Suspension culture of gametophytes of transgenic kelp in a photobioreactor

Transgenic Laminaria japonica gametophytes producing a recombinant tissue-type plasminogen activator (rtPA) protein, which is an effective third-generation thrombolytic agent for acute myocardial infarction (AMI), were cultured in an illuminated bubble column bioreactor. A maximum final dry cell weight of 1120 mg l(-1) was obtained in batch culture with an initial dry cell weight of 126 mg l(-1) and with aeration rate of 1.2 l air min(-1) l(-1) culture, nitrate at 1.5 mM and phosphate at 0.17 mM. The yield of rtPA was 56 mu g g(-1) dry cell wt. This is the first report regarding cultivation of a transgenic macroalga in a bioreactor.

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: enhanced seedling production in tumbled culture

The reuse of holdfasts for regeneration of young seedlings or using wild juvenile plants as the seedling source has played the major role in commercial cultivation of the brown alga Hizikia fusiformis in East Asia over the past 20 years. The possibility of employing zygote-derived germlings for producing seedlings has been discussed in the literature, but has not yet become a reality. Three main obstacles have limited the use of zygotes as a main source of seedlings, (1) the dioecious nature of the algal life cycle which may lead to asynchronous male and female receptacle development and thus different timing of egg and spermatozoa expulsion, (2) the low attachment rate when using zygote-derived germlings with developed rhizoids from wild parental plants for seeding production, and (3) the problem of culturing young germlings in regions where water temperature is high in summer. In this investigation, shifting the timing of receptacle formation earlier than in nature was performed by tumbling the algae in a long-day tank (16-h light per day). Synchronization of egg and spermatozoa expulsion and thereafter fertilization were conducted in indoor tanks. Receptacle formation in constant long days could be shifted by 20 days earlier than in plants cultured on long lines in the open sea, or I month earlier than in plants growing on intertidal rocks. Synchronized expulsion of eggs and spermatozoon led to a high rate of fertilization. This was achieved by tumbling the male and female receptacle-bearing branchlets in the same tank at low density in high irradiance. In two independent trials, a total of 1,400,000 zygote-derived germlings were obtained from 620 g (fresh weight) female sporophytes. The germlings shed from the receptacles were at an identical developmental stage indicating high synchronization of expulsion of eggs and spermatozoon followed by fertilization. Approximately 63% ( +/-9.6%) of the germlings were shed from the receptacle between 16 and 24 It after fertilization and 20% ( +/-11.9%) remained on the receptacle for 3 days after fertilization. Germlings were seeded on string collectors before rhizoids started to elongate and the attachment efficiency was enhanced. Young seedlings reached 800 ( +/-50) mum in length in 25 days at 25 degreesC before they were transferred to open sea cultivation. These results provide the basis of a practical way of seedling production by use of zygote-derived germlings in the commercial cultivation of Hizikia fusiformis. (C) 2004 Elsevier B.V All rights reserved.

New life cycles of Porphyra katadae var. hemiphylla in culture

Porphyra katadae Miura var. hemiphylla Tseng et T. J. Chang, a species distributed around the Liaodong and Shandong Peninsulas of China, produces gametophytes from late winter to early spring. These are monoecious with male and female reproductive tissues in distinct halves or sectors. Vegetative tissues from sectors expected to differentiate into sexual tissue were cultured in the laboratory. Male and female reproductive organs, as well as conchocelis and blades, were differentiated from these tissues. The male and female reproductive tissues were in patches and mixed on the cultured tissue pieces. This was quite different from the wild-type sectored individuals. The F-1 conchospore germlings also produced monospores, carposporangia, spermatangia and conchocelis. These carposporangia and spermatangia were in patches and were mixed on the F-1 fronds. The results imply that P. katadae var. hemiphylla is possibly sex-differentiated rather than sex-determined. This is the first report of such a dimorphic life history in the genus Porphyra.

