Enrichment, isolation, and characterization of 4-chlorophenol-degrading bacterium Rhizobium sp. 4-CP-20

The objectives of this research were to monitor the variations of species in mixed cultures during the enrichment period, isolate species and identify and characterize the pure 4-chlorophenol (4-CP) degrading strains from enriched mixed cultures. Strain Rhizobium sp. 4-CP-20 was isolated from the acclimated mixed culture. The DGGE result indicated that strain Rhizobium sp. 4-CP-20 was undetectable at the beginning but detectable after 2 weeks of enrichment. The optimum growth temperatures for Rhizobium sp. 4-CP-20 were both 36°C using 350 mg l⁻¹ glucose or sodium acetate as the substrate. The optimum pH range for degrading 100 mg l⁻¹ 4-CP was between 6.89 and 8.20. Strain Rhizobium sp. 4-CP-20 could degrade 4-CP completely within 3.95 days, as the initial 4-CP concentration was 100 mg l⁻¹. If the initial 4-CP concentration was higher than 240 mg l⁻¹, the growth of bacterial cells and the activity of degrading 4-CP were both inhibited.     

Improved <Emphasis Type="Italic">in vitro</Emphasis> micropropagation method with adventitious corms and roots for endangered saffron

The objective of this study was to investigate development of an efficient in vitro tissue culture system for saffron (Crocus sativus L.) complete with roots and corms. In indirect organogenesis, Murashige and Skoog (MS) media with 3% (w/v) sucrose, 100 mg L⁻¹ ascorbic acid, and the combination of 0.25 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg L⁻¹ 6-benzylaminopurine (BAP) were best for callus initiation and growth while 1.5 mg L⁻¹ BAP was excellent for high rate of adventitious shoot formation. 1 mg L⁻¹ indole-3-butyric acid (IBA) was more preferable for adventitious corm and root initiation as well as growth. Overall, 64% rooting and 33% corm production rates were achieved in indirect organogenesis. In direct organogenesis, MS medium supplemented with 3 % sucrose, 100 mg L⁻¹ ascorbic acid and 1 mg L⁻¹ BAP was optimum for shoot growth. While 1 mg L⁻¹ IBA was best for adventitious corm formation, 2 mg L⁻¹ IBA promoted adventitious root initiation and growth. Overall, 36% and 57% of explants had corm and contractile root, respectively. The high rates suggest that efficient tissue culture system could be achieved for mass propagation and ex situ conservation of threatened saffron genetic resources.     

Characterisation of a novel thermotolerant yeast, Kluyveromyces marxianus var marxianus: development of an ethanol fermentation process

The fermentation characteristics of the novel, thermotolerant, isolate Kluyveromyces marxianus var marxianus were determined to evaluate its aptitude for use in an ethanol production process. Sustainable growth was not observed under anaerobic conditions, even in the presence of unsaturated fatty acid and sterol. A maximum ethanol concentration of 40 g L⁻¹ was produced at 45°C, with an initial specific ethanol production rate of 1.7 g g⁻¹ h⁻¹. This was observed at ethanol concentrations below 8 g L⁻¹ and under oxygen-limited conditions. The low ethanol tolerance and low growth under oxygen-limited conditions required for ethanol production implied that a simple continuous process was not feasible with this yeast strain. Improved productivity was achieved through recycling biomass into the fermenter, indicating that utilising an effective cell retention method such as cell recycle or immobilisation, could lead to the development of a viable industrial process using this novel yeast strain. Received 14 February 1998/ Accepted in revised form 19 May 1998     

Isolation and characterisation of phenol-degrading <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> MTCC 4996

A novel indigenous Pseudomonas aeruginosa strain (MTCC 4996) isolated from a pulp industrial effluent-contaminated site was capable of degrading phenol up to a concentration of 1,300 mg L⁻¹ within 156 h. Complete degradation was observed at pH values ranging from 6.0 to 10.0 and temperatures from 15 to 45°C, with an optimum pH of 7.0 and optimum temperature of 37°C. At an optimum shaking speed of 100–125 rpm, 100% degradation was observed in 66 h, as compared to 84 h under static conditions. Glucose and peptone at lower concentrations enhanced phenol degradation. The rate of phenol degradation was most sensitive to added Hg. Low concentrations of Fe, Cu, Pb, Zn, and Mn stimulated and enhanced the rate of degradation.     

