Catalogue of algae with high growth rates and energy potential

98 Pages·2011·2.76 MB·English

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Produced by:
National Environmental Research Institute (NERI), Aarhus University, Denmark (WP leader)
Central Salt & Marine Chemicals Research Institute (CSMCRI), India (WP leader)
National Interuniversity Consortium for Ocean Sciences (CoNISMa), Italy
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Catalogue of algae species with high growth rates and energy potential

Deliverable 2.1. WP2.

List of contents:
Algae species of interest for BioWalk4Biofuels..................................................................................4
Growth rate..........................................................................................................................................4
Ulva sp.................................................................................................................................................6
General description......................................................................................................................6
Lifecycle.......................................................................................................................................6
Distribution..................................................................................................................................7
Growth rate..................................................................................................................................8
Uptake of nutrients and CO ........................................................................................................9
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Cultivation..................................................................................................................................10
Protoplast technology.................................................................................................................10
Biogas production......................................................................................................................11
Pros and cons for selecting Ulva for cultivation in Augusta plant.............................................12
Chaetomorpha sp...............................................................................................................................13
General description....................................................................................................................13
Distribution................................................................................................................................13
Lifecycle.....................................................................................................................................13
Growth and production..............................................................................................................14
Uptake of nutrients and CO ......................................................................................................14
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Cultivation..................................................................................................................................14
Protoplast technology.................................................................................................................14
Biogas production......................................................................................................................15
Pros and cons for selecting Chaetomorpha for cultivation in Augusta plant............................15
Gracilaria sp......................................................................................................................................16
General description....................................................................................................................16
Lifecycle.....................................................................................................................................16
Distribution................................................................................................................................17
Growth and production..............................................................................................................18
Uptake of nutrients and CO ......................................................................................................18
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Cultivation..................................................................................................................................18
Protoplast technology.................................................................................................................19
Biogas production......................................................................................................................19
Pros and cons for selecting Gracilaria for cultivation in Augusta plant...................................20
Literature............................................................................................................................................23
Appendix 1. Note on Caulerpa (CMSCRI)........................................................................................35
Appendix 2. Algae from Augusta Bay (CoNISMA)..........................................................................35

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Algae species of interest for BioWalk4Biofuels
The species described in this catalogue are species suitable for the specific cultivation required in
the cultivation facility at Augusta, Sicily.
The aim of the BioWalk4Biofuel project is to use cultivation of algae to treat and clean biowaste
streams, yielding a biomass usable for energy production in the form of biogas. The algae species
selected for cultivation in the facility needs to fulfil the following criteria:
• Fast growth in order to achieve a high biomass production and efficient waste removal
• Capability to grow on biowaste sources such as flue gas and nitrogen rich wastewater
• Efficient uptake and assimilation of the CO and nutrients in the waste streams
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• Fairly easy to keep in cultivation
• Efficiently harvestable
• Suitable for biogas production
• Domestic species in Sicilian waters. No invasive species can be introduced at the risk of the
surrounding marine environment.

At the kick-off meeting in Rome (09.04.2010) the WP2 partners agreed on focusing on the
following fast growing species, primarily belonging to the green algae:
• Ulva sp. (chlorophyceae) including the former Enteromorpha sp. (chlorophyceae)*
• Chaetomorpha sp. (chlorophyceae)
• Gracilaria sp. (rhodophyta)
• Caulerpa sp. (chlorophyceae)
* On the basis of genetic analyses, the genus Enteromorpha has been merged with the Ulva (Hayden et al. 2003) .

The species Caulerpa has since been omitted as subject for cultivation on the basis of its status of
invasive species in the Mediterranean area (Appendix 1. (Note on Caulerpa by CSMCRI).
This catalogue therefore primarily concerns the remaining four species.

