Influence of gelling agents on culture medium gel strength, water availability, tissue water potential, and maturation response in embryogenic cultures of Pinus strobus L.

Summary Maturation of somatic embryos of Pinus strobus L. was evaluated on media containing various types (agars and gellan gum), brands and concentrations of gelling agents in the presence of 80 μM ABA and 0.09 M sucrose. The media were characterized with respect to gel strength, water potential and water availability. Embryogenic tissue and somatic embryos cultured on medium with various concentrations of gellan gum were used to determine their water potential (Ψ). Regardless of the type of gelling agent used, gel strength increased with gelling agent concentration and was critical to the maturation response. High gel strength was associated with reduced water availability from the medium to the cultures. The water potential of gelled maturation medium remained constant between 0.4 and 1.0% gellan gum. It is concluded that the embryogenic tissue was exposed to varying amounts of water at the onset of and during the culture period, and that the amount of water in the culture environment in turn influenced the maturation response. Cotyledonary somatic embryos derived from gellan gum medium of high gel strength had a lower Ψ than somatic embryos matured on medium of lower gel strength. Once somatic embryos developed to the cotyledonary stage on the maturation medium, they were transferred to the germination medium. The germination frequency and the number of morphologically normal germinants were higher for somatic embryos matured on medium of high gel strength. Raising the concentration of the gelling agent in the maturation medium may be an alternative to the use of solutes to restrict water available to the embryogenic cultures.     

Agar yield and quality of Gracilaria chilensis (Gigartinales,Rhodophyta) in tank culture using fish effluents

Tank cultivation of Gracilaria using fish effluents has permitted a production of 48 kg m^–2 yr^–1 and can reduce the dissolved nitrogen loads in the seawater. We report the yield, gel strength, gelling and melting point of agar from Gracilaria cultivated in tanks with seawater previously utilized in intensive, land-based salmon cultures and compared to a control using directly pumped seawater, over a study period of 22 months. The results show that the highest agar yield (20 to 22%) was obtained when Gracilaria was cultivated with pure seawater as compared to the fish effluents. The gel strength, gelling and melting point were higher in the agar obtained from algae cultured with fish effluents. During the spring, the gel strength, gelling and melting point increased in tanks with fish effluents and decreased in tanks with a supply of pure seawater.     

SEASONAL VARIATION OF P CONTENT AND MAJOR N POOLS IN PALMARIA PALMATA (RHODOPHYTA)1

The annual variation of major nitrogen pools, phosphorus, carbon, ash, and thallus water content in relation to seasonal environmental changes was studied in two northern Spanish populations of the edible seaweed Palmaria palmata (Linnaeus) Kuntze. Observed patterns were investigated using Spearman rank order correlation coefficients. There were significant relationships between thallus nutrient content and nitrate and orthophosphate seawater concentration, irradiance, temperature, and wave force. The highest levels of total N and P and nitrogenous compounds were observed during autumn and winter because the thallus stored N‐ and P‐rich compounds in response to high nutrient seawater concentration when growth was limited by low light and temperature. Phycoerythrin and other proteins were the main N reserves. Thallus P content was higher in algae from the eutrophic site. During spring, reduced N and P thallus content and increased ash, water, and C content were observed in the growing fronds. N and P seawater concentrations were undetectable during summer when nutrient reserves were low and growth was reduced and eventually suppressed, suggesting nutrient limiting conditions. Palmaria palmata clearly could take advantage of elevated N and P concentrations to create storage reserves in winter to support early summer growth. This storage response reduced the dependence of algal nutrition on the external nutrient supply and supports the use of pulse fertilization to diminish summer nutrient limitation of cultured algae.     

Vegetative propagation by fragmentation of Gelidium sclerophyllum (Gelidiales,Rhodophyta)

Results of the first phase of production of "seed" for a project of marine culture of species of Gelidiales are presented. Vegetative fragments of different sizes and from different parts of the thallus of Gelidium sclerophyllum from the Mexican tropical Pacific coast were cultured under nine treatments with different N and P concentrations. In treatments without N all fragments died; good vegetative growth was present in all remaining treatments. Growth in length and branch production were favored in medial fragments, and rhizoid production was favored in apices. Growth in length and biomass increase were stimulated by high N concentrations (2–3 mM), and branch and rhizoid production were stimulated by high P concentrations (100–150 µM).     

