Aquaculture nutrition, tập 18, số 5, 2012

Endogenous cellulase activity has been reported in the digestive tract of several fish species indicating that these fish species may be able to utilize cellulose and similar fibrous car

1 2 3,4 1

Fisheries Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan, West Bengal, India; 2 AquacultureLaboratory, Department of Zoology, University of Burdwan, Burdwan, West Bengal, India; 3 Norwegian College ofFishery Science, Faculty of Biosciences, Fisheries and Economics, University of Tromsø, Tromsø, Norway;4 AquacultureProtein Centre (a CoE), Department of Aquatic Medicine and Nutrition, Norwegian School of Veterinary Medicine, Oslo,Norway

Digestion of food depends on three main factors: (i) the

ingested food and the extent to which the food is

suscepti-ble to the effects of digestive enzymes, (ii) the activity of

the digestive enzymes and (iii) the length of time the food

is exposed to the action of the digestive enzymes Each of

these factors is affected by a multitude of secondary

fac-tors The present review highlights the experimental results

on the secondary factor, enzymatic activity and possible

contribution of the fish gut microbiota in nutrition It has

been suggested that fish gut microbiota might have positive

effects to the digestive processes of fish, and these studies

have isolated and identified the enzyme-producing

microbi-ota In addition to Bacillus genera, Enterobacteriaceae and

Acinetobacter, Aeromonas, Flavobacterium, Photobacterium,

Pseudomonas, Vibrio, Microbacterium, Micrococcus,

Staph-ylococcus, unidentified anaerobes and yeast are also

sug-gested to be possible contributors However, in contrast to

endothermic animals, it is difficult to conclude the exact

contribution of the gastrointestinal microbiota because of

the complexity and variable ecology of the digestive tract of

different fish species, the presence of stomach and pyloric

caeca and the relative intestinal length The present review

will critically evaluate the results to establish whether or

not intestinal microbiota do contribute to fish nutrition

KEY WORDS: contribution, digestive tract, enzyme-producing

bacteria, fish, nutrition, review

Received 16 June 2011, accepted 2 January 2012

Correspondence: Arun Kumar Ray, Fisheries Laboratory, Department of

Zoology, Visva-Bharati University, Santiniketan-731 235, West Bengal,

India E-mail: aray51@yahoo.com, arun_ray1@rediffmail.com

Traditionally, digestion is described as the process bywhich food in the gastrointestinal (GI) tract is split intosimpler absorbable compounds performed primarily by thedigestive enzymes However, what happens in the alimen-tary tract is only one part of a continuous process that alsoincludes factors outside the GI tract The traditionalaspects involved in digestion and absorption have beencomprehensively reviewed by several authors (Kapoor et al.1975; Fa¨nge et al 1979; Ash et al 1985; Sheridan 1988;Smith & Halver 1989; Sire & Vernier 1992; Olsen & Ringø1997; Bakke et al 2010) However, these reviews have notfocused on the gut microbiota and their possible influence

on digestibility of nutrients An understanding of the tribution of endosymbionts to digestion requires informa-tion on the relative importance of exogenous (produced bythe GI endosymbionts) and endogenous (produced by thehost) digestive enzymes (Clements et al 1997) Prior to thediscussion of the contribution of the gut microbiota in pro-duction of digestive enzymes, a brief introduction regardingendogenous enzyme activities in fish seems pertinent Theendogenous digestive enzymes, which are secreted to thelumen of the alimentary canal, originate from the oesopha-geal, gastric, pyloric caeca and intestinal mucosa and fromthe pancreas (De Silva & Anderson 1995) The presence ofendogenous digestive enzymes in fish has been reported innumerous studies (e.g Dhage 1968; Kawai & Ikeda 1972;Shcherbina et al 1976; Fagbenro 1990; Das & Tripathi1991; Fagbenro et al 2000) All fish species investigatedper se possess the enzymatic apparatus for hydrolysis andabsorption of simple and complex carbohydrates (Krogdahl

con-et al 2005) Digestive a-amylase has been localizedthroughout the entire GI tract of numerous fish species

(Dhage 1968; Kawai & Ikeda 1972; Chiu & Benitez 1981;

Fagbenro 1990; Sabapathy & Teo 1993; Chakrabarti et al

1995; Kuz’mina 1996; Pe´res et al 1997; Hidalgo et al

1999; de Seixas et al 1999; Fagbenro et al 2000;

Ten-gjaroenkul et al 2000; Alarco´n et al 2001; Fernandez et al

2001) In general, amylase activity in the digestive tract of

omnivorous fish is higher than that of carnivorous fish

(Kitamikado & Tachino 1960; Shimeno et al 1977; Cowey

et al 1989; German et al 2004, 2010), but the activity is

also affected by dietary manipulation (German et al 2004,

2010; Skea et al 2005, 2007) Moreover, it is likely that the

activity differs with the structure of the digestive tract,

developmental stages and ambient temperatures of fish

(Kitamikado & Tachino 1960; Kawai et al 1975; Takeuchi

1991; Cahu & Zambonino Infante 2001; Kamaci et al

2010; Miegel et al 2010) Chitinolytic activity is reported

to be present throughout the GI tract, and high activity is

localized in stomach and pyloric tissue, indicating that

these organs or the diet are the main sources of the

enzymes (e.g Micha et al 1973; Fa¨nge et al 1979; Danulat

& Kausch 1984; Lindsay 1984, 1986; Danulat 1986;

Krog-dahl et al 2005; Ringø et al 2012) Endogenous cellulase

activity has been reported in the digestive tract of several

fish species indicating that these fish species may be able to

utilize cellulose and similar fibrous carbohydrates

(Fag-benro 1990; Das & Tripathi 1991; Szlaminska et al 1991;

Chakrabarti et al 1995; Saha & Ray 1998; Salnur et al

2009) Saha & Ray (1998) observed a diet-dependent

cellu-lase activity both in intestine and hepatopancreas of rohu

(Labeo rohita) fingerlings However, a sharp decline in the

level of cellulase activity was observed in fish fed diets

con-taining the antibiotic tetracycline (active against

Streptococ-cus, Mycoplasma etc.), indicating that cellulase activity in

rohu is contributed largely by the microorganisms present

in the digestive tract The early study of Shcherbina &

Kazlauskiene (1971) proposed that an endogenous cellulase

is secreted into the anterior portion of the digestive tract of

carp (Cyprinus carpio), while the remaining cellulose

absorption takes place in the posterior portion of the

diges-tive tract, indicating the presence of microbial cellulase in

this region Lipase activity has been reported in the gut or

gut contents of most fish species studied, and it seems like

a general rule that most of the intestinal lipase activity if

present is located in the pyloric caeca and the proximal

intestine (Olsen & Ringø 1997) The principal sites for

secretion of endogenous proteases in teleosts are stomach,

pancreas and intestine (De Silva & Anderson 1995) In fish,

adaptive changes in the activity of proteolytic enzymes

have been reported in relation to diet (Kawai & Ikeda

1972; Shcherbina et al 1976; Dabrowski & Glogowski1977; Clements et al 2006; German et al 2010) Althoughintestinal phytase activity has been detected in several fishspecies, it was insufficient for any significant improvement

in phytate hydrolysis in most teleosts (Ellestad et al 2003)

