Efficient use of strong light for high photosynthetic productivity: interrelationships between the optical path,the optimal population density and cell-growth inhibition

The interrelationships between the optical path in flat plate reactors and photosynthetic productivity were elucidated. In preliminary works, a great surge in photosynthetic productivity was attained in flat plate photoreactors with an ultra short (e.g. 1.0 cm) optical path, in which extremely high culture density was facilitated by vigorous stirring and strong light. This surge in net photosynthetic efficiency was associated with a very significant increase in the optimal population density facilitated by the very short optical path (OP). A salient feature of these findings concerns the necessity to address growth inhibition (GI) which becomes increasingly manifested as cell concentration rises above a certain, species-specific, threshold (e.g. 1-2 billion cells of Nannochloropsis sp. ml(-1)). Indeed, ultrahigh cell density cultures may be established and sustained only if growth inhibition is continuously, or at least frequently, removed. Nannochloropsis culture from which GI was not removed, yielded 60 mg(-1) h(-1), yielding 260 mg l(-1) h(-1) when GI was removed. Two basic factors crucial for obtaining maximal photosynthetic productivity and efficiency in strong photon irradiance are defined: (1) areal cell density must be optimal, as high as possible (cell growth inhibition having been eliminated), insuring the average photon irradiance (I(av)) available per cell is falling at the end of the linear phase of the PI(av) curve, relating rate of photosynthesis to I(av), i.e. approximately photon irradiance per cell. (2) The light-dark (L-D) cycle period, which is determined by travel time of cells between the dark and the light volumes along the optical path, should be made as short as practically feasible, so as to approach, as much as possible the photosynthetic unit turnover time. This is obtainable in flat plate reactors by reducing the OP to as small a magnitude as is practically feasible.     

Extracellular polysaccharide production in outdoor mass cultures of Porphyridium sp. in flat plate glass reactors

This work concerns an attempt to develop large scalecultivation of Porphyridium sp. outdoors. Theimpact on cell growth and production of solublesulphated polysaccharides of light-path length (LP)was studied in flat plate glass reactors outdoors. TheLP of the plate reactors ranged from 1.3–30 cm,corresponding to culture volumes of 3–72 L. The sidewalls of all reactors were covered, ensuring similarilluminated surfaces for all reactors. Maximal daytemperature was maintained at 26 ±1 ^°C.Growth conditions of pH (7.5), stirring (withcompressed air) and mineral nutrients, were optimal.Maximal volumetric concentration of the soluble sulfated polysaccharide (1.32 g L^-1) was obtained in winter with the smallest light-pathreactor (1.3 cm ) at a cell density of 1.37 ×10¹¹cells L^-1. Under these conditions, theviscosity of the culture medium was also highest,being inversely proportional to the culture'slight-path. Highest areal concentration of solublepolysaccharides (60 g m^-2) and areal cell density(3.01 × 10¹²m^-2) was recorded in the 20 cmLP reactor, progressively lower values being obtainedas the light path became shorter. A similar patternwas obtained for the areal productivity ofpolysaccharides, the highest being 4.15 g m^-2day^-1 (considering the total illuminated reactorsurface), produced in the 20-cm LP reactor.The main sugar composition (i.e. xylose, galactose andglucose) of the sulfated polysaccharides was similarin all reactors. As viscosity increased with timeduring culture growth, there was a substantial declinein bacterial population. Cultivation throughout mostof the year provided good evidence that a light pathlength of 20 cm in flat plate reactors under theseconditions is optimal for maximal areal solublepolysaccharide production of Porphyridium sp.     

Scale-up of tubular photobioreactors

The effect of the light/dark cycle frequency on theproductivity of algal culture at differentday-averaged irradiance conditions was evaluated forPhaeodactylum tricornutum grown in outdoortubular photobioreactors. The photobioreactor scale-upproblem was analyzed by establishing the frequency oflight–dark cycling of cells and ensuring that thecycle frequency remained unchanged on scale-up. Thehydrodynamics and geometry related factors wereidentified for assuring an unchanged light/dark cycle.The light/dark cycle time in two different tubularphotobioreactors was shown to be identical when thelinear culture velocity in the large scale device(U _LL) and that in the small scale unit (>U _LS)were related as follows:U_LL = \frac f ^9/7 \alpha^8/7 U_LS.Here f is the scale factor (i.e., the ratio oflarge-to-small tube diameters), is afunction of the illuminated volumes in the tworeactors, and `dark' refers to any zone of the reactorwhere the light intensity is less than the saturationvalue. The above equation was tested in continuouscultures of P. tricornutum in reactors with 0.03 mand 0.06 m diameter tubes, and over the workableculture velocity range of 0.23 to 0.50 m s^-1. Thepredicted maximum realistic photobioreactor tubediameter was about 0.10 m for assuring a cultureperformance identical to that in reactors with smaller tubes.     

