Accepted Manuscript

Ulva lactuca from an Integrated Multi-Trophic Aquaculture

(IMTA) biofilter system as a protein supplement in gilthead
seabream (Sparus aurata) diet

M. Shpigel, L. Guttman, L. Shauli, V. Odintsov, S. Harpaz, D.

Ben-Ezra

PII: S0044-8486(17)30063-7
DOI: doi: 10.1016/j.aquaculture.2017.08.006
Reference: AQUA 632770
To appear in: aquaculture
Received date: 10 January 2017
Revised date: 7 August 2017
Accepted date: 10 August 2017

Please cite this article as: M. Shpigel, L. Guttman, L. Shauli, V. Odintsov, S. Harpaz, D.
Ben-Ezra , Ulva lactuca from an Integrated Multi-Trophic Aquaculture (IMTA) biofilter
system as a protein supplement in gilthead seabream (Sparus aurata) diet, aquaculture
(2017), doi: 10.1016/j.aquaculture.2017.08.006

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 ACCEPTED MANUSCRIPT

Ulva lactuca from an Integrated Multi-Trophic Aquaculture (IMTA) biofilter system as a

protein supplement in gilthead seabream (Sparus aurata) diet

Shpigel, M.,1* Guttman, L.,2 Shauli, L.,2 Odintsov, V.,2 Harpaz, S.,3 Ben-Ezra, D.2

1
The Interuniversity Institute for Marine Sciences of Eilat. Eilat 88112, Israel
2
Israel Oceanographic and Limnological Research, National Center for Mariculture, P.O. Box

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1212, Eilat 88112, Israel. shpigelm@gmail.com Tel +972523330111

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2
Agricultural Research Organization, The Volcani Center. Institute of Animal Sciences

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HaMaccabim Road, Rishon LeTsiyon. P.O.B 15159, 7528809, Israel

Abstract
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Protein derived from fishmeal is the most expensive ingredient in fish feeds. Any reduction and
replacement of this ingredient by a less expensive protein source which will produce the same
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growth performances will contribute significantly to the reduction of fish production cost.
Protein-rich U. lactuca, used as biofilter in an IMTA system, was evaluated as a dietary
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ingredient for gilthead seabream (Sparus aurata), at moderate (3-8%) and high (up to
approximately 30%) replacement of fishmeal ratio. Three main elements were considered: 1. The
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total Ulva biomass replaced as a proportion of the total feed biomass. 2. The relative reduction of
fishmeal sources in the feed, and 3. The economic benefit of the fishmeal replacement. Growth
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performances similar to those of fish fed the commercial feed used as control were obtained in
two ways: a) by reducing ingredients of animal source in the feed from 41% to 29.1% of the total
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food biomass, and b) by removing 100% of fishmeal and adding up to 14.6% U. lactuca of the
total food biomass. When 260 g kg-1 of fishmeal was removed entirely and replaced with 291 g
kg-1 of poultry meal and 146 g kg-1 of U. lactuca, the total cost of feed was reduced by $0.25 kg-
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. With a FCR of 1.7, the saving was of $0.45 per 1 kg fish produced. Since in intensive
aquaculture fish feeds represent over 60% of the operating costs, saving close to 10% on the cost
of the feed is economically relevant. In addition, reducing nitrogen loads from the effluents by
Ulva biofilter will also save on water treatment costs, an additional advantage of IMTA system.

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Introduction
Fishmeal constitutes one of the major nutritional sources for fish in aquaculture, and the most
expensive diet component (Muzinic et al., 2006). The growing demand for fishmeal stemming
from the expanding global aquaculture industry has ultimately increased the price of this product,
encouraging the fish industry to look for alternative feed ingredients that are locally available
with an equivalent nutritional value (Hardy, 2010; Tacon et al., 2011). Replacing a major part of
fishmeal with less expensive and more sustainable products such as seaweed is expected to

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considerably reduce the cost of fish production. In addition, numerous studies have reported the

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beneficial effects of seaweed meal in the diet of several fish species. Ascophyllum nodosum

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(Nakagawa et al. 1997), Gracilaria cornea and Gracilaria bursa-pastoris (Valente et al. 2006),
Porphyra (Soler-Vila et al. 2009), and Ulva lactuca (Wassef et al. 2001) added to the feed (up to