Numerous studies have reported diverse microbial munities in the GI tract of carnivorous, herbivorous andomnivorous fish species (e.g Fishelson et al 1985; Rimmer

com-& Wiebe 1987; Clements et al 1989; Cahill 1990; Sakata com-&

Lesel 1990; Clements 1991; Rahmatullah & Beveridge 1993;

Luczkovich & Stellwag 1993; Ringø et al 1995; Ringø &

Gatesoupe 1998; Ringø & Birkbeck 1999; Bairagi et al

2002a; Ramirez & Dixon 2003; Fidopiastis et al 2006;

Izvekova et al 2007; Sun et al 2009; Li et al 2009; rifield et al 2010a; Nayak 2010a) However, surprisingly,the endosymbiotic community and its role in digestion, ofthe dominant aquatic vertebrate herbivore fish, are poorlyinvestigated In the reviews of Stone (2003), Krogdahl et al

Mer-(2005) and Rowland (2009), the topic is either neglected oronly hinted Cahill (1990), Ringø et al (1995), Austin(2006) and Nayak (2010a) presented some information onstudies of exogenous enzyme activity in fish, but a morecomprehensive review is needed as the GI microbiota offish have been reported to produce a wide range ofenzymes; amylase, cellulase, lipase, proteases, chitinase andphytase (Tables 1–6) Furthermore, the role of enzyme-pro-ducing fish gut bacteria as probiotics in enhancement offood digestibility and their effect on gut enzyme activityhas been evaluated through several investigations (Table 7)

In the present review, we addressed the issue to provide

an overview of the information available on the producing microbiota isolated from the GI tracts of fishtogether with a critical evaluation of the results obtained sofar The results cited include works published in well-known as well as minimally circulated journals This is per-formed to indicate that there are numerous interestinginvestigations published on the topic enzyme-producingmicroorganisms isolated from the digestive tracts of fish

enzyme-The gut microbiota of fish is classified as autochthonous orindigenous when they are able to adhere and colonize thehost’s gut epithelial surface or allochthonous, when theyare incidental visitors in the GI tract and are rejected aftersome time without colonizing (Ringø & Birkbeck 1999;

Ringø et al 2003; Kim et al 2007; Merrifield et al 2011)

However, one study has hinted that the allochthonous robiota might be able to ‘colonize’ the area between the

mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic- mic-.

microvilli under special conditions such as stress, when a

peal of effect of mucus occurs (Olsen et al 2005) Based on

the criteria for testing autochthony of microorganisms

reported in the GI tracts of endothermic animals, Ringø &

Birkbeck (1999) proposed some criteria for testing

indige-nous microorganisms in fish: (i) the microorganisms should

be detected in healthy individuals, (ii) colonize early stages

and persist throughout the life cycles, (iii) demonstrated in

both free-living and hatchery-cultured fish, (iv) able to

grow anaerobically and (v) be detected associated with the

epithelial mucosa in the stomach, proximal or distal

intes-tine In addition, several factors such as (i) gastric acidity,

(ii) bile salts, (iii) peristalsis, (iv) digestive enzymes, (v)

immune response and (vi) indigenous bacteria and the

anti-bacterial compounds that they produce are suggested to

influence adhesion and colonization of the microbiota

within the digestive tract (Ringø et al 2003)

The historical data stem from culturing methods of the

fish digestive tracts reported that aerobes or facultative

anaerobes are dominant in the digestive tract of fish (e.g.Trust & Sparrow 1974; Cahill 1990; Sakata & Lesel 1990;Ringø et al 1995; Ringø & Birkbeck 1999; Bairagi et al.2002a; Saha et al 2006) However, these results are based

on culture methods and mainly evaluated aerobes and ultative anaerobes with a subsequent underestimation ofthe obligate anaerobic microbiota and the un-culturablemicrobiota This is clearly demonstrated in numerousrecent publications evaluating the fish gut microbiota byusing molecular methods (e.g Moran et al 2005; Pond

fac-et al 2006; Clements et al 2007; Hovda et al 2007; Liu

et al 2008; Navarrete et al 2009; Ferguson et al 2010;

He et al 2010; Zhou et al.2011) In addition, severalauthors have suggested that electron microscopic (EM)examinations of the GI tract should be included as animportant tool for investigating the microbial ecology ofthe gut ecosystem and determining the presence of autoch-thonous or allochthonous microbiota (e.g Fishelson et al.1985; Clements 1991; Andlid et al 1995; Ringø et al 2003;

Table 1 Amylase-producing bacteria isolated from the digestive tract of fish

Japanese eel and tilapia

Sugita et al (1997)

Aeromonas spp.; Enterobacteriaceae;

Pseudomonas spp.; Flavobacterium spp.

Citrobacter sp.; Enterobacter sp.; Bacillus coagulans

and an uncultured bacterium clone isolated from

the PI of Catla catla Bacillus cereus isolated from

the DI of C catla Bacillus sp isolated from the PI

of Cirrhinus mrigala Bacillus cereus, Citrobacter

freundii and an uncultured bacterium clone isolated

from the DI of C mrigala Bacillus sp isolated from

the DI of Labeo rohita

Bacillus thuringiensis, B cereus, Bacillus sp isolated

from the GI tract of Salmo salar fed control

diet Bacillus subtilis and Acinetobacter sp Isolated

from the GI tract of S salar fed 5% chitin

supplemented diet

Brochothrix sp and Brochothrix thermosphacta isolated

from the GI tract of Atlantic cod fed fish meal,

soybean meal and bioprocessed soybean meal

.

Aquaculture Nutrition 18; 465–492 ª 2012 Blackwell Publishing Ltd

Fidopiastis et al 2006; German 2009; Ghosh et al 2010;

Merrifield et al 2010b, 2011; Harper et al 2011) However,

to the author’s knowledge, only one recent study has used

EM examination related to the gut enzyme-producing

mic-robiota of fish (Ghosh et al 2010) Scanning electron

microscopy (SEM) evaluation revealed that bacteria present

in the GI tract of rohu were rod shaped, probably bacilli,

attached to the intestinal fold associated with mucous As

this topic is underestimated, we recommend that the topic

merits further investigations

It has only been during the last decade that there has been

an improved understanding of the importance of

commen-sal intestinal microbiota in fish intestine Nevertheless, the

first studies on enzyme production by the fish gut bacteria,

to the author’s knowledge, were reported in 1979 (Hamid

et al 1979 and Trust et al.1979) Since then, numerous

studies have been carried out, and an overview of thesestudies is presented in Tables 1–6

Microbial amylase activity in the fish gut has been mented in several studies (Table 1) To the authors’ knowl-edge, occurrence of amylolytic bacteria (strict anaerobesand Aeromonas hydrophila) in the gut of grass carp (Cteno-pharyngodon idella) was first reported by Trust et al

docu-(1979) Later, Lesel et al (1986) demonstrated amylolyticbacteria in the digestive tract of grass carp, but the bacteriawere not characterized and identified In their study ongrass carp, Das & Tripathi (1991) suggested the presence ofamylase-producing bacteria, but no specific informationwas given Gatesoupe et al (1997) reported amylase-pro-ducing Vibrio spp isolated from sea bass (Dicentrarchuslabrax) larvae, but the activity of the gut bacteria wasaffected by diet formulation Sugita et al (1997) detected

Table 2 Cellulase-producing bacteria isolated from the digestive tract of fish

Citrobacter sp.; Enterobacter sp.; Bacillus coagulans and

an uncultured bacterium clone isolated from the PI

of Catla catla Bacillus cereus isolated from the DI of

C catla Bacillus sp isolated from the PI of Cirrhinus

mrigala Bacillus cereus, Citrobacter freundii and an

uncultured bacterium clone isolated from the DI of

C mrigala Bacillus sp isolated from the DI of Labeo rohita

Bacillus thuringiensis, B cereus, Bacillus sp isolated from the

GI tract of Salmo salar fed control diet Bacillus subtilis

and Acinetobacter sp Isolated from the GI tract of

S salar fed 5% chitin supplemented diet.