Morphological evaluation and antioxidant activity of <Emphasis Type="Italic">in vitro</Emphasis>- and <Emphasis Type="Italic">in vivo</Emphasis>-derived <Emphasis Type="Italic">Echinacea purpurea</Emphasis> plants

An effective in vitro protocol for rapid clonal propagation of Echinacea purpurea (L.) Moench through tissue culture was described. The in vitro propagation procedure consisted of four stages: 1) an initial stage - obtaining seedlings on Murashige and Skoog (MS) basal medium with 0.1 mg L⁻¹ 6-benzylaminopurine, 0.1 mg L⁻¹ α-naphthalene acetic acid and 0.2 mg L⁻¹ gibberellic acid; 2) a propagation stage — shoot formation on MS medium supplemented with 1 mg L⁻¹ 6-benzylaminopurine alone resulted in 9.8 shoots per explant and in combination with 0.1 mg L⁻¹ α-naphthalene acetic acid resulted in 16.2 shoots per explant; 3) rooting stage — shoot rooting on half strength MS medium with 0.1 mg L⁻¹ indole-3-butyric acid resulted in 90% rooted microplants; 4) ex vitro acclimatization of plants. The mix of peat and perlite was the most suitable planting substrate for hardening and ensured high survival frequency of propagated plants. Significant higher levels were observed regarding water-soluble and lipid-soluble antioxidant capacities (expressed as equivalents of ascorbate and α-tocopherol) and total pnenols content in extracts of Echinaceae flowers derived from in vitro propagated plants and adapted to field conditions in comparison with traditionally cultivated plants.     

Plant tissue cultures from four Tuscan globe artichoke cultivars

The aim of the study was to examine the possibility of propagating in vitro four of the most common cultivars in Tuscany (central Italy): Terom, Violetto di Toscana, Chiusure and Empolese. The first three belong to the “Violetti” group, while cv Empolese belongs to the “Romaneschi” group. Explants were cultured on an induction medium (IM), which is a modified MS medium consisting of nitrate concentrations reduced by one quarter, 0.8 mg L⁻¹ 6-benzylaminopurine (BA) and 0.2 mg L⁻¹ 3-indole butyric acid (IBA). Explants were then transferred to a proliferation medium (PM) consisting of the same basal medium together with 0.03 mg L⁻¹ BA and 0.05 mg L⁻¹ gibberellic acid (GA₃). A rooting double-phase was then established. The pre-rooting medium (PRM), consisting of a basal MS medium with half strength nitrate concentrations, 0.5 mg L⁻¹ indole-3-acetic acid (IAA) and 1 mg L⁻¹ paclobutrazol (PBZ) was used for two weeks. Over the next four weeks, a rooting medium (MR) was used, consisting of a basal MS medium with 2 mg L⁻¹ β-cyclodextrin and 2 mg L⁻¹ α-naphthaleneacetic acid sodium salt (NAA). The cv Empolese provided the highest number of proliferated explants and rooted plantlets using the method described.     

Long-term cultured callus and the effect factor of high-frequency plantlet regeneration and somatic embryogenesis maintenance in <Emphasis Type="Italic">Zoysia japonica</Emphasis>