Mass culture of Undaria gametophyte clones and their use in sporeling culture

Undaria pinnatifida (Harv.) Sur. is one of the three main seaweed species under commercial cultivation in China. In the mid-1990s the annual production was about 20000 tons dry. The supply of healthy sporelings is key to the success of commercial cultivation of Undaria. Previous studies demonstrated that instead of the zoospore collection method, sporelings can be cultured through the use of gametophyte clones. This paper reports the experimental results on mass culture of clones and sporeling raising in commercial scale. Light had an obvious effect on growth of gametophyte clones. Under an irradiance of 80 mumol m(-2) s(-1) and favorable temperature of 22-25degreesC, mean daily growth rate may reach as high as 37%. Several celled gametophyte fragments were sprayed onto the palm rope frame. Gametogenesis occurred after 4-6 days. Juvenile sporeling growth experiments showed that nitrate and phosphate concentrations of 2.9 10(-4) mol 1(-1) and 1.7 10(-5) mol 1(-1) were sufficient to enable the sporelings to maintain a high daily growth rate. Sporelings can reach a length of 1 cm in a month. Since 1997, extension of the clone technique has been carried out in Shandong Province. Large-scale production of sporelings for commercial cultivation of 14 and 31 hectares in 1997 and 1998 had been conducted successfully.

Primary culture and characteristic morphologies of medulla terminalis neurons in the eyestalks of Chinese shrimp, Fenneropenaeus chinensis

A method for culturing medulla terminalis (MT) neurons in the eyestalk of Chinese shrimp, Fenneropenaeus chinensis, was first established. The neurons showed immediate outgrowth in the culture medium supplemented with glutamine, glucose and antibiotics. The cells grew for about 2-7 days and then sustained for a week or more. At least six types of neurons were distinguished on the basis of size and form of soma and outgrowth pattern of cells. (C) 2003 Elsevier Science B.V. All rights reserved.

A simple and low-cost airlift photobioreactor for microalgal mass culture

A simple, low-cost, and efficient airlift photobioreactor for microalgal mass culture was designed and developed. The reactor was made of Plexiglas, and composed of three major parts: outer tube, draft tube and air duct. The fluid-dynamic characteristics of the airlift reactor were studied. The system proved to be well suited to the mass cultivation of a marine microalga, Chlorella sp. In batch culture, the biomass volumetric output rate of 0.21 g l(-1) d(-1) was obtained at the superficial gas velocity of 4 mm s(-1) in the draft tube.

SOME ASPECTS OF THE GROWTH OF GRACILARIA-TENUISITIPITATA IN POND CULTURE

The effect of temperature, salinity, nitrogen, culture density and depth on the growth of Gracilaria tenuistipitata were investigated between April 1985 and March 1986 in outdoor ponds in Guangxi Province, South China. The mean annual growth rate was 2.4% per day. Under favourable temperatures of 20-30-degrees-C, daily growth rate may reach as high as 3.3%. Salinity had an obvious effect on growth and photosynthesis and growth peaked at 21 parts per thousand, with a broad plateau between 7-27 parts per thousand. Growth experiments showed that a total nitrogen (NH4-N plus NO3-N) concentration of 4 muM was sufficient to enable the plants to maintain a daily growth rate of 2.7%. The best growth of the plant was obtained at a culture density of 0.5-1 kg M-2 and a culture depth of 30 cm in the pond.

Temperature and light tolerance of representative brown, green and red algae in tumble culture revealed by chlorophyll fluorescence measurements

Laminaria japonica, Undaria pinnatifida, Ulva lactuca, Grateloupia turuturu and Palmaria palmata are Suitable species that fit the requirements of a seaweed-animal integrated aquaculture system in terms of their viable biomass, rapid growth and promising nutrient uptake rates. fit this investigation, the responses of the optimal chlorophyll fluorescence yield of the five algal species in tumble Culture were assessed at a temperature range of 10 similar to 30 degrees C. The results revealed that Ulva lactuca was the most resistant species to high temperature, withstanding 30 degrees C for 4 h without apparent decline in the optimal chlorophyll fluorescence yield. While the arctic alga Palmaria palmata was the most vulnerable one, showing significant decline in the optimal chlorophyll fluorescence yield at 25 degrees C for 2 h. The cold-water species Laminaria japonica, however, demonstrated strong ability to cope with higher temperature (24 similar to 26 degrees C) for shorter time (within 24 h) without significant decline in the optimal chlorophyll fluorescence yield. Grateloupia turuturu showed a general decrease in the optimal chlorophyll fluorescence yield with the rising temperature from 23 to 30 degrees C, similar to the temperate kelp Undaria pinnatifida. Changes of chlorophyll fluorescence yields of these algae were characterized differently indicating the existence of species-unique strategy to cope with high light. Measurements of the optimal chlorophyll fluorescence yield after short exposure to direct solar irradiance revealed how long these exposures could be without significant photoinhibition or with promising recovery in photosynthetic activities. Seasonal pattern of alternation of algal species in tank culture in the Northern Hemisphere at the latitude of 36 degrees N was proposed according to these basic measurements.