Brochothrix sp and Brochothrix thermosphacta isolated from

the GI tract of Atlantic cod fed fish meal, soybean meal

and bioprocessed soybean meal.

.

amylase production by the intestinal microbiota in cultured

ayu (Plecoglossus altivelis), common carp (C carpio),

chan-nel catfish (Ictalurus punctatus), Japanese eel (Anguilla

japonica) and tilapia (Oreochromis niloticus) Of the 206

isolates examined, 65 (31.6%) produced  0·01 U

amy-lase mL 1, and they were identified as Aeromonas spp.,

Bacterioidaceae and Clostridium spp In a more recent

study, enumerating the specific enzyme-producing bacterial

community in the gut of nine species of adult freshwater

teleosts, Bairagi et al (2002a) observed higher densities of

amylolytic strains in herbivorous grass carp, common carp

and tilapia (Oreochromis mossambica), but these bacteria

were not characterized and identified Furthermore, the

authors could not detect amylolytic bacterial strains in the

GI tract of carnivorous catfish (Clarias batrachus) and

murrel (Channa punctatus) Amylolytic bacteria (Bacillus

circulans, Bacillus pumilus and Bacillus cereus) have been

documented in the gut of rohu (Ghosh et al 2002)

indicat-ing its possible link with feedindicat-ing habit

Skrodenyte-Arbacˇiauskiene (2007) examined in vitro amylolytic ties of bacteria isolated from the intestinal tract of adultroach (Rutilus rutilus) that feed mainly on mollusks andmacrophytes Of total 60 bacterial strains isolated from theintestinal contents, amylolytically active isolates comprised50%, 65% and 55% of all bacteria isolated from the fore-gut, midgut and hindgut, respectively Of the 34 bacteriaisolated displaying in vitro amylolytic activity, 29 isolatesbelonged to Aeromonas spp However, amylolytic activitywas only detected in bacteria belonging to Enterobacteria-ceae, Pseudomonas and Flavobacterium isolated from theforegut Kar & Ghosh (2008) reported amylase-producingbacteria in the digestive tracts of rohu and murrel, but noinformation was given about their identification Proteaseand cellulase activities were exhibited by all bacterialstrains isolated from rohu and murrel, but amylase produc-tion was poorly detected in strains isolated from murrel.Mondal et al (2008) documented higher densities of amy-lolytic strains in the foregut region of two carps species

activi-Table 3 Protease-producing bacteria isolated from the digestive tract of fish

Enterobacter spp.; Vibrio spp.; Pseudomonas spp.;

Acinetobacter spp.; Aeromonas spp.

freshwater teleosts

Bairagi et al (2002a)

Aeromonas spp.; Enterobacteriaceae; Pseudomonas spp.;

Flavobacterium spp.; Micrococcus sp.

Citrobacter sp.; Enterobacter sp.; Bacillus coagulans and an uncultured

bacterium clone isolated from the PI of Catla catla Bacillus cereus

isolated from the DI of C catla Bacillus sp isolated from the PI of

Cirrhinus mrigala Bacillus cereus, Citrobacter freundii and an

uncultured bacterium clone isolated from the DI of C mrigala.

Bacillus sp isolated from the DI of Labeo rohita

Three species of Indian major carps

Ray et al (2010)

Bacillus thuringiensis, Bacillus cereus, Bacillus sp isolated from the

GI tract of Salmo salar fed control diet Bacillus subtilis and

Acinetobacter sp Isolated from the GI tract of S salar fed 5%

chitin supplemented diet.

Brochothrix sp and Brochothrix thermosphacta isolated from the

GI tract of Atlantic cod fed fish meal, soybean meal and

bioprocessed soybean meal.

.

Aquaculture Nutrition 18; 465–492 ª 2012 Blackwell Publishing Ltd

(Labeo calbasu and Labeo bata) [12.2 9 103

colony-formingunits (CFU) g 1 gut tissue and 11.59 103

CFU g 1 guttissue, respectively] in comparison with the hindgut region

In a more recent study, Mondal et al (2010) isolated

amy-lase-producing Bacillus licheniformis and Bacillus subtilis

from the digestive tract of bata (L bata) Ray et al (2010)

detected a huge population of amylase-producing bacteria

in the GI tract of three Indian major carps, catla (Catla

catla), mrigal (Cirrhinus mrigala) and rohu (L rohita),

where amylase production was considerably higher by the

strains isolated from the proximal intestine of catla and

mrigal, except the strain CF4, isolated from the proximal

intestine of catla A description of the identified bacteria inthe study of Ray et al (2010) is given in Table 1

Cellulose consists of a b-1,4-glycosidic linkages and is mated as the most abundant biomass (1015 metric tons;

esti-Wilson et al 1999) in the world Complete cellulose lysis to glucose demands the action of exoglucanases (alsocalled cellobiohydrolyses), endoglucanases and b-glucosid-ases Exoglucanases (1,4-b-D-glucan cellobio-hydrolase, EC3.2.1.91) are usually active on crystalline cellulose and are

hydro-Table 4 Lipase-producing bacteria isolated from the digestive tract of fish

Agrobacterium; Pseudomonas; Brevibacterium;

Microbacterium; Staphylococcus

Vibrio spp., Acinetobacter spp Enterobacteriaceae,

Pseodomonas spp.

Bacillus thuringiensis, Bacillus cereus, Bacillus sp isolated from

the GI tract of Salmo salar fed control diet Bacillus subtilis

and Acinetobacter sp Isolated from the GI tract of S salar

fed 5% chitin supplemented diet.

Brochothrix sp and Brochothrix thermosphacta isolated from the

GI tract of Atlantic cod fed fish meal, soybean meal and

bioprocessed soybean meal.

N.i* – indicates the presence of microbial lipase; N.i – no information was given; Rainbow trout – Oncorhynchus mykiss; Arctic charr –

Table 5 Phytase- and tannase-producing microorganisms isolated from the digestive tract of fish

identified strains

fish species

Li et al (2008a,b) Bacillus thuringiensis, Bacillus cereus, Bacillus sp isolated

from the GI tract of Salmo salar fed control diet Bacillus

subtilis and Acinetobacter sp Isolated from the GI tract of

S salar fed 5% chitin supplemented diet.

Brochothrix sp and Brochothrix thermosphacta isolated from the

GI tract of Atlantic cod fed fish meal, soybean meal and

bioprocessed soybean meal.