A two-stage solar photobioreactor for cultivation of microalgae based on solar concentrators

A novel two-stage experimental photobioreactor (PBR) with a total volume of 450 L and based uniquely on solar concentrators—linear Fresnel lenses—has been constructed and tested. Daily courses of irradiance, and also its distribution inside cultivation tubes, were studied in two unit types. The supra-high irradiance units in the ‘roof’ achieved a maximum summer value above 6 mmol photon m⁻² s⁻¹, while irradiance in the vertical-facade units was lower than ‘ambient’. In model cultivations, cultures of the cyanobacterium Arthrospira platensis were cultivated at much higher solar irradiances than those usually recorded outdoors in summer, indicating that this organism is resilient to high-irradiance (photoinhibition). Starting from a biomass density of 0.5 g L⁻¹ at optimum temperature, the cultures grew exponentially. A two-stage cultivation process of the green microalga Haematococcus pluvialis was investigated with respect to correlations between photochemical activities and astaxanthin production. The culture was first grown in low-irradiance units, and then exposed to supra-high irradiance when the rate of astaxanthin production was 30–50% higher than in the culture exposed to ‘ambient’ irradiance. Within 4 days, the astaxanthin content reached 3% of dry weight, whereas under ambient irradiance the astaxanthin content was 25% lower.     

Chemical and gelling profile of ice-ice infected carrageenan from Kappaphycus striatum (Schmitz) Doty “sacol” strain (Solieriaceae, Gigartinales, Rhodophyta)

The occurrence of ice-ice disease in farmedKappaphycus spp. has tremendous implication on thecarrageenan industry in the Philippines. To elucidate the effect of thisdiseaseon the carrageenan quality, the chemical profile of carrageenan extracted fromhealthy and diseased Kappaphycus striatumsacol strain, was investigated using ¹³C and¹H NMR, FT-IR and GPC methods of analysis. The native carrageenanextracts were composed predominantly of kappa carrageenan with trace amounts ofiota and 6-O-methylated carrageenan. Structural evidence indicatesdepolymerization of carrageenan from the ice-ice infectedportion of the thallus (whitish and soft part) lowered levels of iota andmethylconstituents. This finding was correlated to its low number average molecularweight (30 kDa) value. Accordingly, appreciable decreases incarrageenan yield, gel strength and viscosity and increase in syneresis indexwere noted. On the other hand, archetype quality of kappa-carrageenan wasobserved from both healthy and uninfected portion of the diseased thalli.     

Artificial cultures of the cyanobacterial lichenPeltigera didactyla (Peltigeraceae) in the natural environment

Soredia of the cyanobacterial lichenPeltigera didactyla were cultivated on soil substratum in the natural environment. Various developmental stages (germination, formation of a soredia field, differentiation of thallus lobes with soralia and fruiting bodies) were attained during a cultivation period of 6–7 months. Our field experiments revealed that the ephemeral pioneer lichenPeltigera didactyla has a short life cycle. Thus this lichen is a well-suited object for further investigations on thallus morphogenesis of lichens with cyanobionts.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

Structure and properties of agar from two unexploited agarophytes from Venezuela

The red seaweeds Gelidiella acerosa and Gracilaria mammillaris growing along the coast of Venezuela were investigated as potential economic sources of agar. Agar extracted from Gracilaria mammillaris accounted for 27% of the algal dry weight but had quite a poor gelling ability because of the presence of alkali-stable sulphate groups on the D-galactose residue. However, the gel strength of its aqueous solutions was considerably enhanced by the addition of potassium, sodium and calcium ions (up to 1N). On the contrary, the galactan from Gelidiella acerosa (yield of about 20% w/w) exhibited quite good properties. In particular, the gel strength was comparable to that of commercial agaroses. Sulphate esters were not detectable by chemical methods and NMR spectroscopy revealed an agarose backbone with a high degree of methylation on both D and L-galactose residues.     

