Chromate reduction by PVA-alginate immobilized <Emphasis Type="Italic">Streptomyces griseus</Emphasis> in a bioreactor

Microbial reduction of toxic Cr⁶⁺ to the less toxic Cr³⁺ is potentially a useful bioremediation process. Among the matrices tested for whole cell immobilization of an efficient chromate-reducing Streptomyces griseus strain, PVA-alginate was the most effective and was used for reduction of Cr(VI) in a bioreactor. Cr⁶⁺ reduction efficiency decreased as Cr⁶⁺ was increased from 2 to 12 mg l⁻¹ but increased with an increase in biomass concentration. However, increasing the flow rate from 2 to 8 ml h⁻¹ did not significantly affect Cr⁶⁺ reduction. The reduction was faster in simulated effluent than in synthetic medium and complete removal of 8 mg Cr⁶⁺ l⁻¹ from effluent and synthetic medium occurred in 2 and 12 h, respectively. Our results indicate that immobilized S. griseus cells could be applied for the large-scale bioremediation of chromate-containing effluents and wastewaters.     

Mineralization of diuron [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] by co-immobilized <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. and <Emphasis Type="Italic">Delftia acidovorans</Emphasis>

Mineralization of diuron has not been previously demonstrated despite the availability of some bacteria to degrade diuron into 3,4-dichloroaniline (3,4-DCA) and others that can mineralize 3,4-DCA. A bacterial co-culture of Arthrobacter sp. N4 and Delftia acidovorans W34, which respectively degraded diuron (20 mg l⁻¹) to 3,4-DCA and mineralized 3,4-DCA, were able to mineralize diuron. Total diuron mineralization (20 mg l⁻¹) was achieved with free cells in co-culture. When the bacteria were immobilized (either one bacteria or both), the degradation rate was higher. Best results were obtained with free Arthrobacter sp. N4 cells co-cultivated with immobilized cells of D. acidovorans W34 (mineralization of diuron in 96 h, i.e., 0.21 mg l⁻¹ h⁻¹ vs. 0.06 mg l⁻¹ h⁻¹ with free cells in co-culture).     

Enhanced phenol degradation by immobilized Acinetobacter sp. strain AQ5NOL 1

A locally isolated Acinetobacter sp. Strain AQ5NOL 1 was encapsulated in gellan gum and its ability to degrade phenol was compared with the free cells. Optimal phenol degradation was achieved at gellan gum concentration of 0.75% (w/v), bead size of 3 mm diameter (estimated surface area of 28.26 mm²) and bead number of 300 per 100 ml medium. At phenol concentration of 100 mg l⁻¹, both free and immobilized bacteria exhibited similar rates of phenol degradation but at higher phenol concentrations, the immobilized bacteria exhibited a higher rate of degradation of phenol. The immobilized cells completely degrade phenol within 108, 216 and 240 h at 1,100, 1,500 and 1,900 mg l⁻¹ phenol, respectively, whereas free cells took 240 h to completely degrade phenol at 1,100 mg l⁻¹. However, the free cells were unable to completely degrade phenol at higher concentrations. Overall, the rates of phenol degradation by both immobilized and free bacteria decreased gradually as the phenol concentration was increased. The immobilized cells showed no loss in phenol degrading activity after being used repeatedly for 45 cycles of 18 h cycle. However, phenol degrading activity of the immobilized bacteria experienced 10 and 38% losses after the 46 and 47th cycles, respectively. The study has shown an increased efficiency of phenol degradation when the cells are encapsulated in gellan gum.     

Enhancement of phenol degradation by free and immobilized mixed culture of Providencia stuartii PL4 and Pseudomonas aeruginosa PDM isolated from activated sludge

Biodegradation of phenol has been investigated using a bacterial consortium consisting of two bacterial isolates; one of them used for the first time in phenol biodegradation. This consortium was isolated from activated sludge and identified as Providencia stuartii PL4 and Pseudomonas aeruginosa PDM (accession numbers KY848366 and MF445102, respectively). The degradation of phenol by this consortium was optimal at pH 7 with using 1500?mg?l^?1 ammonium chloride as a nitrogen source. Interestingly, after optimizing the biodegradation conditions, this consortium was able to degrade phenol completely up to 1500?mg?l^?1 within 58?h. The immobilization of this consortium on various supporting materials indicated that polyvinyl alcohol (PVA)-alginate beads and polyurethane foam (PUF) were more suitable for biodegradation process. The freely suspended cells could degrade only 6% (150?mg?l^?1) of 2500?mg?l^?1 phenol, whereas, the immobilized PVA-alginate beads and the immobilized PUF degraded this concentration completely within 120?h of incubation with degradation rates (q) 0.4839 and 0.5368 (1/h) respectively. Thus, the immobilized consortium of P. stuartii PL4 and P. aeruginosa PDM can be considered very promising in the treatment of effluents containing phenol.     