.

lacking from incomplete cellulose systems Endogluconases

(1,4-b-D-glucan-4-glucanohydrolase, EC 3.2.1.4) are more

active against the amorphous regions of cellulose, and they

can also hydrolyze substituted celluloses, such as

carboxy-methylcellulose (CMC) and hydroxyethyl-cellulose (HEC)

Cellobiohydrolases cleave disaccharide (cellobiose) units

either from non-reducing or reducing ends, whereas

endo-glucanases hydrolyze the cellulose chain internally

b-gluco-sidases (EC 3.2.1.21) are needed to cleave cellobiose and

other soluble oligosaccharides to glucose (Be´guin 1990)

Cellulose is completely hydrolyzed to its constituent

oligo-mers by the cellulase (endogluconase,1,4-b-

D-glucan-4-glu-canohydrolase, EC 3.2.1.4) Thus, many cellulose-eating

animals require the aid of symbiotic microorganisms in

their GI tract to digest cellulose and make the energy in

this compound available to the host (Bergman 1990; Mo

et al.2004; Karasov & Martinez del Rio 2007) Reports on

the existence of cellulase activity in the digestive system of

fish are rare with contradictory result In early studies on

fish, Fish (1951), Barrington & Brown (1957) and Yokoi &

Yasumasu (1964) believed that fish do not posses

endoge-nous cellulase However, cellulase activity has been

reported in several fish species, indicating that fish may be

able to utilize cellulose and similar fibrous carbohydrates

(Chakrabarti et al 1995)

To the author’s knowledge, the first study indicating the

presence of microbial cellulase in the GI tract of fish was

reported in the distal intestine of common carp by bina & Kazlauskiene (1971) Later, Stickney & Shumway(1974) investigated cellulase activity in the stomachs of 62species of elasmobranches and teleost fish Of the 62 speciesstudied, 17 showed cellulase activity One species of fresh-water catfish (channel catfish, I punctatus) demonstratedcellulase activity Channel catfish exposed to streptomycin(Gram-positive bacteria are more susceptible than Gram-negatives) for 24 h showed no cellulase activity whilecontrol fish, not exposed to the antibiotic, continued todemonstrate cellulase activity Based on their results, theauthors hinted that the cellulase activity, at least in

Shcher-I punctatus, was derived from alimentary tract microbiotarather than from cellulase secreting cells within the fish.Stickney (1975) evaluated cellulase activity in a number offreshwater species and concluded that herbivores are unli-kely to have the enzyme, while omnivores and carnivoresmight pick up cellulolytic bacteria from the invertebratesthat harbour the bacteria, which might explain the presence

of the cellulolytic bacteria within the GI tract of carnivorefishes Lindsay & Harris (1980) displayed cellulase activity

in the digestive tract of 138 fish representing 42 species andsuggested that the source of cellulase activity originatesfrom the microbial population, although the authors dis-carded the hypothesis of a stable cellulolytic microbiota infish In a study on catfish (Clarias isheriensis) fed anomnivorous diet, mainly the pond plankton Cyanophycea,

Table 6 Chitinase-producing bacteria isolated from the digestive tract of fish

Acinetobacter sp., Enterobacteriaceae, Flavobacterium sp., Photobacterium

spp., Vibrio spp and a unidentified Gram-negative rod

gray mullet

Sugita et al (1999) Marinobacter lutaoensis, Ferrimonas balearica, Pseudoalteromonas piscicida,

Enterovibrio norvegicus, Grimontia hollisae, Photobacterium damselae spp.

damselae, P leiognathi, P lipolyticum, P phosphoreum, P rosenbergii,

Vibrio campbelli, V chagasii, V fischeri, V fortis, V gallicus, V harveyi,

V natrigenes, V nigripulchritudo, V ordalii, V parahaemolyticus,

V pomeroyi, V ponticus, V proteolyticus, V rumoiensis, V shilonii, V.

tasmaniensis and V tubiashii

Various Japanese costal fishes

Itoi et al (2006)

V iscthyoenteri group type 3

Bacillus thuringiensis, Bacillus cereus, Bacillus sp isolated from the GI tract

of Salmo salar fed control diet Bacillus subtilis and Acinetobacter sp.

Isolated from the GI tract of S salar fed 5% chitin supplemented diet.

Brochothrix sp and Brochothrix thermosphacta isolated from the GI tract

of Atlantic cod fed fish meal, soybean meal and bioprocessed soybean meal.

.

Aquaculture Nutrition 18; 465–492 ª 2012 Blackwell Publishing Ltd

high cellulase activities were detected in both the stomach

and in the proximal and distal parts of the mid intestine

(Fagbenro 1990)

Several studies have reported cellulase-producing bacteria

isolated from the GI tract of fish (Table 2) Lesel et al

(1986) reported cellulolytic gut bacteria in grass carp, but

the bacteria were not characterized and identified In a

study on digestive enzymes in grass carp, cellulase activity

was reported both in hepatopancreas and intestine, and

dietary cellulose level significantly affected the cellulase

activity (Das & Tripathi 1991) The fact that cellulase

activity was reduced to approximately one-third when

tetracycline (effective against Vibrio, Mycoplasma and

Streptococcus) was supplemented to the diet indicates that

the gut microbiota may contribute to the cellulolytic

activ-ity in the intestinal tract of grass carp In a study

evaluat-ing cellulase activity in rohu, Saha & Ray (1998) reported

cellulase-producing bacteria, but they were not

character-ized and identified Abundance of cellulolytic bacteria has

further been documented in the GI tract of grass carp

(Bairagi et al 2002a; Saha et al 2006; Li et al 2009),

common carp and silver carp (Hypophthalmichthys

moli-trix) (Bairagi et al 2002a), rohu (Saha & Ray 1998; Ghosh

et al 2002; Kar & Ghosh 2008; Ray et al 2010), catla and

mrigal (Ray et al 2010), bata (Mondal et al.2008, 2010),

tilapia (Saha et al 2006), murrel (Kar & Ghosh 2008) and

wood-eating catfishes of genus Panaque (Nelson et al

1999) Bairagi et al (2002a), however, failed to isolate

cel-lulolytic bacteria in the GI tract of carnivorous catfish and

murrels In contrast to these results, Kar & Ghosh (2008)

reported the presence of cellulolytic bacteria in murrel

Nel-son et al (1999) isolated several aerobic bacteria from the

guts of wood-eating catfishes that showed the ability to

grow on cellulose and to produce cellulases Nelson and

colleagues also measured cellulases in the fish guts Based

on their results, they concluded that wood-eating catfishes

digested cellulose in their guts with the aid of aerobic

endo-symbiotic microbes Mondal et al (2008) evaluated

enzyme-producing bacteria in the foregut and hindgut

regions of seven freshwater teleosts and quantitatively

assayed the cellulase activity However, the authors did not

identify the isolated strains Ray et al (2010) isolated and

enumerated cellulase-producing autochthonous bacteria in

the proximal and distal intestine of three species of Indian

major carps and identified the most promising strains by

16S rRNA gene sequence analysis Recently, Jiang et al

(2011) investigated the bacterial community in the gut of

grass carp using genomic DNA-based 16S rRNA gene

library The analysis revealed 28 different bacteria species

belonging to seven genera; Vibrio, Acinetobacter, cia, Yersinia, Pseudomonas, Morganella and Aeromonas,respectively All cellulase-producing bacteria isolated fromthe intestine of grass carp belonged to Aeromonas Peixoto