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10%) as partial substitutes for dietary fishmeal, increased the fish growth rate and improved
protein assimilation (Table 1).
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Ulva spp. are green macroalgae that grow in a variety of habitats and on several different
substrates (Bunker et al. 2010). The distribution of Ulva spp. in many climatic and ecological
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conditions and their opportunistic growth make these seaweeds suitable and cost effective for
cultivation practically everywhere (Neori et al. 2000; Msuya and Neori 2002; Mata et al. 2003).
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Ulva spp. have a good vitamin and mineral profile, are especially rich in glutamic and ascorbic
acid, alanine, iron and are also an important source of dietary fibers, mainly soluble (Briand and
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Morand 1997; Boarder and Shpigel 2001; Ortiz et al. 2006; García-Casal et al. 2007; Silva et al.
2015). Most of the studies using Ulva spp. as fishmeal replacement dealt with
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herbivorous/omnivorous freshwater fish (e.g. Nile tilapia, carps and mullets). It has been
observed that when Ulva levels in the diet were up to 10%, red tilapia weight gain, feed
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conversion ratio and specific growth rate improved (Ergun et al. 2009; El-Tawil 2010). In the
majority of these studies, Ulva was obtained from the wild and replaced feed ingredients from
plant sources (soybean, corn starch or maize meal) rather than animal protein (fish or poultry
meal). The nutritional value of seaweeds from the wild, however, is not constant throughout the
year and is both site and season specific. Total protein and lipid levels of Ulva spp. from the wild
were found to range between 8-16% and 0.11-1.4%, respectively (Fleurence 1999; Marinho et al.
2013).

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An Integrated Multi-Trophic Aquaculture (IMTA) system is a practice in which excretions of

one or more organisms are utilized by other cultured organisms from different trophic levels.
In Israel, seaweeds are frequently used in IMTA systems as biofilters of fish pond effluents
(Shpigel and Neori 1996; Neori et al. 2004;), saving the cost of water treatment while being at
the same time a reliable feed source throughout the year. Fresh Ulva spp. have been used as a
food source for macroalgivore invertebrates such as abalone and sea urchins (Shpigel and Neori
1996; Neori and Shpigel 1999; Baorder and Shpigel 2001; Neori et al. 2004; Naidoo et al. 2006).

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IMTA-produced seaweeds generally present higher productivity levels and less variability in

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protein content compared to seaweed from the natural environment due to the continuous steady

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supply of nutrients and the minimal disturbance by grazers and epiphytes (Schuenhoff et al.
2003; Mata et al. 2010; Abreu et al. 2011; Silva et al. 2015). The potential use of Ulva spp. in

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fish feeds depends on the costs involved in their production and harvesting/processing prior to
their inclusion in the fish diets. In the present study, protein-rich U. lactuca used as biofilter in an
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IMTA system was evaluated as a dietary ingredient for gilthead seabream (Sparus aurata), at up
to 30% replacement ratio of fishmeal.
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Material and Methods

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Ulva meal preparation

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U. lactuca was grown in a 60 m3 (20x3x1m) bottom-aerated rectangular pond. The pond was
used as biofilter of the IMTA system established at the NCM in Eilat and described in detail in
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Ben Ari et al. (2014). Briefly, the U. lactuca biofilter pond received the effluents from three
semi-intensive fishponds (40 m3 each) where 1,500 kg of gilthead seabream (Sparus aurata)
ranging between 150–500 g were grown at a stocking density of 10-15kg/m3. The fish were fed a
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feed containing 42% protein at a rate of 2% body weight d−1. U. lactuca was collected, air-dried
in the sun for about 6 days, and then ground to 2.4 mm size particles. U. lactuca proximate
composition (dry weight), including levels of amino acids are shown in Table 2.

Experimental diets
Diet ingredients for both experiments are summarized in Table 3. The diets were prepared as
follows: In the first experiment, Low Ratio Ulva (LRU), U. lactuca replaced 2.6% and 7.8% of

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the total feed biomass (and 6.8% and 23.5% of the fishmeal feed ingredient excluding fish oil) in
each test, respectively. Poultry-meal biomass was kept constant at 150 g kg-1. Fishmeal was
reduced from 260 g kg-1 (in the control) to 234 and 182 g kg-1 for the 2.6% and 7.8% Low Ratio
Ulva (LRU) treatments, respectively, and replaced by U. lactuca (Table 3).
In the second experiment, High Ratio Ulva (HRU), U. lactuca replaced the fishmeal and
comprised 14.6% and 29.1% of the total feed biomass in each test, respectively. Poultry-meal
biomass was increased from 150 g kg-1 in the control feed to 291 g kg-1 and to 218 g kg-1 in the

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two treatments. Crude protein levels in the fish feed ranged between 32 to 37%. In both the LRU

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and the HRU experiments U. lactuca originated from the same culture batch. The diets were

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mixed for 1h in a 25-L batch mixer and 2.4-mm diameter pellets were formed using a laboratory-
model California Pellet Mill with a steam pre-treatment unit. The moist pellets were sun-dried,

