Isolation of algicidal compounds from the red alga <Emphasis Type="Italic">Corallina pilulifera</Emphasis> against red tide microalgae

We determined the structure of two compounds, namely, 5,8,11,14,17-eicosapentaenoic acid (EPA) and di-n-octylphthalate (DnOP), which have algicidal activity against the toxic dinoflagellate, Cochlodinium polykrikoides. The polyunsaturated fatty acid EPA and the anthropogenic DnOP were isolated from the MeOH extract of the red alga Corallina pilulifera. We also found that a commercial EPA has algicidal activity identical to that of the EPA purified from C. pilulifera. At low inoculum (5.0 × 10² cells mL⁻¹), the highest algicidal activity of a commercial EPA exhibited approximately 92.6% algicidal activity after 1 h and 96.8% after 6 h treatment at 6 μg mL⁻¹, respectively. At high inoculum (1.0 × 10⁴ cells mL⁻¹), the strongest algicidal activity of EPA showed 69.5% after 1 h and 75.5% algicidal activity after 6 h treatment at 6 μg mL⁻¹, respectively. However, EPA did not show algicidal activity against several microalgae used in aquaculture such as Pavlova lutheri, Tetraselmis suecica, Isochrysis galbana, and Nannochloris oculata for 6 h treatment at 6 μg mL⁻¹. The algicidal activity of the five red tide strains to EPA (3 μg mL⁻¹) showed about 86.6%, 86.6%, and 67.3% algicidal activity against Skeletonema costatum, Chaetoceros curvisetus, and C. polykrikoides after 1 h treatment at low inoculum (5.0 × 10² cells mL⁻¹), respectively, but not against Prorocentrum minimum and Scrippsiella trochoidea. We concluded that EPA might be useful as a controlling agent of harmful algal blooms.     

Isolation of antifouling compounds from the marine bacterium, <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> SCH0402

Two compounds, 2-hydroxymyristic acid (HMA) and cis-9-oleic acid (COA), were isolated from a chloroform extract of the marine bacterium, Shewanella oneidensis SCH0402. In a spectrophotometer-based chemotaxis assay, HMA completely eliminated the optical density (OD) of Alteromonas marina SCH0401 and Bacillus atrophaeus SCH0408, motile, fouling bacteria, at 100 and 1000 μg ml⁻¹, respectively. COA similarly decreased the OD of A. marina and B. atrophaeus by 100% at 1000 μg ml⁻¹. The commercially available, highly toxic anti-fouling compound, tributyltin oxide (TBTO) never reduced the OD of the target bacteria by 100% even at higher concentration. Instead, all the test bacterial cells were killed at higher than 1000 μg ml⁻¹ of concentration. Both HMA and COA inhibited germination of Ulva pertusa spores completely at 10 and 100 μg ml⁻¹, respectively, while TBTO inhibited germination at 0.01 μg ml⁻¹. However, in field assays, both HMA and COA showed anti-fouling activities as potent as TBTO against a wide range of fouling organisms, including micro- and macro-algae, barnacles, and mussels. The average fouling coverage on the surface of the control panel was 93 ± 6% after 1.5 years but no fouling was observed on the surface of the test panel onto which each compound was applied separately. Thus, bacterial repellent compounds can be used as substitutes for potent toxic anti-fouling compounds, resulting in higher standards of environmental safety without loss of anti-fouling performance.     

Isolation of algal spore lytic C17 fatty acid from the crustose coralline seaweed Lithophyllum yessoense

The algal spore lytic fatty acid of heptadeca-5,8,11-trien (HpDTE: C17:3) was isolated from the crustose coralline seaweed Lithophyllum yessoense. HpDTE, an odd-numbered carbon fatty acid, showed more than 50% lysis at a concentration of 5 μg.mL⁻¹ against the spores of three chlorophyte species, nine rhodophytes, four phaeophytes, and the cells of four phytoplankton species. Lysis activity increased with the number of double bonds and carbon atoms in the fatty acid increased. HpDTE showed a ten-fold stronger activity with a LC₅₀ of 3.1 μg.mL⁻¹ than α-linolenic acid (C18:3).     