Providen-et al.(2011) evaluated the cellulolytic potential of B

subtil-is P6 and Bacillus velesensis P11 originally isolated fromthe midgut of the South American warm water teleosts,pacu (Piaractus esoiptamicus) and piaucom-pinata (Lepori-nus friderici), respectively The authors reported bacterialgrowth and cellulase production (mainly endoglucanases),and the highest residual cellulase activity was reported at

pH values between 7.0 and 9.0

Luczkovich & Stellwag (1993) and Stellwag et al (1995)reported carboxymethylcellulase (CMCase)-producingmicrobes from the intestinal tract of the omnivorous pin-fish (Lagodon rhomboids) Stellwag et al (1995) isolated atotal of 550 anaerobic bacterial strains, 200 from environ-mental samples and 350 isolates from the intestinal tractcontents of seven different pinfish and screened them forCMCase activity The 200 environmental strains revealed

no detectable CMCase activity, whereas 36 of the 350(10.3%) obligate anaerobes recovered from the intestinaltract contents of seagrass-consuming pinfish expressedCMCase activity To understand the taxonomic relation-ships among CMCase-producing strains, the authors con-ducted morphological, physiological and biochemicalcharacterization of 36 strains but did not identify them

In a study on common carp, Kihara & Sakata (2002)showed that intestinal bacteria isolated from the fish wasable to metabolize oligosaccharides commonly found in soyand other beans with the liberation of short-chain fattyacids, carbon dioxide and methane gas Diaz & Espana(2002) reported that the hindgut chamber of the kingangelfish (Holacanthus passer) contained a high populationlevel of microorganisms able to hydrolyze complex carbo-hydrates The hindgut of this fish species is highly vascular-ized indicating absorption in this gut segment Populations

of symbiotic organisms in the gut of most terrestrial brate herbivores play a key role in digestion by breakingdown plant cell walls (cellulose and hemicelluloses) to sim-ple compounds such as short-chain fatty acids (SCFAs)that are taken by the host and are used for energy genera-tion and biosynthesis (Stevens & Hume 1995; Seeto et al.1996) The SCFAs produced are rapidly absorbed from thegut lumen The major SCFA is normally acetate withminor amounts of propionate and butyrate (Stevens &Hume 1995) Acetate produced by microbial fermentationconstitutes an important source of energy to the host(Mountfort et al 2002) Besides their contribution to

verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte- verte-.

Aquaculture Nutrition 18; 465–492 ª 2012 Blackwell Publishing Ltd

energy metabolism, SCFAs perform various physiological

functions SCFAs stimulate cell proliferation in the

intesti-nal epithelium in vivo, while in in vitro, they inhibit cell

proliferation, but they are potent enhancers of gene

expres-sion in cultured cells (Von Engelhardt et al 1989)

Propio-nate is converted to glucose in the liver and may modify

hepatic metabolism Butyrate is the preferred fuel for the

colony epithelial cells (Roediger 1980; Von Engelhardt

et al 1989) It has also been shown that butyrate protects

these cells against agents that lead to cellular differentiation

and may even inhibit tumour growth (Young et al 1994)

Many marine herbivorous fishes contain SCFA

(predomi-nantly acetate) in their hindgut, which indicate microbial

activity (Clements et al 1994; Clements & Choat 1995;

Mountfort et al 2002) Such information is essential to

understand the contribution of gut microorganisms to

con-tribute to the energy needs of the fish Mountfort et al

(2002) estimated the rates of acetate production in the gut

of three species of temperate marine herbivorous fish from

north-eastern New Zealand, viz., Kyphosus sydneyanus,

Odax pullus and Aplodactylus arctidens The rates of

turn-over of acetate were in the same order of magnitude as

those values detected in the intestinal tracts of herbivorous

reptiles and mammals, even though the ectothermic fishes

were held at much lower temperatures (17–23 °C)

How-ever, this result does not support the previous hypothesis

that high temperatures are a prerequisite for efficient

fer-mentation systems to operate in marine herbivores (Kandel

et al 1995) The importance of SCFAs to overall energy

supply and metabolism has not yet been quantified for any

of these herbivores, but it may be substantial (Mountfort

et al.2002) Titus & Ahearn (1988, 1991) reported the

con-centration of SCFAs along the gut of tilapia, O

mossambi-cus, and characterized a specific transport system for

acetate However, in this study, the authors did not

deter-mine the role of SCFA metabolism in the investigated

spe-cies Algae consumed by marine fishes contain much more

complex and different carbohydrates than vascular plants

with mainly cellulose and hemicellulose-based structural

components (Clements et al 2009) In addition to different

sets of secondary metabolites, digestion is achieved in a

dif-fering ionic environment Neither has attracted much

atten-tion by researchers

Cellulase yields appear to depend on a complex

relation-ship involving a variety of factors, like inoculums size

(car-bon source and cellulose quality), pH, temperature,

presence of inducers, medium additives, aeration and

growth time (Immanuel et al 2006) Ray et al (2007)

investigated the optimum environmental and nutritional

conditions required to enhance cellulase production by

B subtilis CY5 and B circulans TP3, originally isolatedfrom the gut of common carp and Mozambique tilapia(O mossambicus), respectively The authors concluded thatsolid-state fermentation was suitable for increased cellulaseproduction by the bacterial strains The strains could read-ily utilize the substrate at 40°C in in vitro culture at pH7.5, and organic nitrogen sources were reported to be moresuitable for optimum cellulase production

Proteases are hydrolytic enzymes that catalyse the totalhydrolysis of proteins in to amino acids Although prote-ases are widespread in nature, microbes serve as a preferredsource of these enzymes because of their rapid growth, thelimited space required for their cultivation and the easewith which they can be genetically manipulated to generatenew enzymes with altered properties that are desirable fortheir various applications (Chu 2007) Bacteria belonging

to Bacillus sp are by far the most important source of eral commercial microbial enzymes (Ferrero et al 1996;

sev-Kumar et al 1999; Sookkheo et al 2000; Singh et al

2001; Gupta et al 2002; Beg & Gupta 2003; Shafee et al

2005; Chu 2007; da Silva et al 2007) Some information isavailable regarding production of proteases by fish gut bac-teria (Table 3)

To our knowledge, the first studies on ing bacteria isolated from the digestive tract of fish, graymullet and grass carp were carried out by Hamid et al

protease-produc-(1979) and Trust et al protease-produc-(1979), respectively Gatesoupe

et al.(1997) displayed that protease activity of gut bacteriaisolated from sea bass larvae was affected by diet formula-tion In this study, all bacteria (Vibrio spp.) isolated fromlarvae fed the compound diet showed amylase activity,while larvae fed Artemia only, 40% of the gut bacteria dis-played protease activity This finding could be related tothe fact that gut microbiota was more diverse when the lar-vae were fed Artemia In a study isolating bacteria fromintestinal contents of Arabesque greenling (Pleurogrammusazonus), one of the isolates showed strong proteolytic activ-ity (Hoshino et al 1997) The isolate was identified togenus Pseudomonas and displayed highest protease produc-tion at 10°C, but the activity decreased with increasingcultivation temperature Morita et al (1998) detected pro-tease activity in the culture medium of Flavobacterium bal-ustinum isolated from salmon (Oncorhynchus keta)intestine The molecular mass of the protease was 70 kDa,and its isoelectric point was close to 3.5, and maximal