Sustainable, High-Yielding Outdoor Mass Cultures of Chaetoceros muelleri var. subsalsum and Isochrysis galbana in Vertical Plate Reactors

Continuous cultures of Chaetoceros muelleri and Isochrysis galbana were grown outdoors in flat plate-glass reactors in which light-path length (LPL) varied from 5 to 30 cm. High daily productivity (13 to 16 g cell mass per square meter of irradiated reactor surface) for long periods of time was obtained in reactors in which the optical path as well as cell density were optimized. 'Twenty centimeters was the optimal LPL, yielding the highest areal productivity of cell mass (g m^–2d^–1), eicosapentaenoic acid, and docosahexaenoic acid, which was identical with that previously found for polysaccharide production of Porphyridium and not far from the optimal LPL affecting maximal productivity in Nannochloropsis species. Relating the energy impinging on a given reactor surface area to the appropriate number of cells showed that the most efficient light dose per cell, obtained with the 20-cm LPL reactor, was approximately 2.5 times lower than the light dose available per cell in the 5-cm LPL reactor, in which a significant decline in areal cell density accompanied the lowest areal output of cell mass. The most effective harvesting regimen was in the range of 10% to 15% of culture volume harvested daily and replaced with fresh growth medium, resulting in a sustainable culture density of 24 × 10⁶ and 28 × 10⁶ cells/ml of C. muelleri and I. galbana, respectively.     

Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101

This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone–butanol–ethanol, ABE) at elevated levels. This organism expresses high levels of amylases when grown on starch. C. beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27–29 g l⁻¹. C. beijerinckii BA101 has been characterized for both substrate and butanol inhibition. Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g l⁻¹. The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors. Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C. beijerinckii BA101, and solvent production as high as 165 g l⁻¹ has been achieved. Immobilization of C. beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g l⁻¹ h⁻¹. Recent economic studies employing C. beijerinckii BA101 suggested that butanol can be produced at US$0.20–0.25 lb⁻¹ by employing batch fermentation and distillative recovery. Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices. Journal of Industrial Microbiology & Biotechnology (2001) 27, 287–291. Received 12 September 2000/ Accepted in revised form 27 January 2001     

Heterotrophic high-cell-density fed-batch and continuous-flow cultures of <Emphasis Type="Italic">Galdieria sulphuraria</Emphasis> and production of phycocyanin

Production of biomass and phycocyanin (PC) were investigated in highly pigmented variants of the unicellular rhodophyte Galdieria sulphuraria, which maintained high specific pigment concentrations when grown heterotrophically in darkness. The parental culture, G. sulphuraria 074G was grown on solidified growth media, and intensely coloured colonies were isolated and grown in high-cell-density fed-batch and continuous-flow cultures. These cultures contained 80–110 g L⁻¹ biomass and 1.4–2.9 g L⁻¹ PC. The volumetric PC production rates were 0.5–0.9 g L⁻¹ day⁻¹. The PC production rates were 11–21 times higher than previously reported for heterotrophic G. sulphuraria 074G grown on glucose and 20–287 times higher than found in phototrophic cultures of Spirulina platensis, the organism presently used for commercial production of PC.     

Effect of liquid circulation velocity and cell density on the growth ofParietochloris incisa in flat plate photobioreactors

For more accurately describing the durations of the light and the dark phases of microalgal cells over the whole light-dark cycle, and probing into the relationship between the liquid circulation time or velocity, the aeration rate and cell density, a series of experiments was carried out in 10 cm light-path flat plate photobioreactors. The results indicated that the liquid flow in the flat plate photobioreactor could be described by liquid dynamic equations, and a high biomass output, higher content and productivity of arachidonic acid, 70.10 gm⁻²d⁻¹, 9.62% and 510.3 mg/L, respectively, were obtained under the optimal culture conditions.     

