Probiotics in Marine Larviculture

Probiotics in marine larviculture
Niall G. Vine1, Winston D. Leukes2 & Horst Kaiser1

1
Department of Ichthyology & Fisheries Science, Rhodes University, Grahamstown, South Africa and 2Department of Biochemistry, Microbiology
& Biotechnology, Rhodes University, Grahamstown, South Africa
Correspondence: Niall G. Vine, Department Abstract

of Ichthyology & Fisheries Science, PO Box 94,
Rhodes University, Grahamstown 6140,
Owing to the problem of antibiotic resistance and subsequent reluctance of using
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South Africa. Tel.: 127 46 6038415/6; fax: antibiotics, the use of probiotics in larviculture is becoming increasingly popular.
127 46 6224827; e-mail: n.vine@ru.ac.za or During the early stages of development, manipulation of the larval digestive system
niall_vine@hotmail.com seems possible through the addition of probiotics either through the culture water
or via the livefood. Well-studied probiotics used in human medicine and terrestrial
Received 24 August 2005; revised 7 December agriculture have proved to be successful in aquaculture and therefore reduce the
2005; accepted 8 December 2005. need for extensive biosafety trials. The selection of probiotics requires various in
First published online 21 March 2006.
vitro screening experiments, which assay for the production of antagonist
compounds, their growth in and attachment to fish intestinal mucus, and the
doi:10.1111/j.1574-6976.2006.00017.x
production of other beneficial compounds such as vitamins, fatty acids and
Editor: Simon Cutting
digestive enzymes. Further information regarding probiont suitability can be
obtained from its identification, interaction with livefood and host pathogenicity.
Keywords Finally, pilot-scale in vivo tests need be performed, after which a production
probiotic; probiont; aquaculture; larviculture; cost–benefit analysis to determine its economic viability needs to be undertaken.
selection criteria.
Introduction duced by one protozoan that stimulated the growth of

another’, which was expanded on from an agricultural
Combined with the problem of antibiotic contamination of perspective by defining it as ‘a live microbial feed supple-
aquaculture facilities and livestock, the indiscriminate ment which beneficially affects the host animal by improv-
worldwide use of antibiotics in aquaculture has led to the ing its intestinal microbial balance’ (Fuller, 1989). The above
development of drug-resistant bacteria that are becoming definition was broadened to include ‘mono- or mixed
increasingly difficult to control and eradicate (Aoki & cultures of live microorganisms . . .’ (Havenaar & Huis
Watanabe, 1973; Aoki, 1975; Aoki et al., 1980; Hayashi int’Veld, 1992) and Salminen et al. (1999) suggested it
et al., 1993; DePaola, 1995; Bruun et al., 2000; Sahul & include ‘microbial cell preparations or components of
Balasubramanian, 2000; van der Waaij & Nord, 2000; microbial cells’. Gatesoupe (1999) redefined probiotics for
Miranda & Zemelman, 2002). Consequently, certain anti- aquaculture as ‘microbial cells that are administered in such
biotics such as chloramphenicol have been banned in some a way as to enter the gastrointestinal tract and to be kept
countries (Robert et al., 1995; FAO, 2002). As a result of alive, with the aim of improving health’. The definition of
resistant bacterial strains becoming more prevalent and Gatesoupe will be used in this review as it focuses on the oral
difficult to treat, alternative methods of controlling the delivery of the probiont and its ability to improve the health
microbial environment are being investigated. One of the of the host as a result of its presence in the digestive tract.
methods gaining recognition for controlling pathogens Whereas vaccines require the participation of host cells to
within the aquaculture industry is the use of beneficial or elicit positive effects, probiotics protect their host against
probiotic bacteria (Ring & Gatesoupe, 1998; Gatesoupe, neighbouring or invading pathogens by interfering with
1999; Ring & Birkbeck, 1999; Verschuere et al., 2000; their cellular functions. Probiotics may protect their host
Irianto & Austin, 2002b). from pathogens by producing metabolites that inhibit the
In aquaculture, the term ‘probiotics’ is often loosely used colonization or growth of other microorganisms or by
to describe a microbial formulation responsible for biocon- competing with them for resources such as nutrients or
trol or bioremediation. The word probiotics comes from the space (Ouwehand et al., 1999a, 2001; Forestier et al., 2001;
Greek ‘pro bios’ meaning ‘for life’. The original definition Pinchuk et al., 2001; Fiorillo et al., 2002; Mukai et al., 2002;
(Lilly & Stillwell, 1965) of probiotics was ‘substances pro- Servin & Coconnier, 2003; Vine et al., 2004a, b).

c 2006 Federation of European Microbiological Societies FEMS Microbiol Rev 30 (2006) 404–427
Published by Blackwell Publishing Ltd. All rights reserved
Probiotics in marine larviculture 405
Although the addition of potentially probiotic microor- Munilla-Moran et al., 1990). With a reduction in the use of
ganisms to culture water in larval fish systems is a means of antibiotics, the use of probiotics to help control or manip-
biocontrol, it is possible that some may be ingested and have ulate the microflora associated with aquatic larval culture
a probiotic effect on the host animal. This is often not has become increasingly popular; see the review (mainly on
investigated (Nogami & Maeda, 1992; Jory, 1998; Moriarty, probiotics in aquatic invertebrates) by Gomez-Gil et al.
1998; Ruiz-Ponte et al., 1999; Verschuere et al., 1999; (2000). A number of general review articles on the use of
Douillet, 2000b; Chythanya et al., 2002) as is considered a probiotics in aquaculture are also available (see Ring &
method of improving water quality rather than of enhancing Gatesoupe, 1998; Ring & Birkbeck, 1999; Gatesoupe, 1999;
the intestinal microflora of the animals living in the water. Skjermo & Vadstein, 1999; Verschuere et al., 2000; Irianto &
Rengpipat et al. (2000) fed probiotic Bacillus S11 to tiger Austin, 2002b).
shrimp (Penaeus monodon) and found viable probiotic in Previous reviews have looked at the use and potential

the intestinal tract. Although the probiont had no adverse application of probiotics in aquaculture in general; this
effect on water quality and improved shrimp health when review focuses on the use of probiotics during the larval
the crustaceans were challenged with luminescent bacteria stages, particularly of fish. The aims of this paper are (1) to
(Rengpipat et al., 1998), it was not clear whether this was present information on the interaction between probiotics
due to a probiotic or biocontrol effect. and aquatic larvae in the culture environment, (2) to
To optimize the requirements for the species being identify probiotic selection criteria and constraints for the
farmed, intensive aquaculture conditions should be carefully use of probiotics in marine fish larviculture, and (3) to
controlled. Microorganisms that can be added to an inten- suggest future research objectives regarding the selection
sive fish culture system have until recently been poorly and use of probiotics in aquaculture.
available. A range of microorganisms are now available to
seed biofilters, improve water quality or provide a food
The larval rearing environment -- the
source for livefood organisms. However, the large-scale
relationship between microorganisms and
supply of probiotic bacteria is uncommon as product
larvae
development has been slow owing to lack of expertise, and
paucity of research and knowledge about their mode of In aquaculture, exposure to a diverse bacterial microflora is
action. Similarly, after more than 20 years of research, the limited by the resources available. Therefore, the gastro-
preventative mechanism of probiotics on gut disturbances intestinal flora usually resembles the microflora initially
in humans is still not completely understood (Gismondo present in the rearing water, microalgae and livefood (Tana-
et al., 1999). Little is known about the establishment and somwang & Muroga, 1989; Munro et al., 1994; Ring et al.,
action of the microflora, particularly during the larval stages 1996; Gatesoupe, 1999; Riquelme et al., 2001). Verschuere
of aquatic organisms (Olafsen, 2001). et al. (1997) showed that the development of the microbial
Although many studies of probiotics in aquaculture have community in Artemia populations is influenced by both
been published in the past decade, the research has generally deterministic and stochastic factors. Deterministic factors
been of an applied nature with few discussions on the include salinity, temperature, feed quality, etc., while organ-
bacterial mode of action. In the fields of medical and isms in the right place at the right time to enter the habitat
agricultural probiotic research, unequivocal evidence of the and to proliferate, comprise the stochastic factors. The
exact mode of action has been demonstrated (Fuller, 1989). combination of controllable and chance factors determines
In particular, the use of lactic acid bacteria (Perdigon et al., the composition of the resulting microflora. This makes
1995; Salminen et al., 1998; Kontula, 1999; Tannock, 1999) research on probiotics for larviculture both interesting and
and aspects regarding knowledge of the organism’s biology, challenging.
culture and suitability have been of benefit to aquaculture Upon hatching, most marine fish larvae possess a sterile
(Gildberg & Mikkelsen, 1998; Ring & Gatesoupe, 1998; (Ring et al., 1996), immature digestive system (Govoni
Nikoskelainen et al., 2003). As antibiotics become less et al., 1986; Timmermans, 1987; Hansen et al., 1992; Roo
popular for controlling the aquatic microflora in hatcheries, et al., 1999), which is colonized by the egg flora at the time
the concept of treating the early stages of aquatic animals of hatching (Hansen & Olafsen, 1990) or through water-
with probiotics has become increasingly popular. Initial borne bacteria as the larvae drink water to osmoregulate
research focused on the potential use of probiotics during (Ring et al., 1996; Reitan et al., 1998; Olafsen, 2001) or
the adult and juvenile phases of aquatic organisms (Limsu- begin feeding (Munro et al., 1993; Bergh et al., 1994; Huys
wan & Lovell, 1981; Lovell, 1981; Lovell & Limsuwan, 1982; et al., 2001). Ring et al. (1996) found that the intestinal
MacDonald et al., 1986) while research into the use of microflora of turbot larvae was strongly influenced by the
probiotics in aquatic larviculture began in the late 1980s addition of Vibrio pelagius to the rearing water. Similarly,
(Castagna et al., 1989; Nicolas et al., 1989; Gatesoupe, 1990; 70% of the intestinal microflora of cod larvae comprised
FEMS Microbiol Rev 30 (2006) 404–427