Temperature tolerance of young sporophytes from two populations of Laminaria japonica revealed by chlorophyll fluorescence measurements and short-term growth and survival performances in tank culture

The cold-water subtidal brown alga Laminaria japonica has been commercially fanned in the Far East and has been on top of all marine-fanned species in terms of farming area and annual output worldwide. The successful trials of transplantation of young sporophytes from the north to the south in winter along the Chinese coast in the 1950s led to the spreading of cultivation activities down to a latitude of 25-26 degrees N. Up to today, nearly 50% of the annual output of this farmed alga, as a cold-water species, comes from the sub-tropical south in China. The demand to have high-temperature-tolerant strains/ecotypes in farming area calls for a practical method to judge and select the desired parental plants for breeding programs and for seedling production. In this paper, we report our results on using chlorophyll fluorescence measurement and short-term growth performance in tank culture to estimate the temperature tolerance of offspring from two populations, Fujian Farmed Population (FFP) sampled from Fujian province (latitude: 25-26 degrees N) in subtropical area and Qingdao Wild Population (QWP) sampled from Qingdao (latitude: 36 degrees N). Contrary to what has been usually thought, the results revealed that offspring from Qingdao wild population in the north showed better performance both in short-term growth and survival rates and in optimal quantum efficiency (F-v/F-m) when exposed to higher temperature (20-25 degrees C). This result was further confirmed by fluorescence quenching analysis. QWP distributed along the southern distribution limit at a latitude of 36 degrees N in the Pacific west coast is thus taken as a more ideal one than the fanned population in subtropical region as a source of parental plants for breeding high-temperature-tolerant varieties. (c) 2006 Elsevier B.V. All rights reserved.

Dynamic changes of total bacteria and Vibrio in an integrated seaweed-abalone culture system

Polyculture of seaweeds alongside fed animal aquaculture is an environmentally friendly means of avoiding eutrophication problem both in land-based and sea-based monoculture systems. Many aspects of such polyculture systems have been described, but little attention has been given to the impact of live seaweeds on the microbiological properties of the water that connects the algae and animals. In this investigation, the Pacific red alga Gracilaria textorii was cultured in a recirculated dual tank system (150 L) with the juvenile abalone Haliotis discus hannai. Dynamic changes of total bacteria (TB) and total Vibrio (TV) in the water of polyculture and monoculture systems were evaluated. Results revealed that (1) level of TB in the polyculture was constantly higher than in the monoculture over a 6.5-day period. While levels of TV in the polyculture was detected to be constantly lower than in the monoculture, (2) integration of G. textorii in the abalone culture changed the Vibrio compositions in the water as judged by the changes of bacteria colony types; (3) application of artificial diet led to dramatic increase of the levels in TB and TV in both systems at 12 h after application in the 24-h test and resulted in selective propagation of Vibrio in the water in the monoculture system; (4) polyculture of G. textorii with juvenile abalone in combination with feeding with live algal diet helped to maintain low levels of TV and the balance of the Vibrio composition; (5) living biomass of G. textorii was effective in preventing propagation of two purified Vibrio strains (V alginolaticus and V logei) in the water. These results provide a general basis of the dynamic changes of levels in TB and TV in a seaweed-abalone polyculture system with or without artificial diet in tanks. (c) 2005 Elsevier B.V All rights reserved.