In this study, we have demonstrated that Zoysia japonica callus induced from mature seeds can produce high frequencies of plant regeneration and somatic embryogenesis, even following a prolonged period of subculturing. Initial callus cultures were induced from mature seeds of Japanese lawngrass (Z. japonica Steud.) incubated on a medium containing major N₆ medium salts, minor Murashige and Skoog (MS) medium salts, and modified MS medium organic elements supplemented with 3 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01–0.02 mg L⁻¹ 6-benzyladenine. Compact callus were selected and subcultured monthly on a medium containing 2 mg L⁻¹ 2,4-D, 0.5 mg L⁻¹ kinetin, 500 mg L⁻¹ casein hydrolysate, 500 mg L⁻¹ proline, and 500 mg L⁻¹ myoinositol. Callus maintained in vitro for 18 mo could be induced to regenerate plantlets with a frequency of >90%. By contrast, 36-mo-old callus cultures failed to produce normal shoot regeneration. However, the addition of CuSO₄ to the subculture media maintained >90% regeneration frequencies in such long-term callus cultures. Histological observations revealed that plant regeneration occurred both through somatic embryogenesis and organogenesis pathways. The ability to sustainable regeneration in long-term callus cultures will be valuable to the program of genetic transformation and somaclonal variant selection.     

Efficient biocatalytic production of <Emphasis Type="SmallCaps">d</Emphasis>-4-hydroxyphenylglycine by whole cells of recombinant <Emphasis Type="Italic">Ralstonia pickettii</Emphasis>

The first establishment of a homologous expression system in the host Ralstonia pickettii CGMCC1596 using the compatible broad-host-range plasmid pWB5 is described. When whole cells of the recombinant strain R. pickettii MMYY01 (CGMCC1596/pYY05) were used as the biocatalyst to transform dl-4-hydroxyphenylhydantoin (dl-HPH) to d-4-hydroxyphenylglycine (d-HPG), the conversion rate reached 94 % in first 9 h, at a production rate of 2.8 g L⁻¹ h⁻¹, with the rapid reduction of the intermediate [N-carbamoyl-2-(4-hydroxyphenyl)glycine], compared with 80 % in >50 h at a rate of 0.5 g L⁻¹ h⁻¹ for the CGMCC1596. The stability of the recombinant plasmid pYY05 is sufficient for its application in industrial batch fermentation. An alternative strategy for the conversion of dl-HPH to d-HPG by resting CGMCC1596 cells and heterologous DCase expressed by E. coli is discussed.     

Aerobic degradation of 3-chlorobenzoic acid by an indigenous strain isolated from a polluted river

An indigenous strain of Pseudomonas putida capable of degrading 3-chlorobenzoic acid as the sole carbon source was isolated from the Riachuelo, a polluted river in Buenos Aires. Aerobic biodegradation assays were performed using a 2-l microfermentor. Biodegradation was evaluated by spectrophotometry, chloride release, gas chromatography and microbial growth. Detoxification was evaluated by using Vibrio fischeri, Pseudokirchneriella subcapitata and Lactuca sativa as test organisms. The indigenous bacterial strain degrades 100 mg l⁻¹ 3-chlorobenzoic acid in 14 h with a removal efficiency of 92.0 and 86.1% expressed as compound and chemical oxygen demand removal, respectively. The strain was capable of degrading up to 1,000 mg of the compound l⁻¹. Toxicity was not detected at the end of the biodegradation process. Besides initial concentration, the effect of different factors, such as initial pH, initial inoculum, adaptation to the compound and presence of other substrates and toxic related compounds, was studied.     

Single-culture aerobic granules with <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis>

Aerobic granules are cultivated by a single bacterial strain, Acinetobacter calcoaceticus, in a sequencing batch reactor (SBR). This strain presents as a good phenol reducer and an efficient auto coagulator in the presence of phenol, mediated by heat-sensitive adhesins proteins. Stable 2.3-mm granules were formed in the SBR following a 7-week cultivation. These granules exhibit excellent settling attributes and degrade phenol efficiently at concentrations of 250–2,000 mg l⁻¹. The corresponding phenol degradation rate reached 993.6 mg phenol g⁻¹ volatile suspended solids (VSS) day⁻¹ at 250 mg l⁻¹ phenol and 519.3 mg phenol g⁻¹ VSS day⁻¹ at 2,000 mg l⁻¹ phenol concentration. Meanwhile, free A. calcoaceticus cells were fully inhibited at phenol >1,500 mg l⁻¹. Denaturing gradient gel electrophoresis fingerprint profile demonstrated no genetic modification in the strain during aerobic granulation. The present single-strain granules showed long-term structural stability and performed high phenol degrading capacity and high phenol tolerance. The confocal laser scanning microscopic test revealed that live A. calcoaceticus cells principally distributed at 200–250 μm beneath the outer surface, with an extracellular polymeric substance layer covering them to defend phenol toxicity. Autoaggregation assay tests demonstrated the possibly significant role of secreted proteins on the formation of single-culture A. calcoaceticus granules.     

