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Culture of the Terrestrial Cyanobacterium, Nostoc Flagelliforme (Cyanophyceae), Under Aquatic Conditions1

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Culture of the Terrestrial Cyanobacterium, Nostoc Flagelliforme (Cyanophyceae), Under Aquatic Conditions1 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Xiamen University Institutional Repository J. Phycol. 39, 617–623 (2003) CULTURE OF THE TERRESTRIAL CYANOBACTERIUM, NOSTOC FLAGELLIFORME (CYANOPHYCEAE), UNDER AQUATIC CONDITIONS1 Kunshan Gao2 and Changpeng Ye Marine Biology Institute, Science Center, Shantou University, Shantou, Guangdong, China 515063, and Institute of Hydrobiology, The Chinese Academy of Science, Wuhan, Hubei, China 430072 Both colonies and free-living cells of the terres- Russia, Somalia, and the United States). It has been trial cyanobacterium, Nostoc flagelliforme (Berk. & used as a herbal ingredient in China and in Japan and Curtis) Bornet & Flahault, were cultured under as a food delicacy by the Chinese for hundreds of years. aquatic conditions to develop the techniques for the Increased market demands that are completely depen- cultivation and restoration of this endangered re- dent on collection from natural populations and over- source. The colonial filaments disintegrated with exploitation are lessening the N. flagelliforme resources their sheaths ruptured in about 2 days without any and seriously damaging vegetation in its growing areas desiccating treatments. Periodic desiccation played with deterioration of the environment, causing sand- an important role in preventing the alga from de- storms of increasing scale (You 2000), the effects of composing, with greater delays to sheath rupture which have even been recognized in Japan (Zhang with a higher frequency of exposure to air. The bacte- and Lu 1999). Therefore, N. flagelliforme should be rial numbers in the culture treated with seven periods cultivated to meet its market demand and to conserve of desiccation per day were about 50% less com- the endangered resource and the environment. pared with the cultures without the desiccation treat- A number of studies have been performed on the ment. When bacteria in the culture were controlled, ecology, physiology, biochemistry, and culture of Nos- the colonial filaments did not disintegrate and main- toc species (Dodds et al. 1995, Gao 1998, Potts 2000). tained the integrity of their sheath for about 20 days Studies showed that N. flagelliforme, after rehydration, even without the desiccation treatments, indicating required light and potassium to reactivate photo- the importance of desiccation for N. flagelliforme to synthesis (Gao et al. 1998a, Qiu and Gao 1999), prevent them from being disintegrated by bacteria. showed unaffected photosynthetic efficiency until af- On the other hand, when free-living cells obtained ter 50% of water loss (Gao et al. 1998b), and de- from crushed colonial filaments were cultured in liq- manded higher CO2 concentrations to maintain posi- uid medium, they developed into single filaments tive net photosynthesis when highly desiccated with sheaths, within which multiple filaments were (water loss Ͼ70%) (Qiu and Gao 2001). When sub- formed later on as a colony. Such colonial filaments merged, this alga can use bicarbonate as a photo- were developed at 15, 25, and 30Њ C at either 20 or synthetic inorganic carbon source (Gao and Zou ␮mol photonsؒm؊2ؒs؊1; colonies did not develop at 2001). The bicarbonate transport in cyanobacteria 60 ␮mol photonsؒm؊2ؒs؊1, though this light level re- is thought to involve an Naϩ-dependent process 180 sulted in the most rapid growth of the cells. Condi- (Bhaya et al. 2000). -tions of 60 ␮mol photonsؒm؊2ؒs؊1 and 25Њ C appeared Methods of cultivating this alga have been exam to result in the best colonial development and faster ined both in laboratory and in field. When the alga growth of the sheath-held colonies of N. flagelliforme collected from nature was cultured in an aqueous me- when cultured indoor under aquatic conditions. dium, it grew little and then disintegrated (Zhu et al. Key index words: blue-green algae; colonial filament; 1982, Qian et al. 1989); when exposed to air with en- cyanobacteria; desiccation; Nostoc flagelliforme; sheath riched CO2 and periodic watering, it grew by 31% in 15 days (Gao and Yu 2000). The daily net photosyn- ؒ disintegration; single filament ␮ thetic production varied by 132–1280 mol CO2 g Abbreviation: PFD, photon flux density (dry wt)Ϫ1, or 0.64%–6.14% (dry wt.) when N. flagelli- forme was cultured in air with varied levels of CO2 and water supply (Qiu and Gao 2002). However, field ob- The terrestrial cyanobacterium, Nostoc flagelliforme, servation recognized only 6% elongation over a year as previously described (Gao 1998), is an economic- (Dai 1992). These studies presented important data ally significant, terrestrial, macroscopic, filamentous in estimating the growth of N. flagelliforme but have species that is distributed on arid or semiarid steppes not led to any successful cultivation. of northwestern parts of China (also in Algeria, This