Agrobacterium tumefaciens-mediated transformation of Lesquerella fendleri L., a potential new oil crop with rich lesquerolic acid

A protocol was developed for regeneration and Agrobacterium-mediated genetic transformation of Lesquerella fendleri. Calli were first induced from hypocotyls and cotyledons on MS plus 0.5 mg l⁻¹ BA, 1 mg l⁻¹ NAA and 1 mg l⁻¹ 2,4-D, then co-cultivated for 2–3 days in darkness on MS supplemented with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹ NAA and 100 μmol l⁻¹As together with Agrobacterium tumefaciens strain EHA105/pCAMBIA1301 that harbored genes for uidA (GUS) and hygromycin resistance. Following co-cultivation, calli transfected by A. tumefaciens were transferred to MS with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹NAA, 500 mg l⁻¹ Cef and 10 mg l⁻¹ hygromycin and cultured for 10 days, then the hygromycin was increased to 20 mg l⁻¹ on the same medium. After 4 weeks the resistant regenerants were transferred to MS with 0.5 mg l⁻¹BA, 0.2 mg l⁻¹ NAA, 500 mg l⁻¹ Cef and 25 mg l⁻¹ hygromycin for further selections. Transgenic plants were confirmed by polymerase chain reaction analysis, GUS histochemical assay and genomic Southern blot hybridization. With this approach, the average regeneration frequency from transfected calli was 22.70%, and the number of regenerated shoots per callus was 6–13. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for improvement of this Lesquerella species.     

The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of <Emphasis Type="Italic">Gentiana kurroo</Emphasis> (Royle)

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated in terms of their efficiency in producing viable cultures and regenerating whole plants. With liquid culture, cell division occurred in only a low number of the protoplasts isolated, and no plant regeneration was successful. Cell division occurred within 2 or 3 days in case of agarose solidified media. After 10 days of culture, the number of dividing cells was the highest with modified MS medium in which NH₄NO₃ was replaced with 3.0 g l⁻¹ glutamine. The best results were obtained with agarose bead cultures: plating efficiency was 68.7% and 58.1% for protoplasts isolated from cotyledon and hypocotyl derived suspensions, respectively. The results were achieved with using medium containing 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ dicamba + 0.1 mg l⁻¹ NAA + 80 mg l⁻¹ adenine sulfate. Protocalluses transferred on the following composition of plant growth regulators: 0.5 mg l⁻¹ 2,4-D + 1.0 mg l⁻¹ kinetin or 1.0 mg l⁻¹ kinetin + 0.5 mg l⁻¹ GA₃ + 80.0 mg l⁻¹ adenine sulfate developed in embryogenic cultures. However, the best embryo production occurred with the first one. Later embryos were transferred to half-strength MS mineral salts to promote plants formation. Flow cytometry studies revealed increased amounts of DNA in about one third of the regenerants.     

Plantlet regeneration from protoplast-derived embryogenic calli of Crocus cancellatus

Plant regeneration from protoplast culture of Crocus cancellatus was investigated using regenerable embryogenic calli obtained from shoot meristem culture on LS (Linsmaier and Skoog, 1965) medium containing 4 mg l⁻¹ kinetin and 1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Protoplasts were isolated directly from embryogenic calli. The best protoplast growth was found on those embedded in Ca-alginate beads and cultured with nurse cells in MS (Murashige and Skoog, 1962) medium supplemented with 2 mg l⁻¹ kinetin, 1 mg l⁻¹ 2,4-D and 100 mg l⁻¹ ascorbic acid at 25 °C in darkness. After 4–5 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads, but the protoplasts without immobilization in Ca-alginate beads showed very low cell division. Growth of the microcalli in the medium with nurse cells was much better than in the medium without nurse cells. Transferring beads onto half strength MS medium supplemented with 0.2 mg l⁻¹ kinetin and 0.1 mg l⁻¹ 2,4-D, increased the growth of embryogenic calli. Somatic embryo development was observed either on half strength MS medium growth regulator free or with 1 mg l⁻¹ abscisic acid. Matured embryos germinated on half strength MS medium containing 25 mg l⁻¹ of gibberelic acid. Plantlet formation was obtained on half strength MS medium containing 1 mg l⁻¹ 6-benzyladenine and 1 mg l⁻¹ α-naphthaleneacetic acid at 20 °C in a 16/8 h light/dark cycle. This revised version was published online in June 2006 with corrections to the Cover Date.     