Effects of phosphate and nitrate supply on productivity,agar content and physical properties of agar ofGracilaria strain G-16S

Gracilaria strain G-16S was cultured in various phosphorus (P) supply rates with low or high nitrogen (N) supply to determine the effects of nutrient supply on its productivity, agar content and physical properties of the agar. Productivity was reduced after four weeks of growth in zero P supply as plants reached 0.07% P tissue content (critical level), with fragmentation of these plants by six weeks (0.05% P; minimum viable level). Native agar content was higher in low P and high N, or low N conditions. Agar content appeared to increase with decreasing P under high N supply. This increase was not apparent with alkali treatment prior to extraction. Agar gel strength was greatly increased by alkali treatment. The highest gel strengths were obtained under high N supply at all P supply rates except zero P, and under low N supply at 12 M P week^–1. Native agar gel strengths showed a similar pattern on a lower scale. Melting temperatures were higher in agars with higher gel strengths. Dynamic gelling temperatures were generally high for alkali-treated agar, with agar from plants grown in zero P supply showing a slightly elevated gelling temperature. Melting and gelling temperatures of native agars with the highest gel strengths were in the same range as bacteriological agar. These results show that P and N supply affects productivity, agar content and agar physical properties, but the tradeoffs between a slightly higher agar quantity under nutrient limitation and higher agar quality under nutrient-replete conditions seem to favor the latter.     

Effect of temperature on yield and night biomass loss in Spirulina platensis grown outdoors in tubular photobioreactors

Outdoor experiments carried out in Florence, Italy (latitude 43.8° N, longitude 11.3° E), using tubular photobioreactors have shown that in summer the average net productivity of a Spirulina platensis culture grown at the optimal temperature of 35 °C was superior by 23% to that observed in a culture grown at 25 °C. The rates of night biomass loss were higher in the culture grown at 25 °C (average 7.6% of total dry weight) than in the one grown at 35 °C (average 5%). Night biomass loss depended on the temperature and light irradiance at which the cultures were grown, since these factors influenced the biomass composition. A net increase in carbohydrate synthesis occurred when the culture was grown at a low biomass concentration under high light irradiance or at the suboptimal temperature of 25 °C. Excess carbohydrate synthesized during the day was only partially utilized for night protein synthesis.     

Problems in developing the biotechnology of algal biomass production

Summary The effects of environmental conditions (solar irradiance and temperature) and population density on the production of Spirulina biomass with brackish water are reported for cultures grown in outdoor ponds. Higher specific growth rates were observed at lower population densities. Lower growth rates were associated with limitation by light in dense cultures under optimum conditions in the summer. Seasonal variation in productivity was observed. In summer, light was the limiting factor, whereas in winter the low daytime temperature appeared to constitute the major limitation. The oxygen concentration in the culture can serve as a useful indicator of limiting factors and can also be used to estimate the extent of such limitations.     

Water relations in culture media influence maturation of avocado somatic embryos

Application of transformation and other biotechnological tools in avocado (Persea americana Mill.) is hampered by difficulties in obtaining mature somatic embryos capable of germination at an acceptable rate. In this work, we evaluated the effect of different compounds affecting medium water relations on maturation of avocado somatic embryos. Culture media were characterized with respect to gel strength, water potential and osmotic potential. Improved production of mature somatic embryos was achieved with gelling agent concentrations higher than those considered standard. The osmotic agents such as sorbitol and PEG did not have positive effects on embryo maturation. The number of w-o mature somatic embryos per culture was positively correlated with medium gel strength. Gel strength was significantly affected by gelling agent type as well as by gelling agent and PEG concentration. Medium water potential was influenced by sorbitol concentration; incorporation of PEG to a culture medium did not affect medium water potential. The highest maturation results were achieved on a medium gelled with 10 g l⁻¹ agar. Moreover, these somatic embryos had improved germination rates. These results corroborate the role of water restriction as a key factor controlling maturation of somatic embryos.     

Sodium polyacrylamide polyacrylate, a gelling agent in diets for cabbage looper, omnivorous looper and western avocado leafroller