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and stored at -20°C until utilization. AN
Experimental protocols design
Two feeding trials were carried out at the National Center for Mariculture (NCM) in Eilat
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(Israel) for 111 days (December 13, 2013 - April 14, 2014) and for 141 days (October 26, 2014 -
March 16, 2015). Unfiltered Red Sea water (40±0.5 ppt salinity) from an inlet located 300 m
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offshore at a depth of 20 m was pumped into twelve 220-liter polyethylene cylindrical tanks with
a steep conical bottom. The water (400 l h-1) entered each tank at the top perpendicular to the
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tank wall and exited from a central outlet at the bottom, through an external vertical stand-pipe.
Water flow rate was measured daily and, if necessary, adjusted. This design created a continuous
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circular flow and concentrated the faecal matter on the bottom of the tanks. Each tank was
stocked with 15 gilthead seabream (Sparus aurata) of 10.5 0.45g and 20 gilthead seabream of
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7.1 2.1g in the LRU and HRU experiment respectively (144-152 g initial total biomass per
tank). The fish were acclimated to the experimental tanks and diets for 14 days. They were fed
the experimental pelleted diets throughout the day (08:00-16:00) by means of belt-feeders. Daily
feeding level was calculated according to fish weight and water temperature (Lupatsch and Kissil
1998). Water temperatures, oxygen levels and pH were recorded at 08:00 and 14:00 with
portable devices, OxyGuard® and Checker-Hanna Instruments. In the first experiment, water
temperature (measured at 14:00) gradually increased in all the tanks from 21±2.3oC in
December, 2013 to 23±1.5oC in April, 2014. pH decreased from 8.6±0.11 in December, 2013 to

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8.4±0.12 in April, 2014. Throughout the experiment, oxygen levels measured at 08:00 ranged
between 89% and 93%; while at 14:00, oxygen levels decreased and ranged between 75% and
83%. In the second experiment, pH was constant at 8.5±0.2 while water temperature gradually
decreased in all tanks from 25.5±1.3oC in October 2014 to 21.5±1.5oC in March 2015.
Throughout the second experiment, oxygen saturation levels at 08:00 ranged between 90% and
95%; while at 14:00 oxygen levels decreased and ranged between 80% and 85%. In both
experiments no significant differences in these abiotic parameters were observed among all the

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fish tanks.

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Calculations
The following parameters were measured and calculated at the end of the experiment: Specific

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growth rates (SGR) and yield, food conversion ratio (FCR), protein productive value (PPV),
survival, and food biochemical composition.
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The specific growth rate (SGR) was calculated as: SGR = 100 x [ln (WT/W0)] / t,
where W0 = initial biomass, WT = final biomass with t expressing the days of culture in the
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experimental set. Yield was calculated as the difference between initial and final weights and
expressed in units of g m−2 day−1.
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Food conversion ratio (FCR) was calculated as:

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FCR = Fg / (WT/W0),

Where Fg=feed administered (g); W0= initial mean wet weight (g);
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WT=final mean wet weight (g).

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Protein efficiency ratio (PER) was calculated as:

PER = wet weight gain (g)/crude protein consumed (g).

Protein productive value (PPV) was calculated as:

PPV = protein gain (g)/crude protein consumed (g) x 100.

Analytical methods
The biochemical composition (proteins, carbohydrates, lipids) of U. lactuca and fish feeds were
measured at the end of each experiment. Nitrogen levels were determined using the Kjeldahl

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method (AOAC International 1980), and protein content was calculated using 6.25 as N-Protein
factors (Jones 1931). Crude carbohydrate was determined using the phenol-sulfuric acid method
(Dubois et al. 1956) after boiling the sample in 0.5 mol L–1 sulfuric acid for 1 h. Crude lipid was
measured after chloroform-methanol extraction (Folch et al. 1957). Samples were homogenized
with a high-speed homogenizer for 5 min and lipid was determined gravimetrically after
separation and vacuum drying. Ash was calculated after incineration of the samples for 24 h at
550°C in a muffle furnace. Dry matter was calculated by weight loss after 24 h drying at 105°C.

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Amino acid composition of the Ulva was measured in a certified pharmaceutical laboratory

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“Aminolab” (Ness-Ziona, Israel).