Chlamydomonas reinhardtii: duration of its cell cycle and phases at growth rates affected by temperature

Synchronized cultures of the green alga Chlamydomonas reinhardtii were grown photoautotrophically under a wide range of environmental conditions including temperature (15–37°C), different mean light intensities (132, 150, 264 μmol m⁻² s⁻¹), different illumination regimes (continuous illumination or alternation of light/dark periods of different durations), and culture methods (batch or continuous culture regimes). These variable experimental approaches were chosen in order to assess the role of temperature in the timing of cell division, the length of the cell cycle and its pre- and post-commitment phases. Analysis of the effect of temperature, from 15 to 37°C, on synchronized cultures showed that the length of the cell cycle varied markedly from times as short as 14 h to as long as 36 h. We have shown that the length of the cell cycle was proportional to growth rate under any given combination of growth conditions. These findings were supported by the determination of the temperature coefficient (Q ₁₀), whose values were above the level expected for temperature-compensated processes. The data presented here show that cell cycle duration in C. reinhardtii is a function of growth rate and is not controlled by a temperature independent endogenous timer or oscillator, including a circadian one.     

Growth physiology of a marine nitrogen-fixing cyanobacterium (Nodularia harveyana) in outdoor culture

The performance ofNodularia harveyana, a N₂-fixing cyanobacterium isolated from seawater, has been studied outdoors in two different culture systems: open pond (OP) and tubular photobioreactor (TPR). The productivity in both devices was influenced by areal density. The maximum yield obtained was 12.0 g (d.wt) m^–2 day^–1 in OP and 14.0 g (d.wt) m^–2 day^–1 in TPR in August, corresponding to the highest solar radiation received. In a month-long experiment with the cyanobacterium cultivated in TPR at high circulation speed, a net increase in productivity was obtained over that at low circulation speed. The influence of temperature on the productivity of the cultures grown in open ponds and tubular photobioreactors has been investigated. The higher productivity obtained in TPR compared to OP was attributed to its better controlled temperature conditions. In outdoor culture the maximum nitrogenase activity did not coincide with the maximum light intensity, but occurred in early afternoon. The amount of carbohydrate accumulated during the day probably influenced the rate of dark nitrogenase activity and its duration in the night.     

Investigation of poly ( β-L-malic acid) production by strains of Aureobasidium pullulans

 Eight strains of the genus Aureobasidium obtained from culture collections were tested for their capability to produce poly(β-L-malic acid) (PMA). Four of the tested strains showed positive results. The most productive strain, A. pullulans CBS 591.75, was used to study the production of PMA in stirred-tank reactors. It was found that PMA was mainly produced in the late exponential phase, and the production related positively to glucose consumption. At the beginning of the fermentation the pH increased from 4.0 to about 7.0; subsequently the pH decreased and remained stable at around 3.0–3.5 for several days. Temperatures higher than 25^°C were detrimental to PMA production and cell growth. PMA production and cell growth at 20^°C and 25^°C exhibited no significant differences. PMA production and cell growth were studied under pH-controlled fermentation (at pH 2.0, 4.0, 5.5). The highest PMA production occurred at pH 4.0. PMA production was reduced at pH 2.0 although quite reasonable cell growth occurred at this pH value. Under optimized conditions 9.8 g PMA/l was produced during 9 days of fermentation in the stirred-tank reactors with an overall yield of 0.11 g PMA/g glucose. A procedure for the isolation of PMA and its separation from the other components of the fermentation broth was developed. The isolated PMA was characterized by ¹H and ¹³C-NMR spectroscopy as well as by infrared absorption spectroscopy. Gel-permeation chromatography revealed a relative molecular mass of approximately 3000–5000 by comparison with polyethylene glycol standards. Received: 13 February 1996/Received revision: 25 April 1996/Accepted: 1 May 1996     

Variation in some photosynthetic characteristics of microalgae cultured in outdoor thin-layered sloping reactors