c2006 Federation of European Microbiological Societies
406 N.G. Vine et al.
Lactobacillus plantarum when exposed to the bacteria in the et al., 1996; Gatesoupe, 1997; Gatesoupe et al., 1997; Ring
larval rearing water compared with 1% in unexposed tanks & Vadstein, 1998). It has been suggested that the efficacy of
(Strm & Ring, 1993). probiotics is likely to be highest in the host species from
The composition of the colonizing microflora is deter- where they were isolated (Verschuere et al., 2000). However,
mined by the interaction between bacteria (Ring & Birk- the human probiotic Lactobacillus rhamnosus enhanced
beck, 1999) and available nutrients in the form of partially survival of rainbow trout (Nikoskelainen et al., 2003)
digested compounds, mucus or secretions of the gastro- challenged with furunculosis (Nikoskelainen et al., 2001a).
intestinal tract (Fänge & Grove, 1979). Colonization of the Some probiotics used for humans and terrestrial animals
gut generally increases at the onset of exogenous feeding, have shown promise in aquaculture species (Nikoskelainen
resembling the microflora of the livefood as opposed to that et al., 2001a, b, 2003; Carnevali et al., 2004). Although not a
of the surrounding environment (Muroga et al., 1987; major part of the normal microflora of aquatic animals

Tanasomwang & Muroga, 1989; Bergh et al., 1994; Munro (Ring, 2003), lactic acid bacteria have been intentionally
et al., 1994,1993). For example, Nicolas et al. (1989) showed introduced into Atlantic salmon (Salmo salar) (Gildberg
that nonfeeding turbot larvae retained an internal micro- et al., 1995), rainbow trout (Oncorhynchus mykiss) (Nikos-
flora similar to that of the water and not that of the rotifers. kelainen et al., 2001a, 2003), turbot (Scopthalmus maximus)
Colonization of the turbot gut appears to occur in two steps: (Gatesoupe, 1994), Atlantic cod (Gadus morhua) (Strm &
first, low levels of bacteria (about 5 102 larva 1) are found Ring, 1993; Gildberg & Mikkelsen, 1998) and pollack
during the first 4 days, then, between 5 and 16 days after (Pollachius pollachius) (Gatesoupe, 2002). However, within
hatching, the bacteria reach concentrations of ‘a few days’ after adding the probionts they were no longer
5 104 larva 1 (Ring et al., 1996; Ring & Vadstein, 1998). detectable in the gastrointestinal tract of Atlantic salmon or
Reasons for this are unclear. It could be related to an increase rainbow trout (Joborn et al., 1997). The reasons why lactic
in attachment sites suitable for bacteria as a result of the acid bacteria are seldom isolated from larvae is probably due
histological and functional development of the larvae to different preferred incubation temperatures and periods,
(Ring et al., 1995) and improved internal environmental and the absence of glucose from the growth medium (Ring
conditions for bacterial growth (Ring & Vadstein, 1998). et al., 1995). Another reason may be due to the rapid gut
Both marine and freshwater fishes have been shown to evacuation rate of larvae, which possibly flushes the slow-
have a specific indigenous microflora (Horsley, 1977; Sakata, growing microorganisms before they are able to attach and
1990; Ring & Gatesoupe, 1998). Whereas humans and colonize. The use of probiotics derived from the aquatic
terrestrial farm animals tend to have an intestinal microflora environment and used in terrestrial animals has yet to be
dominated by Gram-positive obligate or facultative anae- demonstrated. Because of the infancy of the field, informa-
robes (Gatesoupe, 1999), that of aquatic animals consists tion pertaining to the survival and continued effect of these
mainly of Gram-negative aerobic, obligate anaerobic and terrestrial probiotics in the aquatic environment is limited
facultative anaerobic bacteria, the composition of which (Gatesoupe, 1999).
may change with environmental stresses (Ring & Strm, In larvae, the development of the stomach, foregut,
1994; Ring et al., 1997; Kennedy et al., 1998), diet (Munro midgut and hindgut becomes compartmentalized in terms
et al., 1993; Douillet & Langdon, 1994; Ring & Strm, 1994; of pH, enzyme type and activity (Govoni et al., 1986; Gisbert
Gildberg et al., 1995; Ring et al., 1997) and fish age (Bergh et al., 1998; Ribeiro et al., 1999a, b; Gallagher et al., 2001),
et al., 1994; Prayitno & Latchford, 1995; Olafsen, 2001). The and thus for the probiotic to be of benefit it must reach and
most common members of the microflora of healthy marine maintain itself in the part where its effect is to be applied. An
fish are Vibrio spp., Pseudomonas spp. and Acinetobacter spp. inability to thrive in a particular part of the digestive tract is
(Muroga et al., 1987; Nicolas et al., 1989; Tanasomwang & also unlikely to benefit the host. It is therefore important
Muroga, 1989; Mac & Fraile, 1990; Bergh et al., 1994; Munro that when screening potential probionts, an understanding
et al., 1994; Gatesoupe et al., 1997), while Aeromonas spp., of how probiotics act within the host is required.
Plesiomonas spp., Pseudomonas spp. and members of the There is a paucity of knowledge in our understanding of
family Enterobacteriaceae dominate the microflora of fresh- the mode of action of probiotics (Klaenhammer & Kullen,
water species (Ugajin, 1979; Sugita et al., 1988, 1991a; 1999; Tannock, 1999). Klaenhammer & Kullen (1999)
Sakata, 1990; Ring et al., 1995). A variety of microorgan- attributed this to three factors: (1) the complexities of the
isms have been used as probiotics to improve the growth or microbial activity in the gastrointestinal tract and the
survival of aquatic larval species (Table 1). competition and interaction between the species that influ-
Aquatic candidate probionts for larviculture have been ence the microecology of their environment are poorly
isolated from adults (Olsson et al., 1992; Gildberg et al., understood; (2) confusion over identification, activity and
1997; Gram et al., 1999; Rengpipat et al., 2000; Riquelme viability of the probiotics, which has contributed to mis-
et al., 2000; Gullian et al., 2004) and healthy larvae (Ring identification of cultures; (3) individual probiotic strains

Table 1. Intestinal probiotics used in fish and shellfish larviculture and the effect on their host
Microbe Host species Effect on host Reference
Molluscs
Roseobacter sp. (strain Pecten maximus Short-term improvement in survival Ruiz-Ponte et al. (1999)
BS107)
Arthrobacter sp. (strain 77) Argopecten Produces inhibitory compounds. Replaced resident microflora within Riquelme et al. (2000)
purpuratus 24 h
Strains 11 and C33 Argopecten Added with microalgae and colonized digestive tract Avendano & Riquelme
purpuratus (1999)
Vibrio sp. 33, Pseudomonas Argopecten Compared with antibiotic treatment, the addition of probiotics Riquelme et al. (2001)
sp. 11 and Bacillus sp. purpuratus increased number of eyed larvae
(strain B2)

Strain CA2 Crassostrea Enhanced growth Douillet & Langdon (1994)
gigas
Aeromonas media (strain Crassostrea Reduced mortality when challenged with Vibrio tubiashii Gibson et al. (1998)
A199) gigas
Crustaceans
Vibrio alginolyticus Litopenaeus Survival improved after challenge with Vibrio parahaemolyticus Garriques & Arevalo (1995)
vannamei
Lactobacillus sporogenes Macrobrachium Fed as bio-encapsulated probiotic via Artemia. Improved growth rate Venkat et al. (2004)
rosenbergii and feed efficiency ration of postlarvae
Bacillus S11 Penaeus Postlarvae survival was higher when challenged with Vibrio harveyi. Rengpipat et al. (2000)
monodon (PL- Probiont provided cellular and humoral immune defence responses
10)
VKM-124 Penaeus sp. Reduced mortality by controlling pathogenic viruses Maeda et al. (1997)
Pseudoalteromonas undina
Bacterial strains F3 and PM- Portunus Improved crab larval survival. PM-4 repressed growth of Vibrio spp. in Nogami & Maeda (1992)
4 trituberculatus culture water
Fish
Bacillus toyoi Scophthalmus Improved turbot larval growth when added to disinfected rotifers Gatesoupe (1989)
maximus
Bacillus strain IP5832 spores Scophthalmus Improved weight gain of larvae and reduced mortality after Gatesoupe (1991b)
maximus challenging with Vibrio
Streptococcus Scophthalmus Added to rotifer cultures which were fed to turbot larvae. Larval Gatesoupe (1991c)
thermophilus, Lactobacillus maximus survival increased
helveticus and Lactobacillus
plantarum
Vibrio pelagius Scophthalmus Higher larval survival than controls when added alone or in Ring & Vadstein (1998)
maximus combination with Aeromonas caviae
Vibrio mediterranei Q40 Scophthalmus Reduced colonization of gut by opportunistic microflora Huys et al. (2001)
strain maximus
Vibrio alginolyticus Scophthalmus Presence of probiotic correlated with better survival Gatesoupe (1990)
maximus
Lactobacillus plantarum and Scophthalmus Improved survival after challenge with Vibrio sp. Gatesoupe (1994)
Carnobacterium sp. maximus
Vibrio (strain E) Scophthalmus Improved survival and growth after challenge with Vibrio splendidus Gatesoupe (1997)
maximus
Carnobacterium divergens Scophthalmus Beneficial effect of Carnobacterium divergens on survival Ring (1999)
and Vibrio pelagius maximus inconclusive from in vivo study
Streptococcus lactis and Scophthalmus Sixfold increase in survival of larvae fed rotifers enriched with Garcia de la Bande et al.
Lactobacillus bulgaricus maximus probionts (1992)
Roseobacter strain 27-4 Scophthalmus Improved larval survival over first 5 days Hjelm et al. (2004)
maximus
Roseobacter spp. Scophthalmus Displayed in vitro antagonism towards Vibrio anguillarium Hjelm et al. (2004b)
maximus
Lactobacillus plantarum Gadus morhua Opportunistic colonization reduced by 70% Strm & Ring (1993)
Carnobacterium divergens Gadus morhua Improved survival after challenge with Vibrio anguillarum Gildberg et al. (1997)
Vibrion salmonicida strain Hippoglossus Larval survival increased (to 72.8%) compared with control (58.2%) Ottesen & Olafsen (2000)
hippoglossus after 32 days posthatch