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Statistical analyses

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Data were analyzed using one-way analysis of variance (ANOVA) and Tukey‟s multiple
significant difference tests using the software program (Prism-6). Differences were regarded as
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significant at P<0.05. Mean values of SGR, FCR, PER and PPV were compared.
Averages of the abiotic parameters were compared using non-parametric matched pairs. No
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transformations were performed on the data. Figures are presented as average values ± STD.
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Results
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Low Ratio Ulva (LRU) experiment

Fish performances are summarized in Table 4. After 111 days, survival rate in all the
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experimental tanks ranged between 98.3-100%. Average final weights of the fish were
63.1±7.69, 63.70±9.12 and 62.3±8.38 g for the 2.60% Ulva diet, 7.80% Ulva diet and the control
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treatments, respectively. Final yields were 1.14±0.02, 1.06±0.04 and 1.11±0.03 kg for the 2.60%
Ulva diet, 7.80% Ulva diet and the control treatments, respectively. SGRs were 1.98±0.03,
1.99±0.05, 1.98 ±0.02 for the 2.60% Ulva diet, 7.80% Ulva diet and the control treatments,
respectively. FCRs were 1.51±0.01, 1.619±0.04 and 1.5±0.03 for the 2.60% Ulva diet, 7.80%
Ulva diet and the control treatments, respectively. PERs were 2.67±0.04, 2.47± 0.09 and
2.58±0.07 for the 2.60% Ulva diet, 7.80% Ulva diet and the control treatments, respectively.
PPVs were 48±0.7, 45±1.7 and 47±1.3 for the 2.60% Ulva diet, 7.80% Ulva diet and the control

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treatments, respectively. No significant differences in SGR, FCR, PER, and PPV were observed
among all the tanks of fish undergoing the same treatments.

High Ratio Ulva (HRU) experiment

Fish performances are summarized in Table 4. After 141 days, survival rate in all the tanks
ranged between 95.6-100%. Average final weights of the fish were 79.9±1.91, 75±1.33 and
81.8±4.83 g for the 14.6% Ulva diet, 29.1% Ulva diet and the control treatments, respectively.

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Final yields were 1.04±0.04, 0.94±0.06 and 1.03±0.05 kg for the 14.6% Ulva diet, 29.1% Ulva

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diet and the control treatments, respectively. SGRs were 1.82±0.03, 1.78±0.01 and 1.81±0.06 for

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the 14.6% Ulva diet, 29.1% Ulva diet and the control treatments, respectively. Final weight and
SGR were significantly (P<0.05) lower in the 29.1% Ulva treatment. FCRs were 1.68±0.0,

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1.85±0.0 and 1.74±0.15 for the 14.6% Ulva diet, 29.1% Ulva diet and the control treatments,
respectively. In all the tested parameters no significant differences were observed between 14.6%
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Ulva diet and the control treatments. In the 29.1% Ulva diet, final weight and SGR were
significantly (P<0.05) lower.
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PERs were 1.75±0.05, 1.69±0.11 and 1.56±0.13 for the 14.6% Ulva diet, 29.1% Ulva diet and
the control treatments, respectively. PPV were 31.6±0.09, 30.5±1.9 and 28±2.4 for the 14.6%
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Ulva diet, 29.1% Ulva diet and the control treatments, respectively. No significant differences in
PER and PPV were observed among all the tanks of fish undergoing the same treatments.
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Discussion
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The IMTA-produced U. lactuca represents multiple advantages for the aquaculture sector by
considerably reducing nitrogen loads in the effluents (a significant source of environmental
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pollution), saving water treatment costs, and turning into an additional valuable crop (Stuart and
Shpigel 2009; Holdt and Edwards 2014). In earlier studies, Ulva spp. replaced plant feed
ingredients and not fishmeal. Most of the tested fish were freshwater herbivorous species (Table
1). Only few studies deal with Ulva spp. replacing fishmeal in diets for marine fish (Nakagawa et
al. 1987, 1993; Mustafa et al. 1995). In evaluating Ulva lactuca as a source of animal protein
replacement, we took into consideration three relevant points: 1. The total Ulva biomass replaced
as a proportion of the total feed biomass. 2. The relative reduction of fishmeal in the feed and, 3.

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The potential economic benefit of the fishmeal replacement. In general, it was found that by
replacing 100% of the fishmeal by poultry meal and U. lactuca, while keeping the level of all
other ingredients constant, S. aurata performances were similar to those of fish fed the control
feed containing fishmeal.

By reducing ingredients of animal source in S. aurata feed from 41% to 29.1% of the total food
biomass, removing 100% of the expensive fishmeal, adding up to 14.6% U. lactuca originated

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from the IMTA system and 14.1% poultry meal (of the total food biomass), performances (SGR,

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survival, FCR) similar to those of fish fed the commercial control feed were obtained.