Ingredient substitutions for agar or carrageenan in artificial diets for lepidopterous larvae were evaluated. A sodium polyacrylamide polyacrylate gelling agent, Water-Lock G-400, was compared with agar for solidifying diets of the cabbage looper, Trichoplusia ni (Hübner), omnivorous looper, Sabulodes aegrotata (Gueneé), and the western avocado leafroller, Amorbia cuneana Walsingham. Water-Lock G-400 is considerably less expensive than agar or carrageenan, it does not require heating to gel, and it is made from a dependable supply of agricultural starch. Sabulodes aegrotata pupae from larvae reared on agar were significantly heavier than those reared on Water-Lock G-400. However, the remainder of the variables observed (larval survival, developmental times, fecundity, sex ratio and pupal weights for A. cuneana and T. ni) were all independent of diet. Decreasing the total amount of ingredients in a Water-Lock-G-400 diet produced fewer and smaller T. ni pupae than did an agar diet when the larvae were reared using a mass production protocol. However, these lower values were still above acceptable levels for mass production and reduced the cost of diet production by 65%.Further ingredient substitutions impaired larval development (i.e., replacing pinto beans with soy protein or combinations of soy protein and -cellulose), while others did not (i.e., excluding pinto beans and reducing water content and replacing pinto beans with -cellulose or corn cob grits). Moisture control by adding corn cob grits to diets increased the yield of pupae, while removing water from diets increased survivorship and the yield of pupae.     

Production ecology of Gelidium

Available data on determinants of production in species of Gelidium suggest several general patterns. Species diversity is higher in tropical latitudes, whereas in temperate latitudes the size of the fronds is larger, the species are ecologically dominant and commercially viable. Typically, the species occur on rocky substrate, often on coralline crusts, associated with rapid water movement and arranged in successive belts that can extend down to 25 m depth. Yields vary among species, to a maximum of 2.0 kg m^–2 y^–1. Growth and production in many species can best be explained by complex interactions between irradiance and nutrients. Temperature can interact synergistically with irradiance, while water movement compensates for nutrient limitations. Increased water movement or the addition of nutrients can prevent, to an extent, bleaching by high light and high temperature. Available data suggest the existence of at least eight biological factors affecting predicted productivity of Gelidium crops: morphology, age of the frond, thallus part, reproductive state, seasonality, crop density, life history phase and geographic and ecological origin of the species. At least four events can remove or destroy Gelidium crops: extreme low tides, storms, grazing and careless harvesting. Only the last-named factor has been analyzed over more extensive experimental periods.     

Seasonal changes in agar characteristics of two populations ofPterocladia capillacea in Gran Canaria,Spain

Agar characteristics ofPterocladia capillacea were examined seasonally at two intertidal populations exposed to different wave energy on the northern rocky shore of Gran Canaria Island. Plants were collected monthly from August 1991 to July 1992. Agar yield, gel strength, melting and gelling temperature and chemical properties such as sulphate and pyruvate content were measured. Percent epiphytism was determined on both populations, together with the changes in biomass as dry weight. Specimens in the sheltered habitat were larger and more epiphytized than ones in the exposed area. There was a clear seasonal change in agar characteristics in both populations. Agar yields decreased in late spring and early summer, although in the sheltered habitat fluctuations were more erratic. Gel strength increased in winter, reaching a maximum in December–February. No significant differences were found in agar yield, gel strength or melting and gelling temperatures, but there was a difference between fresh to dry weight ratio. The role of the exposure degree as a possible environmental factor responsible for this behavior is discussed. Agars ofPterocladia capillacea from Canary Islands show characteristics for industrial use.Author for correspondence     

Biosorption of Chromium,Cadmium, and Cobalt from Aqueous Solution by Immobilized Living Cells of Chryseomonas luteola TEM 05

Abstract

In this work, the potential use of the immobilized cells of Chryseomonas luteola TEM 05 for the removal of Cr⁺⁶, Cd⁺² and Co⁺² ions from aqueous solutions was investigated. The living cells of C. luteola TEM 05 were firstly entrapped both in carrageenan and chitosan coated carrageenan gels and then used in biosoption of the metal ions in batch reactors at pH 6.0, 25°C, in 100 mg L^?1 of each metal solution. Besides this, a process of competitive biosorption of these metal ions was also described and compared to single metal ion adsorption in solution. According to the immobilization results, the replacement of KCl by KCl-chitosan as gelling agent improved the mechanical strength and thermal stability of the gel. In addition, the C. luteola TEM 05 immobilized carrageenan-chitosan gel system was quite more efficient for the fast adsorption of metal ions from aqueous solution than the carrageenan gels without biomass.     

Characterization of kappa-carrageenan gels used for immobilization of Bacillus firmus.