Alginate immobilization of recombinant Escherichia coli whole cells harboring l-arabinose isomerase for l-ribulose production

Recombinant Escherichia coli whole cells harboring Bacillus licheniformis l-arabinose isomerase (BLAI) were immobilized with alginate. The operational conditions for immobilization were optimized with response surface methodology. Optimal alginate concentration, Ca²⁺ concentration, and cell mass loading were 1.8% (w/v), 0.1 M, and 44.5 g L⁻¹, respectively. The interactions between Ca²⁺ concentration, alginate concentration, and initial cell mass were significant. After immobilization of BLAI, cross-linking with 0.1% glutaraldehyde significantly reduced cell leakage. The half-life of immobilized whole cells was 150 days, which was 50-fold longer than that of free cells. In seven repeated batches for l-ribulose production, the productivity was as high as 56.7 g L⁻¹ h⁻¹ at 400 g L⁻¹ substrate concentration. The immobilized cells retained 89% of the initial yield after 33 days of reaction. Immobilization of whole cells harboring BLAI, therefore, makes a suitable biocatalyst for the production of l-ribulose, particularly because of its high stability and low cost.     

Somatic embryogenesis of <Emphasis Type="Italic">Gentiana cruciata</Emphasis> (L.): Histological and ultrastructural changes in seedling hypocotyl explant

Summary Structure and ultrastructure changes that occurred during tissue culture of upper explants of hypocotyl (adjacent to cotyledons) of 10-d-old seedlings of Gentiana cruciata were studied. The explants were cultured on Murashige and Skoog induction medium supplemented with 1.0 mg l⁻¹ dicamba +0.1 mg l⁻¹ naphthaleneacetic acid +2.0 mg l⁻¹ benzyladenine +80.0 mg l⁻¹ adenine sulfate. The initial response of the explant and callus formation were ultrastructurally analyzed during the first 11 d of culture. After 6–8 wk, various methods were employed to collect evidence of indirect somatic embryogenesis. After 48 h of culture, the earliest cell response was cell division of epidermis and primary cortex. There were numerous disturbances of karyo- and cytokinesis, leading to formation of multinuclear cells. With time, the divisions ceased, and cortex cells underwent strong expansion, vacuolization and degradation. About the 6th day of culture, callus tissue proliferated and the initial divisions of vascular cylinder cells were observed. Their division appeared normal. Cells originating from that tissue were small, weakly vacuolated, with dense cytoplasm containing active-looking cell organelles. Numerous divisions occurred in the vascular cylinder, which led to its expansion and the formation of embryogenic callus tissue. During the 6–8th wk of culture, in the proximal end of the explant, masses of somatic embryos were formed from outer parts of intensively proliferating tissue.     

The use of TTC reduction assay for assessment of Gentiana spp. cell suspension viability after cryopreservation