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The high variations (5-15%) of the optimal biomass amount of Ulva meal replacement
determined in earlier studies (summarized in Table 1) in geographically diverse regions are quite

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obviously due to the differences in fish species, food origin, feeding physiology (herbivores vs.
carnivores), Ulva species (U. pertusa, U. rigida and U. lactuca), fish size, and climatic
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(temperature) conditions. In addition, the amount of replaced Ulva spp. ratios was represented in
all works as a percentage of the total feed biomass rather the relative reduction of animal feed
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sources. Furthermore, protein levels of Ulva spp. in these studies, ranged between 8-26% (Diler
et al. 2007; Güroy et al. 2007; Azaza et al. 2008; Ergün et al. 2009; Güroy D. 2011). Although
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the alga biomass replacements in at least some works were similar to ours, such a variable
contribution of Ulva spp. as a protein replacement source in each study cannot provide a
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comparable protein contribution to the feed. For the same reason, the wide range of the protein
levels of the fishmeal (25-55%) replaced by Ulva makes the performances of the fish among all
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the published data not comparable.

In our study, fish performances were altogether better in the LRU than in the HRU treatments.
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SFG and FCR in the LRU were 30% higher, while in both experiments fish SGR and FCR were
similar to those observed in the control. Therefore, the differences in fish performances between
LRU and HRU experiments do not seem to be due to U. lactuca ratio in the feed. Since the
experiments were carried out under the same conditions, difference in cohort robustness and
different initial weight (7.1g vs. 10.5g) might explain the disparities in fish performances
between the two treatments.
Partial substitution by Ulva spp. can induce positive effects on growth, feed utilization, body
composition, and resistance to stress and diseases (Diler et al. 2007; Ergün et al. 2009; Pereira et

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al. 2012; Güroy et al. 2013; Silva et al. 2015). However, seaweeds can also contain substances
that may be toxic or have an anti-nutrient activity, and may thus contribute to the reduction in
nutritional quality and have negative effect on fish growth (Francis et al. 2001; Azaza et al. 2008;
Oliveira et al. 2009). Sáez et al. (2012) reported that Ulva spp. meal contains anti-nutritive
substances able to inhibit digestive proteases in S. aurata juveniles. In addition, replacement of
fishmeal with ingredients of plant origin such as soy protein, wheat gluten, and corn gluten meal
showed poorer performances in sea bream (Kissil and Lupatsch 2004; Yldirim 2009) and for the

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Atlantic salmon (Krogdahl et al. 2015). In both our experiments, no negative effects on survival,

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SGR, FCR, PPV and PER were observed. This may be due to the fact that S. aurata is not

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susceptible to intestinal disturbances since U. lactuca is part of its natural diet (Wassef et al.
1985). Similarly, Mustafa et al. (1995) showed that the carnivore Red Sea Bream (Pagrus

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major) are able to ingest algal meal including Ulva.
Although total protein levels (32-37%) in our feed was relatively low, juveniles S. aurata (7-70
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g) performances were very similar to those observed in other studies using commercial feeds
with higher (42-46%) protein levels (Lupatsch and Kissil 1998; Ökte 2002; Schuenhoff et al.
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2003; Venou et al. 2003; Shpigel et al. 2016). For example, SGR of 13g S. aurata fed a 45%
crude protein diet ranged between 1.44-1.66 and PER ranged between 1.27-1.47 (Venou et al.
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2003). These results are similar to those obtained in our study while using a 32-37% crude
protein diet. A study by Nakagawa et al. (1993) supports the use of protein-reduced diet of only
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35% protein for feeding black sea bream (Acanthopagrus schlegeli), revealing a PER of 1.21-
1.44%.
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Ensuring an adequate amino acid profile for growth is crucial when formulating diets involving
the inclusion of plant protein sources (NRC 1993). Deficiencies and unbalances in amino acid
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profiles have lead to stunted growth in several fish species in whose diets plant proteins were
incorporated (Dias et al. 2005; Goda et al. 2007; Silva et al. 2010). Azaza et al. (2008) reported
possible deficiencies in phenylalanine, methionine, cysteine, and threonine in diets containing U.
rigida meal. However, high levels of arginine and histidine were found in previous studies of U.
lactuca (Wahbeh 1997; Boarder and Shpigel, 2001). For example, arginine level in our U.
lactuca was much higher (12% of protein) comparing to arginine levels in poultry meal,
fishmeal, (3.8-6.4 % of protein; FAO, 2001) and Ulva from the wild (4.5%, Wassef et al. 1985).
Although in our work the amino acid composition was measured only in the IMTA-Ulva based

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diets and not in all other diets, similar growth performances of fish suggest a balanced amino
acids composition in all experimental feeds. In other words, replacement of 35% of animal meal
ingredients with U. lactuca originating from IMTA system and the addition of the relatively
inexpensive poultry from 150 g to 291g kg-1 to the diets can successfully fulfill the amino acid
requirements without having negative effects on fish performances.
Protein from fishmeal is the most expensive ingredient in fish feeds. Any reduction of this
ingredient and replacement by a less expensive protein source yielding the same fish

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performances will contribute to the reduction of production cost.