study aimed to develop the techniques for the Czechoslovakia, France, Mexico, Mongolia, Morocco, cultivation of this economic but endangered cyanobac- terium. First, the algal filaments (colonies) were directly cultivated under aquatic conditions and effects of desic- 1 Received 29 January 2002. Accepted 19 February 2003. cation during the culture estimated. The methods of 2 Author for correspondence: e-mail [email protected]. producing the “seeds” (free-living cells or developed fil- 617 618 KUNSHAN GAO AND CHANGPENG YE aments) and the process of formation of N. flagelliforme Five colonial filaments (7–10 cm long, 0.1 cm wide) were colonies from unicells were then investigated. placed in 400 mL of aerated medium in a 500-mL flask main- tained in a plant growth chamber (EF7, Conviron, Winnipeg, Manitoba, Canada). During culture, filaments were taken out materials and methods of the media and desiccated at frequencies varying from zero to Materials and treatments of samples. Nostoc flagelliforme Born et seven times for 30 min each time or at varied time intervals of Flash was collected in 1999 from Guyuan prefecture of Ning 0–3.5 h for one exposure per day. There were five duplicates Xia Province and stored dry at room temperature before use in for each exposure. The desiccating treatments were carried out experiments. Colonial filaments were selected from the alga on a super-purifier clean bench (4 Foot Purifier Clean Bench, mats and rinsed with distilled water to avoid further contamina- Labconco, Kansas City, MO, USA) at 30Њ C and 15 ␮mol pho- tion before culture under bacteria-exposed or controlled con- tonsؒmϪ2ؒsϪ1. The wet samples lost 90%–95% or 93%–97% of ditions. The surfaces of selected filaments for the bacteria-con- their water after each 30-min or 3.5-h exposure, respectively. The trolled culture were sterilized by submerging them in 75% time when the sheath of the filament was ruptured was defined alcohol for 30–60 s and then rinsing five to seven times with as disintegration time; sheath rupture was examined microscopic- aseptic water (Chen and Cai 1988). This treatment reduced the ally (BX 50, Olympus, Tokyo, Japan). The number of bacteria in number of bacteria in the initial phase of culture by 95%–97% cultures with different treatments was then determined accord- compared with the control treatment. Physiological activity of ing to Zu et al. (1993). Culture medium (0.2 mL) was striped the colonial filaments was reactivated by maintaining them in on a brewis-peptone-agar plate with a sterilized glass rod. Bacte- BG-11 medium (Stanier et al. 1971) at 25Њ C and a photon flux rial colonies on each plate were counted after 24 h incubation .density (PFD) of 40 ␮mol photonsؒmϪ2ؒsϪ1 for 9–10 h (Gao et al. at 37Њ C. Triplicate plates were made for each treatment 1998a). The cultures were aerated directly with ambient air or in- Culture of free-living cells. Free-living cells were obtained from directly through a filter (0.2 ␮m pore size, Cole-Parmer, Vernon colonial filaments of N. flagelliforme. A colonial filament, after Hills, IL, USA) for the bacteria-exposed and control treatments, sterilization and physiological reactivation as described as respectively. above, was sandwiched between two sterilized slides, crushed Culture of natural colonial filaments. The colonial filaments slightly on the clean bench, and washed into sterilized HGZ -were cultured at 25Њ C and 140 ␮mol photonsؒmϪ2ؒsϪ1 (14:10-h medium. Unicell or trichomes were separated from the super light:dark cycle) in HGZ medium (Song and Liu 1996), which natant and transferred into 300 mL HGZ medium and cultured has soil broth in addition to the otherr components of BG -11 at 15, 25, and 30Њ C at three levels, respectively, of PFD, 20, 60, -and, and gave rise to better growth in preparative experiments. and 180 ␮mol photonsؒmϪ2ؒsϪ1 (14:10-h light:dark cycle). Trip Fig. 1. Sheath integrity (a) and sheath rupture (b, disintegration) in Nostoc flagelliforme. CULTURE OF A TERRESTRIAL CYANOBACTERIUM 619 Fig. 2. Effects of desiccation time (A, one exposure per day) and fre- quency (B, 0.5 h for each exposure) on the disintegration (sheath rupture) of Nostoc flagelliforme colonies under bac- teria-controlled or -exposed aquatic conditions. Data are means of five sam- ples Ϯ SD. licate cultures were maintained under each of the nine condi- appeared to have played an important role in prevent- tions of light and temperature. ing alga disintegration. In bacteria-controlled cul- Determination of growth rate. To investigate the growth rate, a single colony or filaments with sheath formed from the unicells tures, the colonial filaments of N. flagelliforme disinte- were transferred into flasks with 200 mL medium and cultured grated in about 20 days independent of desiccation .(at 20, 60, or 180 ␮mol photonsؒmϪ2ؒsϪ1 and 25Њ C for 13 days. frequency and time (Fig. 2 The resulting biomass was collected on a filter paper, dried at The number of bacteria in the cultures differed Њ 80 C for 12 h, and then cooled in a desiccator before weighing with different treatments (Table 1).
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