Production of Cold-Adapted Amylase by Marine Bacterium <Emphasis Type="Italic">Wangia</Emphasis> sp. C52: Optimization,Modeling, and Partial Characterization

The aim of this work was to optimize the fermentation parameters in the shake-flask culture of marine bacterium Wangia sp. C52 to increase cold-adapted amylase production using two statistical experimental methods including Plackett–Burman design, which was applied to find the key ingredients for the best medium composition, and response surface methodology, which was used to determine the optimal concentrations of these components. The results showed starch, tryptone, and initial pH had significant effects on the cold-adapted amylase production. A central composite design was then employed to further optimize these three factors. The experimental results indicated that the optimized composition of medium was 6.38 g L⁻¹ starch, 33.84 g L⁻¹ tryptone, 3.00 g L⁻¹ yeast extract, 30 g L⁻¹ NaCl, 0.60 g L⁻¹ MgSO₄ and 0.56 g L⁻¹ CaCl₂. The optimized cultivation conditions for amylase production were pH 7.18, a temperature of 20°C, and a shaking speed of 180 rpm. Under the proposed optimized conditions, the amylase experimental yield (676.63 U mL⁻¹) closely matched the yield (685.60 U mL⁻¹) predicted by the statistical model. The optimization of the medium contributed to tenfold higher amylase production than that of the control in shake-flask experiments.     

Continuous cultures of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> mt-2 overcome catabolic function loss under real case operating conditions

The long-term performance and stability of Pseudomonas putida mt-2 cultures, a toluene-sensitive strain harboring the genes responsible for toluene biodegradation in the archetypal plasmid pWW0, was investigated in a chemostat bioreactor functioning under real case operating conditions. The process was operated at a dilution rate of 0.1 h⁻¹ under toluene loading rates of 259 ± 23 and 801 ± 78 g m⁻³ h⁻¹ (inlet toluene concentrations of 3.5 and 10.9 g m⁻³, respectively). Despite the deleterious effects of toluene and its degradation intermediates, the phenotype of this sensitive P. putida culture rapidly recovered from a 95% Tol⁻ population at day 4 to approx. 100% Tol⁺ cells from day 13 onward, sustaining elimination capacities of 232 ± 10 g m⁻³ h⁻¹ at 3.5 g Tol m⁻³ and 377 ± 13 g m⁻³ h⁻¹ at 10.9 g Tol m⁻³, which were comparable to those achieved by highly tolerant strains such as P. putida DOT T1E and P. putida F1 under identical experimental conditions. Only one type of Tol⁻ variant, harboring a TOL-like plasmid with a 38.5 kb deletion (containing the upper and meta operons for toluene biodegradation), was identified.     

Effect of eight benthic diatoms as feed on the growth of red abalone (Haliotis rufescens) postlarvae

The growth rate of abalone post larvae of Haliotis rufescens fed ad libitum with a benthic monoalgal diatom culture maintained as monocultures on a semi-commercial scale, was evaluated and correlated with the biochemical composition of the diatoms. The cell size (7.0 × 4.0 μm to 21.0 × 7.5 μm), protein percentage (7.42% to 13.66%), and ash content (49.03% to 59.61%) were different among diatom strains; lipid percentage, nitrogen free extract, and energy content (Kcal g⁻¹) were similar among diatom strains. The values of essential and non-essential amino and fatty acids composition differed among diatom strains. Differences in the abalone shell length and orthogonal analyses revealed postlarval growth was dependent on the quality of the food source. Postlarvae abalone displaying the longest shell lengths were fed Nitzschia thermalis var. minor and Amphiprora paludosa var. hyalina (1,712.0 ± 61 μm and 1,709 ± 67 μm, respectively), followed by Navicula incerta (1,413.3 ± 43 μm). The fatty acid content of benthic diatoms and abalone growth rate were not correlated.     

Mutagenic and Genotoxic Effects of <Emphasis Type="Italic">cis</Emphasis>-(Dichloro)tetraammineruthenium(III) Chloride on Human Peripheral Blood Lymphocytes

Chemotherapeutic agents play an important role in cancer treatment mostly due their systemic action on human organism allowing access to liquid tumors and even metastases. Among these drugs, ruthenium compounds have been showing promising results to treat tumors and represent an important development of new antitumor therapy. This study presents the evaluation of cis-(dichloro)tetraammineruthenium(III) chloride, cis-[RuCl₂(NH₃)₄]Cl, genotoxic effects using human peripheral blood lymphocytes cultured in vitro. Mitotic index (MI), chromosome aberrations (CA), and DNA damage using the comet assay were analyzed. MI in human peripheral blood lymphocyte cultures treated with 1, 10, 100, and 1,000 μg mL⁻¹ cis-[RuCl₂(NH₃)₄]Cl were 5.9%, 4.6%, 3.9%, and 0%, respectively. Doxorubicin chloridate was used as the positive control. CA derived from 1, 10, and 100 μg mL⁻¹ concentrations were defined as spontaneous when compared with the negative control, and at the concentration of 1,000 μg mL⁻¹, the cell cycle was inhibited (IM = 0%). Results obtained for the comet assay using cis-[RuCl₂(NH₃)₄]Cl suggest that this compound has no genotoxic activity against cultured human peripheral blood lymphocytes.     