Table 1. Continued.
Microbe Host species Effect on host Reference
Strains PB 52 and 4:44 Hippoglossus Both bacteria were capable of colonizing the gut by addition to the Makridis et al. (2001)
hippoglossus water and attachment to rotifers. No improvement in survival
Vibrio strains PB 1-11 and Hippoglossus No difference in gut bacterial CFUs or growth between controls and Makridis et al. (2001)
PB 6-1 hippoglossus bacteria bioencapsulated in Artemia franciscana
Mixture of Pseudomonas Hippoglossus Improved larval survival and reproducibility of treatment Skjermo & Vadstein (1999)
and Cytophaga/ hippoglossus
Flavobacterium
Microbiologically matured Hippoglossus Improved survival of yolk-sac larvae Vadstein et al. (1993)
water hippoglossus
Bacillus no. 48 Centropomus Reduced Vibrio spp. in the microflora Kennedy et al. (1998)

undecimalis
Pediococcus acidilactici and Pollachius Addition of P. acidilactici with Artemia improved growth of larvae Gatesoupe (2002)
Saccharomyces cerevisiae pollachius
VKM-124 Sparus auratu Reduced infection with various pathogenic viruses Maeda et al. (1997)
Pseudoalteromonas undina,
Lactobacillus fructivorans Sparus auratu Reduced larval mortality between days 39 and 66 posthatch Carnevali et al. (2004)
(AS17B) and L. plantarum
(906)
Arnobacterium sp. (strain Oncorhynchus Detected in intestine 10 days after being administered Robertson et al. (2000)
K1) mykiss fry
Carnobacterium sp. Strain 1
Oncorhynchus Fed with pellet diet. No adverse affect on survival Joborn et al. (1997)
mykiss fry
Streptococcus faecium M74 Cyprinus carpio Improved growth and food conversion ratio in fry over 6 weeks Bogut et al. (1998)
Kocuria AP4 Amphiprion Improved clownfish larval survival Vine (2004)
percula
Pseudoalteromonas AP5 Amphiprion Reduced Vibrio spp. in the culture water microflora Vine (2004)
percula
have been proposed but have yet to be tested in expensive humoral and/or cellular immune response, (2) modification
large-scale in vivo trials. In the past few years, interest in of the metabolism of bacterial pathogens by changing their
probiotic formulations for human and animal use has enzyme levels, and (3) competitive exclusion either through
increased and can be attributed to the realization that the production of inhibitory compounds that are antagonistic
concept can be validated. towards pathogens, or by competing for nutrients, attach-
ment sites or oxygen.
The enhancement of nonspecific immunity by probiotics
Modes of action in humans is well recognized (Gill, 2003) and their effect on
An understanding of the mechanisms probiotics use to stimulating cytokine production, which regulates the pro-
compete either with other probiotics or with pathogens is duction of T cells, has been documented in some animal
important when designing a protocol for their selection. The species (Maassen et al., 2000; Perdigon et al., 2002). Larval
selection criteria for human probiotics have focused on fish have a poorly developed immune system, relying
methods of processing and production, biosafety considera- primarily on their nonspecific immune response (Vadstein,
tions and taking into account the part of the body where the 1997). In larvae exposed to high bacterial concentrations, a
organism is active (Huis int’ Veld et al., 1994). For aqua- greater amount of mucus-producing saccular cells has been
culture purposes, subjects for consideration have been the found, suggesting that nonspecific defence mechanisms
evaluation of the probiotic’s ability to out-compete patho- against invaders were stimulated by the augmented micro-
genic strains (Vine et al., 2004a), assessment of the probio- flora (Ottesen & Olafsen, 2000).
tic’s pathogenicity, evaluation of the probiotic in vivo and Irianto & Austin (2002a) showed that after feeding trout
production cost considerations (Gomez-Gil et al., 2000). with probiotics for 2 weeks, stimulation of cellular immu-
The common modes of action available to probionts as nity was detected with an increase in lysozyme activity and
suggested by Fuller (1987) include (1) the stimulation of the in the number of erythrocytes, macrophages and

lymphocytes. Panigrahi et al. (2004) also fed trout a pellet action. Antibiotics are chemical substances usually pro-
containing the probiotic Lactobacillus rhamnosus JCM1136, duced as secondary metabolites that, although created in
resulting in an increased level of the nonspecific immune small quantities, inhibit or kill other microorganisms (Brock
response. Stimulation of the immune system by probiotics & Madigan, 1997). Other inhibitory compounds produced
has also been shown in carp (Stosik & Szenfeld, 1996) and by bacteria include organic acids, hydrogen peroxide (Ring
shrimp (Penaeus monodon) (Rengpipat et al., 2000). A & Gatesoupe, 1998; Vazquez et al., 2005), carbon dioxide
paucity of information is available regarding the ability of (Gill, 2003) and siderophores (Braun & Braun, 2002;
microorganisms to stimulate the immune response of fish Yoshida et al., 2002). Bacteriocins are proteinacious agents
larvae (Olafsen & Hansen, 1992). Therefore, to gain a better produced by bacteria to inhibit or kill other bacteria. They
understanding of the possible modes of action used by are ribosomally synthesized unlike antibiotics, which are
probiotics, screening for stimulation of the immune re- synthesized by other mechanisms (Brock & Madigan, 1997).

sponse system should be performed. The genes encoding the proteins involved in processing the
The ability of probiotics to modify the metabolism and bacteriocin are often carried out by plasmids or transposons
enzyme levels of the other intestinal bacteria has been tested (Brock & Madigan, 1997), suggesting that the ability to
in terrestrial animals. For example, probiotic yeast cultures produce bacteriocins can be transferred between bacteria.
provided to dairy cattle were shown to influence ruminal Siderophores are iron-complexing chemicals secreted by
microbial metabolism by increasing microbial protein pro- bacteria and fungi (Braun & Braun, 2002). Bacteria that
duction (Miller-Webster et al., 2002). Therefore, research produce siderophores have highly specific transport proteins
should be performed to test the use of probiotics to in their outer membrane and are therefore capable of
beneficially manipulate the metabolism of the microflora in utilizing Fe31, which is otherwise insoluble in the surround-
aquatic organisms. ing medium (Braun & Killmann, 1999). Siderophore-pro-
Quorum-sensing in bacteria is a process whereby bacteria ducing bacteria can survive in nutrient-poor environments
communicate using excreted signalling molecules, thereby (Mazoy et al., 1992; Braun & Braun, 2002). Gatesoupe
allowing a population of bacteria to regulate gene expression (1997) showed that the siderophore-producing ‘Vibrio E’
collectively and subsequently to control behaviour on a increased the resistance of turbot larvae against pathogenic
community-wide scale (Henke & Bassler, 2004). A link Vibrio splendidus and improved larval growth. The omission
between virulence factor expression and quorum sensing of ferrous salt in the diet of sea-bass larvae had no negative
has been demonstrated in Aeromonas salmonicida and Aero- effect on larval growth or survival (Gatesoupe et al., 1997),
monas hydrophila (Swift et al., 1997). Bacteria have been which suggests that the establishment of siderophore-pro-
found which block the quorum sensing systems of their ducing bacteria could help to control the growth of those
bacterial competitors by producing enzymes which inacti- opportunistic pathogens that have a low iron uptake. The
vate the signal compound (Defoirdt et al., 2004). Screening addition of an efficient iron-chelating probiont to inhibit
for microorganisms that produce these enzymes has im- pathogens must be done with caution as iron limitation may
mense potential for disease control in aquaculture. cause a reverse effect, inducing the expression of side-
If a probiotic excludes a pathogen by producing antag- rophores in the pathogen and thereby aiding its develop-
onistic extracellular metabolites, it is possible that the ment in the host (Gram et al., 2001).
pathogen will eventually develop resistance to the metabo- The production of antagonistic or inhibitory compounds
lites. It is therefore important that a probiotic has different against any other microflora in vitro is no guarantee that the
ways of out-competing pathogens in order to grow. These potential probiotic will be effective in vivo (Gibson et al.,
include (1) production of antagonistic compounds, (2) 1998; Ring & Gatesoupe, 1998; Gram et al., 2001). Addi-
advantageous growth characteristics, i.e. a short lag period tionally, the lack of effectiveness may be a result of selective
and a short doubling time, (3) attachment ability to intest- ingestion by the host (Prieur, 1983; Riquelme et al., 2000) or
inal mucus and (4) production of other compounds bene- death of the probiotic microbe in the digestive tract. Ruiz-
ficial to the host. These four criteria can be used for the in Ponte et al. (1999) found a strain of Roseobacter sp. that only
vitro screening of suitable probiotic candidates (see Fig. 1). produced an inhibitory compound in the presence of
foreign cells. This suggests that in vitro antagonism studies
using mono-cultured probiotic supernatant instead of
Antagonistic compounds
whole cells may inadvertently result in potential antimicro-
For the purpose of this review, antagonistic compounds are bial-producing probionts going undetected.
defined as chemical substances produced by bacteria that are Bacteria with antagonistic activity against other micro-
toxic or inhibitory towards other microorganisms. These organisms were present in low quantities (2% of the total
substances may be produced as either primary or secondary microflora) in the larval rearing environment of the Chilean
metabolites and therefore have different modes of inhibitory scallop, Argopecten purpuratus (Riquelme et al., 1997), but

