at 25°C with light irradiation intensity of 24 mol m^–2 s^–1 on solidified B5 media supplemented with 0.5 mg 6-benzylaminopurine l^–1, 10 mg gibberellin l^–1, 800 mg casein hydrolysate l^–1 and 20 g sucrose l^–1. After 30 d culture, the biomass reached 15.5 g dry wt callus l^–1 medium and its PEG content was 10.7% (w/w). The PeG content was 42%–127% higher than those in explants.     

Mixed-acid fermentation and polysaccharide production by Lactobacillus helveticus in milk cultures

Lactobacillus helveticus grown in milk with pH control at 6.2 had a slower growth rate (=0.27 h^–1) and produced less exopolysaccharide (49 mg l^–1) but increased lactic acid production (425 mM) compared to cultures without pH control (=0.5 h^–1, 380 mg exopolysaccharide l^–1, and 210 mM lactate), respectively. Both cultures displayed a mixed-acid fermentation with formation of acetate, which is linked not only to citrate metabolism, but also to alternative pathways from pyruvate.     

High density culture of the freshwater rotifer,Brachionus calyciflorus

The freshwater rotifer, Brachionus calyciflorus is one of the live food organisms used for the mass production of larval fish. In this study possibility of obtaining high density cultures of the freshwater rotifer B. calyciflorus were investigated. The two culture systems used differed in their air and dissolved oxygen supplies using three temperatures in each case: 24, 28 and 32 °C. Rotifers were batch-cultured using 5 l-vessels and fed with the freshwater Chlorella. The growth rate of rotifers significantly increased with an increase in temperature. The maximum density of the rotifers with air-supply at 24 °C, 6500 ind. ml^–1, was significantly lower than those cultured at 28 and 32 °C, i.e. 8600 and 8100 ind. ml^–1, respectively. Dissolved oxygen levels decreased with time and ranged from 0.8 to 1.4 mg l^–1 when the density of freshwater rotifer was the highest at each temperature. The highest density (19200 ind. ml^–1) of freshwater rotifer was obtained in cultures with a supply of oxygen at 28 °C. Densities of 13500 and 17200 ind. ml^–1 were found at 24 and 32 °C, respectively. Levels of NH₃-N increased with time and a dramatic increase of NH₃-N was observed at high temperatures. Levels of NH₃-N at 24, 28 and 32 °C were 13.2, 18.5 and 24.5 mg l^–1, respectively. These levels coincided with the highest rotifer density at each of the three temperatures. When rotifers were cultured with an oxygen-supply and pH was adjusted to 7, the maximum density of rotifer reached 33500 ind. ml^–1 at 32 °C . These results suggested that high density culture of freshwater rotifer, B. calyciflorus could be achieved under optimal conditions with DO value of exceeding 5 mg l^–1 and NH₃-N values of lower than 12.0 mg l^–1.     

Production of inulooligosaccharides from inulin by a novel endoinulinase from Xanthomonas sp.

A novel inulinolytic microorganism, Xanthomonas sp. produced an endoinulinase, to be used for inulooligosaccharide (IOS) formation from inulin, at an activity of 11 units ml^–1 (1.2 mg protein ml^–1). The endoinulinase was optimally active at 45°C and pH 6.0. Batchwise production of IOS was carried out by the partially purified endoinulinase with a maximum yield of about 86% on a total sugar basis with 10 g inulin l^–1. The major IOS components were DP (degree of polymerization) 5 and 6 with trace amount of smaller oligosaccharides.     

Temporal variability and production of Euterpina acutifrons (Copepoda: Harpacticoida) in the Cananéia Lagoon estuarine system,São Paulo,Brazil

Diel and seasonal variations in abundance, population structure, biomass and production rate of the harpacticoid copepod Euterpina acutifrons were studied in the Cananéia Lagoon estuarine system, São Paulo, Brazil. Zooplankton samples were collected at 4-h intervals during multiple 24-h periods, from February 1995 to January 1996. Copepodites and adults of E. acutifrons were present in the plankton throughout the year (temperature, 18.6–29.4 °C; salinity, 4.5–33.0 psu; chlorophyll-a concentration, 1.32–20.42 g l^–1). Abundance of E. acutifrons showed considerable diel variations. On most sampling dates, higher abundances were recorded at times when salinity was higher. Biomass varied from 0.044 ± 0.046 (daily mean ± SD) to 5.264±3.425 mg C m^–3. The estimated production rates (minimum ± SD–maximum ± SD) were 0.034±0.035–4.95±3.25 (Ikeda-Motoda model), 0.035±0.036–5.123±3.347 (Huntley-Lopez model), and 0.016±0.017–2.101±1.372 mg C m^–3 d^–1 (Hirst-Sheader model).     