Growth and carrageenan quality of <Emphasis Type="Italic">Kappaphycus striatum</Emphasis> var. <Emphasis Type="Italic">sacol</Emphasis> grown at different stocking densities,duration of culture and depth

Kappaphycus striatum var. sacol was grown in two separate studies: (1) at two stocking densities, and (2) at four different depths, each for three different durations of culture (30, 45 and 60 days) in order to determine the growth rate of the seaweed and evaluate the carrageenan content and its molecular weight. The results demonstrated that stocking density, duration of culture and depth significantly (P < 0.01) affected the growth rate, carrageenan content and molecular weight of K. striatum var. sacol. Decreasing growth rate was observed at both stocking densities and at four depths as duration of culture increased. A lower stocking density (500 g m⁻¹line⁻¹) showed a higher growth rate for the shortest durations, i.e. 30 days, as compared to those grown at a higher density. Likewise, decreasing growth rate was observed as depth increased, except at 50 cm after 60 days of culture. A 45-day culture period produced the highest molecular weight at both stocking densities (500 g m⁻¹line⁻¹ = 1,079.5 ± 31.8 kDa, 1,000 g m⁻¹line⁻¹ = 1,167 ± 270.6 kDa). ‘Sacol’ grown for 30 days at 50 cm (1,178 kDa) to 100 cm (1,200 kDa) depth showed the highest values of molecular weight of carrageenan extracted. The results suggested that K. striatum var. sacol is best grown at a stocking density of 500 g m⁻¹line⁻¹, at a depth of 50–100 cm, and for a duration of 30 days in order to provide the highest growth rate, carrageenan content and molecular weight.     

Growth and survival performance of the gametophyte of Gigartina skottsbergii (Rhodophyta, Gigartinales) under defined nutrient conditions in laboratory culture

Gigartina skottsbergii is a commercially important carrageenan producer that has been suffering severe extraction pressure in Chile’s Magellan Region and Cape Horn Archipelago since 1998. In order to create baseline information for its cultivation and repopulation, we studied the effects of agricultural fertilizers on growth of G. skottsbergii early developmental stages. The culture media utilized were: a) seawater + Bayfoland, b) seawater + Superphosphate, c) seawater + Urea, d) seawater + Provasoli and e) seawater as a control. The culture conditions were: a) 12L:12D photoperiod; b) temperature 8 ± 1°C and c) irradiance at 45 μmol photons m⁻² s⁻¹. After 60 days, higher relative growth rates between treatments were observed; the treatments that included Bayfoland and Provasoli showed greater growth (382 ± 55 and 378 ± 50 μm, respectively,) compared to Superphosphate (88 ± 16 μm), control (78 ± 10 μm) and Urea (70 ± 11 μm) treatments, after 81 days. The Urea treatment and the control had inhibitory effects on G. skottsbergii germlings growth and survival, as evidenced by progressive loss of pigmentation and death after 60 days. These results showed that Bayfoland was an excellent alternative to develop cultures.     

Palm oil mill effluent (POME) cultured marine microalgae as supplementary diet for rotifer culture

Malaysia is the world’s leading producer of palm oil products that contribute US$ 7.5 billion in export revenues. Like any other agro-based industries, it generates waste that could be utilized as a source of organic nutrients for microalgae culture. Present investigation delves upon Isochrysis sp. culture in POME modified medium and its utilization as a supplement to Nanochloropsis sp. in rotifer cultures. The culture conditions were optimized using a 1 L photobioreactor (Temp: 23°C, illumination: 180 ∼ 200 μmol photons m⁻²s⁻¹, n = 6) and scaled up to 10 L outdoor system (Temp: 26–29°C, illumination: 50 ∼ 180 μmol photons m⁻²s⁻¹, n = 3). Algal growth rate in photobioreactor (μ = 0.0363 h⁻¹) was 55% higher compared to outdoor culture (μ = 0.0163 h⁻¹), but biomass production was 1.3 times higher in outdoor culture (Outdoor = 91.7 mg m⁻²d⁻¹; Photobioreactor = 69 mg m⁻²d⁻¹). Outdoor culture produced 18% higher lipid; while total fatty acids (FA) was not significantly affected by the change in culture systems as both cultures yield almost similar concentrations of fatty acids per gram of sample (photobioreactor = 119.17 mg g⁻¹; outdoor culture = 104.50 mg g⁻¹); however, outdoor cultured Isochrysis sp. had 26% more polyunsaturated fatty acids (PUFAs). Rotifers cultured in Isochrysis sp./ Nanochloropsis sp. (1:1, v/v) mixture gave similar growth rate as 100% Nanochoropsis sp. culture (μ = 0.40 d⁻¹), but had 45% higher counts of rotifers with eggs (t = 7, maximum). The Isochrysis sp. culture successfully lowered the nitrate (46%) and orthophosphate (83%) during outdoor culture.     