Large-scale production of tannase using the yeast <Emphasis Type="Italic">Arxula adeninivorans</Emphasis>

Tannase (tannin acyl hydrolase, EC 3.1.1.20) hydrolyses the ester and depside bonds of gallotannins and gallic acid esters and is an important industrial enzyme. In the present study, transgenic Arxula adeninivorans strains were optimised for tannase production. Various plasmids carrying one or two expression modules for constitutive expression of tannase were constructed. Transformant strains that overexpress the ATAN1 gene from the strong A. adeninivorans TEF1 promoter produce levels of up to 1,642 U L⁻¹ when grown in glucose medium in shake flasks. The effect of fed-batch fermentation on tannase productivity was then investigated in detail. Under these conditions, a transgenic strain containing one ATAN1 expression module produced 51,900 U of tannase activity per litre after 142 h of fermentation at a dry cell weight of 162 g L⁻¹. The highest yield obtained from a transgenic strain with two ATAN1 expression modules was 31,300 U after 232 h at a dry cell weight of 104 g L⁻¹. Interestingly, the maximum achieved yield coefficients [Y(P/X)] for the two strains were essentially identical.     

Establishment of an <Emphasis Type="Italic">Agrobacteriuim</Emphasis>-mediated cotyledon disc transformation method for <Emphasis Type="Italic">Jatropha curcas</Emphasis>

Jatropha curcas contains high amounts of oil in its seed and has been considered for bio-diesel production. A transformation procedure for J. curcas has been established for the first time via Agrobacterium tumefaciens infection of cotyledon disc explants. The results indicated that the efficiency of transformation using the strain LBA4404 and phosphinothricin for selection was an improvement over that with the strain EHA105 and hygromycin. About 55% of the cotyledon explants produced phosphinothricin-resistant calluses on Murashige and Skoog (MS) medium supplemented with 1.5 mg l⁻¹ benzyladenine (BA), 0.05 mg l⁻¹ 3–indolebutyric acid (IBA), 1 mg l⁻¹ phosphinothricin and 500 mg l⁻¹ cefotaxime after 4 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.05 mg l⁻¹ IBA, 0.5 mg l⁻¹ gibberellic acid (GA3), 1 mg l⁻¹ phosphinothricin and 250 mg l⁻¹ cefotaxime, and about 33% of the resistant calli differentiated into shoots. Finally, the resistant shoots were rooted on 1/2 MS media supplemented with 0.3 mg l⁻¹ IBA at a rate of 78%. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. 13% of the total inoculated explants produced transgenic plants after approximately 4 months. The procedure described will be useful for both, the introduction of desired genes into J. curcas and the molecular analysis of gene function.     

Effects of Artemisinin Derivative on the Growth Metabolism of <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis> BF5 Based on Expression of Thermokinetics

The toxic effects of artesunate and dihydroartemisinin on the growth metabolism of Tetrahymena thermophila BF5 were studied by microcalorimetry. The results showed that: (1) low concentrations of artesunate (≤1 mg L⁻¹) and dihydroartemisinin (≤ 2 mg L⁻¹) promoted the growth metabolism of T. thermophila BF5, whereas high concentrations of artesunate (1–60 mg L⁻¹) and dihydroartemisinin (2–60 mg L⁻¹) inhibited its growth; (2) the half inhibition concentrations IC₅₀ of artesunate and dihydroartemisinin were 17.5817 and 9.5089 mg L⁻¹, respectively. It was concluded that the inhibition of dihydroartemisinin was stronger than that of artesunate.     