Plasmid mediated silver resistance in Acinetobacter baumannii

Acinetobacter baumannii BL88, an environmental isolate, was resistant to 13 metals and 10 antibiotics. Plumbagin cured resistance to silver, cadmium, antimony, streptomycin and ampicillin at varying frequencies. However, only silver resistance transferred (1 × 10^–6 recepient^–1) to Escherichia coli K12 during conjugation. Correspondingly there was transfer of a 54 kb plasmid (pUPI199) from A. baumannii BL88. The plasmid transformed E. coli DH5 cells at a frequency of 1 × 10^–8 recepient^–1. The growth rate of E. coli DH5; (pUPI199) was slower as compared with E. coli DH5. Plasmid pUPI199 was 76 and 9.6% stable in the host A. baumannii BL88 in the presence and absence of selection pressure, respectively. A. baumannii BL88 was found to accumulate and retain silver whereas E. coli DH5 (pUPI199) effluxed 63% of the accumulated silver ions.     

Synthesis of carotenoids by<Emphasis Type="Italic"> Rhodotorula rubra</Emphasis> GED8 co-cultured with yogurt starter cultures in whey ultrafiltrate

Two cultures, a yeast (Rhodorula rubra GED8) and a yogurt starter (Lactobacillus bulgaricus 2–11+Streptococcus thermophilus 15HA), were selected for associated growth in whey ultrafiltrate (WU) and active synthesis of carotenoids. In associated cultivation with the yogurt culture L bulgaricus 2–11+S. thermophilus 15HA under intensive aeration (1.3 l^–1min^–1 air-flow rate) in WU (45 g lactose l^–1), initial pH 5.5, 30 °C, the lactose-negative strain R. rubra GED8 synthesized large amounts of carotenoids (13.09 mg l^–1 culture fluid). The carotenoid yield was approximately two-fold higher in association with a mixed yogurt culture than in association with pure yogurt bacteria. The major carotenoid pigments comprising the total carotenoids were -carotene (50%), torulene (12.3%) and torularhodin (35.2%). Carotenoids with a high -carotene content were produced by the microbial association 36 h earlier than by Rhodotorula yeast species. No significant differences were notd in the ratio between the pigments synthesized by R. rubra GED8+L. bulgaricus 2–11, R. rubra GED8+S. thermophilus 15HA, and R.rubra GED8+yogurt culture, despite the fact that the total carotenoid concentrations were lower in the mixed cultures with pure yogurt bacteria.     

Phytotoxic activity of selected water-soluble metabolites of Fusarium against Lemna minor L. (Duckweed)

Phytotoxicity and inhibitory effects of the fusarial toxins fumonisin B₁ (FB₁) [m.p. 103–105 °C], fusaric acid [m.p. 106–107 °C], butenolide (4-acetamido-4-hydroxy-2-butenoic acid lactone) [116–117 °C], 9, 10-dihydroxyfusaric acid [m.p. 150–155 ° C], and moniliformin on chlorophyll synthesis in the aquatic macrophyte Lemna minor (duckweed) were examined. FB₁ proved to be most active, reducing the growth of L. minor fronds and their ability to synthesize chlorophyll by 53% and 59%, respectively, at 0.7 g/ml. The growth rate of L. minor was reduced 59% by 6.7 g/ml fusaric acid, 62% by 66.7 g/ml butenolide, and 22% by 66.7 g/ml 9,10-dihydroxyfusaric acid. Moniliformin was the least phytotoxic to L. minor, with only a 16% suppression of growth rate and a 54% reduction in chlorophyll at 66.7 g/ml.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Decolorization of azo dyes by Rhodobacter sphaeroides