Evaluation of Zostera detritus as a potential new source of zosteric acid

Detritus of the seagrasses Zostera noltii and Z. marina collected on the beaches of Arcachon Lagoon (France) over a 3-year period was screened as a new source of zosteric acid (ZA). This natural sulphated phenolic acid is a high value-added product capable of preventing settlement of marine organisms and protecting crops from fungal diseases. The seasonal variation of the ZA content was quantified in methanolic and aqueous crude extracts using high-performance liquid chromatography. The concentration found ranged from 65 to 456 μg g⁻¹ dry wt for Z. noltii and 51–692 μg g⁻¹ dry wt for Z. marina, respectively. This is the first report of ZA in Zostera noltii. Detrital leaves of Zostera have never before been screened for their bioactive substances. These results show that this low cost, very abundant and renewable, but heretofore unused, marine resource has potential as a source of a rare and naturally occurring bioactive product.     

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods

The tissue distribution and ontogeny of Na⁺/K⁺-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na⁺/K⁺-ATPase with the polyclonal antibody α (H-300) raised against the human α1-subunit of Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na⁺/K⁺-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na⁺/K⁺-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na⁺/K⁺-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 ± 32.4 μmol g_FM⁻¹ h⁻¹) than in those of L. vulgaris (31.8 ± 3.3 μmol g_FM⁻¹ h⁻¹). S. officinalis gills and pancreatic appendages achieved activities of 94.8 ± 18.5 and 421.8 ± 102.3 μmol_ATP g_FM⁻¹ h⁻¹, respectively. High concentrations of Na⁺/K⁺-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO₂) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.     

Synchronous fluorescence determination and molecular modeling of 5-Aminosalicylic acid (5-ASA) interacted with human serum albumin

In this paper, we proposed a new method for the determination of either human serum albumin (HSA) or 5-Aminosalicylic acid (5-ASA) by synchronous fluorescence spectra and examined the interaction between them using the molecular modeling method under simulative physiological conditions. The optimum conditions of synchronous fluorometric determination of HSA were investigated and the method was successfully applied to the determination of 5-ASA added to serum, urine, and saliva samples. The linear range of the determination of HSA and 5-ASA were 1.60 – 414 μg mL⁻¹ and 0.76 –22.95 μg mL⁻¹, the detection limits were 0.552 μg mL⁻¹ and 0.38 μg mL⁻¹, respectively. In addition, the effect of various common ions on the determination of HSA with 5-ASA was also discussed at room temperature. Figure The salicylic acid moiety is located within the binding pocket. The ring of 5-ASA was inserted in the hydrophobic cavity of site I, and it is important to note that the residue ARG-218 and the trptophan residue of HSA (Trp214) are in close proximity to the ring of 5-ASA suggesting the existence of hydrophobic interaction between them.     

Effects of reduced diameter of bag cultures on content of essential fatty acids and cell density in a continuous algal production system