Growth rate
Growth rates of algae depend on the environmental conditions such as nutrient concentrations, light
availability, temperature and water turbulence. Many green algae are opportunistic fast growing
species, proliferating at high nutrient concentrations and relatively high incoming light. Certain
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species can under optimal conditions grow into massive populations, so-called “green tides”. This
phenomenon is observed along many coast lines in particular in Europe, South America and China.
Documented growth rates vary not only between species, but also within the species, and growth
rates obtained under optimal conditions in laboratory scale are most likely difficult to achieve in
larger scale experiments or cultivation systems.
In the following growth rates, production potential, nutrient removal capacity, biogas production
potential as well as considerations for cultivation and harvest are described for species within the
three selected genera.
At the end of the report, various data – from own experiments as well as from the existing literature
– are compiled in a table (Table 2) as well as data on biochemical composition of a number of
species is investigated by CSMCRI is included (Table 3).

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Ulva sp.
Chlorophyceae, Ulvaceae.

General description
Ulva sp. is a genus of marine and brackish water green algae. It is edible and often called 'Sea
Lettuce'. Distinction of species of Ulva have traditionally been based on morphological, anatomical
and cytological characteristics such as shape, size, presence or absence of dentation, thickness, cell
dimensions and number of pyrenoids. Many studies have shown that these characteristics can be
highly variable within species, varying with age, reproductive state, wave exposure, tidal factors,
temperature, salinity, light and biological factors such as grazing. In recent years developmental
patterns in culture, reproductive details and the apparent inability of species to interbreed have been
used to evaluate species concepts based on morphological and anatomical characteristics.
Species with hollow, one-layered thalli were formerly included in Enteromorpha, but it is widely
accepted now that such species should be included in Ulva. The thallus of ulvoid species is flat and
blade-like and is composed of two layers of cells. There is no differentiation into tissues; all the
cells of the plant are more or less alike except for the basal cells, which are elongated to form
attachment rhizoids. Each cell contains one nucleus and has a cup-shaped chloroplast with a single
pyrenoid (www.AlgaeBase.org).

Lifecycle
Ulva undergoes a very definite alternation of generations (Figure 1). Biflagellate isogametes are
formed by certain cells of the haploid, gametangial plant. These are liberated and fuse in pairs to
form a diploid zygote which germinates to form a separate diploid plant called the sporophyte; this
resembles the haploid gametangial plant in outward appearance. Certain cells of the sporophyte
undergo meiosis and form zoospores in sporangia; these zoospores are quite different to the gametes
in that they form quadriflagellate zoospores (with 4 flagella). The zoospores are released, swim
around for a time, settle and germinate to form the haploid gametangial thallus. Note that the
haploid gametes are capable of settling and germinating without fusion to form a haploid thallus
directly. Most Ulva populations reproduce by this form of parthenogenesis and sexual reproduction
is not very common.
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In mass cultures of Ulva, sporulation may occur in response to seasonal environmental cues and
reduce the biomass of vegetative thalli by more than half within a few days. It has been attempted to
prevent the uncontrolled mass sporulation by applying “artificial moon-shine” in order to break the
hypothesised semi-lunar sporulation system of Ulva (EU-project SEAPURA report
(www.cbm.ulpgc.es/seapura/SeapuraAnnRepY2PartA.pdf). However, this attempt was not
successful (Lüning et al. 2008). Recently, a swarming inhibitor (SWI) excreted by the algae, was
isolated and described in two of Ulva species (U. lactuca and U. mutabilis) (Wichard and Oertel
2010).