Rhodobacter sphaeroides AS1.1737 decolorized more than 90% of several azo dyes (200 mg dyes l^–1) in 24 h. The optimal culture conditions were: anaerobic illumination (1990 lx), peptone as carbon source, temperature 35–40 °C and pH 7–8. Intracellular crude enzyme from this strain had azoreductase activity, optimized temperature as 45–50 °C, and decolorization kinetics which were consistent with a ping-pong mechanism.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation and development of herbicide-resistant sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> species hybrids) using axillary buds

Direct regeneration from explants without an intervening callus phase has several advantages, including production of true type progenies. Axillary bud explants from 6-month-old sugarcane cultivars Co92061 and Co671 were co-cultivated with Agrobacterium strains LBA4404 and EHA105 that harboured a binary vector pGA492 carrying neomycin phosphotransferase II, phosphinothricin acetyltransferase (bar) and an intron containing -glucuronidase (gus-intron) genes in the T-DNA region. A comparison of kanamycin, geneticin and phosphinothricin (PPT) selection showed that PPT (5.0 mg l^–1) was the most effective selection agent for axillary bud transformation. Repeated proliferation of shoots in the selection medium eliminated chimeric transformants. Transgenic plants were generated in three different steps: (1) production of putative primary transgenic shoots in Murashige-Skoog (MS) liquid medium with 3.0 mg l^–1 6-benzyladenine (BA) and 5.0 mg l^–1 PPT, (2) production of secondary transgenic shoots from the primary transgenic shoots by growing them in MS liquid medium with 2.0 mg l^–1 BA, 1.0 mg l^–1 kinetin (Kin), 0.5 mg l^–1 -napthaleneacetic acid (NAA) and 5.0 mg l^–1 PPT for 3 weeks, followed by five more cycles of shoot proliferation and selection under same conditions, and (3) rooting of transgenic shoots on half-strength MS liquid medium with 0.5 mg l^–1 NAA and 5.0 mg l^–1 PPT. About 90% of the regenerated shoots rooted and 80% of them survived during acclimatisation in greenhouse. Transformation was confirmed by a histochemical -glucuronidase (GUS) assay and PCR amplification of the bar gene. Southern blot analysis indicated integration of the bar gene in two genomic locations in the majority of transformants. Transformation efficiency was influenced by the co-cultivation period, addition of the phenolic compound acetosyringone and the Agrobacterium strain. A 3-day co-cultivation with 50 M acetosyringone considerably increased the transformation efficiency. Agrobacterium strain EHA105 was more effective, producing twice the number of transgenic shoots than strain LBA4404 in both Co92061 and Co671 cultivars. Depending on the variety, 50–60% of the transgenic plants sprayed with BASTA (60 g l^–1 glufosinate) grew without any herbicide damage under greenhouse conditions. These results show that, with this protocol, generation and multiplication of transgenic shoots can be achieved in about 5 months with transformation efficiencies as high as 50%.Abbreviations BA 6-Benzyladenine - CaMV Cauliflower mosaic virus - GUS -Glucuronidase - Kin Kinetin - NAA -Naphthaleneacetic acid - Nos Nopaline synthase - nptII Neomycin phosphotransferase II - PCR Polymerase chain reaction - PPT Phosphinothricin - YEP Yeast extract and peptone     

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Tolerance of high temperature and antibiotic resistance

Forty one strains ofRhizobium phaseoli were screened for the ability to multiply at high temperatures on yeast extract-mannitol agar. Most strains were tolerant of 30°C, eight strains were tolerant of 45°C and two of 47°C although the rate of multiplication was reduced at 45–47°C. The high temperature-tolerant strains were isolated from Kenyan soils and were fast-growing. Seven of the eight strains tolerant of 45–47°C lost their infectiveness after incubation at high temperature but four strains tolerant of 40°C remained infective after incubation at that temperature.Thirty six strains were resistant to 200 g ml^–1 streptomycin sulphate and 29 strains to 200 g ml^–1 spectinomycin dihydrochloride. Eight strains were resistant to both antibiotics each at 200 g ml^–1. Two of the double-labelled antibiotic-resistant mutants lost their infectiveness onPhaseolus vulgaris. The response to acidity was unaltered and two of the mutants showed a decrease in temperature tolerance. The doublelabelled mutants were recoverable from two Kenyan soils.     