Docosapentaenoic acid (C22:5, ω-3) production by Pythium acanthicum

The physiological roles of omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid have been investigated in detail and microbial strains producing these polyunsaturated fatty acids have been characterised. It has recently been suggested that docosapentaenoic acid may have an important role, especially in infant nutrition, and that its positive health effects have been overlooked. Docosapentaenoic acid (C22:5, ω-3) production by a strain of Pythium acanthicum ATCC 18660 was thus investigated. Optimum conditions for growth of P. acanthicum ATCC 18660 and docosapentaenoic acid production were: pH 6.0, temperature 20°C and incubation time, 10 days. Among different saccharides and complex nitrogen sources tested, glucose and sodium glutamate were preferred carbon and nitrogen sources, respectively. Maximum biomass content (10.4 g L⁻¹) and docosapentaenoic acid yield (49.9 mg L⁻¹) were obtained in 10 days. An increase in docosapentaenoic acid volumetric yields to 108–110 mg L⁻¹ was obtained when linseed oil was used to supplement glucose or soy flour-containing medium. Batch feeding of additional glucose or linseed oil further enhanced the docosapentaenoic acid volumetric yield to 132 mg L⁻¹ and 125 mg L⁻¹, respectively, in 14 days. The specific production of docosapentaenoic acid in preliminary experiments ranged from 1.0–5.0 mg g⁻¹ biomass. As conditions were optimised, docosapentaenoic acid specific production titers were generally in the range of 4.0–5.5 mg g⁻¹ and increases in docosapentaenoic acid volumetric production could be attributed to increased biomass production. The limited improvement obtained by modifying culture conditions indicates that increasing volumetric yields of docosapentaenoic acid by modifying culture conditions appears to represent a significant barrier to commercialisation of such a process and suggests a more fundamental manipulation of metabolism and physiology is required. Received 06 November 1997/ Accepted in revised form 10 January 1998     

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.     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.     

Isolation and Characterization of a Carbendazim-Degrading Rhodococcus sp. djl-6

Bacterium djl-6, capable of degrading carbendazim, was isolated by continuous enrichment culture originating from carbendazim-treated soil. The isolate was identified as Rhodococcus sp. according to its phenotypic features, physiologic and biochemical characteristics, and phylogenetic analysis. The strain could use carbendazim as sole carbon or nitrogen source. It showed a high average degradation rate of 55.56 mg · L⁻¹ · d⁻¹ in M9 medium amended with carbendazim. High-pressure liquid chromatography–mass spectrometry (HPLC-MS) analysis showed the presence of 2-aminobenzimidazole, benzimidazole, and an unknown metabolite with molecular ions (M⁺) of m/z 104.8 and 118.5. The degradation in the isolate djl-6 seems to be initiated with the cleavage of the methyl carbemate side chain, resulting in the formation of 2-aminobenzimidazole and benzimidazole. This is the first report of the intermediates benzimidazole and 2-aminobenzimidazole found together in the culture filtrate of pure bacterium.     

Development of an efficient tissue culture protocol for callus formation and plant regeneration of wetland species <Emphasis Type="Italic">Juncus effusus</Emphasis> L.

Wetland species mat rush (Juncus effusus L.) is an important economic plant, but no information is available regarding plant regeneration, callus induction, and its proliferation from in vitro seed grown plantlets. The present study investigates the effects of growth regulator combinations and medium innovation on tissue culture system of five mat rush varieties. Addition of N6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in Murashige and Skoog (MS) medium showed significantly positive effect on callus proliferation, plant regeneration, and its multiplication compared to the medium devoid of BA. The highest callus induction frequency (80.95%, 90.48%, 75.40%, 70.83%, and 83.33%) was observed in MS medium containing 0.5 mg L⁻¹ (2.2 μM) BA in Yinlin-1, Nonglin-4, Gangshan, Taicao, and Taiwan green, respectively. Various growth regulator combinations with successive subculture (medium replacement) were found essential to develop organogenic calluses and to regenerate shoots. The combination of 0.1 mg L⁻¹ BA (0.4 μM) and 2 mg L⁻¹ 2,4-D (9.0 μM) in MS medium was found best for callus proliferation for all the varieties under trial. The plant regeneration required two steps involving successive medium replacements as well as optimal hormonal balances. Successful plant regeneration (over 70%) was observed only by transferring the organogenic callus from regeneration medium I [MS medium containing 0.5 mg L⁻¹ BA (2. μM) and 1.0 mg L⁻¹ kinetin (KT; 4.6 μM)] to the regeneration medium II [MS medium containing 0.5 mg L⁻¹ BA (2.2 μM), 1.0 mg L⁻¹ KT (4.6 μM) and 3.0 mg L⁻¹ indoleacetic acid (IAA; 17.1 μM)]. Our results confirmed the importance of the ratio of auxin (IAA) to cytokinin (BA and KT) in the manipulation of shoot regeneration in J. effusus L. The maximum plant survival frequency and multiplication rates (90.97% and 5.40 and 94.23% and 8.25) were recorded in the presence of 0.5 mg L⁻¹ BA (2.2 μM) in the 1/2 MS multiplication medium for the varieties of Nonglin-4 and Taicao, respectively. About 100% survival rate was also observed for all the varieties in soil conditions. The efficient plant regeneration system developed here will be helpful for rapid micropropagation and further genetic improvement in J. effusus L.     