Fig 1. Lifecycle of Ulva

Distribution
Ulva has a cosmopolitan distribution in salt and brackish waters. It is known to form dense mass
populations in shallow nutrient rich areas (green tides), and cause local nuisance, when washed up
and decaying on the beaches. The green tides are described since the 1970’s in Europe and South
America. The washed up material has been attempted to use for compost, ie. (Wosnitza and
Barrantes 2006) as well as biogas, ie. (Charlier et al. 2007). In recent years, reports of massive
green tides are described along the Chinese coast line, particularly in the Qingdao region. The green
tides are caused by nutrient effluents from sewage, agri- and aquaculture.
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Growth rate
Regarding the growth rates of Ulva there are numerous studies from nature as well as from
cultivation experiments on different size scales. The general picture of Ulva species is that they
have relatively high growth rates compared to other algae, in nature as well as in cultivation
facilities. In nature, growth rates of up to 35% have been reported for the Ulva type species
(holotype) U. lactuca (Pedersen and Borum 1996). In the Mediterranean relatively low growth rates
of U. rigida, <10% d-1, are reported from eutrophicated sites (de Casabianca et al. 2002). In
laboratory scale high growth rates have been achieved with several Ulva species i.e.: U. curvata
(Kützing) De Toni, 52% wet wt.day−1 in short-term laboratory studies (Duke et al. 1989a; Duke et
al. 1989b), U. fasciata Delile, 36% wet wt.day−1 (Lapointe and Tenore 1981). Within this project,
experiments for determining the growth rates of selected species of Ulva have been carried out by
CMSCRI (Figure 2): The RGR was 30.2±1.13% for Ulva reticulata, 25.3±0.14% for U. taeniata,
19.6 ± 1.06% for U. fasciata, 25.85±2.31% for U. lactuca and 17.75±1.16% for Monostroma.
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30
1) 25
%d- 20
(
R 15
G
R 10
5
0
U. retic ulata U. ta e niata U. fa sciata U.la ctu c a M o n o stro m a

Figure 2. Results from growth measurements CMSCRI (Relative growth rates ±SD, n=3).