In this study, aimed at a biochemical and physical characterization of kappa-carrageenan gels used for entrapment of Bacillus firmus NRS 783 (a superior producer of an alkaline protease), effects of carrageenan concentration, gelation temperature, initial cell loading, and strength of the curing agent (KCl) on the properties of cell-free and cell-laden gels were examined. The physical properties of the differently prepared gels that were examined included density, free volume fraction, mechanical strength, and change in gel volume during gel curing. The biochemical characteristics studied included viability of gel-entrapped cells, cell leakage from cell-laden gels, and cell penetration into cell-free gels. For the range of carrageenan contents investigated [between 2% and 5% (w/v)], the mechanical strength of the gels with/without KCl curing was observed to increase with an increase in carrageenan content of gels. The mechanical strength of each gel increased substantially upon extensive curing. Free volume fractions in excess of 0.8 were observed for all gels. Of cells that were viable prior to immobilization, 90-92% remained viable after formation and extensive curing of gels for cell-gel mixtures prepared at 45 degrees C. Attempts at prolonged storage of cell-laden gel beads at 0 degrees C as stock cultures of immobilized B. firmus were unsuccessful due to a significant decline in cell viability during such storage. On the basis of the cell leakage studies, the average pore sizes of 2%, 3%, 4%, and 5% gels were deduced to increase in the following order of carrageenan content (w/v): 4%, 3%, 2%, and 5%. Commensurate with the decrease in the average pore size (or the increased tightness of the gels) with increasing carrageenan content, both the extent of cell leakage and the extent of net cell penetration decreased with increasing carrageenan content for the first three gels. Owing to non-uniform distribution of free space and much larger pores, the extent of net cell penetration in 5% carrageenan gels was considerably low, while the extent of cell leakage in 5% carrageenan gels was an order of magnitude greater than the extents of cell leakage in the other three gels.     

Effect of ammonium on growth and carrageenan content in Kappaphycus alvarezii (Gigartinales,Rhodophyta)

Kappaphycus alvarezii, cultured in a region deficient in nitrogen with intermittent application of 10 mM ammonium at three-day intervals for 1 hour, attained a daily growth rate of 4.6%, which was considered to be the optimum growth rate. Under such conditions, the C/N ratio was 29 with a carrageenan content of 58% and gel strength of 45–70 g cm^–2. Gel strength of carrageenan was high when nitrogen content was high and vice versa.     

Optimized Shoot Regeneration System for the Commercial Korean Radish ‘Jin Ju Dae Pyong’

We have investigated the usefulness of hypocotyl (cultured on N1B2 medium) and cotyledon explants (on CR medium: Murashige and Skoog (MS) medium, 3% sucrose, 20 μm benzylaminopurine, pH 5.8) for the regeneration of shoots of the Korean radish ‘Jin Ju Dae Pyong’. The importance of ethylene (indirectly), polyamines and gelling agent were studied in both media. Although the addition of ethylene-inhibitors and silver nitrate to the culture media were beneficial towards shoot regeneration and agar-based treatments (0.8% w/v) being superior in shoot production compared to agarose treatments (0.4% w/v), both explants responded differently in culture. Hypocotyls cultured in the presence of silver nitrate or aminoethoxyvinylglycine (AVG) regenerated significantly (p < 0.05) more shoots compared to N1B2 medium alone; supplementation of 20 μM AVG to N1B2 medium gave optimal shoot production (40% of explants regenerating shoots). The addition of 10 μM AVG to CR medium produced maximum shoot regeneration from cotyledon explants (60% producing shoots). Plants derived from 3-month-old cultures produced greater seed weights, larger leaves and greater genetic variability (50–80% of cells having 20–40 chromosomes) compared to seed-derived (85–90% diploid) and plants from 1-month-old cultures (78–88% diploid). Our results show, that if prolonged culture of explants is avoided, a large number of phenotypically-normal plants can be produced, which in turn, could be utilized in the genetic improvement of radish.     

Production of pigments byMonascus purpureus in solid culture

Summary The effect of physical and nutritional factors on the production of pigments byMonascus purpureus FRR 2190 was studied using cultures grown on both rice and a synthetic medium that was solidified with carrageenan and extruded into rice-like particles. Pigment yield was highly sensitive to physical parameters. Optimal pigment formation in rice cultures occurred at an initial pH of 6 and an initial moisture content of 56%. Lower moisture contents led to a large decrease in pigment concentration. Red and yellow pigment production on solidified gel media was increased up to three-fold compared to that of liquid cultures of the same medium composition, particularly when peptone was used as the sole nitrogen source. Solid state rice cultures gave the highest pigment yields.