Dynamics of oxygen evolution and biomass production during cultivation of Agardhiella subulata microplantlets in a bubble-column photobioreactor under medium perfusion

Cell and tissue cultures derived from macrophytic marine red algae are potential platforms for unique secondary metabolites. This work presents the first successful bioreactor cultivation study of an in vitro tissue culture derived from a macrophytic marine red alga. Specifically, the photosynthetic growth characteristics of a novel microplantlet suspension culture established from the macrophytic marine red alga Agardhiella subulata were studied. A bubble-column bioreactor with external illumination (43 microE m(-2) s(-1), 10:14 LD photoperiod), liquid medium perfusion, and 3800 ppm CO(2) in the aeration gas provided sufficient light and nutrient delivery for sustained growth of the microplantlet suspension at 24 degrees C and pH 8. Microplantlets, which consisted of shoot tissues of 3-5 mm length branching out from a common center, were not friable in a bubble-aerated suspension of about 1100 plantlets per liter. Since the microplantlet tissues were not friable, only batch and fed-batch cultivation modes were considered. Batch cultivation was phosphate-limited in ASP12 artificial seawater medium. However, cultivation at a medium perfusion rate of 20% per day avoided phosphate limitation and extended the growth phase to provide plantlet mass densities exceeding 14 g FW L(-1) (3.7 g DW L(-1)) after 50 days of cultivation if the suspension was not sampled. The specific oxygen evolution rate vs cultivation time profile possessed a significant pulse within the 14 days following inoculation and then leveled off at longer times. In recognition of this nonexponential growth pattern, a new photobioreactor growth model was developed that used the oxygen evolution rate vs time profile to predict the biomass growth curve in perfusion culture. Model predictions agreed reasonably with the measured growth curves.     

Some effects of plant growth regulators on tissue cultures of the marine red alga Agardhiella subulata (Gigartinales,Rhodophyta)

We examined whether auxins and cytokinins, either singly or in combination, stimulate cell division in tissue cultures of a red seaweed. Our experimental model consisted of filamentous and callus-like growths that developed from cross-sectional discs cut from young branches of Agardhiella subulata. Plant growth regulators were added to the medium to give combinations of an auxin with a cytokinin over a range of concentrations (1 µg L^–1 –10 mg L^–1). Several mixtures of auxins and cytokinins, as well as some single auxins, cytokinins and phenolics, stimulated cell division and growth in the tissue cultures beyond that of controls. The treatments that were effective included: phenylacetic acid/zeatin; phenylacetic acid/6-benzylaminopurine; -naphthaleneacetic acid/zeatin; 2,4,5-trichlorophenoxyacetic acid/6-benzylaminopurine; and indoleacetic acid/kinetin. High concentrations of cytokinins (i.e. 10 mg L^–1) inhibited the regeneration of plants in some of the cell cultures. These results provide further evidence that growth regulators can be used for the tissue culture of seaweeds and for the study of developmental phenomena in these plants.     

Ultrahigh-cell-density culture of a marine green alga Chlorococcum littorale in a flat-plate photobioreactor

To test the feasibility of CO₂ remediation by microalgal photosynthesis, a modified type of flat-plate photobioreactor [Hu et al. (1996) Biotechnol Bioeng 51:51–60] has been designed for cultivation of a high-CO₂-tolerant unicellular green alga Chlorococcum littorale. The modified reactor has a narrow light path in which intensive turbulent flow is provided by streaming compressed air through perforated tubing into the culture suspension. The length of the reactor light path was optimized for the productivity of biomass. The interrelationship between cell density and productivity, as affected by incident light intensity, was quantitatively assessed. Cellular ultrastructural and biochemical changes in response to ultrahigh cell density were investigated. The potential of biomass production under extremely high CO₂ concentrations was also evaluated. By growing C. littorale cells in this reactor, a CO₂ fixation rate of 16.7 g CO₂ l⁻¹ 24 h⁻¹ (or 200.4 g CO₂ m⁻² 24 h⁻¹) could readily be sustained at a light intensity of 2000 μmol m⁻² s⁻¹ at 25 °C, and an ultrahigh cell density of well over 80 g l⁻¹ could be maintained by daily replacing the culture medium. Received: 20 October 1997 / Received revision: 19 December 1997 / Accepted: 24 January 1998     