Bioactive hydroxyphenylpyrrole-dicarboxylic acids from a new marine Halomonas sp.: production and structure elucidation

The new marine Halomonas sp. strain GWS-BW-H8hM (DSM 17996) was found to produce 3-(4′-hydroxyphenyl)-4-phenylpyrrole-2,5-dicarboxylic acid (HPPD-1) and 3,4-bis(4′-hydroxy- phenyl)pyrrole-2,5-dicarboxylic acid (HPPD-2). In initial cultivations using marine broth, only low contents of these compounds have been isolated. Improving the conditions and growing the strain on artificial seawater supplemented with tryptone 10 g l⁻¹, yeast extract 5 g l⁻¹, l-tyrosine 0.6 g l⁻¹, glycine 1 g l⁻¹, and glucose 6 g l^-1, the growth-associated HPPD-1 and HPPD-2 production of a 40-l batch cultivation reached the amounts of 47 mg l⁻¹ and 116 mg l⁻¹, respectively, after 65 h. Both compounds showed potent anti-tumor-promoting activities.     

Effects of salinity on cell growth and docosahexaenoic acid content of the heterotrophic marine microalga Crypthecodinium cohnii

The effects of salinity on cell growth and docosahexaenoic acid (DHA) content of three marine microalgal strains, Crythecodinium cohnii ATCC 30556, C. cohnii ATCC 50051 and C. cohnii RJH were investigated. The lag phases of the three strains increased with increasing salinity in Porphyridium medium. The specific growth rate of C. cohnii ATCC 30556 was the highest at 9 g L⁻¹ NaCl while the other two strains had their highest specific growth rates at 5 g L⁻¹ NaCl. The highest cell dry weight concentrations of 2.51 g L⁻¹ and 1.56 g L⁻¹ were achieved at 9 g L⁻¹ NaCl for C. cohnii ATCC 30556 and ATCC 50051, respectively, while the highest dry weight concentration of 2.49 g L⁻¹ was achieved at 5 g L⁻¹ NaCl for C. cohnii RJH. The highest cell growth yield coefficient on glucose was 0.5 g g⁻¹ for both C. cohnii ATCC 30556 and C. cohnii RJH and 0.45 g g⁻¹ for C. cohnii ATCC 50051. All three strains responded to the change of salinity by modifying their cellular fatty acid compositions. At 9 g L⁻¹ NaCl, C. cohnii ATCC 30556 had the highest total fatty acid content and DHA (C22:6) proportion. In contrast, C. cohnii ATCC 50051 and C. cohnii RJH had the highest DHA content at 5 g L⁻¹ NaCl. C. cohnii ATCC 30556 and ATCC 50051 had the highest DHA yield (131.55 and 68.24 mg L⁻¹ respectively) at 9 g L⁻¹ NaCl while C. cohnii RJH had the highest DHA yield (128.83 mg L⁻¹) at 5 g L⁻¹ NaCl. Received 27 May 1999/ Accepted in revised form 27 August 1999