1 Acquisition of strains
from healthy animals
In vitro tests
Production of antimicrobial Growth characteristics

compounds in mucus
2
Attachment to Production of other

intestinal mucus beneficial compounds

Suitable candidates
Organism
3
identification
Safe to humans
4 Pathogenecity/toxicity test
Not pathogenic/toxic to host

5 Choice in route of delivery
Addition to rearing water Attachment to livefood
Efficacy in Attachment to
culture water livefood
Yes
7 Can probiont be re-isolated 6 Pilot-scale in vivo 9 On-going strain

from intestinal tract? experiments testing
Yes Improves growth/survival
8
Production cost-
benefit analysis Still effective
Economically viable
PROBIOTIC
Fig. 1. Proposed research protocol for the selection of intestinal probiotics in marine larviculture (with acknowledgements to Verschuere et al., 2000).
Explanatory note for numbers in parentheses: Suitable probiotics should ideally be derived from healthy individuals (1), preferably of the species into
which they are to be introduced. As a result, problems regarding pathogenicity towards the host organism are reduced. After initial isolation there are
likely to be a large number of bacteria available for screening. The aim of the in vitro tests (2) is to reduce rapidly the initially available large pool of
candidate probionts to a more acceptable number for further testing (see text for further information regarding the order in which the tests should be
conducted). Probiotic candidates can then be identified (3) using microbiological/phenotypic tests or, more accurately, using 16S rRNA gene sequence
techniques. Each identified organism needs to be carefully researched and assessed regarding its potential pathogenicity/toxicity towards humans.
Small-scale pathogenicity/toxicity tests on the host organism can then be conducted further to eliminate some probionts (4). Once suitable candidates
have been selected based on the in vitro results, the route of delivery should be tested. The choice of probiotic delivery (5) may be influenced by certain
factors related to the biology of the fish species, for example the time required from hatching to the start of exogenous feeding. A long time may favour
adding the probiotic to system water with the aim of increasing the exposure of the larvae to the probiotic. In cases where the water in the larval rearing
system is frequently being replaced, attachment of probionts to livefood may be preferable. The prolonged efficacy of the candidate probiont in the
larval rearing system needs to be confirmed before adding it to the water can be recommended. Similarly, experiments testing the attachment of the
candidate probiotics to livefood organisms should be conducted to ensure that sufficient candidate probionts attach and are therefore available to the
larvae when feeding on the livefood. Final validation can only be performed in vivo (6). If a suitable candidate shows good potential in vivo and further
upscaling and production is anticipated, a cost–benefit analysis needs to be performed (7). This should include aspects such as product formulation,
packaging and the dosing recommendations. To be considered a true ‘intestinal’ probiotic, the organism must have been isolated from the intestinal
tract of the larvae (8). If not, it cannot be discounted that larval growth and survival may been improved due to some other factors such as improved
water quality rather than the probiont’s ability to exclude opportunistic or pathogenic bacteria. Finally, a probiont that is economically viable to produce
can be considered to be marketable. Owing to the problem of strain degeneration, ongoing pilot-scale in vivo experiments should be conducted to test
the reliability of probiont efficacy (9).

may contribute up to 21% in microalgae monocultures results confirm suggestions by other authors (Sugita et al.,
(Lodeiros et al., 1991 cited in Avendano & Riquelme, 1997; Robertson et al., 2000) that growth in vitro should not
1999). Once these bacteria enter the gastrointestinal tract, be viewed independently as the candidate probiotic bacteria
they can be found throughout the digestive tract (MacDo- were each capable of excluding different pathogens. For
nald et al., 1986). The activity of antimicrobial metabolites example, the candidate probiotic with the longest lag-period
from the sterile supernatant of the probiotic Pseudomonas inhibited five of seven pathogens although its ranking index
fluorescens AH2 was still effective in vitro after 7 days (Gram was low, whereas the two highest-ranking probiotics showed
et al., 2001). combined antagonism to three pathogens (Vine et al.,
Antagonism may not only be limited to other bacteria. 2004a).
Maeda et al. (1997) isolated a bacterial strain of Pseudoalter-
omonas undina, VKM-124, which had vibrio-static activity
Attachment to mucus

and inhibited the appearance of the cytopathic effect on
prawn epithelioma papillosum cyprini cells. When added to In humans, it appears that attachment and the production
prawn (Penaeus sp.) and sea bream (Sparus aurata) larval of antimicrobial compounds by lactic acid bacteria are the
tanks, P. undina VKM-124 improved larval survival (com- critical factors in excluding pathogens (Reid, 1999; Gill,
pared with a nontreated control), by reducing the infection 2003). This appears to be similar in fish; Ring et al. (1998)
of the larvae with Sima-aji Neuro Necrosis Virus (SJNNV), suggested that attachment of lactic acid bacteria to the
Baculo-like viruses and Irido virus. It is possible that in vivo mucus layer may serve as the first barrier of defence against
the probiotic activated the immune system of the exposed invading pathogenic bacteria. Attachment is therefore re-
organism, thereby reducing the viral infection. Further garded as a prerequisite for colonization (Olsson et al., 1996;
studies should be conducted to confirm whether a reduction Joborn et al., 1997; Ring & Gatesoupe, 1998) and is
in viral count is due to direct antagonism or via the important in the stimulation of the host’s immune system
stimulation of the immune system. (Collins & Gibson, 1999; Ouwehand et al., 1999b; Schiffrin
& Blum, 1999). The superior ability of bacterial pathogens
to attach has been related to virulence (Wilson & Horne,
Growth in mucus
1986; Bruno, 1988) and is considered the first step of
Bacteria may only produce metabolites during the station- bacterial infection (Bengmark, 1998). Research has been
ary growth phase (Monaghan et al., 1999), which may not conducted on the ability of probiotics to attach to the
occur in the gut due to constant flushing. Isolating bacteria intestinal mucus of humans (Beachey, 1981; Kirjavainen
that produce antimicrobial metabolites is common practice, et al., 1998; Ouwehand et al., 1999c, 2000a, b, 2001; Juntu-
but few experiments have determined at what stage of nen et al., 2001) and fish (Krovacek et al., 1987; Olsson et al.,
growth the bacteria produce the metabolites and whether 1992; Vazquez et al., 1997; Andlid et al., 1998; Rinkinen
the bacteria are able to compete for attachment sites et al., 2003; Vine et al., 2004b). Although probiotics may
(Vanbelle et al., 1990). Thus, in vitro studies may create a have beneficial effects on host species from which they have
false impression of the ability of probiotics to inhibit not been isolated, attachment ability is not necessarily host/
pathogens in vivo. Any inability to compete for attachment probiont-species-specific but rather dependent on the bac-
sites on the mucus of the gut wall suggests that these bacteria terial strain (Rinkinen et al., 2003). Therefore, potential
may not multiply sufficiently fast to compensate for being probionts should be tested for their ability to adhere to
flushed from the mucus during gut evacuation. mucus in vitro before large-scale trials are attempted, as the
Screening for organisms with antagonistic abilities to- candidate probiotic may be transient in vivo and conse-
wards pathogens may produce a large number of candidate quently not contribute to the health of the host organism.
probiotics. A simple method for eliminating candidate Attachment to mucus may not be the only invasion
probionts with similar growth characteristics is by growing strategy employed by pathogens, as was the case with Vibrio
them in standard microbiological media and comparing anguillarum O2, which exhibited poor attachment to mucus
their growth profiles (Vine et al., 2004a). Bacteria with but was highly virulent (Olafsen, 2001). The human probio-
similar antagonistic abilities and growth profiles can be tic Lactobacillus reuteri did not produce antimicrobial sub-
assumed to be of equal potential, thereby reducing the pool stances, yet in vitro it inhibited the binding of pathogens to
of candidate probionts to be tested further. Vine et al. receptor sites (Mukai et al., 2002). Competition for attach-
(2004a) suggested a ranking index whereby candidate pro- ment may not necessarily be due to competitive exclusion
bionts grown in vitro in fish intestinal mucus were ranked but may also be due to specific inhibitors (Rojas & Conway,
according to the growth profile characteristics, lag-period 1996) such as those produced by Lactobacillus fermentum,
and specific growth rate. The method would enable the which inhibited the adhesion of Escherichia coli to porcine
rapid screening of candidate probiotics; however, their ileal mucus (Ouwehand & Conway, 1996). An advantage of