Settling abalone veliger larvae in a free-swimming microalgal culture

In the current abalone hatchery in China, insufficient diatoms on vertically placed corrugated pvc plates at later stage often could not support the growth of postlarvae up to the stage that they can feed on live macroalgae. As a result, stripping the spats (35 mm) off by anaesthetization and switching the diet from live diatoms to artificial powdered diet in combination has to be performed in most of the abalone farms. This manipulation normally leads to more than 50% mortality. Here we report the direct use of the unicellular green alga Platymonas helgolandica Kylin var. tsingtaoensis as a potential alga to be used to settle the veliger larvae of the Pacific abalone Haliotis discus hannai and to feed the postlarvae. Settlement rate of 2-day-old veliger larvae in mono culture of P helgolandica could be as high as 92% ( +/- 4.2%) on day 10 in small scale trials, higher than that in the selected benthic diatom strain (53.6% +/- 12.7%) when settled in the water in which bacteria propagation was controlled by treatment of 2 ppm of benzylpenicillinum calcium and streptomycin sulfate. Postlarvae fed solely on P. helgolandica or the selected benthic diatom Navicula-2005-A grew at rates of 40.1 ( +/- 1.9) and 45.8 (+/- 13.4) mu m day(-1), respectively, when raised at 22 degrees C until day 50 postfertilization. P. helgolandica was shown to have distinct diurnal settling rhythm characterized with a peak of settled cells in the middle of the night for cell division and a peak of free-swimming cells in the middle of the day. High density of attached P. helgolandica cells on the inner surface of the culture facility in the night fits the nocturnal feeding behavior of the abalone spats. Judged by the promising larvae settling rate, growth and survival rates of the postlarvae fed with this alga, the free-swimming micro-green alga P. helgolandica constitutes a potential species for settling the veliger larvae and for supporting the growth of postlarvae as well. (c) 2006 Published by Elsevier B.V.

Growth, survival and immune activity of scallops, Chlamys farreri Jones et Preston, compared between suspended and bottom culture in Haizhou Bay, China

We examined the growth, survival and immune response of the scallop, Chlamys farreri, during a 1-year period in deep water of Haizhou Bay. Scallops were cultured using two methods: (1) in lantern nets at a 5 m depth and (2) in a bottom culture system (sleeves) on the seabed at about a 25 m depth. Shell heights, meat dry weight and immune activities in the haemolymph (superoxide dismutase and myeloperoxidase) were measured bimonthly or quarterly from July 2007 to June 2008. Survival was measured at the end of the study and environmental parameters in the experimental layers were monitored during the experiment. The growth and immune activities of scallops were lower when the water temperature was high, which was consistent with the main mortality occurring in summer. The growth and immunity of scallops were higher in the suspended culture than in the bottom culture during the experiment, with the exception of shell growth during the last study period. Survival of scallops in the suspended culture (54.6 +/- 12.3%) was significantly lower than that in the bottom culture (86.8 +/- 3.5%) at the end of this study. We conclude from our results that the high mortality of C. farreri can be prevented by culturing them in a bottom culture system before November of the first year, and then transferring them to a suspended culture to improve scallop production.

Bioremediation potential of the macroalga Gracilaria lemaneiformis (Rhodophyta) integrated into fed fish culture in coastal waters of north China

Fed fish farms produce large amounts of wastes, including dissolved inorganic nitrogen and phosphorus. In China, fish mariculture in coastal waters has been increasing since the last decade. However, there is no macroalgae commercially cultivated in north China in warm seasons. To exploit fish-farm nutrients as a resource input, and at the same time to reduce the risk of eutrophication, the high-temperature adapted red alga Gracilaria lemaneiformis (Bory) Dawson from south China was co-cultured with the fish Sebastodes fuscescens in north China in warm seasons. Growth and nutrient removal from fish culture water were investigated in laboratory conditions in order to evaluate the nutrient bioremediation capability of G. lemaneiformis. Feasibility of integrating the seaweed cultivation with the fed fish-cage aquaculture in coastal waters of north China was also investigated in field conditions. Laboratory seaweed/fish co-culture experiments showed that the seaweed was an efficient nutrient pump and could remove most nutrients from the system. Field cultivation trials showed that G. lemaneiformis grew very well in fish farming areas, at maximum growth rate of 11.03% day(-1). Mean C, N, and P contents in dry thalli cultured in Jiaozhou Bay were 28.9 +/- 1.1%, 4.17 +/- 0.11 % and 0.33 +/- 0.01 %, respectively. Mean N and P uptake rates of the thalli were estimated at 10.64 and 0.38 mu mol g(-1) dry weight h(-1), respectively. An extrapolation of the results showed that a 1-ha cultivation of the seaweed in coastal fish fanning waters would give an annual harvest of more than 70 t of fresh G. lemaneiformis, or 9 t dry materials; 2.5 t C would be produced, and simultaneously 0.22 t N and 0.03t P would be sequestered from the seawater by the seaweed. Results indicated that the seaweed is suitable as a good candidate for seaweed/fish integrated mariculture for bioremediation and economic diversification. The integration can benefit economy and environment in a sustainable manner in warm seasons in coastal waters of north China. (c) 2005 Elsevier B.V. All rights reserved.