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U. lactuca from IMTA systems contains high protein levels ranging between 26-37% and

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relatively high lipid levels for macroalgae, ranging between 2-2.75% (Neori et al. 2000;
Schuenhoff et al. 2003; Valente et al. 2006; Mata et al. 2010; Silva et al. 2015). These protein

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levels, are much higher than those found in Ulva spp. collected from the wild, ranging between
8.0 and 16.0% protein (Diler et al. 2007; Güroy B.K. et al. 2007; Güroy D. et al. 2011).
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The potential use of high protein Ulva spp. in fish feeds depends on costs prior to its inclusion in
fish diets, i.e. production, harvesting and processing of this seaweed. Our results may have
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considerable economic importance for fish farmers. The price of fishmeal in 2017 ranged
between $1.2-1.4 kg-1, while the price of poultry meal ranged between $0.2-0.4 kg-1 (Matmor
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Inc., Central Feed Mill, Israel prices). In the LRU treatment, by replacing 78 g kg-1 fish meal
(60% protein) with the same biomass of U. lactuca (34% protein), the total cost of feed
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containing U. lactuca (in Israeli prices) was $0.10 kg-1 cheaper: with a FCR of 1.5 the saving
was $0.15 per 1 kg fish produced. In the HRU treatment, by removing 100% of the fishmeal,
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replacing 260g kg-1 of fishmeal with 291 g kg-1 poultry meal and 146 g kg-1 U. lactuca, the total
cost of feed containing U. lactuca was reduced by $0.25 kg-1: with a FCR of 1.7 the saving was
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$0.45 per 1 kg fish produced. Based on previous results by Ben-Ari et al. (2014) estimated
production price of U. lactuca (DW) at the NCM- IMTA system averaged $0.25 kg-1 (DW),
when an intermittent aeration regime is applied. Kaushik et al. (2004) reported that replacement
of up to 100% fishmeal of the feed diet biomass of the European seabass Dicentrarchus labrax
by corn and wheat gluten meal did not affect fish performances. However, in that diet, total
crude protein was kept at 45-50%, the amount of fish oil increased by 75% and l-Lysine was
added to the diet. Consequently, the price of the diets with and without the fishmeal was similar:
650±10-euro t-1. To the best of our knowledge, this is first time that IMTA-produced Ulva has

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been proven to be suitable to replace 100% fishmeal for juvenile (up to about 70g) S. aurata.
High protein U. lactuca replaced up to 14.6% of the fish feed biomass and 35% of the animal
source biomass without any apparent negative effect on fish performances. Since intensive
aquaculture fish feeds represent over 60% of the operating costs (Webster et al. 1999), a saving
close to 10% on the cost of this ingredient is economically significant.

Acknowledgments

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The authors are grateful to Dr. Angelo Colorni and Ms. Mikhal Ben-Shaprut for useful

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comments and suggestions. This work was supported by MERC US-AID Foundation TA-MOU-

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06-M25-053.

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scale system. Aquaculture 312, 77-87.

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Ulva Food PER

Fish species What was replaced Fish Performance Protein/lipid Protein/lipid References
levels (%) levels (%) SGR
Black sea bream U. pertusa replaced fish No difference in fish performance

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ND 35/6.6% ND Nakagawa, et al., 1987
Acanthopagrus schlegeli meal up to 15% up to 10% Ulva rep.
Black sea-bream
Acanthopagrus schlegeli
U. pertusa replaced 15%
fish meal
No difference in fish performance
up to 10% Ulva rep.
ND
I P
35/6.6%
1.21-1.44%
ND`
Nakagawa et al., 1993

Japanese flounder
Paralichthys olivaceus
ND
Growth rate improved up to 2%
Ulva sp. rep.
ND
C R ND ND
Xu et al., 1993
(abstract)

Red sea bream

Pagrus major
U. pertusa replaced 5%
fishmeal
U
No difference in fish performance S
ND 40/4.6%
1.36
3.52% d-1
Mustafa et al., 1995

Sea bream
Sparus aurata
Sea bass
ND
A
Both species‟ performance N
improved up to 10% Ulva sp. rep.
17.4/2.5% ND ND Wassef, 2005
Dicentrarchus labrax
Sea bass U. rigida replaced soybean M
No different in fish performance up 30/14% 1.03-1.06
Dicentrarchus labrax

Nile tilapia
meal up to 10%

U. rigida replaced fish meal

E D
to 10% Ulva rep.

