How to grow Ulva in lab Turner, Pollock and Brown. Environmental

How to grow Ulva in lab
Turner, Pollock and Brown. Environmental Pollution 157 (2009) 2314–2319
Samples of U. lactuca were collected from intertidal rock pools at Wembury, a protected beach 7 km to the SE
of Plymouth, during June and July 2008, and were transported to the laboratory in sealed plastic bags containing
local sea water. Samples were cleaned and then acclimatised for about three days in laboratory sea water in
aerated 10 L polyethylene tanks at 15 � 1 �C and under fluorescent lighting (250 mmol m�2 s�1
photosynthetic active radiation) for 12 h per day. As required, discs of 14 mm diameter were cut from the
central portions of thalli with the sharpened end of a polyethylene cylinder and acclimatised for a further 24 h.
Ahmad, Surif, Omar, Rosli and Nor. 2011. UMTAS.
Ulva reticulata used in this study was collected at the small man made Island near the Penang Bridge (05° 21’
N; 100° 20’ E) during a low tides. In the laboratory, the samples were rinsed and washed thoroughly with the
sea water. The sample was then placed in an aquarium containing 30ppt seawater and acclimatized for 3 days
under the following conditions: light intensity of 50 μmol photons m-2s-1 , photoperiod 12L: 12 h D and aerated.
Experiments were performed in an aquarium of 30cm (L) x 15 cm (W) x 15 cm (H). 21 aquaria were used in
this experiment and each of the aquariums containing 4L artificial seawater (ASW) and eight grams (8g) of U.
reticulata and all of the aquaria were aerated for 24 hours. Six different nutrient combinations (and one control)
were used to test the effect of different nutrient loading on the growth and chlorophyll (chlorophyll a and b)
content of U. reticulata. The nutrient concentration and the nutrient combination for the culture media used are
as follows: i) 50μM NH4, ii) 50μM NH4+ + 10μM PO43-, iii) 50μM NH4+ + 50μM NO3-, iv) 50μM NO3-, v) 50μM
NO3+ 10μM PO43- and vi) 10μM PO43- and iiiv) ASW as a control. The experiment was run for a week, nutrient
uptake was measured one hour after the samples were placed in the aquarium and specific growth rate (SGR)
and pigment content was determined after seven days.
Callow, Callow, Pickett-Heaps and Wetherbee. 1997. J. Phycol. 33, 938-947
Spore release. All quantitative data in this paper were obtained from E. mmpressa (L.) Grev. Reproductive
thalli were collected from Wembury, Devon, England (latitude 50"18' N; 4"02' W) a few days before spring
tides. Excess water was squeezed from the material, which was washed thoroughly in sterile seawater before
wrapping in absorbent paper and transporting back to the labo- ratory in a cool box. On the day following
collection, individual thalli were transferred into glass tubes to which 3-5 mI, of sterile natural seawater was
added. Sterile seawater (W-irradiated, 10 pm and 1 pm filtered, Clare [1996]) was used throughout.
Spores released from individual thalli were checked microscop ically for the presence of two or four flagella.
Spores with 4 flagella (zoospores) from separate thalli were pooled and stored on ice until sufficient numbers
had been collected for an experiment (1-2 h). Storage on ice prevented settlement and also provided an effective
gentle method of concentrating spores because spores gravitate toward the bottom of the container. Spore
suspensions were removed from the ice and allowed to warm up to 20" C before using in experiments. This
procedure had no effect on the rate of subsequent settlement compared with freshly re- leased spores.
Biflagellate spores were typed as female (+) gametes, male (-) gametes, or asexual biflagellate zoospores. Drops
of gamete suspension from individual thalli were mixed to check for gamete fusion. Gametes of the same sex
were pooled. Spores with two flagella that failed to fuse with either female (+) or male ( - ) gametes were
designated as biflagellate zoospores.
Standardization of spore suspensions. A plot of absorbance at 660 nm vs. spore number obtained from
bemocytometer counts re- vealed a linear relationship up to 5 X 10" spores.mL-' (R2= 0.919), although there
was variability between different batches and types of spores. The concentration of spores for experiments was
based on absorbance readings, but the absolute number of spores in the highest concentration was checked later
by hemo- cytometer counts of preserved (0.1% osmium tetroxide) spore suspensions. The actual concentrations
from hemocytometer counts are given in the figure legends of individual experiments.