c 2006 Federation of European Microbiological Societies
competitive exclusion based on enhanced attachment is that such bacteria at a level higher than that of Saccharomyces
unlike antibiotics, the factors that inhibit pathogen binding cerevisiae-fed rotifers (Watanabe et al., 1992). In fish, lipids
do not necessarily kill the pathogen, thereby exerting less produced by intestinal microbes (Ring et al., 1992a;
selective pressure on the pathogen to evolve resistance (Reid Shirasaka et al., 1995) have contributed significantly to the
et al., 2001). diet of Arctic charr (Salvelinus alpinus) (Jostensen et al.,
1990; Ring et al., 1992a) and turbot (Scopthalmus max-
imus) (Ring et al., 1992b), and in the tilapia (Oreochromis
Production of other beneficial compounds
niloticus) they have physically altered the morphology of the
digestive system (Kihara & Sakata, 1997).
Beneficial dietary compounds
The bacterial production of highly polyunsaturated fatty
Bacteria that produce various vitamins as secondary meta- acids (HPUFAs) in the larval digestive tract is unknown. A

bolites have been isolated from fish intestine. Vitamin B12 is strain of Shewanella putrefaciens, isolated from the intestine
produced by different bacteria isolated from the microflora of Pacific mackerel (Scomber japonicus), produced eicosa-
of a variety of fish species, including carp (Cyprinus carpio) pentaenoic acid (EPA) as the sole HPUFA. The contribution
(Kashiwada et al., 1970; Sugita et al., 1990, 1991a), channel of EPA amounted to 24–40% of the total fatty acid in the cell
catfish (Ictalurus punctatus) (Limsuwan & Lovell, 1981), (Yazawa, 1996). Rotifers cultured with S. putrefaciens and
various tilapia species (Lovell & Limsuwan, 1982; Sugita then separated from the bacteria lost 48% of their EPA
et al., 1991a) and rainbow trout (Oncorhychus mykiss) content after 6 h and 80% after 24 h (Watanabe et al., 1992).
(Sugita et al., 1991b). Fish species that require dietary To ensure that high levels of fatty acids are available to the
vitamin B12 do not appear to have these vitamin B12- larvae, feeding of EPA and docosahexaenoic acid (DHA)-
producing bacteria, whereas those that do, do not require enriched rotifers to larvae should be immediate or possibly
additional dietary B12 (Sugita et al., 1991a). Whether the include addition of the HPUFA-producing bacteria to the
introduction of vitamin B12-producing bacteria into fish culture water.
species requiring the vitamin would reduce the need for its
inclusion in the diet warrants further investigation.
Beneficial digestive enzymes
Unlike most animals, fish are unable to synthesize ascor-
bic acid from glucuronic acid (Merchie et al., 1997) and are Probiotics may provide their larval host with digestive
therefore dependent on an adequate supply through the enzymes. The digestive capability of the larval digestive
feed. Ascorbic acid is involved in the formation of cartilage system develops and changes as the larva grows (Govoni
and fibrous tissue, and vitamin C deficiencies may result in et al., 1986; Cahu & Zambonino-Infante, 1995; Ribeiro et al.,
broken back syndrome (Merchie et al., 1997). Production of 1999b; Gawlicka et al., 2000; Zambonino-Infante & Cahu,
vitamin C by the intestinal microbes of fish has yet to be 2001). Bacteria isolated from fish digestive systems have
investigated, but the dietary contribution of vitamin K by been shown to digest chitin (Hamid et al., 1979; MacDonald
the intestinal microflora of immature brook trout (Salveli- et al., 1986), starch (Hamid et al., 1979; MacDonald et al.,
nus fontinalis) has been shown (Poston, 1964). 1986; Gatesoupe et al., 1997), protein (Hamid et al., 1979;
Carotenoids can act as antioxidants, scavenging lipid- Gatesoupe et al., 1997), cellulose (Erasmus et al., 1997;
damaging free radicals. Identification of bacteria producing Bairagi et al., 2002) and lipids (Gatesoupe et al., 1997; Vine,
these pigments is simplified as the colonies are bright yellow 2004) in vitro. Sea bass (Dicentrarchus labrax) larvae fed live
or orange when grown on agar media. Brightly pigmented yeast (Debaryomyces hansenii CBS 8339) incorporated in
bacteria generally produce more such metabolites than pale their diet showed increased activity and concentrations of
strains (Holmstrom et al., 2002). This may help in the initial mRNA trypsin and lipase (Tovar-Ramirez et al., 2004).
screening of potential probiotics selected on the basis of Contrary to these findings, the giant bacterium Epulopis-
their ability to produce these beneficial metabolites. cium fishelsoni reduced the activity of the digestive enzymes
Marine bacteria have been shown to contain between 36 in the surgeonfish Acanthurus nigrofuscus, particularly of
and 56% crude protein (dry weight) (Brown et al., 1996). The protease and amylase (Pollak & Montgomery, 1994).
amino acid profile was similar to that of Saccostrea commer- The microflora of juvenile Dover sole, Solea solea, had a
cialis spat, suggesting that the bacteria could act as a protein greater proportion of substrate-degrading bacteria than
source for the developing bivalve (Brown et al., 1996). adults (MacDonald et al., 1986). It is therefore possible that
The production of short-chain fatty acids by intestinal these microorganisms contribute to their host by enhancing
bacteria has been shown to contribute to the nutrition of the their ability to digest and assimilate nutrients. The degrada-
host in humans (Batt et al., 1996) and fish (Clements, 1997), tion of these comparatively unattractive complex molecules
but the effect on aquatic larvae has not been investigated. may release compounds that could be beneficial to the host
Nutritive fortification of rotifers has been achieved with (MacDonald et al., 1986). The importance of digestive

enzymes produced by bacteria remains unclear (MacDonald 1994; Harboe et al., 1994; Theisen et al., 1998; Liltved &
et al., 1986; Pollak & Montgomery, 1994) and the contribu- Cripps, 1999) followed by incubation with the probiotic
tion of ‘introduced’ or probiotic bacteria has yet to be (Gatesoupe, 1991a, 1997, 2002; Gatesoupe & Lesel, 1998;
investigated. Makridis et al., 1998). In addition, a positive effect of
Probiotics used for terrestrial animals or adult fish should probiotics on livefood cultures has been documented (Bo-
be resistant to bile (Nikoskelainen et al., 2001b) if they are to gaert et al., 1993; Shiri Harzevili et al., 1998; Douillet,
persist in the digestive tract. Selection criteria for probiotics 2000a, b; Gatesoupe, 2002; Orozco-Medina et al., 2002;
for larvae differ from those for adult fish in that initially the Villamil et al., 2003) as has the transfer of these bacteria to
pH of the larval digestive system is alkaline (Tanaka et al., larval fish (Gatesoupe, 1997; Gatesoupe & Lesel, 1998;
1996; Ronnestad et al., 2000). Therefore, the probiotic is not Makridis et al., 1998, 2001).
required to move through an acidic environment and, unlike The delivery method to the larvae should be tested and

a probiont designed for adult fish, does not need to be refined in a sequence of studies before testing on a large scale
resistant to acid and bile. Because the larval digestive tract is in vivo. Some probiotics may be able to attach to live food.
immature at hatching, the gall bladder has yet to develop and Marine fish larvae, in particular, require live food as many
subsequently bile is not secreted until later during develop- do not accept artificial diets during the early stages of larval
ment (Govoni et al., 1986). Therefore, probiotics being development (Lavens et al., 1995; Hart & Purser, 1996;
selected for use during the early stages of larval development Yufera et al., 2000). If probiotics can be administered via
do not need to be screened for this characteristic. livefood, their application in marine fish larviculture could
be simplified.
Interaction with livefood