Cell density and fatty acid (FA) content of Pavlova lutheri and Chaetoceros muelleri were analysed in a continuous algal production system (250-L bags) with reduced diameter. The cell density and FA content and composition in the algal production system were determined in replicate bags over a period of 5 weeks. The results showed that the cell density and essential FAs increased during the experiment for both species. After 5 weeks the mean cell numbers had increased to 6.0 ± 0.3 × 10⁶ cells mL⁻¹ in the P. lutheri bags and 6.0 ± 0.4 × 10⁶ cells mL⁻¹ in the C. muelleri bags. The content of total FAs increased significantly (p < 0.05) in all of the bags during the experiment. At the end of the experiment the mean total FA content were 2.7 ± 0.3 pg cell⁻¹ in the P. lutheri bags and 1.8 ± 0.1 pg cell⁻¹ in the C. muelleri bags. Maximum total FA content registered was 3.0 pg cell⁻¹ in one of the P. lutheri bags. The content of the essential FAs (ARA, EPA, DHA) increased over time in both of the species. At the end of the experiment the content of EPA (0.6 ± 0.1 pg cell⁻¹) and DHA (0.3 ± 0.0 pg cell⁻¹) were highest in the P. lutheri bags, while ARA (0.1 ± 0.0 pg cell⁻¹) was highest in C. muelleri. EPA and DHA constituted 22% and 11%, respectively, of total FA content in P. lutheri, while ARA constituted 6% of total FA content in C. muelleri. The results from this experiment indicate that flagellates such as P. lutheri perform better in narrow bags with improved light conditions, while diatoms like C. muelleri perform better in wider bags under light limitation. Implications for bivalve hatcheries are discussed.     

The characteristics of nutrient removal and inhibitory effect of Ulva clathrata on Vibrio anguillarum 65

To investigate the ecological effect of macroalgae on de-eutrophication and depuration of mariculture seawater, the variation of dissolved inorganic nitrogen (DIN) and phosphate (DIP), the amount of Vibrio anguillarum, and total heterotrophic bacteria in Ulva clathrata culture, as well as on the algal surface, were investigated by artificially adding nutrients and V. anguillarum strain 65 from February to April 2006. The results indicated that U. clathrata not only had strong DIN and DIP removal capacities, but also showed a significant inhibitory effect on V. anguillarum, although not reducing the total heterotrophic bacteria. Vibrio anguillarum 65 dropped from 5∼8 × 10⁷ cfu mL⁻¹ to 10 cfu mL⁻¹ (clone-forming units per mL) in 10 g L⁻¹ of fresh U. clathrata culture within 2 days; i.e., almost all of the Vibrios were efficiently eradicated from the algal culture system. Our results also showed that the inhibitory effect of U. clathrata on V. anguillarum strain 65 was both DIN- and DIP-dependent. Addition of DIN and DIP could enhance the inhibitory effects of the algae on the Vibrio, but did not reduce the total heterotrophic bacteria. Further studies showed that the culture suspension in which U. clathrata was pre-cultured for 24 h also had an inhibitory effect on V. anguillarum strain 65. Some unknown chemical substances, either released from U. clathrata or produced by the alga associated microorganisms, inhibited the proliferation of V. anguillarum 65.     

Cultivation of the green alga, Codium fragile (Suringar) Hariot, by artificial seed production in Korea

Codium fragile (Suringar) Hariot is an edible green alga farmed in Korea using seed stock produced from regeneration of isolated utricles and medullary filaments. Experiments were conducted to reveal the optimal conditions for nursery culture and out-growing of C. fragile. Sampling and measurement of underwater irradiance were carried out at farms cultivating C. fragile at Wando, on the southwestern coast of Korea, from October 2004 to August 2005. Growth of erect thalli and underwater irradiance were measured over a range of depths for three culture stages. During the nursery cultivation stage (Stage I), growth rate was greatest at 0.5 m depth (0.055 ± 0.032 mm day⁻¹), where the average midday irradiance over 60 days was 924 ± 32 μmol photons m⁻² s⁻¹. During the pre-main cultivation stage (Stage II), the greatest growth rate occurred at a depth of 2 m (0.113 ± 0.003 mm day⁻¹) with an average irradiance of 248 ± 116 μmol photons m⁻² s⁻¹. For the main cultivation stage (Stage III) of the alga, thalli achieved the greatest increase in biomass at 1 m depth (7.2 ± 1.0 kg fresh wt m⁻¹). These results suggest that optimal growth at each cultivation stages of C. fragile could be controlled by depth of cultivation rope.     

Biofiltering efficiency, uptake and assimilation rates of Ulva clathrata (Roth) J. Agardh (Clorophyceae) cultivated in shrimp aquaculture waste water