Antibacterial bromophenols from the marine red alga Rhodomela confervoides

Two bromophenols, together with three known compounds, were isolated from the methanolic extract of the marine alga, Rhodomela confervoides. By means of MS and NMR spectroscopic analyses, they were identified as 3-bromo-4-[2,3-dibromo-4,5-dihydroxyphenyl] methyl-5-(hydroxymethyl) 1,2-benzenediol (1) and 3-bromo-4-[2,3-dibromo-4,5-dihydroxyphenyl] methyl-5- (ethoxymethyl) 1,2-benzenediol (2). Three known compounds were also isolated, namely 3-bromo-4-[2,3-dibromo-4,5-dihydroxyphenyl] methyl-5-(methoxymethyl) 1,2-benzenediol (3), 4,4'- methylenebis [5,6-dibromo-1,2-benzenediol] (4) and bis (2,3-dibromo-4,5-dihydroxybenzyl) ether (5). Compound 5 was the most active against five strains of bacteria with the MIC less than 70 microg/ml, while compounds 2, 3 and 4 exhibited moderate activity.     

Stable isotope labelling method for studies of saccharide metabolism in Agardhiella subulata

Stable isotopes are preferable in many ways to radioactive isotopes for metabolic studies designed to elucidate biosynthetic pathways. We have developed the methodology to utilize ¹³C-labelled compounds in tracer studies of saccharide metabolism in the red algae. Cultures of Agardhiella subulata were pulse-chase labelled with ¹³C0₂ and ¹²C0₂. Gas chromatography/mass spectrometry (GC-MS) and ¹³C-NMR provided for positive identification of labelled carbohydrate metabolites. In addition, GC-MS can be used to profile the monosaccharide composition of algal species and combined GC-MS and ¹³C-NMR can disclose which carbon(s) is (are) labelled and the extent of labelling. In ¹³C0₂ incubated plants, the label is clearly detected in floridoside and floridean starch. After chasing the labelled alga with ¹²CO₂ for three days or storing the pulse-chase labelled alga in darkness for 6 days, labels disappeared from both floridoside and starch and the contents of these two carbohydrates became very low. More detailed biochemical analysis is being continued to identify labelled cell wall polysaccharides and/or their precursors.     

Studies on R-phycoerythrins from two Antarctic marine red algae and a mesophilic red alga

R-phycoerythrin was purified from two benthic red algae, Iridaea cordata and Phyllophora antarctica, obtained growing at ?2°C under thick sea ice off the coast of Antarctica. For the I. cordata protein, the molecular mass was 245,000 Da, and its secondary structure was 60% α helix, 17% β sheet, 16% turn, and 7% other. The light-harvesting faculties of the I. cordata protein resembled those of R-phycoerythrins from mesophilic red algae and were distinctive from the novel R-phycoerythrin from P. antarctica. Deconvolution of the visible absorption spectrum of R-phycoerythrin from I. cordata indicated a minimum of five component bands having maxima at 568, 558, 534, 496, and 481?nm. R-phycoerythrins from the mesophilic Porphyra tenera and psychrophilic Phyllophora antarctica had the same five bands. The protein from Phyllophora antarctica obtained its unique spectrum from a more intense component at 482?nm, and a less intense band at 533?nm. This change was probably produced by a replacement of phycoerythrobilin by phycourobilin. A temperature study of the circular dichroism CD was obtained for R-phycoerythrin from I. cordata from 4 to 80°C. Laser time-resolved fluorescence studies on R-phycoerythrin showed bilin to bilin energy transfer with a 60.2-ps lifetime, which should occur by the Förster resonance. The similarities in spectra between the proteins from I. cordata and Porphyra tenera and the different spectrum for the protein from Phyllophora antarctica show that only particular antarctic habitats require unique R-phycoerythrins.     