The development of a probiotic selection
For most cultured marine fish species the most suitable prey protocol for marine fish larvae
items at first feeding are rotifers (Sorgeloos et al., 1995; van
der Meeren & Naas, 1997). Manipulation of the external In vitro screening
microflora of the livefood which is then fed to the larvae has
A bottleneck in the isolation of suitable probiotics for
potential application in the delivery of probiotics. The
aquaculture has been the lack of a clearly defined experi-
bacterial flora of rotifers is approximately 5 103 bacteria
mental protocol. The proposed research protocol shown in
per individual (Munro et al., 1993; Skjermo & Vadstein,
Fig. 1 is represented as a flow diagram involving a series of
1993). Attempts to feed rotifers with a considerably higher
experiments that could be conducted for the selection of
bacterial load to turbot larvae have proven unsuccessful
larval probiotics. By systematically conducting in vitro tests
(Nicolas et al., 1989; Munro et al., 1999). The amount of
on a large number of potential probiotics, less-promising
probiotic cells that adhere to the livefood depends on the
candidates can be excluded, thereby reducing the number of
probiont, duration of exposure and the state (dead or alive)
in vivo trials required to validate the effectiveness of the
of the livefood organism (Gomez-Gil et al., 1998). As the
probiont. Ranking the four in vitro experiments described
livefood’s bacterial load increases it may reach levels and/or
earlier as criteria for the selection of larval probiotics must
micro-communities may develop that may negatively affect
be performed with caution. For example, the in vitro
the health of the host larvae. For example, Olsen et al. (1999)
production of antagonistic compounds may not necessarily
found that bacterial overloading of 4-day-old Artemia fed to
be more important than attachment to mucus when applied
halibut larvae resulted in poorer larval growth.
in vivo. Experiments investigating the production of antag-
It is well documented that a change in diet selects for a
onistic compounds cannot test the candidate probiont
different bacterial community (Shiranee et al., 1993; Ring
against all potentially competitive bacteria. Therefore, a low
& Strm, 1994; Olsen et al., 2000). For example, in Arctic
production of antagonistic compounds by a particular
charr (S. alpinus), alteration of dietary fatty acids resulted in
candidate probiotic may be the result of the researcher’s
a major change in contribution of the lactic acid bacterial
choice of pathogens against which it was tested. It is there-
flora (Ring et al., 1998). Large numbers of Vibrio spp. in
fore recommended that researchers, screening for the pro-
the rearing water and larval intestine are usually attributed
duction of antagonistic compounds, use as many species of
to the presence of Artemia (Verschuere et al., 1999; Olsen
pathogenic bacteria as possible.
et al., 1999, 2000; Eddy & Jones, 2002), which diminish as
the fish are weaned onto a formulated diet (Blanch et al.,
Organism identification
1997). Through microbial manipulation, livefood can be
treated to act as a vector for probiotics. Manipulation As not all initially isolated bacteria are to be used as
includes the disinfection of the rotifers (Munro et al., 1999; probionts, identification of candidate probiotics would not
Rombaut et al., 1999) or Artemia (Abreu-Grobois et al., need to be performed until after further screening using

in vitro tests. After the putative probiotic has been tested Not all ‘pathogenic’ bacteria are necessarily harmful.
in vivo and found to be beneficial, it should ideally be Opportunistic species such as Vibrio alginolyticus have been
identified down to strain level (EFSA, 2005a), using either isolated from the intestine of apparently healthy larvae
16S rRNA gene sequence or fatty-acid profile techniques (Munro et al., 1993), and this species has been successfully
(Buyer, 2002). Identification of the candidate probiont can used as a probiotic in algal production (Gomez-Gil et al.,
provide potentially useful information regarding its culture 2002), shrimp (Garriques & Arevalo, 1995; Vandenberghe
requirements, pathogenicity and hence suitability as a et al., 1999) and fish culture (Austin et al., 1995). Similarly,
candidate probiont. If the particular strain does not exhibit Aeromonas hydrophila has been used as a probiont in rain-
any pathogenesis in the target organism but is a known bow trout (Irianto & Austin, 2002a). It is hypothesized that
human or aquatic pathogen, approval by the relevant food in the absence of virulence expression, factors that could
and drug administration may be difficult or even impossi- contribute to pathogenicity, like growth rate or attachment

ble, thereby ruling it out as a candidate for further develop- ability, may influence the microflora to the benefit of its
ment. Data on the bacteria may also assist in the screening of host.
bacterial transference of antibiotic resistance (EFSA, 2005b).
Although the use of antibiotics in aquaculture has become
Route of delivery
less popular, they are still used in humans and therefore the
possibility of transferring resistance from the microflora of To maximize the competitive advantage of added probiotics,
cultured organisms to human bacteria should be prevented. early delivery seems to be best (Ring et al., 1996; Ring
Because prolonged exposure of obligate anaerobic bacter- & Vadstein, 1998; Gatesoupe, 1999) as bacteria colonizing
ia to oxygen results in cell death, probiotics should ideally be the intestine before first feeding may be able to persist
aerobic or facultative anaerobes. The aerobic conditions among the autochthonous microbiota (Hansen & Olafsen,
under which probionts are applied to larvae via the livefood, 1999; Olafsen, 2001; Carnevali et al., 2004). Opportunistic
or in the culture water, suggests that obligate anaerobes are pathogens are commonly introduced along with mass
unlikely to thrive. Additionally, sustaining an anaerobic cultured livefood owing to concentrated feeding levels
bacterial culture requires more technical expertise and combined with the rapid growth rate of the bacterial
equipment and is more expensive than culturing aerobic microflora (Skjermo & Vadstein, 1993), underlining the
bacteria. It is possibly due to the technical considerations concept that probiotic delivery should occur during the
required for working with anaerobes that their use as early stages of larval development prior to exogenous
probiotics has been limited, although various anaerobic feeding.
potential probiotics have been identified for use in aqua- Exposing the larvae to probiotic concentrations greater
culture (Dixon et al., 2001; Ramirez & Dixon, 2003). than those occurring naturally in the water increases the
Delivery of anaerobic probiotics to fish larvae may be chance of probiotics being ingested by the larvae either
logistically problematic and should be carefully considered directly or via the livefood. As a result, the probiotic is able
before further product development is undertaken. better to establish itself in the mucus and epithelium of the
digestive tract (Strm & Ring, 1993; Ring et al., 1996;
Ring & Vadstein, 1998; Makridis et al., 2000a; Huys et al.,
Pathogenicity/toxicity of candidate probionts
2001), and potentially suppress the attachment of patho-
A probiotic must not be pathogenic or toxic to its host. This genic bacteria. This may be particularly beneficial in larvae
can be determined by small-scale challenge tests of the host with a poorly developed immune system (Vanbelle et al.,
species using short-term baths in the bacterial suspension or 1990) as subsequent pathogenic bacterial exposure is sup-
direct addition to the culture water. If a microorganism is to pressed by established probiotics (Gatesoupe, 1994). The
be used as a probiotic for livefood organisms, tests should health of the host is thereby enhanced by preventing or
also be performed on the larvae to which they will be fed reducing the proliferation of pathogens or opportunistic
(Verschuere et al., 2000). Irianto & Austin (2002b), working bacteria, and the latter rather than primary or specific
on trout probiotics, selected four out of 11 bacteria which pathogens contribute most to larval mortality under culture
were deemed harmless to the fish following intraperitoneal conditions (Vadstein et al., 1993; Munro et al., 1995; Huys
or intramuscular injections. et al., 2001). Another method of manipulating the intestinal
As lactic acid bacteria are intrinsically resistant to anti- microflora is the use of microbiologically matured seawater,
biotics (Salminen et al., 1998), potential pathogens would be which compared with membrane-filtered or disinfected
difficult to control. Therefore, in humans, candidate pro- water, has led to an improvement in turbot larval growth
biotics from the genus Lactobacillus are rigorously screened (Vadstein et al., 1993; Skjermo et al., 1997). This supports
for pathogenicity before being passed for human trials the hypothesis that a mature, established microflora, which
(Harty et al., 1994). is dominated by K-selected species, is able to confer

protection on the larvae by reducing the proliferation of r- resulting microflora has not been investigated. It is possible
selected species. that certain probiotics may initially change the microflora to
Livefood organisms such as rotifers and Artemia are filter- the host’s advantage but in doing so may prevent other
feeders and are also known to graze bacteria (Vadstein et al., autochthonous microorganisms from becoming established,
1993; Skjermo & Vadstein, 1999; Olsen et al., 2000). Their thus ultimately harming the host. Long-term studies on the
ability to act as vectors for the delivery of probiotics to fish effect of probiotics on juvenile growth and survival should
larvae (Makridis et al., 1998, 2000a, b; Gatesoupe, 2002) therefore be conducted.
allows for the manipulation of the livefood’s bacterial
community through the addition of probiotics. This in turn Production cost--benefit analysis
reduces the number of opportunistic (r-selected species) or
pathogenic bacteria from entering the larvae and colonizing If the probiotic is to be commercialized, an economic
analysis of the potential commercial production is required

the intestinal tract.
Loading of probiotic bacteria on the livefood should be upon successful completion of the in vivo trials. Investiga-
optimized to maximize probiont delivery while maintaining tions into the economic viability of different product
palatability and the livefood organisms’ movement capabil- formulations, packaging options and dosing recommenda-
ities. For example, turbot larvae refused to eat rotifers with tions would need to be completed.
high bacterial loads of up to 105 bacteria per rotifer (Nicolas Upon successfully completing the screening stages dis-
et al., 1989). cussed above, the microorganism can be called a probiotic.
For species with large, well-developed larvae (such as the To ensure that the probiotic remains viable, ongoing strain
salmonids), incorporation of the probiotic into the artificial testing is required, including aspects of storage and efficacy.
feed is another possible delivery route. For example, Arno-
bacterium sp. (strain K1) incorporated into the artificial diet Strain degeneration
of rainbow trout (Oncorhynchus mykiss) fry was detected in It has been noted that the ability of probionts to inhibit the
the intestine 10 days after administration (Robertson et al., originally tested pathogens diminishes after storage (Vine,
2000). 2004). Strain degeneration should be tested to prevent the
introduction of nonviable probionts. Other studies (Nikos-
In vivo validation kelainen et al., 2001b; Ring et al., 2004) have shown that the
ability of probiotics to produce antimicrobial metabolites,
Based on the definition of probiotic used in this review, a which inhibit the growth of other microorganisms, can be
researcher must be able to reisolate the probiotic from the lost due to storage and subculturing.
intestinal tract of the larvae. If not, it cannot be discounted Many microbes produce extracellular and potentially
that larval growth and survival may have been improved due beneficial metabolites only if they are essential for their
to other factors, for example improved water quality, rather survival (Pirt, 1985). In most cases, culturing bacteria on
than the ability of the probiont to exclude opportunistic or general nutrient media provides the organism with all the
pathogenic bacteria intestinally. essential nutrients to grow and reproduce. However, the
It has been suggested (Verschuere et al., 2000) that ability of bacteria to produce these metabolites can be lost
candidate probiotics should be tested by challenging them with successive generations (Kashket & Cao, 1995). Further
in vivo through the addition of a representative pathogen. research is required regarding the problem of strain degen-
However, larval mortality related to the composition of the eration, particularly its prevention or reduction and, more
microflora can usually be attributed to opportunistic rather importantly, its reversal, which would allow for the testing of
than pathogenic bacteria (Olafsen, 2001). Additionally, as degenerated candidate probionts.
with the choice of pathogens for the in vitro antagonism
experiment, in vivo experiments cannot test all possible
pathogens (usually testing fewer than in the in vitro tests),
Probiotic selection constraints
making it difficult to draw conclusions about the tested
Dosage and dosing frequency
pathogens when they may not occur as part of the normal or
natural larval system microflora. Therefore, for the selection If probiotics are to persist and make any contribution to the
of probiotics for larviculture, we feel that the in vivo indigenous microflora, their introduction would need to be
challenge experiment is not necessary unless the probiotic on a regular basis and/or at a concentration higher than that
has been chosen specifically to out-compete a particular of the already established microbial community (Verschuere
pathogen. et al., 2000).
Although probiotics are usually tested for their ability to The dose–effect relationship must be carefully deter-
exclude particular pathogens, their long-term effect on the mined so as to avoid overdosing, which may result in lower