The growth, biofiltering efficiency and uptake rates of Ulva clathrata were studied in a series of outdoor tanks, receiving waste water directly from a shrimp (Litopenaeus vannamei) aquaculture pond, under constant aeration and two different water regimes: (1) continuous flow, with 1 volume exchange a day (VE day^-1) and (2) static regime, with 1 VE after 4 days. Water temperature, salinity, pH, dissolved inorganic nitrogen (DIN), phosphate (PO₄), chlorophyll-a (chl-a), total suspended solids (TSS), macroalgal biomass (fresh weight) and tissue nutrient assimilation were monitored over 12 days. Ulva clathrata was highly efficient in removing the main inorganic nutrients from effluent water, stripping 70–82% of the total ammonium nitrogen (TAN) and 50% PO₄ within 15 h. Reductions in control tanks were much lower (Tukey HSD, P < 0.05). After 3 days, the mean uptake rates by the seaweed biomass under continuous flow were 3.09 mg DIN g DW day⁻¹ (383 mg DIN m⁻² day⁻¹) and 0.13 mg PO₄ g DW day⁻¹ (99 mg PO₄ m⁻² day⁻¹), being significantly higher than in the static regime (Tukey HSD, P < 0.05). The chl-a decreased in seaweed tanks, suggesting that U. clathrata inhibited phytoplankton growth. Correlations between the cumulative values of DIN removed from the water and total nitrogen assimilated into the seaweed biomass (r = 0.7 and 0.8, P < 0.05), suggest that nutrient removal by U. clathrata dominated over other processes such as phytoplankton and bacterial assimilation, ammonia volatilization and nutrient precipitation.     

Fed-batch production of unsaturated medium-chain-length polyhydroxyalkanoates with controlled composition by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

Unsaturated medium-chain-length polyhydroxyalkanoates (MCL-PHA) were produced at a productivity of 0.63–1.09 g PHA l⁻¹ h⁻¹ with final PHA content ranging from 42.6 to 55.8% in single-stage, carbon-limited, fed-batch fermentations of Pseudomonas putida KT2440. A mixture of nonanoic acid (NA) and 10-undecenoic acid (UDA⁼) was fed exponentially to control growth rate. Varying the specific growth rate (0.14 h⁻¹ vs. 0.23 h⁻¹) at similar substrate feed ratios (NA:UDA⁼ = 5:1) had little effect on the final PHA content and relative composition. However, decreasing the NA:UDA⁼ ratio decreased the final amount of PHA produced from 56% with NA:UDA⁼ = 5.07:1 to only 42% at NA:UDA⁼ = 2.47:1. The molar fraction of all 3-hydroxyalkanoate monomers in the PHA product was relatively constant throughout each fermentation, indicating that the final product was homogeneous rather than a mixture of different copolymers. A linear relationship between unsaturation of the PHA produced and unsaturation of the carbon feed was found, which demonstrates the feasibility of producing unsaturated MCL-PHAs with controlled polymeric composition in a fed-batch process.     

Carbon-limited fed-batch production of medium-chain-length polyhydroxyalkanoates from nonanoic acid by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

Pseudomonas putida KT2440 grew on glucose at a specific rate of 0.48 h⁻¹ but accumulated almost no poly-3-hydroxyalkanoates (PHA). Subsequent nitrogen limitation on nonanoic acid resulted in the accumulation of only 27% medium-chain-length PHA (MCL-PHA). In contrast, exponential nonanoic acid-limited growth (μ = 0.15 h⁻¹) produced 70 g l⁻¹ biomass containing 75% PHA. At a higher exponential feed rate (μ = 0.25 h⁻¹), the overall productivity was increased but less biomass (56 g l⁻¹) was produced due to higher oxygen demand, and the biomass contained less PHA (67%). It was concluded that carbon-limited exponential feeding of nonanoic acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective method of producing MCL-PHA. Nitrogen limitation is unnecessary.     

Somatic embryogenesis and plant regeneration in oil palm using the thin cell layer technique

An efficient procedure has been developed for inducing somatic embryogenesis and regeneration of plants from tissue cultures of oil palm (Elaeis guineensis Jacq.). Thin transverse sections (thin cell layer explants) of different position in the shoot apex and leaf sheath of oil palm were cultivated in Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented with 0–450 μM picloram and 2,4-D with 3.0% sucrose, 500 mg L⁻¹ glutamine, and 0.3 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel. Embryogenic calluses were evaluated 12 wk after inoculation. Picloram (450 μM) was effective in inducing embryogenic calluses in 41.5% of the basal explants. Embryogenic calluses were maintained on a maturation medium composed of basal media, plus 0.6 μM NAA and 12.30 μM 2iP, 0.3 g L⁻¹ activated charcoal, and 500 mg L⁻¹ glutamine, with subcultures at 4-wk intervals. Somatic embryos were converted to plants on MS medium with macro- and micronutrients at half-strength, 2% sucrose, and 1.0 g L⁻¹ activated charcoal and gelled with 2.5 g L⁻¹ Phytagel.