.

activity towards azocasein was at 40°C and from pH 7 to

9 Skrodenyte-Arbacˇiauskiene (2000) determined the role of

the symbiotic gut microbiota of roach (R rutilus) in

pro-tein decomposition, as well as the dependence of

proteo-lytic enzyme activity upon environmental pollution in two

differently polluted lakes This research revealed that the

enzyme activity of the fish intestinal microbiota is

depen-dent upon the ecological state of environment Ghosh et al

(2002) suggested from their in vitro studies on

enzyme-pro-ducing microbiota that B circulans Lr 1.1, B pumilus Lr

1.2 and B cereus Lr 2.2, originally isolated from the

ali-mentary tract of rohu fingerlings, were ‘good’ producers of

proteolytic enzymes, although the enzyme activity was not

quantified Bairagi et al (2002a) quantified the proteolytic

activity in bacterial strains isolated from nine freshwater

teleosts and reported highest activity in bacterial strain

TP3A isolated from the gut of omnivorous tilapia

How-ever, the authors did not identify the promising strains

Bel-chior & Vacca (2006) isolated a psychrotrophic bacterium,

identified as Pseudoalteromonas sp., from the intestinal tract

of hake (Meluccius hubbsi) and reported that the protease

activity at 7°C was lower than at 22 °C Kar & Ghosh

(2008) isolated and enumerated heterotrophic bacteria from

the GI tracts of rohu and murrel to evaluate the importance

of the GI microbiota in fish nutrition Their study revealed

a distinct correlation between the enzyme-producing

bacte-ria and feeding habit of the host fish Maximum population

level of proteolytic bacteria was detected in the carnivorous

C punctatus compared with the herbivorous L rohita

Mondal et al (2008) reported highest proteolytic activity in

strain CH22, isolated from the hindgut region of

detritivo-rous carp (L calbasu) while Ray et al (2010) documented

highest proteolytic activity by strains isolated from the

dis-tal intestine of all the three species of Indian major carps

studied Esakkiraj et al (2009) reported extracellular

prote-ase production by B cereus isolated from the intestine of

brackish water fish (Mugil cephalus) in shake-flask

experi-ment using different preparations of tuna-processing waste

such as raw fish meat, defatted fish meat, alkali hydrolyzate

and acid hydrolyzate as nitrogen source The authors

fur-ther tested the effect of temperature, pH, different carbon

sources and surfactants on protease production by the

bac-terial strain Among the tuna preparations tested, defatted

fish meat supported the maximum protease production, and

3% concentration of the same was reported to be optimum

for maximizing the protease production Among the carbon

sources, galactose aided higher protease production than

the other tested carbon sources, and a concentration of

1.5% galactose was optimum to enhance the protease

production The halotolerancy of B cereus for protease duction indicated that 3% of sodium chloride was optimum

pro-to yield maximum protease Among the surfactants tested,protease production was highest when Triton X100 wasadded to the medium compared with other surfactants, andoptimum protease production was recorded when 0.8%Triton X100 was added

Mondal et al (2010) analysed gut microbiota in bataand revealed that amylolytic strains were present in higherpopulation levels in the foregut region, whereas the cellulo-lytic and proteolytic populations exhibited maximum densi-ties in the hindgut region Maximum amylase, cellulase andprotease activities were exhibited by bacterial strainsbelonging to B licheniformis BF2 and B subtilis BH4, iso-lated from the foregut and hindgut, respectively Subse-quently, Mondal et al (unpublished data) conducted anexperiment to determine the optimum culture conditionsfor extracellular protease production by these two bacterialstrains Based on their experimental findings, the authorsconcluded that pH, temperature and nitrogen sources playthe most crucial role in protease production by B licheni-formisBF2 and B subtilis BH4 The protease produced byboth strains is thermophilic and the production was opti-mized under solid-state fermentation conditions Further-more, beef extract was reported to be more suitable foroptimum protease production among the organic nitrogensources than inorganic sources

Many bacterial lipases are extracellular enzymes and areclassified into three types according to their specificity: (i)non-specific lipases, (ii) 1,3-specific lipases and (iii) fattyacid-specific lipases (Macrane et al 1984) The non-specificand 1,3-specific lipases catalyse the hydrolysis of triglycer-ide (TAG) to free fatty acids and glycerols, while fattyacid-specific lipases catalyse the removal of a specific fattyacid from the TAG molecule, preferentially removing cis–

D9 – monounsaturated fatty acids The production of terial lipases is influenced by temperature, ratio of nitrogen

bac-to carbon, inorganic salts and oxygen In general, bacteriallipase synthesis is stimulated by lard, butter, olive oil andfatty acids (Finnerty et al 1989) The gut microbiota cantheoretically act on lipolysis in two different ways: (i) bycontribution to TAG breakdown through bacterial actionand (ii) by changing pancreatic lipase secretion or inactivat-ing it by bacterial proteases (Ringø et al 1995)

The microbial breakdown of dietary lipids to free fattyacids may improve the absorption efficiency of lipids in the

.

Aquaculture Nutrition 18; 465–492 ª 2012 Blackwell Publishing Ltd

GI tract To detect lipolytic activity, initially, a variety of

methods were investigated using cultures on solid agar

plates containing TAG and PC substrates; (i) indication of

lipolytic activity was primarily detected based on the

method of Hamid et al (1979) This method involves the

assessment of enzyme activities of intestinal bacteria, on

agar plates, on the basis of liquification of starting

sub-strate (ii) The addition of calcium chloride (0.001% w/v)

to the basic medium– precipitation of free fatty acids with

calcium – gives a ‘white or opaque’ zone around colonies

containing lipolytic bacteria (Sigurgisladottir et al 1993)

(iii) The addition of Nile blue indicator (1.0%) to the basic

medium indicates the presence of lipolytic bacteria by a

blue ring around colonies (Mckenzie 1994) Lipase activity

can also be assayed using 1% Tween 80 or 0.75%

tributy-rin (Jensen 1983; Ando et al.1991)