No difference in fish performance

29.5/1.4%
ND
1.4-1.6
Valente et al., 2006

Oreochromis niloticus up to 15%

P T up to 10% Ulva rep.

8/0.15% 40/5.4-7.7%
0.7-1.1% d-1
1-1.3
Guroy et al., 2007

Common carp
Cyprinus carpio meal starch

C E
U. rigida replaced wheat Weight gain and SGR increase up to
5% Ulva rep.
8/0.15% 40/8.2%
2.2-2.9% d-1
Diler et al., 2007

C
Nile tilapia U. rigida replaced soybean No difference in growth rates up to 1.29-2.02
16.41/2% 28/7% Azaza et al., 2008
Oreochromis niloticus meal up to 30% 20% Ulva rep. 1.99-2.33% d-1

Nile tilapia
Oreochromis niloticus
A
U. rigida replaced corn
starch
Weight gain and SGR increase up to
5% Ulva rep.
9.9/0.11%
40.5/10-20% 1.35-1.71
0.81-0.91% d-1
Ergun et al., 2009

Rainbow trout U. lactuca replaced wheat Reduction in growth and feeding 1.49
10%/ND 45.5/9/11% Yildirim et al., 2009
Oncorhynchus mykiss meal up to 10% utilization 1.45% d-1

Nile tilapia Ulva sp. replaced wheat Weight gain and SGR increase up to ND 25/7.2% 2.39-2.55 El-Tawil, 2010

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Oreochromis niloticus flour and wheat bran up to 15% Ulva rep.

25% (fish meal was 3.33-3.53% d-1
increased)
No difference in growth rates up to 2.86
Nile tilapia Ulva sp. from IMTA
10% Ulva rep. 27%/ND 34/2.6% Marinho et al., 2013
Oreochromis niloticus replaced fishmeal up to 20%
2.78% d-1

P T 3.4-3.7

I
Nile tilapia Improve growth rate with increasing
ND 15%/ND 32%/ND Natify et al., 2015
Oreochromis niloticus Ulva sp. up to 10% rep. 1.2-1.4% d-1

Nile tilapia
Oreochromis niloticus
Ulva sp. from IMTA
replaced fishmeal up to 10%
No effect on growth rate with
increasing Ulva sp. up to 10% rep.
C
26.7/2.7% R35/7.5%
1.91
1.22 % d-1
Silva et al., 2015

Sea bream
Sparus aurata
U. lactuca from IMTA
replaced /fish meal
No effect on fish performance up to
14.9% rep.
U S
34/2.74% 34/9%
1.56-2.67
1.43-1.99% d-1
This study

A N
Table 1. Publications related to Ulva spp. as a dietary ingredient for fish meal. (ND: not determined; rep=replacement)

M
E D
P T
C E
A C

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Table 2. Composition and proximate analyses of the diets containing different levels of U. lactuca

December 13-April 14 October 14-March 15

Exp. 1(111 d)- LRU Exp. 2 (141 d)-HRU

Diet - Pellet 2.4 mm U. lactuca U. lactuca Control U. lactuca U. lactuca

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Ulva from total food ingredients (%) 2.60% 7.80% 14.60% 29.10%

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Ingredients g/1000g g g g g g

CR
Fishmeal 234 182 260 0 0

Poultry meal 150 150 150 291 218

US
Ulva lactuca (34 % protein) 26 78 0 146 291

Wheat flour 151.2 151.2 151.2 124.4 51.6

AN
Wheat gluten 80 80 80 80 80

Soybean protein
M

120 120 120 120 120

Corn gluten 110 110 110 110 110

ED

Soya bean 105 105 105 105 105

Fish oil 2.5 2.5 2.5 2.5 2.5

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Soya oil 2.5 2.5 2.5 2.5 2.5

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Vitamin Minerals mix 18.8 18.8 18.8 18.8 18.8

Total diets 1000 g

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Total meal from animal source (g) 384 332 410 291 218

Total meal from animal + Ulva (g) 410 410 437 509

Ulva ratio from total animal meal

biomass (%) 6.8% 23.5% 50.2% 133.5%

Dry matter [%] 89 89 93 89 89

Crude protein [%] 35 34 37 34 32

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Crude lipid [%] 9.5 9.5 11.5 10.5 10.5

Ash [%] 17.5 17.5 11 17.5 17.5
Energy (MJ kg-1) 14.5 14 19.4 13.5 13.0
Vitamins and mineral mixed was according to Kissil and Lupatsch, 2004; Energy was calculated according the results of Kissil
and Lupatsch, 2004; and Schlosser et al., 2005.

Soya bean meal (48% Protein) =roasted.

Soy protein (70% Protein) = protein concentrate.