Decolorization of sulfonated azo dyes with two photosynthetic bacterial strains and a genetically engineered Escherichia coli strain

Sulfonated azo dyes were decolorized by two wild type photosynthetic bacterial (PSB) strains (Rhodobacter sphaeroides AS1.1737 and Rhodopseudomonas palustris AS1.2352) and a recombinant strain (Escherichia coli YB). The effects of environmental factors (dissolved oxygen, pH and temperature) on decolorization were investigated. All the strains could decolorize azo dye up to 900 mg l⁻¹, and the correlations between the specific decolorization rate and dye concentration could be described by Michaelis–Menten kinetics. Repeated batch operations were performed to study the persistence and stability of bacterial decolorization. Mixed azo dyes were also decolorized by the two PSB strains. Azoreductase was overexpressed in E. coli YB; however, the two PSB strains were better decolorizers for sulfonated azo dyes.     

Efficient <Emphasis Type="Italic">in vitro</Emphasis> bulblet regeneration from immature embryos of endangered <Emphasis Type="Italic">Sternbergia fischeriana</Emphasis>

Sternbergia fischeriana is an endangered geophyte and therefore in vitro micropropagation of this plant will have great importance for germplasm conservation and commercial production. Bulb scale and immature embryo explants of S. fischeriana were cultured on different nutrient media supplemented with various concentrations of plant growth regulators. Immature embryos produced higher number of bulblets than bulb scales. Large numbers of bulblets were regenerated (over 80 bulblets/explants) from immature embryos on Murashige and Skoog (MS) medium supplemented with 4 mg l^–1 6-benzylaminopurine (BA) and 0.25 mg l^–1 -naphthaleneacetic (NAA) or 2 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D) after 14 months of culture initiation. Regenerated bulblets were kept at 5 °C for 5 weeks and then transplanted to a potting mixture.     

Biodegradation of Methyl red by Galactomyces geotrichum MTCC 1360

Galactomyces geotrichum MTCC 1360 can decolorize triphenylmethane, azo and reactive high exhaust textile dyes. At shaking condition this strain showed 100% decolorization of a toxic azo dye Methyl red (100 m gl⁻¹) within 1 h in deionized water at 30 °C. The degradation of Methyl red was possible through a broad pH (3–12) and temperature (5–50 °C) range. Glucose and mycelium concentration had increased the decolorization rate, but the addition of 1 gl⁻¹ molasses in deionized water made decolorization possible in only 10 min. Induction in the NADH–dichloro phenol indophenol (NADH–DCIP) reductase, Malachite green reductase, laccase and lignin peroxidase (Lip) activities were observed in the cells obtained after complete decolorization, showing that there is direct involvement in the degradation of Methyl red. The absence of N-N′-dimethyl-p-phenylenediamine (DMPD) in 5 °C, 2-aminobenzoic acid (ABA) in 50 °C and both the compounds in 30 °C sample have shown the differences in the metabolic fate of Methyl red at different temperatures. The untreated dye at 300 mg l⁻¹ concentration showed 88% germination inhibition in Sorghum bicolor, whereas it was 72% in Triticum aestivum. There was no germination inhibition for both the plants by Methyl red metabolites at 300 mg l⁻¹ concentration.

The scientific relevance of the paper

The azo dye Methyl red (100 mg l⁻¹) was decolorized by G. geotrichum MTCC 1360 within 1 h at shaking condition in deionized water. This organism could decolorize Methyl red at wide pH and temperature ranges. Decolorization time was reduced to 10 min by the addition of molasses to deionized water. There was induction in laccase and Lip, NADH–DCIP reductase and Malachite green reductase activities. The metabolic fate of Methyl red changes with temperature which can be evidenced by the formation of 2-ABA at 5 °C, N-N′-DMPD at 50 °C and both the compounds were absent at 30 °C. Phytotoxicity showed that metabolites of dye had induced shoot and root length of both the tested plants.     