Sulfur and N-methylformamide from the marine red alga Erythrophyllum delesserioides

Elementant S (0.2 % dry wt) and N-methylformamide (0.2 % dry wt) were isolated from the marine red alga Erythrophyllum delesserioides.     

Metabolic flux analysis of halogenated monoterpene biosynthesis in microplantlets of the macrophytic red alga Ochtodes secundiramea

The bioprocess engineering of marine macroalgae (i.e. seaweeds) for the production of secondary metabolites is an emerging area of marine biotechnology. One novel system is the biosynthesis of halogenated monoterpenes by "microplantlet" suspension cultures derived from the red alga Ochtodes secundiramea. This biosynthetic platform has three principal components: elaboration of myrcene from geranyl diphosphate (GPP); bromonium-ion promoted halogenation of myrcene to 10E-bromomyrcene, 3-chloro-10E-bromo-alpha-myrcene, and 3,10E-dibromomyrcene; bromonium-ion promoted cyclization of myrcene to Apakaochtodene B. In this study, a metabolic flux analysis on halogenated monoterpene biosynthesis was performed. To facilitate this effort, a "bromine free" cell line of O. secundiramea microplantlets was developed where biohalogenation was temporarily disabled but myrcene biosynthesis was still enabled. This cell line was cultivated within an airlift photobioreactor under nutrient medium perfusion. Halogenated monoterpene biosynthesis was "turned on" by coordinated addition of bromide and vanadate (a co-factor for vanadium bromoperoxidase) to the perfusion medium. From these experiments, the effects of bromide and vanadate delivery on the metabolic flux of each metabolite were determined. Bromination of myrcene at its Delta(6-10) olefinic bond was the dominant branch of the bioreaction network, whereas chlorination steps in the pathway were "weakly rigid". This study represents the first application of metabolic engineering principles to the analysis and manipulation of secondary metabolism in macrophytic marine organisms.     

Sesquiterpenes from the marine red alga Laurencia distichophylla

A new cuparene-type sesquiterpene, isolaurenisol, has been isolated and identified from the New Zealand red alga Laurencia distichophylla. Major differences in the chemical composition of two morphologically indistinguishable samples of L. distichophylla are noted.     

Terpenoids from marine red alga Laurencia pinnatifida

A new halogenated chamigrene named pinnatifenol, a rearranged chamigrene derivative a diterpenoid have been isolated from the ethyl acetate fraction of marine red alga Laurencia pinnatifida. Their structures were elucidated by spectroscopic means.     

Purification and characterization of a new lectin from the red marine alga Hypnea musciformis

A lectin from the red marine alga Hypnea musciformis (HML) was purified by extraction with 20 mM PBS, precipitation with 70% saturated ammonium sulphate, ion-exchange DEAE-Cellulose chromatography and RP-HPLC. The 9.3 kDa polypeptide agglutinates erythrocytes from various sources and shows oligomerization tendencies under certain MALDI-TOF/MS conditions. Preliminary N-terminal sequencing and biological assays strongly suggest that the HML may belong to a new class of algae lectins.     

Antiplasmodial halogenated monoterpenes from the marine red alga Plocamium cornutum

In our continuing search for antimalarial leads from South African marine organisms we have examined the antiplasmodial organic extracts of the endemic marine red alga Plocamium cornutum (Turner) Harvey. Two new and three known halogenated monoterpenes were isolated and their structures determined by standard spectroscopic techniques. The 3,7-dimethyl-3,4-dichloro-octa-1,5,7-triene skeleton is common to all five compounds. Interestingly, compounds bearing the 7-dichloromethyl substituent showed significantly higher antiplasmodial activity toward a chloroquine sensitive strain of Plasmodium falciparum.     