In outdoor thin-layer sloping reactors algae are batch cultured and harvested at biomass concentrations of about 15 g (dw) I^-1 whereafter a portion is used as inoculum for the next cycle. Light saturation curves of the oxygen evolution (PII curves) of the algae were measured using diluted aliquots of suspension taken from the reactors. The maximum specific photosynthetic rates (P ^B _max) and the light intensity at the onset of saturated photosynthesis (I _k ) decreased whilst the maximum specific photosynthetic efficiency ( ^B ) increased with an increase in the biomass concentration, during the production cycle. These differences reflect transition from light- to dark-acclimated state of the algae that occurs as a result of an increase of the suspension concentration during the production cycle. During these experiments the thin-layered smooth sloping cultures (TLSS, culture depth 5–7 mm) had higher photosynthetic rates per volume than the thin-layered baffled sloping cultures (TLBS, culture depth 5–15 mm). This was ascribed to the higherP ^B _max values of the algae grown in the TLSS cultures, allowing them to utilise high incident irradiancies more effectively. However, the areal productivity of the TLBS was higher than the TLSS indicating a higher photosynthetic efficiency of the TLBS reactors. The specific productivity decreased rapidly with an increase in the biomass concentration, but the yield remained linear during the batch production cycle, even at high areal densities.     

Productivity correlated to photobiochemical performance of <Emphasis Type="Italic">Chlorella</Emphasis> mass cultures grown outdoors in thin-layer cascades

This work aims to: (1) correlate photochemical activity and productivity, (2) characterize the flow pattern of culture layers and (3) determine a range of biomass densities for high productivity of the freshwater microalga Chlorella spp., grown outdoors in thin-layer cascade units. Biomass density, irradiance inside culture, pigment content and productivity were measured in the microalgae cultures. Chlorophyll-fluorescence quenching was monitored in situ (using saturation-pulse method) to estimate photochemical activities. Photobiochemical activities and growth parameters were studied in cultures of biomass density between 1 and 47 g L⁻¹. Fluorescence measurements showed that diluted cultures (1–2 g DW L⁻¹) experienced significant photostress due to inhibition of electron transport in the PSII complex. The highest photochemical activities were achieved in cultures of 6.5–12.5 g DW L⁻¹, which gave a maximum daylight productivity of up to 55 g dry biomass m⁻² day⁻¹. A midday depression of maximum PSII photochemical yield (F _v/F _m) of 20–30% compared with morning values in these cultures proved to be compatible with well-performing cultures. Lower or higher depression of F _v/F _m indicated low-light acclimated or photoinhibited cultures, respectively. A hydrodynamic model of the culture demonstrated highly turbulent flow allowing rapid light/dark cycles (with frequency of 0.5 s⁻¹) which possibly match the turnover of the photosynthetic apparatus. These results are important from a biotechnological point of view for optimisation of growth of outdoor microalgae mass cultures under various climatic conditions.     

Sp1 polymorphism in collagen I α1 gene is associated with osteoporosis in lumbar spine of Mexican women

The Sp1 binding site polymorphism in collagen type I alpha 1 gene (COLIA1) has been associated with osteoporosis (OP) and bone mineral density (BMD). The aim of this study was to explore the association of this polymorphism with OP and BMD in the Mexican population by polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP) procedure. Allelic and genotypic frequencies from the Sp1 polymorphism were determined in 100 women with OP, 100 women without OP and 500 subjects from general Mexican population (GMP). Distribution of Sp1 polymorphism was in Hardy–Weinberg equilibrium. In spite of population structure due to racial mix in Mexican population, associations with OP were demonstrated. The frequency of “s” allele was significantly higher in women with OP (35%) than in women without OP (11%; P < 0.00001). Interestingly, “ss” genotype, was exclusive of women with OP and was associated with low BMD (0.588 ± 0.077 g/cm²) in contrast to “SS” genotype (0.733 ± 0.039 g/cm²; P = 0.0001). This work confirms the association of Sp1 polymorphism with low BMD and OP in Mexican population and make sure to use Sp1 as a genetic marker for OP in our population.     

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.     

Fine root vertical distribution and temporal dynamics in mature stands of two enset (Enset ventricosum Welw Cheesman) clones