Trust et al (1979) reported lipolytic activity in bacterial

isolates (strict anaerobes and A hydrophila) isolated from

the GI tract of grass carp Later, Mckenzie (1994)

demon-strated the production of extracellular lipolytic enzymes by

mixed cultures of bacteria originally isolated from the

digestive tract of turbot (Scophthalmus maximus), rainbow

trout (Oncorhynchus mykiss) and piranha (Serrasalmus

nat-tereri) Ringø et al (1995) reported that some bacterial

stains isolated from the proximal and distal intestine of

Arctic charr (Salvelinus alpinus L.) were able to degrade

0.75% tributyrin as substrate The authors suggested that

the isolated gut bacteria (Agrobacterium, Pseudomonas,

Brevibacterium, Microbacterium and Staphylococcus) might

contribute to nutritional processes in Arctic charr

Gate-soupe et al (1997) reported that the majority of the isolates

with lipase activity isolated from sea bass larvae belonged

to Vibrio spp., but 25% of the gut isolates showing lipase

activity were classified as Acinetobacter, Enterobacteriaceae

and Pseudomonas Furthermore, dietary formulation seems

to affect the bacterial lipase activity, but in contrast to the

finding that highest amylase and protease activities were

reported by gut bacteria isolated from larvae fed the

com-pound diet, the proportion of lipase active bacteria was

highest when larvae were fed Artemia

Ringø & Birkbeck (1999) put forward the hypotheses

that gut bacterial phospholipase may be beneficial for

lar-val growth and survilar-val However, to the authors’

knowl-edge, less information is available regarding bacterial

phospholipase in relation to fish nutrition (Gatesoupe et al

1997; Henderson & Millar 1998) In the study of

Gate-soupe and co-authors, extracellular phospholipase activity

was reported in approximately 95% of the strains isolated

from the gut of 20-day-old sea bass larvae fed compound

diets in contrast to only 40% of the gut microbiota of emiafed fish Henderson & Millar (1998) suggested that aVibrio sp originally isolated from the GI tract of Arcticcharr, later suggested to belong to Shewanella baltica based

Art-on 16S rRNA gene sequence analysis (B Landfald, persArt-onalcommunication), produce a phospholipase B capable ofhydrolyzing both intact phospholipids and intact lysophos-pholipids However, based on the scattered informationavailable, we cannot conclude that gut bacteria make a sig-nificant contribution to the overall phospholipase activity infish gut Bairagi et al (2002a) detected lipolytic bacteria inthe gut of nine freshwater teleosts, and maximum popula-tion density (5.09 103

bacterial cells g 1) was reported insilver carp (H molitrix) Unfortunately, the authors did notidentify the lipase-producing strains Readers with specialinterest on microbial lipase production are referred to therecent comprehensive review of Treichel et al (2010)

Phytate forms compounds with a large number of minerals(K, Mg, Ca, Zn, Fe and Cu) and also forms complexeswith proteins and amino acids, thereby reduces bioavail-ability of minerals and decrease digestibility of proteins inmost animals because of lack of intestinal phytase (Pointill-art et al 1987) Phytases have a wide distribution in plants,microorganisms and in some animal tissues (Vohra & Sat-yanarayana 2003) Endogenous phytase activity has beenreported in hybrid tilapia (O niloticus9 O aureus)(LaVorgna 1998) and striped bass (Morone chrysops9Morone sexatilis) (Ellestad et al 2003) Industrial produc-tion of phytase currently utilizes the soil fungus, Aspergil-lus, on which considerable research has been conducted(Ullah et al 1999) However, because of some properties,such as substrate specificity, resistance to proteolysis andcatalytic efficiency, bacterial phytases may be a real alter-native to the fungal enzyme (Konietzny & Greiner 2004)

To our knowledge, few reports have considered phytaseactivity by gut bacteria from freshwater teleosts Roy et al

(2009) identified two phytase-producing strains, LF1 andLH1 isolated from L rohita as B licheniformis Khan &

Ghosh (2011) evaluated phytase-producing bacteria in 14freshwater teleosts, and two promising strains isolated from

L bata and Gudusia chapra were identified as B subtilisand Bacillus atrophaeus, respectively Khan et al (2011)isolated an efficient phytase-producing strain CC 1.1 from

C catla and identified it as Rhodococcus sp MTCC 9508

on the basis of phenotypic characterization Apart fromthese limited information in freshwater teleosts, Li et al

.

(2008a,b) documented that several marine yeast strains

iso-lated from the gut of sea cucumber (Holothuria scabra) and

marine fish (Hexagrammos otakii and Synecogobius hasts)

had the ability to produce large amount of extra-cellular

phytase and opined that such marine yeasts might play

important role in degradation of phytate within the guts of

marine animals

Tannin is a substance found in many different plants

Tan-nin is notably found naturally occurring in grapes, tea

leaves and oak (Liener 1980; Francis et al 2001; Gatlin

et al.2007; Krogdahl et al 2010; Ghosh & Ray 2011) The

word tannin comes from the historical practice of using

the tannin found in oak bark to tan leather, although in

the modern world, synthetic is usually used for this

pur-pose instead It is well known that tannins are toxic and

bacteriostatic compounds (Scalbert 1991) However,

tann-ase (tannin acyl hydroltann-ase, EC 3.1.1.20) is produced by a

group of tolerant microorganisms, such as fungi, yeast and

bacteria (Lekha & Lonsane 1997) Lewis & Starkey (1969)

reported degradation of hydrolyzable tannin by an aerobic

bacterium, Achromobacter sp., while Deschamps et al (1980)

isolated 15 bacterial strains belonging to the genera Bacillus,

Staphylococcusand Klebsiella able to degrade tannins

Supplementary feeds may contain tannin-like compounds

as there is a thrust to substitute fish meal in aquafeed with

the less expensive and protein-rich plant ingredients for

economic fish production in most of the developing

coun-tries (Mukhopadhyay & Ray 1996; Becker & Makkar

1999) Consequently, a relevant question is; do fish also

contain tannase-producing autochthonous microbiota in

their gut? However, less information of tannase-producing

bacteria isolated from fish gut is available because of lack

of studies carried out on this topic To the authors’

knowl-edge, only one study has documented findings on this issue

Mandal & Ghosh (2010) reported existence of

autochtho-nous tannase-producing microbiota, both bacteria and

yeasts in the intestines of 10 fresh water teleosts Maximum

number of tannase-producing microorganisms were

re-ported in the hindgut regions, but appreciable amount of

tannase-producing microorganisms were also detected in the

mid gut regions In this study, the tannase-producing

micro-biota were dominated by different species of yeasts; Pichia

sp and Candida spp Tannin-degrading microbiota detected

in fish gut may offer some ecological advantage enabling

them to overcome the antinutritional effects of plant

tan-nins However, this issue merits further investigation

Xylanases (Endo-1,4-b-xylanase, or XYNII, EC 3.2.1.8) aregroups of enzymes that depolymerize xylan molecules intoxylose units used by microbial populations as a primarycarbon source (Nath & Rao 2001) Xylanase consists of

190 amino acids and has a molecular weight of 21 kDa.The enzymatic hydrolysis of xylan, a major hemicellulosecomponent of agro-industrial residues, is advantageous forthe recovery of hexose and pentose sugars to be used asraw materials in a wide number of biotechnologicalapplications Many microorganisms including bacteria(Nakamura et al.1994; Yang et al 1995; Gupta et al.2000;Balakrishnan et al 2002; German & Bittong 2009; Azeri

et al 2010), actinomycetes (Ball & Mccarth 1989;Techapun et al 2001; Tuncer et al 2004) and filamentousfungi (Taneja et al 2002; Angayarkanni et al 2006; Sudan