efficacy while increasing costs, or conversely, underdosing, Two doses of probiotic-bioencapuslated Artemia were in-
which reduces the efficacy of the probiont. For the purpose sufficient to influence predictably the intestinal species
of administering probiotics, a dose can be defined as the composition of halibut larvae over a 10-day period (Makri-
concentration (in number of probiotic cells mL 1) that is dis et al., 2001).
available to the aquatic host. At this dose, they can be added
directly to the water, attached to the livefood or incorpo- Shelf-life and storage
rated into the artificial diet. Additional doses may be
When bacteria are cultured under artificial conditions, i.e.
required to maintain the desired probiotic concentration in
with an excess of nutrients and without competition from
the culture water, the frequency of which may depend on the
other organisms, they may lose the ability to produce
probiotic species, stage of fish development, diet, culture
compounds that would otherwise have been produced
conditions and desired probiotic concentration.
under stressful conditions. For example, subcultures of the

It has been suggested that aquaculture systems cannot
industrially cultured Gram-positive, spore-forming Clostri-
support bacterial concentrations greater than 106 cells mL 1
dium acetobutylicum are known to lose their ability to
(Maeda, 1994). Lactobacillus rhamnosus at a dose of
produce the acids for which they are cultured (Kashket &
109 CFU g 1 of feed protected rainbow trout from experi-
Cao, 1995). This problem of strain degeneration is impor-
mentally induced furunculosis (Nikoskelainen et al., 2001a)
tant when considering Gram-negative candidate probionts,
and (Gomez-Gil et al., 2000) found that the concentration of
as unlike some Gram-positive bacteria they are not endo-
added bacteria rarely exceeded 107 cells mL 1 culture water
spore-forming. Therefore, to maintain viable cultures,
but that it decreased over 72 h, at a rate depending on the
Gram-negative candidate probionts require frequent sub-
species. Doses between 104 and 106 cells mL 1 in the culture
culturing on solid or liquid media.
water of strain 77 (Arthrobacter sp.) were capable of displa-
Because the production of inhibitory metabolites de-
cing the resident microflora of larval Chilean scallop, Aero-
pends on the media upon which the bacteria are cultured
monas purpuratus, after 24 h of incubation (Riquelme et al.,
(Olsson et al., 1992), culture methods and media which
2000). In Crassostrea gigas, addition of CA2 bacteria at a
eliminate or reduce the loss of these selective criteria need to
culture environment concentration of 105 cells mL 1 in-
be used. Nikoskelainen et al. (2001b) found that the ability
creased larval growth (Douillet & Langdon, 1994). Roseo-
of three lactic acid probionts to produce the antimicrobial
bacter 27-4 inoculated at 107 cells mL 1 improved turbot
compounds antagonistic towards fish pathogens diminished
(Scopthalmus maximus) larval survival better than concen-
over time. Although it is possible that the lack of antimicro-
trations of 103–105 cells mL 1 (Hjelm et al., 2004a). Chal-
bial production was due to subculturing, the authors
lenge experiments in which juvenile rainbow trout were
attributed it in part to different screening methods. Ring
exposed to the probiotic Pseudomonas fluorescens AH2 at a
et al. (2004) suggested that the ability of probiotics to
concentration of 105 cells mL 1 followed by exposure to
produce metabolites which inhibit the growth of other
Vibrio anguillarum at 104–105 cells mL 1 in the system water
microorganisms can be lost due to storage and subculturing.
reduced mortality (Gram et al., 1999). Therefore, although
Therefore, ongoing in vivo testing may ensure that the
culture conditions, probiotic and fish species may have an
efficacy of the probiotic remains at a level beneficial to the
influence, a dose delivering between 104 and 106 cells mL 1
larvae (Fig. 1).
to the total culture volume may be sufficient in introducing
Some Gram-positive organisms have the advantage of
a probiotic capable of dominating the intestinal microflora.
forming endospores, thereby making culturing and long-
A single dose may be used for probiotics capable of
term storage easier. A simple and rapid means of culturing,
surviving within the gut and attaching to the mucus and/or
storing and administering probiotics to larvae would be
intestine. However, because doses higher than 106 cells mL 1
preferred by fish farmers; however, with many candidate
may be unnecessarily high (Maeda, 1994), regular dosing at
probiotics being Gram-negative, sterile laboratory techni-
lower concentrations may be required to ensure the bacteria
ques, equipment and skills are required to ensure product
persist in the water and subsequently the intestinal tract. By
quality, all at extra expense. Therefore, Gram-negative
their sustained presence, periodic additions may also assist
probiotics which require inexpensive equipment and simple
in maintaining the desired microflora at levels that inhibit
techniques would be of great benefit to the industry.
the development of pathogenic or opportunistic bacteria.
After 24 h of exposure of the probiotic ‘strain 77’ at
concentrations of 104 and 106 cells mL 1, almost 100% of
Nonbacterial sources of probiotics
the gut of Chilean scallop larvae was dominated by the Yeasts have been identified which exhibit probiotic charac-
probiotic (Riquelme et al., 2000). Probiotics were main- teristics. With our vast knowledge of yeasts, particularly
tained in the gut by providing them every few days at a their biology, genetics and safety issues, their potential as
concentration of 5 103 cells mL 1 (Riquelme et al., 2001). probiotics is good.

Yeasts are not affected by antibiotics. This is advantageous Synbiotics are the combination of pre- and probiotics which
in probiotic preparations used for preventing disturbances are added together in an effort to improve the action of the
in the normal microflora in the presence of antibacterial probiotic synergistically (Bielecka et al., 2002). Owing to the
metabolites. Strains of Saccharomyces cerevisiae and Debar- improved growth of the probiont, it would be more compe-
yomyces hansenii isolated from salmonids (Andlid et al., titive and thereby reduce the growth of pathogens and
1999) have been shown to attach and grow in fish intestinal subsequently their ability to attach. The concept of prebio-
mucus (Vazquez et al., 1997; Andlid et al., 1998). A strain of tics raises the question of the role of diet to improve
Saccharomyces boulardii has been successfully used as a probiotic efficacy. Management of the gut microflora in
probiotic for Artemia nauplii, conferring resistance against humans is possible through dietary manipulation (Walker &
a pathogenic Vibrio sp. (Patra & Mohamed, 2003), although Duffy, 1998; Fooks et al., 1999). Research is required
the ability to transfer resistance to fish or shrimp larvae was regarding the effect of dietary ingredients on the persistence