Quantification of the role of fine roots in the biological cycle of nutrients necessitates understanding root distribution, estimating root biomass, turnover rate and nutrient concentrations, and the dynamics of these parameters in perennial systems. Temporal dynamics, vertical distribution, annual production and turnover, and nitrogen use of fine roots (≤2 mm in diameter) were studied in mature (5-year-old) stands of two enset (Ensete ventricosum) clones using the in-growth bag technique. Live fine root mass generally decreased with increasing depth across all seasons except the dry period. Except for the dry period, more than 70% of the fine root mass was in the above 0-20 cm depth, and the fine root mass in the upper 0–10 cm depth was significantly higher than in the lowest depth (20–30 cm). Live fine root mass showed a seasonal peak at the end of the major rainy season but fell to its lowest value during the dry or short rainy season. The difference between the peak and low periods were significant (p ≤ 0.05). Fine root nitrogen (N) use showed significant seasonal variation where the mean monthly fine root N use was highest during the major rainy season. There were significant effects on N use due to depths and in-growth periods, but not due to clones. Enset fine root production and turnover ranged from 2,339 to 2,451 kg ha⁻¹ year⁻¹ and from 1.55 to 1.80 year⁻¹, respectively. Root N return, calculated from fine root turnover, was estimated at 64–65 kg ha⁻¹ year⁻¹. Fine root production, vertical distribution and temporal dynamics may be related to moisture variations and nutrient (N) fluxes among seasons and along the soil depth. The study showed that fine root production and turnover can contribute considerably to the carbon and nitrogen economy of mature enset plots.     

Cell cycle propagation is driven by light–dark stimulation in a cultured symbiotic dinoflagellate isolated from corals

Endosymbiosis is an intriguing plant–animal interaction in the dinoflagellate–Cnidaria association. Throughout the life span of the majority of corals, the dinoflagellate Symbiodinium sp. is a common symbiont residing inside host gastrodermal cells. The mechanism of regulating the cell proliferation of host cells and their intracellular symbionts is critical for a stable endosymbiotic association. In the present study, the cell cycle of a cultured Symbiodinium sp. (clade B) isolated from the hermatypic coral Euphyllia glabrescens was investigated using flow cytometry. The results showed that the external light–dark (L:D) stimulation played a pivotal role in regulating the cell cycle process. The sequential light (40–100 μmol m⁻² s⁻¹ ~ 12 h) followed by dark (0 μmol m⁻² s⁻¹ ~ 12 h) treatment entrained a single cell cycle from the G₁ to the S phase, and then to the G₂/M phase, within 24 h. Blue light (~450 nm) alone mimicked regular white light, while lights of wavelengths in the red and infrared area of the spectrum had little or no effect in entraining the cell cycle. This diel pattern of the cell cycle was consistent with changes in cell motility, morphology, and photosynthetic efficiency (F _v /F _m ). Light treatment drove cells to enter the growing/DNA synthesis stage (i.e., G₁ to S to G₂/M), accompanied by increasing motility and photosynthetic efficiency. Inhibition of photosynthesis by 3-(3, 4-dichlorophenyl)-1, 1-dimethyl-urea (DCMU) treatment blocked the cell proliferation process. Dark treatment was required for the mitotic division stage, where cells return from G₂/M to G₁. Two different pools of adenylyl cyclase (AC) activities were shown to be involved in the growing/DNA synthesis and mitotic division states, respectively. Communicated by Biology Editor Dr Michael Lesser     

Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can produce the ketocarotenoid astaxanthin under heterotrophic culture conditions. Here we report the growth-associated biosynthesis of astaxanthin in this biotechnologically important alga. With glucose as sole carbon and energy source, C. zofinginesis grew fast in the dark with rapid exhaustion of nitrogen and carbon sources from media, leading to a high specific growth rate (0.034 h⁻¹). Cultures started at a cell concentration of about 3.4 × 10⁹ cells l⁻¹ reached, after 6 days, standing biomass values of 1.6 × 10¹¹ cells or 8.5 g dry weight l⁻¹. Surprisingly, the biosynthesis of astaxanthin was found to start at early exponential phase, independent of cessation of cell division. A general trend was observed that the culture conditions benefiting cell growth also benefited astaxanthin accumulation, indicating that astaxanthin was a growth-associated product in this alga. The maximum cell dry biomass and astaxanthin yield were 11.75 g l⁻¹ and 11.14 mg l⁻¹ (about 1 mg g⁻¹), simultaneously obtained in the fed-batch culture with a combined glucose–nitrate mixture addition, which were the highest ever reported in dark-heterotrophic algal cultures. The possible reasons why dark-heterotrophic C. zofingiensis could produce astaxanthin during the course of cell growth were discussed.     