Isolation and characterization of a marine magnetotactic spirillum axenic culture QH-2 from an intertidal zone of the China Sea

Magnetotactic bacteria (MTB) are ubiquitous in aquatic habitats. Because of their fastidious requirements for growth conditions, only very few axenic MTB cultures have been obtained worldwide. In this study, we report a novel marine magnetotactic spirillum axenic culture, designated as QH-2, isolated from the China Sea. It was able to grow in semi-solid or liquid chemically defined medium. The cells were amphitrichously flagellated and contained one single magnetosome chain with an average number of 16 magnetosomes per cell. Phosphate and lipid granules were also observed in the cells. Both rock magnetism and energy-dispersive X-ray spectroscopy characterizations indicated that the magnetosomes in QH-2 were single-domain magnetites (Fe3O4). QH-2 cells swam mostly in a straight line at a velocity of 20-50 mu m/s and occasionally changed to a helical motion. Unlike other magnetotactic spirilla. QH-2 cells responded to light illumination. As a consequence of illumination, the cells changed the direction in which they swam from parallel to the magnetic field to antiparallel. This response appears to be similar to the effect of an increase in [O-2]. Analysis of the QH-2 16S rRNA sequence showed that it had greater than 11% sequence divergence from freshwater magnetotactic spirilla. Thus, the marine QH-2 strain seems to be both phylogenetically and magnetotactically distinct from the freshwater Magnetospirillum spp. studied previously. (C) 2010 Elsevier Masson SAS. All rights reserved.

A new three-phase culture method for Manila clam, Ruditapes philippinarum, farming in northern China

Studies on reproduction, hatchery management, and culture of Manila clams Ruditapes philippinarum were carried out in an attempt to optimize their culture conditions and techniques. Results from these studies led to the development of a three-phase culture method for Manila clam farming in northern China. The key components of the new method were: 1) early spawning and over-wintering indoors (greenhouse); 2) optimized larval culture conditions and techniques; 3) juvenile rearing in shallow, fertilized nursery ponds; 4) optimized stocking size and density and substrate for mudflat grow out. Broodstock were maturated indoors for a month from early April to early May. Primarily because of higher water temperatures in the greenhouse the clams spawned more than one month earlier than in the natural environment. From May to July, juveniles were reared for 1-2 months indoors to a size of 2.0-3.0 mm in shell length before being moved to outdoor, pre-disinfected, nursery ponds. Juveniles were then reared in the nursery ponds for one month to about 1.0 cm before being transferred to the mudflat for grow out. Juvenile clams in nursery ponds grew considerably faster than in the natural environment probably because of higher temperatures and more abundant natural food. During grow out, the clams were reared for 4-7 months until they reached a market size (3.0-3.3 cm). Juveniles produced after August were over-wintered in the greenhouse in which the water temperature was about 3 degrees C higher than that of the outdoor environment. Juveniles grew at an average rate of > 20 mu m day(-1), while in the natural environment no growth was observed during winter because of low temperatures. Juveniles in the greenhouse grew to 2-3 mm by the following March before being moved into outdoor nursery ponds. The three-phase culture method not only shortened the production period from spawn to market size from 24-36 months to about 10-14 months, but also prolonged the spawning season from 2 to 7 months, resulting in increased production of seed and market-size clams. Compared with the traditional method, the new method could increase the yield of market-size clams by 10-11 times, and increase the profit per ha mudflat by as much as 124 times and the profit per kg market-size clams produced by 13 times. (c) 2006 Elsevier B.V. All rights reserved.