Hiraoka and Oka. J Appl Phycol (2008) 20:97–102
Thalli of U. prolifera were collected from 5 km above the estuary of the Yoshino River, Tokushima, Shikoku
Island, Japan in November 1999. It has been reported that algae growing in the Yoshino River have an asexual
life history involving biflagellate asexual zoids (Hiraoka et al. 2003). A high rate of synchronous zoid formation
in this natural strain of U. prolifera was induced by cutting the thalli into small fragments (Dan et al. 2002). One
to two hundred thallus fragments of 1–2 mm length were rinsed with autoclaved seawater and transferred to a
Petri dish contain- ing 30 mL of autoclaved seawater. The dish was placed in an incubator at 20°C and set to a
12:12 L:D cycle using cool white fluorescent light (100 μmol photons m−2s−1). Under these culture conditions,
thallus fragments can produce zoids within several days. Zoids released from mature fragments were collected
by their phototactic response and concentrated. The density of the zoid suspension was measured using a
haemacytometer. Several droplets of the zoid suspension were placed in a Petri dish containing 20 mL of PES
medium (Provasoli 1968), and adjusted to a density of more than 104 zoids per 1 mL medium. The Petri dish
was returned to the incubator and cultured as before. Under these conditions, germlings grew at high density on
the bottom of the Petri dish, and attached to one another to form aggregations. The aggregations were scraped
off the dish with tweezers and torn into numerous small clusters of germlings using an electric mixer fitted with
stainless steel blades. These “germling clusters” were then placed in a glass flask containing 500 mL PES
medium and cultured with aeration under the same conditions as above, allowing them to drift freely with the
current in the vessel. Once the germling clusters attained a length of 1 mm or more, they were ready for mass
cultivation in outdoor tanks and were maintained as a stock in glass bottles. In order to generate a continuous
supply of clonal germling clusters, a smaller subset of the stock germling clusters were cultured until they had
grown to a length of more than 20 cm. Well-developed thalli raised in this way were then fragmented for
induction of zoid production and the production of germling clusters as before. This culture process was
repeated to produce a large stock of clonal germling clusters. Germling clusters were maintained at 10–15°C
with a 12:12 L:D cycle under cool white fluorescent light (10 μmol photons m−2s−1). Under these conditions,
the clusters survived for more than one year and thallus growth was not observed. Germling clusters could then
be transferred to outdoor tanks for cultivation using DSW. The schemes for production of germling clusters in
the laboratory and in outdoor tanks are summarized in Fig. 1.
Pinchettia, Fernandez, Diez and Reina. 1998. J App Phyc 10 (4), 383-389
Ulva rigida C. Agardh was collected from Taliarte harbour, east coast of Gran Canaria (Canary Islands) and
cultivated in 750 L aerated tanks at a density of 2.5 g L−1. Nitrogen enriched seawater at turnover rates of 8 vol
d−1 was pumped from a 2000-m3 tank with approximately 40 t of gilthead seabream (Sparus aurata). Daily
concentrations of NH4+ and NO3− + NO2− ranged between
0.06– 0.15 mg L−1,
respectively. Maximum irradiance levels were 1975 ± 98 μmol photon m−2 s−1 and the water temperature
ranged between 20 and 24 ◦C. Relative growth rates μ (% d−1) were calculated according to the equation μ =
100 ln (Wt /W0)/t (D’Elia & De- boer, 1978), where W0 = initial fresh weight (FW), Wt = final fresh weight,
and t = time in days.
After one month, algae were transferred (under the same physical conditions) to running seawater without any
(NH4+ + NO3− + NO2−
con- centrations ≤ 3 μm). After 14 d, when
photosynthetic rates reached values lower than 2.0 μmol O2 g−1 FW min−1 and strong bleaching was observed,
algae were returned (early in the morning) to the initial nitrogen enriched seawater to study recovery.