Effect of light/dark cycles on wastewater treatments by microalgae

Chlorella kessleri was cultivated in artificial wastewater using diurnal illumination of 12 h light/12 h dark (L/D) cycles. The inoculum density was 10⁵ cells/mL and the irradiance in light cycle was 45 μmol m² s⁻¹ at the culture surface. As a control culture, another set of flasks was cultivated under continuous illumination. Regardless of the illumination scheme, the total organic carbon (TOC) and chemical oxygen demand (COD) was reduced below 20% of the initial concentration within a day. However, cell concentration under the L/D lighting scheme was lower than that under the continuous illuminating scheme. Thus the specific removal rate of organic carbon under L/D cycles was higher than that under continuous illumination. This result suggested thatC. kessleri grew chemoorganotrophically in the dark periods. After 3 days, nitrate was reduced to 136.5 and 154.1 mg NO₃ ⁻-N/L from 168.1 mg NO₃ ⁻-N/L under continuous illumination and under diurnal cycles, respectively. These results indicate nitrate removal efficiency under continuous light was better than that under diurnal cycles. High-density algal cultures using optimized photobioreactors with diurnal cycles will save energy and improve organic carbon sources removal.     

High throughput culture and gametogenesis induction of <Emphasis Type="Italic">Laminaria japonica</Emphasis> gametophyte clones

In anticipation of the application of a new sporeling-raising method using gametophyte clones to Laminaria commercial cultivation in China, techniques of mass culture and gametogenesis induction of L. japonica gametophyte clones were developed, as a mass of fertile gametophytes is a prerequisite for sporeling-raising with the new method. Gametophyte clones which were subjected to fed-batch culture exhibited a classical logistic growth curve. Growth rates decreased gradually after 2 months of culture, and were negatively correlated to cell density. UNOVA also showed that only cell density has a significant effect on the growth of gametophyte clones under the experimental conditions. Based on the dynamics models revealed, a culture strategy only directed at the control of cell density was adopted. By this strategy, a total of 36 kg wet weight from an initial weight of 0.75 kg was achieved after 3 months culture in 100 20-L bottles. The final average density reached 24 g L⁻¹. For the subsequent gametogenesis induction, amplificatory male and female gametophyte clones were cut, mixed and cultured in bottles under the same conditions used in amplification except for a change of photoperiod from continuous irradiance to 10 h light: 14 h dark cycle. Egg discharge occurred 10 days after the mixed culture and increased gradually with the culture duration. Most gametophytes gave rise to sporophytes 20 days after induction. Large-scale culture of gametophyte clones and gametogenesis induction for commercial cultivation in 2003–2005 have been conducted successfully.     

南亚热带3种阔叶树种人工幼龄纯林及其混交林碳贮量比较

如何通过优化造林模式来提高人工林生态系统碳贮量已受到广泛关注。以南亚热带8年生格木(Erythrophleum fordii)纯林(PE)、红锥(Castanopsis hystrix)纯林(PC)、米老排(Mytilaria laosensis)纯林(PM)及格木×红锥×米老排混交林(MECM)生态系统为研究对象,对其碳贮量及其分配特征进行了比较研究。结果表明:格木、红锥和米老排不同器官平均碳含量分别为512.4—561.7 g/kg,474.2—553.4 g/kg和512.8—556.3 g/kg。相同树种不同器官之间碳含量差异显著(P0.05)。各器官碳含量的平均值大小顺序为格木(539.3 g/kg)米老排(532.7 g/kg)红锥(515.3 g/kg)。不同林分间,灌木层、草本层和凋落物层碳含量均以米老排纯林最高,混交林(MECM)居次,红锥纯林和格木纯林最低;不同林分之间的土壤碳含量差异显著(P0.05),0—10cm,10—30cm,30—50cm和50—100cm土壤碳含量均以米老排纯林最高,红锥纯林居次,格木纯林和混交林(MECM)土壤碳含量最低。生态系统碳贮量大小顺序为米老排(308.0 t/hm2)混交林(182.8 t/hm2)红锥纯林(180.2 t/hm2)格木纯林(135.2 t/hm2),相同组分不同林分间以及相同林分的不同组分间均存在显著差异(P0.05),但混交林与红锥纯林间碳贮量总量无显著差异(P0.05)。造林模式对人工林碳贮量及其分配有显著影响,营建混交林有利于红锥和格木地上碳的累积,不利于土壤碳的固定,而营建纯林既有利于米老排生物量碳的吸收,也有利于土壤碳的固定。因而,对碳汇林造林模式的选择,应根据树种固碳特性而定。