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Table 3. Composition and proximate analyses of the diets containing different levels of U. lactuca

December 13-April 14 October 14-March 15

Exp. 1(111 d)- LRU Exp. 2 (141 d)-HRU

Diet - Pellet 2.4 mm U. lactuca U. lactuca Control U. lactuca U. lactuca

Ulva from total food ingredients (%) 2.60% 7.80% 14.60% 29.10%

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Ingredients g/1000g g g g g g

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Fishmeal 234 182 260 0 0

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Poultry meal 150 150 150 291 218

Ulva lactuca (34 % protein) 26 78 0 146 291

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Wheat flour 151.2 151.2 151.2 124.4 51.6

Wheat gluten 80 80 80 80 80
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Soybean protein 120 120 120 120 120
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Corn gluten 110 110 110 110 110

Soya bean 105 105 105 105 105

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Fish oil 2.5 2.5 2.5 2.5 2.5

Soya oil
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2.5 2.5 2.5 2.5 2.5

Vitamin Minerals mix 18.8 18.8 18.8 18.8 18.8

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Total diets 1000 g

Total meal from animal source (g) 384 332 410 291 218
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Total meal from animal + Ulva (g) 410 410 437 509

Ulva ratio from total animal meal

biomass (%) 6.8% 23.5% 50.2% 133.5%

Dry matter [%] 89 89 93 89 89

Crude protein [%] 35 34 37 34 32
Crude lipid [%] 9.5 9.5 11.5 10.5 10.5
Ash [%] 17.5 17.5 11 17.5 17.5

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Energy (MJ kg-1) 14.5 14 19.4 13.5 13.0

Vitamins and mineral mixed was according to Kissil and Lupatsch, 2004; Energy was calculated according the results of Kissil
and Lupatsch, 2004; and Schlosser et al., 2005.

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Table 4. Performances (survival, SGR, FCR, PER, PPV, AE) of gilthead seabream (Sparus aurata)
using U. Lactuca biofilter from IMTA system as a dietary protein supplement (20 fish t-1, 142 gr t-1
total biomass in the LRU experiment; 15 fish t-1, 150 gr t-1 total biomass in the HRU experiment;
mean values in the same row with different letters are significantly different P<0.05).
December 13-April October 14-March 15
14 (111 d) (141 d)

Exp. 1 Low Ratio Exp. 2 High Ratio

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Ulva (LRU) Ulva (HRU)

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U. Cont U. Cont
Diet - Pellet 2.4 mm U. lactuca lactuca rol lactuca U. lactuca rol

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Ulva ratio from animal meal
ingredients (%) 6.4% 19.0% 50% 135%

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Ulva ratio from total food
ingredients (%) 2.6% 7.8% 14.6% 29.1%
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M

N=3

Initial Weight 7. ±2. 7. ±2. 7. ±2. 10 ± 10 ±0. 10 ±0.7

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[gr] 1 09 1 09 1 09 .5 0.45 .4 10 .9 4

63 ±7. 63 ±8. 62 ±8. 79 ±1.9 75 ±1. 81 ±4.8

.1 69 .7 12 .3 38 .9 0b .0 33a .8 3b
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Final Weight [gr]

Biomass gain [kg 1. ±0. 1. ±0. 1. ±0. 1. ±0.0 0. ±0. 1. ±0.0

tank-1] 1 02 1 04 1 03 0 4 9 06 0 5
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1. ±0. 1. ±0. 1. ±0. 1. ±0.0 1. ±0. 1. ±0.0

SGR [% d-1] 98 03 99 05 98 02 44 3b 40 01a 43 6b
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1. ±0. 1. ±0. 1. ±0. 1. ±0.0 1. ±0. 1. ±0.1

FCR 5 01 6 04 5 03 7 5 8 13 7 5

98 ±1. 10 10 10 97 ±3. 95 ±3.8

Survival [%] .3 15 0 0 0 .8 85 .6 5

PER 2. ±0. 2. ±0. 2. ±0. 1. ±0.1 1. ±0.1 1. ±0.1

67 04 47 09 58 07 75 45 69 1 56 3

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Highlights

 Ulva produced in an IMTA system was proven to be suitable to replace (in

combination with poultry meal) 100% of fishmeal in diets for juvenile marine fish (S.
aurata).
 By removing 100% of the fishmeal (replacing 260gr kg-1 of fishmeal by 291 gr kg-1
poultry meal and 146 gr kg-1 U. lactuca, S. aurata performances were similar to

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those of fish fed the control feed containing fishmeal.

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 Juvenile S. aurata show no difficulty in digesting and utilizing U. lactuca protein.

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 By replacing 100% of the fishmeal the total cost of feed was reduced by $0.25 kg-1.

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