Biology of Moina mongolica (Moinidae,Cladocera) and perspective as live food for marine fish larvae: review

Moina mongolica, 1.0-1.4 mm long and 0.8 mm wide, is an Old World euryhaline species. This paper reviewed the recent advances on its autecology, reproductive biology, feeding ecology and perspective as live food for marine fish larviculture. Salinity tolerance of this species ranges from 0.4–1.4 to 65.2–75.4. Within 2–50 salinity, Moina mongolica can complete its life cycle through parthenogenesis. The optimum temperature is between 25 °C and 28 °C, while it tolerates high temperature between 34.4 °C and 36.0 °C and lower temperature between 3.2 °C and 5.4 °C. The non-toxic level of unionised ammonia (24 h LC₅₀) for M. mongolica is <2.6 mg NH₃–N l^–1. Juvenile individuals filter 2.37 ml d^–1 and feed 9.45×10⁶ algal cells d^–1, while mature individuals filter 9.45 ml d^–1 and consume 4.94×10⁶ algal cells d^–1. At 28 °C, M. mongolica reaches sex maturity in 4 d and gives birth once a day afterward; females carry 7.3 eggs brood^–1 and spawn 2.8 times during their lifetime. A variety of food can be used for M. mongolica culture including unicellular algae, yeast and manure, but the best feeding regime is the combination of Nannochloropsis oculata and horse manure. Moina mongolica reproduces parthenogenetically during most lifetime, but resting eggs can be induced at temperature (16 °C) combined with food density at 2000–5000 N. oculata ml^–1. The tolerance to low dissolved oxygen (0.14–0.93 mg l^–1) and high ammonia makes it suitable for mass production. Biochemical analyses showed that the content of eicospantanoic acid (20:53) in M. mongolica accounts for 12.7% of total fatty acids, which is higher than other live food such as Artemia nauplii and rotifers. This cladoceran has the characteristics of wide salinity adaptation, rapid reproduction and ease of mass culture. The review highlights its potential as live food for marine fish larvae.     

Bioconversion of linoleic acid into conjugated linoleic acid during fermentation and by washed cells of Lactobacillus reuteri

Conjugated linoleic acid (CLA) was produced at 300 mg l^–1 after 24 h culture of Lactobacillus reuteri in de Man–Rogosa–Sharpe medium containing 0.9 g linoleic acid (LA) l^–1 and 1.67% (v/v) Tween 80. CLA was mainly located in the extracellular space of the cells. Washed cells previously grown on LA were less active than unadapted washed cells in converting LA into CLA. Most of the CLA transformed by washed L. reuteri cells was located in cells or associated with cells. CLA production by washed L. reuteri cells was most efficient in conversion with 0.45 g LA l^–1 at pH 9.5 and 37°C for 1 h.     

Production of a recombinant, immunogenic protein, P64k, of Neisseria meningitidis in Escherichia coli in fed-batch fermenters

P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, codifying for this protein, was cloned in Escherichia coli and the P64k protein was expressed in Escherichia coli K12 W3110 under the control of the tryptophan promoter. The recombinant bacteria were grown in batch or fed-batch cultures. P64k was expressed as an intracellular soluble form at about 40% of the total cellular protein. A final productivity of 215 mg l^–1 h^–1 and 11 g cell dry wt l^–1 were obtained when the fed-batch culture conditions were optimised, compared to 30% of total protein, and a productivity of 76 mg l^–1 h^–1 and 5.1 g cell dry wt l^–1 in batch cultivation.     

Arsenic precipitation in the bioleaching of enargite by Sulfolobus BC at 70 °C

Enargite (Cu₃AsS₄) was leached at 70°C by Sulfolobus BC in shake-flasks. The highest copper dissolution (52% after 550 h of leaching) was obtained with bacteria and 1 g l^–1 ferric ion. In the absence of ferric ion, Sulfolobus BC catalyzes the bioleaching of enargite through a direct mechanism after adhesion onto the mineral surface. In ferric bioleaching, arsenic precipitated as ferric arsenate and arsenic remained associated to the solid residues, preventing the presence of a high dissolved arsenic concentration in the leaching solution. About 90% inhibition of bacterial growth rate and activity was observed for dissolved arsenic concentrations above 600 mg l^–1 for As(III) and above 1000 mg l^–1 for As(V). Arsenic-bearing copper ores and concentrates could be leached by Sulfolobus BC in the presence of ferric iron due to the favourable precipitation of arsenic ion as ferric arsenate, avoiding significant bacterial inhibition.