This study was aimed at improving the 2,3,5-triphenyl-tetrazoliumchloride (TTC) reduction test for initial assessment of cell survival after cryopreservation. Experiments were carried out on three embryogenic cell suspensions of different ages: 9-year-old Gentiana tibetica (King ex Hook. F.), 2-year-old G. kurroo (Royle), and 1-year-old G. cruciata (L.). The suspensions were maintained in MS medium supplemented with 1.0 mg 1⁻¹ 3,6-dichloro-o-anisic acid, 0.1 mg 1⁻¹ naphthaleneacetic acid, 2.0 mg l⁻¹ 6-benzylaminopurine, 80.0 mg 1⁻¹ adenine sulphate and 0.09 M sucrose. Four weeks before freezing, part of the tissue was subcultured to the same medium with sucrose concentrations elevated from 0.09 M (3%sMS) to 0.175 M (6%sMS) or 0.26 M (9%sMS). In freezing treatments without cryoprotection, tissue was plunged directly into liquid nitrogen (LN) or cooled gradually. In freezing treatments with cryoprotection, the cells were pretreated with 1 M sucrose, or with 0.4 M sorbitol + 0.25 M proline or + 0.08 M DMSO, or with vitrification solution (PVS2). Encapsulation was another variant. TTC reduction activity was spectrophotometrically assessed immediately, 1, 3, 5, 24 and 48 h after thawing. Cells without cryoprotection were lethally damaged, but TTC reduction activity in those cells ranged from 6.5% (tissue from 3%sMS) to 73 % (tissue from 9%sMS) directly after thawing. Formazan production was reduced to zero after 24 h. The TTC test showed 50% formazan content immediately after thawing of DMSO-protected G. tibetica tissue, but only 22.47% after 24 h and 2.9% after 48 h. Ultrastructural analysis of those cells showed lethal damage in many of them. For the PVS2 treatment, the formazan content was similar in samples analyzed directly after thawing and 24 h later. Cells treated with PVS2 did not show structural disturbances. Encapsulated cell aggregates of G. cruciata treated with concentrations of sucrose increasing up to 1 M produced 2.6 times more formazan. When applied at least 48 h after thawing, the TTC test can reflect cell viability and can be used to compare the effectiveness of cryoprotectant performance and freezing protocols, but it must be carefully evaluated, with appropriate controls.     

Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method

Shoot tips obtained from in vitro Rosa plants (three cultivars) were successfully cryopreserved by a combined droplet vitrification method and subsequently shoots regenerated. The excised shoot tips (1–4 mm long) were incubated in a liquid MS medium supplemented with 2.5 mg l⁻¹ thiamine, 0.2 mg l⁻¹ biotin, 0.2 mg l⁻¹ pyridoxine, 0.25 mg l⁻¹ 6-benzylaminopurine (BAP), 0.5 mg l⁻¹ gibberellic acid (GA₃) and 0.08 M sucrose, for 24 h. Following that incubation shoot tips were pre-cultured in this MS medium containing 0.1 till 1.0 M sucrose for 24 and 48 h, respectively. Pre-cultured shoot tips were dehydrated with concentrated PVS2 cryoprotective solution for 10–30 min at room temperature, prior to a direct plunge in liquid nitrogen. After rapid rewarming in the above mentioned liquid medium shoot tips were plated on a modified MS medium (5 g l⁻¹ agar) supplemented with vitamins and plant growth regulators as mentioned above for regrowth. Cryopreserved shoot tips resumed growth within 10 days and regenerated shoots within 3 weeks. The highest numbers of regrowing shoot tips were 64.44% for cv. Kardinal, 67.73% for cv. Fairy and 57.57% for cv. Maidy.     

Heavy metal resistant <Emphasis Type="Italic">Distigma proteus</Emphasis> (Euglenophyta) isolated from industrial effluents and its possible role in bioremediation of contaminated wastewaters

Summary The alga, Distigma proteus, isolated from industrial wastewater showed tolerance against Cd²⁺ (8.0 μg/ml), Cr⁶⁺ (12 μg/ml), Pb²⁺ (15 μg/ml) and Cu²⁺ (10 μg/ml). The metal ions slowed down the growth of the organism after 4–5 days of exposure. The reduction in cell population was 90% for Cu²⁺, 84% for Cd²⁺, 71% for Cr⁶⁺, and 63% for Pb²⁺ after 8 days of metal stress. The order of resistance to heavy metal, in terms of reduction in the cellular population, was Cu²⁺ > Cd²⁺ > Cr⁶⁺ > Pb²⁺. Chromium- and cadmium-processing capabilities of the alga were worked out for its potential use as a bioremediator of wastewater. The reduction in the amount of Cr⁶⁺ after 2, 4, 6 and 8 days of algal culture containing 5.0 μg Cr⁶⁺ ml⁻¹ of culture medium was 77, 85, 92 and 97%, respectively. Distigma could also remove 48% Cd²⁺after 2 days, 68% after 4 days, 80% after 6 days and 90% after 8 days from the medium. The heavy metal uptake ability of Distigma can be exploited for metal detoxification and environmental clean-up operations.     