Robertson-Andersson (2003), showed that there is a decrease in standard growth rate (SGR) when
scaling up tank sizes (Bolton et al. 2009), which is reflected i.e. in the following studies: Pilot scale
cultivation growth rates of up to18.7% d-1 and 9.2 % d-1 are reported for biomass densities of 1 and
4 kg’s, respectively (U. lactuca) (Bruhn et al. 2011). Growth rates for U. fenestrata Postels et
Ruprecht under experimental conditions were 16% wet wt.day−1 (Bjornsater and Wheeler 1990).
This is lower than those obtained for U. lactuca, 18.6% wet wt.day−1 (Neori et al. 1991). In a large
scale cultivation experiment in Japan growth rates of up to 55% d-1 were reported for U. prolifera
cultivated in 500 l tanks with supply of nutrient rich deep sea water. This cultivation method was
based on production of “germling clusters” (Hiraoka and Oka 2008).
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Uptake of nutrients and CO
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There are a number of studies treating the uptake of nutrients of Ulva in nature as well as in
cultivation in connection to integrated multitrophic aquaculture (IMTA) or waste water treatment.
The N removal efficiency of Ulva in co-cultivation with fin fish under different TAN loads (NH +
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NH ) proved highest under high or medium TAN loads as compared to low or very high loads (up
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to 65%), whereas the areal uptake of N was highest under very high TAN loads (up to 7.4 g N m-2
d-1) (Msuya and Neori 2008). Uptake rates of NH of 50-390 µmol h-1 g DW-1 are reported (Neori et
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al. 1991). In co-cultivation with abalone, Ulva was removing on average 80.8% (between 24.3%
and 99.5%) of the NH in the effluent water. However, the NH concentrations were relatively low
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(<34 µM) (Bolton et al. 2009). In Greece on the island of Ios, experiments using Ulva to remove
phosphate from waste water achieved an average efficiency of 35% phosphate removal with an
average concentration of 100µM P. The algae would take up 2.13 µmol PO 3− g−1 DW h−1 in the
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day time, with a significantly lower uptake during night (Tsagkamilis et al. 2010). NERI has
initiated experiments with cultivation of U. lactuca on fresh, as well as degassed, liquid pig manure.
The manure was diluted to approximately 300µM NH (300-400 times dilution) and both types of
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manure supported as high growth rates as inorganic N in the form of NO and even higher than NH
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(average of 30-35 % g FW d-1) (Nielsen, et al. in prep).
Generally, there is a co-limitation of growth by light and N, meaning that in order to utilise high N
concentrations and achieve high growth rates, incoming irradiance must be high (Lapointe and
Tenore 1981). Surge uptake of NH is reported for many macroalgae including Ulva (Pedersen and
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Borum 1997). Thus, addition of NH in pulses can give macroalgae an advantage in competition
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with fouling microalgae, as well as potentially lowering the evaporisation of NH .
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Various species of Ulva are reported to be able to utilise carbon (C) in the form of CO as well as in
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the form of HCO - (Gao and Mckinley 1994). Regarding pH, dissolved inorganic carbon (DIC) and
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CO , the growth rate of Ulva is documented to decline at pH values above 7.5-8 (Frost-Christensen
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and Sand-Jensen 1990), and at pH values below 7 (NERI, unpublished). Cultivation of U. lactuca
using flue gas as source of DIC has been carried out by NERI, documenting the capability of Ulva
to utilise the CO from the flue gas as C source and increasing growth rates by up to 21% as
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compared to aeration with atmospheric air. Addition of flue gas was controlled by the pH of the
growth media, keeping the pH in the range of 7.5 (Bruhn, et al. in prep). This increase in growth
rate by flue gas addition is also demonstrated in one red algae (Gracilaria cornea) and one green
microalgae (Chlorella vulgaris) (Douskova et al. 2009; Israel et al. 2005).
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Cultivation
Ulva has been cultivated successfully for biomass production in connection to aquaculture and
bioenergy purposes (Bruhn et al. 2011; Msuya and Neori 2008; Robertson-Andersson et al. 2008;
Ryther et al. 1984). Several cultivation methods are tested and described ranging from different size
ponds to different size raceways. Also non energy intensive cultivation forms have been tested
(Ryther et al. 1984). Production rates ranging from 74 T DW ha-1 y-1 (18.8 g DW m-2 d-1) for energy
intensive systems to 25 T DW ha-1 y-1 (6.8 g DW m-2 d-1) in non-energy intensive systems are
reported (Ryther et al. 1984). Similar areal yields, and even higher, have been attained in South
Africa in IMTA of U. lactuca and abalone (19.71-26.1 g DW m-2 d-1). Ulva was here cultivated in
raceways (Bolton et al. 2009). In IMTA with fin fish biomass yields of up to 376 g FW m-2 d-1 is
reported from Israel (Msuya and Neori 2008). Annual yields of 45 T DW ha-1 y-1 (up to 38.8 g DW
m-2 d-1) are described for locations as far north as Denmark (Bruhn et al. 2011).
Yields are directly correlated to incoming light with higher irradiance supporting a higher areal
biomass density (Bruhn et al. 2011).

Protoplast technology
There are numerous species within the Ulva genus that have been proven successful for protoplast
propagation as well as for genetic engineering (for review see Reddy et al, 2008). Enzyme based
methods for producing a large number of viable protoplasts has been developed for several Ulva
species and Ulva has successfully been seeded from protoplast cultures onto cultivation nets and
ropes (CSMCRI, unpublished). An alternative cultivation strategy based on cultivation of germling
clusters has been proven successful using the species U. fasciata (Figure 3) (Hiraoka and Oka
2008).
Table 1. Yield of viable protoplasts from various Ulva species (CSMCRI, unpublished).

Species Protoplasts yield
(cells /g fresh wt.)
U. beytensis 2.47 ± 1.6 ×106
U. fasciata 1.85 ± 0.62× 106
U. lactuca 4.0 ± 0.45× 108
U. linza 3.75 ± 1.45 ×106
U. ovata 1.2 ± 1.20 ×105
U. reticulata 1.4 ± 0.7× 107
U. taeniata 1.4 ± 0.78 ×107
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