not tested. In sea bass (Dicentrarchus labrax) larvae, Debar- and efficacy of probiotics in fish.
yomyces hansenii CBS 8339 exhibited a probiotic effect, The ease of probiotic delivery to the larval gastrointestinal
improving larval development by promoting intestinal tract suggests that the ability of a probiotic to attach to
maturation and increasing the ability to absorb nutrients livefood could be used as a selection criteria for probiotics
(Tovar-Ramirez et al., 2004). used in larviculture. It could be hypothesized that coloniza-
The use of greenwater is common in the larval rearing of tion of the larval intestine would be faster if more of the
many marine fish species (Reitan et al., 1997; Dhert et al., same bacteria were initially taken in by the larvae. Therefore,
1998). Bacteria may extend the stationary phase of the algae the introduction of large numbers of probiotic bacteria
(Avendano & Riquelme, 1999), which is ideal for larval during early larval stages could increase the percentage
rearing as the culture of many species utilizes the stagnant contribution of the probiotic in the overall microflora.
water technique. The presence of microalgae in the culture Further research is needed to test this hypothesis.
water has been shown to influence the composition of the Whether probionts can improve resistance against infec-
surrounding microflora, often reducing the number of tions through conferred resistance should also be tested.
potentially pathogenic or opportunistic bacteria (Douillet Fish larvae do not have the ability to develop specific
& Langdon, 1994; Avendano & Riquelme, 1999; Eddy & immunity during the early stages of development (Ellis,
Jones, 2002; Gomez-Gil et al., 2002). The presence of algae 1988) and therefore rely on passive immunization from
in seabass larval-rearing water has been shown in vivo to maternal antibodies (Vadstein, 1997). In humans, adminis-
increase the production of larval digestive enzymes at both tration of live bacteria has been shown to change the
pancreatic and intestinal levels (Cahu et al., 1998). Apart immune response (Pelto et al., 1998; Spanhaak et al., 1998),
from the known beneficial effects of algae on larval survival suggesting research should be conducted to test if aquatic
(i.e. provides contrast to aid feeding, food for livefood) the broodstock exposed to probiotics would confer resistance to
use of greenwater during the early stages of larval rearing the larvae via the egg.
may also benefit the larvae by extending the stationary Genetic manipulation of beneficial strains has been
growth phase of the algae, controlling the culture water suggested for human probionts (Reid et al., 2001; Steidler,
microflora and improving digestive enzyme production. 2003). Bacterial resistance to disease can be transferred to
other strains or species by moving genetic material between
cells via plasmids or conjugative transposons (Salminen
Future research et al., 1998; Teuber et al., 1999). Therefore, using plasmids,
The concept of prebiotics, generally nondigestible oligosac- genes could be inserted into candidate probionts that
charides that stimulate the growth and colonization of produce or increase antimicrobial or beneficial compounds,
probiotic bacteria, is gaining interest in the human health enhance attachment ability, or improve their ability to
food industry (Walker & Duffy, 1998; Rastall & Maitin, utilize limited energy resources. For example, an engineered
2002). An initial dose of probiotics followed by continuous strain of interleukin-10-secreting Lactococcus lactis has been
addition of a prebiotic may extend persistence by the used to treat chemically induced inflammatory bowel dis-
probiotic, particularly if it is not indigenous to the host ease in mice (Steidler et al., 2000). Care is required in the use
species’ normal microflora. of genetically modified organisms, particularly of microor-
Prebiotics can also perform the role of antiadhesive ganisms that are capable of rapidly establishing themselves
oligosaccharides, which prevent the adhesion of pathogens in the natural environment. Therefore, testing methods for
to intestinal cells and mucin (Zopf & Roth, 1996). When genetically modified probiotics must be uncompromising
formulated specifically for the ingestible probiotic, prebio- with strict attention to biological safety (Steidler, 2003).
tics could prove useful by ensuring that the growth of the Until 2003, there were no known genetically modified
probiotic exceeds the flushing rate of the digestive tract. probiotics in use in human medicine (Steidler, 2003).

Until the advent of PCR, detection and enumeration of through the epithelial mucosa to infect otherwise sterile
the probiotic and surrounding microflora was commonly tissues. Experiments should be performed to exclude the
performed using general plating techniques (it still is). likelihood that a probiont causes translocation of bacteria
However, because only a small percentage of heterotrophic and/or bacterial products (such as endotoxin) that may
bacteria can be cultured (Fang et al., 1996; Olafsen, 2001) result in intraabdominal infections.
these techniques may therefore not provide data representa- If the mode of action of the probiotic is the production of
tive of the microflora community. The ability to examine an antimicrobial metabolite, it is possible that if isolated, it
quickly the community interaction between probiotics and may have other uses. Marine organisms have been targeted
other microorganisms could be made possible with the use as sources of novel bioactive drugs and potential anticancer
of flow cytometry or denaturing gradient gel electrophor- agents (Riguera, 1997). Process intensification through the
esis. The species and their contribution are easily deter- use of bioreactors to scale-up the production of novel

mined, thereby rapidly providing researchers with marine antibiotics (Marwick et al., 1999) and techniques to
information upon which the success of the probiotics can isolate and identify these compounds (Riguera, 1997) have
be compared. been developed. Therefore, the isolation and identification
Identifying the various modes of action could be per- of probiotics may have far-reaching consequences in terms
formed using different techniques, each aimed at helping of their contribution to both terrestrial agriculture and
identify a particular mechanism. A probiont may use human health.
various mechanisms to remain in the digestive tract and
therefore modes of action are not mutually exclusive. Testing
whether antagonism, for example through the production of
Concluding remarks
antimicrobial metabolites, contributes to the success of the Although there are many examples of microorganisms used
probiotic requires extensive screening for antimicrobial for biocontrol, typically used in shrimp or pond culture,
compounds and an understanding of their characteristics. there are currently only a few commercial ‘intestinal’ pro-
This could be achieved using nuclear magnetic resonance or biotics used specifically during the larval stages of aquatic
other similar techniques. Once the bacteria have colonized organisms. Many of the probiotics discussed in this review
the larval digestive tract, the larvae could be sampled and the have been tested in small- or pilot-scale facilities, but it is not
composition of the intestinal microflora determined. Un- clear how many have been tested on a commercial scale. The
exposed larvae would serve as controls from which the total general lack of empirical data on the large-scale testing of
microflora could be determined in the absence of the intestinal probiotics therefore warrants urgent investigation.
probiotic. In the larvae exposed to the probiont, the survival The potential benefits of developing probionts are very
of the predominant microflora (other than the probiotic) good. Because of space limitation in many hatcheries better
could be tested against the antagonistic compounds pro- larval survival does not necessarily equate to higher produc-
duced by the probiont. The difference between the antagon- tion. Where increased survival is of benefit to hatcheries the
ism towards the microflora of exposed and control larvae number of broodstock required to produce the required
would provide a partial indication of the success of each number of larvae can be reduced. Broodstock is expensive to
antagonistic compound in reducing the microflora. maintain and obtain, for example in the case of shrimp, and
Competitive exclusion by the probiont through competi- therefore an increase in larval survival means that fewer
tion for attachment sites could be assessed in vivo. To broodstock animals are required for production. Vibrio
provide a means of quantification, the probiotic would need salmonicida increased halibut (Hippoglossus hippoglossus)
to be labelled using immunocytochemical techniques and larval survival by 14.6% (Ottesen & Olafsen, 2000), which
then added to the larval culture system. Once incorporated equates to either an increase in production or an equivalent
in the larval gut, repeated larval gut samples could be reduction in the number of broodstock required to produce
prepared for transmission electron microscopy analysis and the same number of larvae.
the contribution of labelled bacteria (probiotics) attached to The low numbers of commercial intestinal probionts for
the intestinal mucus could be determined. This method aquatic larviculture can (in no particular order) be attrib-
would also provide information regarding the region of uted to (1) the infancy of the field of research, which only
probiont attachment, which may provide further clues as to gained momentum in the late 1990s; (2) the use of Gram-
the activity of potentially beneficial compounds in that negative, nonspore-forming probiotics, which make scaling
region. For example, attachment confined to the stomach up the process difficult; (3) the fact that methods of
area or where the pH is low suggests that enzymes with high delivering the probiotics to the larvae have not been
activity at low pH may dominate. This technique could also optimized; (4) the inconsistent nature of in vivo experiment
be used to investigate the possibility of bacterial transloca- results, particularly on a large scale; (5) that the potential
tion, the migration of bacteria from the intestinal lumen effects of the probiont on fish health during grow-out has

not been examined; and (6) a possible hesitancy from Bergh O, Naas KE & Harboe T (1994) Shift in the intestinal
industry, which until recently has stood to lose more during microflora of Atlantic halibut (Hippoglossus hippoglossus)
the R&D stages. As these points are addressed over the next larvae during first feeding. Can J Fish Aquat Sci 51: 1899–1903.
few years, it seems reasonable to assume that commercially Bielecka M, Biedrzycka E & Majkowska A (2002) Selection of
available larval probiotics will soon enter the aquaculture probiotics and prebiotics for synbiotics and confirmation of
market. their in vivo effectiveness. Food Res Int 35: 125–131.
Blanch AR, Alsina M, Simn M & Jofre J (1997) Determination of
bacteria associated with reared turbot (Scophthalmus
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The Deutscher Akademischer Austauschdienst (DAAD), the bacteria on the growth of the rotifer Brachionus plicatilis in
National Research Foundation and SANCOR (South Afri-

culture. Book of abstracts of World Aquaculture ‘93 (Carrillo M,
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Probiotics in Marine Larviculture

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Probiotics in marine larviculture

Niall G. Vine1, Winston D. Leukes2 & Horst Kaiser1

Correspondence: Niall G. Vine, Department Abstract

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Introduction duced by one protozoan that stimulated the growth of

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FEMS Microbiol Rev 30 (2006) 404–427

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FEMS Microbiol Rev 30 (2006) 404–427

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FEMS Microbiol Rev 30 (2006) 404–427

Production of antimicrobial Growth characteristics

Attachment to Production of other

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Not pathogenic/toxic to host

Addition to rearing water Attachment to livefood

7 Can probiont be re-isolated 6 Pilot-scale in vivo 9 On-going strain

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FEMS Microbiol Rev 30 (2006) 404–427

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Interaction with livefood

FEMS Microbiol Rev 30 (2006) 404–427

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FEMS Microbiol Rev 30 (2006) 404–427

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FEMS Microbiol Rev 30 (2006) 404–427

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FEMS Microbiol Rev 30 (2006) 404–427

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FEMS Microbiol Rev 30 (2006) 404–427

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FEMS Microbiol Rev 30 (2006) 404–427

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FEMS Microbiol Rev 30 (2006) 404–427

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FEMS Microbiol Rev 30 (2006) 404–427

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