Simplified clonal multiplication of mulberry using liquid shake culture

Organogenesis was induced in callus derived from mature zygotic embryos of six families of loblolly pine (Pinus taeda L.) within 24 weeks of culture. Elongation of adventitious buds was achieved on TE medium supplemented with 0.5 mg l⁻¹ indole-3-butyric acid (IBA) and 2 mg l⁻¹ 6-benzyladenine (BA). The most suitable medium for root formation proved to be TE medium supplemented with 0.1 mg l⁻¹ IBA, 1 mg l⁻¹ BA, and 0.5 mg l⁻¹ gibberellic acid (GA₃). One hundred and sixty-nine regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1:1:1) soil mixture, and 98 plantlets survived in the field. This revised version was published online in June 2006 with corrections to the Cover Date.     

Continuous phenol biodegradation in a simple packed bed bioreactor of calcium alginate-immobilized <Emphasis Type="Italic">Candida tropicalis</Emphasis> (NCIM 3556)

Phenol biodegradation in a continuous system of immobilized Candida tropicalis NCIM 3556 was studied. The bioreactor was simple, it had a feed inlet from the bottom and the effluent outlet from top, no supplementary oxygen was supplied, the reactor was operated continuously for 116 days. Initially the column was run continuously with a feed concentration of 2 g l⁻¹ for 42 days whence a degradation of >97% was achieved. The feed concentration was then increased to 3 g l⁻¹, for which a ~80% biodegradation was sustained for 90 days after which there was a steady decrease in the performance. When the phenol degradation was reduced to ~50% in 116 days, the reactor was stopped. The efficiency of free cells recycled every 24 h and immobilized cells were compared; it was estimated that repeated reuse of free cells in batch mode gave an overall efficiency of 0.102 g phenol degradation g⁻¹ cell wet weight in 12 days. In contrast, the immobilized system of the same biomass had a longer working lifetime of ~4 months indicating an efficiency of 3.72 g phenol g⁻¹ cell wet wt.     

Metal resistance and removal by two strains of the green alga, <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> Beijerinck,isolated from Laguna de Bay,Philippines

Two strains of Chlorella vulgaris Beijerinck isolated from two different sites in Laguna de Bay, Philippines, were studied for their resistance and ability to remove four metal ions, i.e., Cu²⁺, Cr⁶⁺, Pb²⁺, and Cd²⁺ added separately in BG-11 growth medium. The growth of the two strains was severely inhibited at 2 mg.L⁻¹ of Cu²⁺, 5 mg.L⁻¹ of Cr⁶⁺, 8 mg.L⁻¹ of Pb²⁺, and 10 mg.L⁻¹ of Cd²⁺. However, the two strains exhibited different EC₅₀ values for the same metal ion. The WB strain had a significantly higher resistance (p < 0.01) for Cd²⁺ and Cr⁶⁺ compared with the SB strain, while the SB strain had significantly higher resistance (p < 0.01) for Cu²⁺ compared with the WB strain. On the other hand, the two strains behaved differently in their capacity to remove the metal ions in BG-11 medium containing 1.0 mg.L⁻¹ of the three metal ions, except for Cu²⁺, which was added at 0.1 mg.L⁻¹. The WB strain showed the highest removal of Cd²⁺ at 70.3% of total, followed by Pb²⁺ at 32%, while the SB strain exhibited the highest removal of Pb²⁺ at 48.7% followed by Cd²⁺ at 40.7% of the total. Both strains showed the least removal of Cr⁶⁺ at 28% and 20.8% of the total for the WB and SB strains respectively. The percentage removal for Cu²⁺ was 50.7% and 60.8% for the WB and SB strains respectively. After 12 days of incubation, both strains showed that a greater percentage of the metal ions removed were accumulated intracellularly than adsorbed at a ratio of at least 2:1. Both strains manifested the same cytological deformities, like a loss of pyrenoids at 10 mg.L⁻¹ in all four metal ions. Discoloration and disintegration of chloroplasts were observed at 1.0 mg.L⁻¹ in Cu²⁺ and 5 mg.L⁻¹ in Cr⁶⁺. The nonrelease of autospores from the mother cells was observed at 10 mg.L⁻¹ in Cu²⁺ and Cr⁶⁺. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Ca<Superscript>2+ </Superscript>and Cu<Superscript>2+ </Superscript>supplementation increases mannitol production by <Emphasis Type="Italic">Candida magnoliae</Emphasis>

Supplementation with CaCl₂·2H₂O (50 mg l⁻¹) or CuSO₄·5H₂O (10 mg l⁻¹) improved mannitol production by Candida magnoliae by 14.5 and 18.6% (25 and 32 g/L), respectively. When used in combination, they acted synergistically: Ca²⁺ decreased the intracellular concentration of mannitol 30%, whereas Cu²⁺ increased the intracellular activity of mannitol dehydrogenase 1.6-times more than control. Ca²⁺ probably works by altering the permeability of cells to mannitol, whereas, Cu²⁺ increases the activity of an enzyme responsible for mannitol biosynthesis.     

Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens

Three white-rot basidiomycetes, Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens, were cultivated on a liquid medium supplemented with l-phenylalanine, a precursor for benzaldehyde (bitter almond aroma) and benzyl alcohol. Remarkable amounts of benzaldehyde (587 mg l⁻¹) were found in cultures of B. adusta. Immobilization of this fungus on polyurethane foam cubes allowed an 8.3-fold increase of the production of benzaldehyde and a 15-fold increase of the productivity as compared with non-immobilized cells. Aryl-alcohol oxidase activity was only detected in B. adusta. This activity was also significantly enhanced in immobilized cells, suggesting that it plays an important role in benzaldehyde biosynthesis. Conversely, consistent amounts of benzyl alcohol (340 mg l⁻¹ for B. adusta and I. benzoinum and 100 mg l⁻¹ for D. squalens) were produced by the three fungi when immobilized. Laccase activity was found only in the strains I. benzoinum and D. squalens. This activity was markedly enhanced in free cells cultures. Immobilization of the fungi did not promote benzyl alcohol production by comparison with free cell cultures (500 mg l⁻¹). Received: 10 December 1996 / Received revision: 17 February 1997 / Accepted: 22 February 1997     

Lactic acid production from sugar-cane juice by a newly isolated Lactobacillus sp.

A newly isolated sucrose-tolerant, lactic acid bacterium, Lactobacillus sp. strain FCP2, was grown on sugar-cane juice (125 g sucrose l⁻¹, 8 g glucose l⁻¹ and 6 g fructose l⁻¹) for 5 days and produced 104 g lactic acid l⁻¹ with 90% yield. A higher yield (96%) and productivity (2.8 g l⁻¹ h⁻¹) were obtained when strain FCP2 was cultured on 3% w/v (25 g sucrose l⁻¹, 2 g glucose l⁻¹ and 1 g fructose l⁻¹) sugar-cane juice for 10 h. Various cheap nitrogen sources such as silk worm larvae, beer yeast autolysate and shrimp wastes were also used as a substitute to yeast extract.     

Efficient production of ectoine using ectoine-excreting strain

Halophilic bacteria strain Halomonas salina DSM 5928 was found to excrete ectoine, suggesting its potential in the development of a new method of ectoine production. We performed HPLC and LC–MS analyses that showed that Halomonas salina DSM 5928 excreted ectoine under constant extracellular osmolarity. Medium adopting monosodium glutamate as a sole source of carbon and nitrogen was beneficial for ectoine synthesis. The total concentration of ectoine was not affected by NaCl concentration in the range 0.5–2 mol l⁻¹. The total concentration of ectoine and productivity in a 10-l fermentor with 0.5 mol l⁻¹ NaCl were 6.9 g l⁻¹ and 7.9 g l⁻¹ d⁻¹, respectively. These findings show that Halomonas salina DSM 5928 efficiently produces ectoine at relatively low NaCl concentration. This research also indicates the potential application of free or immobilized cells for continuous culture to produce ectoine.     

Decolorization of 1:2 metal complex dye Acid blue 193 by a newly isolated fungus, Cladosporium cladosporioides

Summary A fungus Cladosporium cladosporioides isolated from coal sample as a decolorizing microorganism. It decolorized five different azo and triphenylmethane dyes like acid blue 193, acid black 210, crystal violet, reactive black B(S) and reactive black BL/LPR both on solid and in liquid broth medium. Culture broth of this fungus decolorized completely 100 mg of acid blue 193 l⁻¹ in 8 days. The extracellular enzyme of Cladosporium cladosporioides decolorized acid blue 193 on repeated addition to a total (out of 700 mg l⁻¹) concentration of 564 mg l⁻¹ within 168 h without significant decline in the activity, showing the resistant property of Cladosporium cladosporioides to a high concentration of the dye. The optimal temperature 40 °C, pH 5.6 and sugar concentration of 4% required for decolorization of acid blue 193. Cladosporium cladosporioides showed manganese peroxidase activity with 41 U l⁻¹, laccase activity with 1413 U l⁻¹ and lignin peroxidase activity was negligible after day 8 of incubation.