Ethylene evolution in marine algae and a proteinaceous inhibitor of ethylene biosynthesis from red alga

Fronds of marine algae, especially green alga, Codium latum,and red alga, Porphyra tenera, evolved a quantity of ethylenewhen IAA was exogenously applied, while brown alga, Padina arborescens,evolved only a little. Propionic acid, when added together withIAA, noticeably enhanced IAA-induced ethylene evolution in P.tenera and P. arborescens. This evolution was also enhancedby added acrylic acid in P. arborescens but not in P. tenera.It was promoted by methionine, though only at a high concentration(0.1 M), in P. tenera but not in P. arborescens. The rate ofethylene evolution was highest at 12?C among the incubationtemperatures tested of 5, 12 and 15?C. The conversion of ¹⁴C-3-methionineto radioactive ethylene in P. tenera was remarkably inhibitedby a proteinaceous inhibitor from P. tenera. ¹Present address: Division of Environmental Biology, NationalInstitute for Environment, Yatabe, Ibaraki, Japan. (Received May 27, 1976; )     

Primary structures of two hemagglutinins from the marine red alga, Hypnea japonica

As the first examples among marine algal hemagglutinins, the primary structures of two hemagglutinins, named hypnin A-1 and A-2, from the red alga Hypnea japonica, were determined by Edman degradation. Both hemagglutinins were single-chain polypeptides composed of 90 amino acid residues including four half-cystines, all of which were involved in two intrachain disulfide bonds, Cys(5)-Cys(62) and Cys(12)-Cys(89). Hypnin A-1 and A-2 had calculated molecular masses of 9146.7 and 9109.7 Da which coincided with determined values, 9148 and 9109 Da, by electrospray ionization-mass spectrometry, respectively. Both hemagglutinins only differed from each other at three positions; Pro(19), Arg(31) and Phe(52) of hypnin A-1 as compared with Leu(19), Ser(31), and Tyr(52) of hypnin A-2. Approximately 43% of total residual numbers consisted of three kinds of amino acids: serine, glycine and proline. The hemagglutination activities were lost by reduction and alkylation of the disulfide bonds. The nature of the small-sized polypeptides, including disulfide bonds, may contribute to the extreme thermostability of the hemagglutinins. Sequence having overall similarity to hypnin A-1 or A-2 was not detected in databases. Unexpectedly, however, hypnins contained a motif similar to the alignment of the C-terminal conserved amino acids within carbohydrate-recognition domains of C-type animal lectins. Furthermore, interestingly, the hemagglutination activities were inhibited by a protein, phospholipase A-2 besides some glycoproteins, suggesting that hypnins may possess both a protein-recognition site(s) and a carbohydrate-recognition site(s).     

Self-cycling fermentation in a stirred tank reactor

Self-cycling fermentations (SCFs) were conducted in a stirred tank apparatus using Bacillus subtilis and Acinetobacter calcoaceticus. The systems were very stable and the experiments lasted through many cycles. The variation of parameters such as biomass and doubling time from cycle to cycle was small. The stirred tank reactor (STR) allowed a much better control of the working volume in the fermentor from cycle to cycle, compared to the cyclone column, and it was not necessary to make periodic corrections.The production of surfactin from B. subtilis was achieved without extending the cycle time. The harvested broth at the end of each cycle was allowed to remain in a secondary vessel, at ambient temperature, before being collected. It is exhaustion of the limiting nutrient which causes an increase in dissolved oxygen (DO). At this point, the computer, which constantly monitors the DO, triggered the harvesting sequence to end the cycle. Thus, the mature culture in the secondary vessel experienced appropriate conditions for the production of the secondary metabolite. Meanwhile, the next batch of cells was being grown in the primary reactor.The response of a gas analyzer on the effluent paralleled that of the DO measurements in the fermentor. These data for oxygen and carbon dioxide exhibited less noise than the DO readings. Either would be a more reliable parameter for feedback control of the SCF because the problem of fouling of the DO probe after extended runs of many cycles would be eliminated. (c) 1993 John Wiley & Sons, Inc.