& Bajaj 2007) have been reported to produce xylanase.Some wood-eating or xylivorous insects require the aid ofsymbiotic microorganisms in their GI tracts to digest cellu-lose and make the energy in this compound available tothe host (Prins & Kreulen 1991; Mo et al 2004) However,reports on production of xylanase by fish gut endos-ymbionts are scanty German & Bittong (2009) reportedb-xylosidase activity in the microbial extracts of threewood-eating catfish (Panaque nocturnus, Hypostomus pyrin-eusi and Panaque cf nigrolineatus) and one detritivorouscatfish (Pterygoplichthy disjunctivus) The enzyme activitieswere reported to be different between the intestinal fluidand the microbial extract and the activities ofb-xylosidaseslightly increased, although not significantly, towards thedistal intestine The catfish species examined by German &Bittong (2009) did not show any b-xylosidase activity intheir gut walls and hadlow b-xylosidase activities in theirmicrobial extracts, which decreased distally in the digestivetract They, however, opined that low and variable cellulaseand xylanase activities observed in the catfish, and the lack

of any consistent pattern of activity along the guts of thefish, these enzymes are most likely produced by microbesingested by the fish with detritus rather than produced by aresident endosymbiotic community Whether the fish gutcontains any autochthonous xylanase-producing microbiotarequires further investigation

Chitin consists of a b-1,4-linked N-acetylglucosamine dues and is estimated as the second most abundant bio-mass (1013 metric tons; Jolles & Muzzarelli 1999) in the

resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi- resi-.

Aquaculture Nutrition 18; 465–492 ª 2012 Blackwell Publishing Ltd

world after cellulose and forms a major structural

compo-nent of many organisms, including fungi, crustaceans,

mol-luscs, coelenterates, protozoan and green algae (Rinaudo

2006; Khoushab & Yamabhai 2010) Chitin is completely

hydrolyzed to its constituent oligomers and monomer by

the binary chitinase enzyme system: chitinase (EC 3.2.1.14)

and b-N-acetylglucosaminidase (EC 3.2.1.30) (Takiguchi

1995)

The general method to evaluate chitinolytic bacteria can

be enumerated on casein chitin agar with the following

composition: 0.2% casein, pancreatic digest (Difco,

Lawr-ence, KS, USA), 0.8% colloidal chitin, 1.5% technical grade

agar, made up with 75% aged filtered sea water and pH

adjusted to 6.7 Hydrolysis of chitin can also be measured

on ZA agar plates containing 0.5% chitin and

phenolphtha-lein diphosphate sodium salt (0.01%) according to the

method of Cowan (1974) Screening of chitinolytic bacteria

can also be carried out using 1/20 PYBG agar plates

con-taining 0.2% colloidal chitin (Itoi et al 2006)

To our knowledge, the first study reporting chitin

destruction by bacteria was conducted by Benecke (1905)

who reported the isolation of Bacillus chitinovorus from the

polluted waters of Kiel harbour An overview of the studies

relevant to the present review is presented in Table 6

Hamid et al (1979) reported in their study with gray

mul-let, that bacteria belonging to the genera Enterobacter,

Vib-rio and Pseudomonas had the capacity to degrade chitin,

while Sakata et al (1980) reported chitinase-producing

bac-teria (Aeromonas and Vibrio) isolated from the GI tract of

tilapia MacDonald et al (1986) reported in their study on

gut microbiota of Dover sole (Solea solea L.) that strains

belonging to Acinetobacter (1), Enterobacteriaceae (4),

Pho-tobacterium (6) and Vibrio spp (48) were able to degrade

chitin In a study on tilapia (Sarotherodon niloticus), Sakata

& Koreeda (1986) reported that gut bacteria isolated from

intestinal contents belonging to Plesiomonas shigelloides

and A hydrophila decompose chitin and the authors related

the finding to the feeding habitat (ponds) of the fish Sugita

et al.(1999) reported that Aeromonas caviae, A hydrophila,

A jandaei, A sobria and A veroni isolated from common

carp, crucian carp and gray mullet displayed chitinase

activity Based on their results, the authors put forward the

hypothesis that in chitin digestion Aeromonasspecies have

similar status in the digestive tract of freshwater fish as

Vibrio species in marine fish This hypothesis was

con-firmed in a study on various Japanese costal fish species

where chitin-rich organisms such as crustaceans and

proto-zoa are a major part of the food and that 99% of the 361

isolates belonging to the family Vibrionaceae were capable

of decomposing colloidal chitin (Itoi et al 2006) In thestudy of Sugita & Ito (2006), the authors reported thatalmost all isolates (98.8%) isolated from the Japaneseflounder intestine were chitinolytic and the gut isolateswere identified as Vibrio fischeri, Vibrio harveyi and theVibrio scophthalmi – Vibrio ichthyoenteri group As thePCR amplification technique for chiA gene seems to be use-ful in detecting chitinolytic bacteria associated with thedigestive tract of fish (Sugita & Ito 2006), we recommendthat this technique is used in future studies evaluatingchitinase-producing bacteria from fish

In comparison with the numerous available reports on theaerobic enzyme-producing microbiota from fish gut, infor-mation on anaerobic enzyme-producing microbiota isscarce (Trust et al 1979; Sugita et al 1997; Ramirez &

Dixon 2003) From our point of view, this is a paradox asanaerobic bacteria are probably the most important con-tributors to fish nutrition (Clements et al 1997, 2006,2009) Trust et al (1979) reported that 14 of 150 strictanaerobes gut isolates of grass carp displayed lipase activitywhen tested on tributyrin agar Furthermore, the authorsreported similar frequency of strict anaerobes with amylase,protease and casease activity Sugita et al (1997)documented that 56% of the gut anaerobes producedamylase, whereas only 20% of the aerobes had this ability

More than 50% of Aeromonas, Bacteroidaceae andClostridiumstrains produced amylase efficiently, while Acinet-obacter, coryneforms, Enterobacteriaceae, Moraxella, Plesio-monasand Streptococcus strains did not Ramirez & Dixon(2003) isolated obligate anaerobic intestinal bacteria from thefreshwater angelfish (Pterophyllum scalare), oscars (Astrono-tus ocellatus) and the marine southern flounder (Paralichthyslethostigma) Clostridium was recovered from southern floun-der, while both Clostridium and Gram-negative generaincluding Fusobacterium, Bacteroides and Porphorymonaswere recovered from oscars and angelfish The authors docu-mented enzyme activities of acid and alkaline phosphatases,C4 and C8 esterases, C14 lipases, arylamidases and glycosid-ases by the anaerobic bacteria The Gram-negative generapossessed enzymes for the breakdown of carbohydrates,while Clostridium had the capability for breakdown of pro-teins All genera produced phosphatases possibly for absorp-tion of nutrients In addition to the enzymatic contribution, ithas been suggested that anaerobic bacteria can contribute tofish nutrition by supplying VFAs (Clements et al 1997)

.

Although numerous studies have indicated that the major

microbial colonizers in the GI tract of fish are bacteria (for

review, see Cahill 1990; Sakata & Lesel 1990; Ringø et al

1995; Spanggaard et al 2000; Pond et al 2006; Nayak

2010a), yeast has also been reported to colonize within the

GI tract of some fish species (Va´zquez-Jua´rez et al 1994,

1997; Andlid et al 1995, 1998; Gatesoupe 2007; Kutty &

Philip 2008) Yeasts are ubiquitous microorganisms which

can grow in various environments where organic substrates

are available (Gatesoupe 2007)

Yeast may be generally considered commensal in fish

gut, and possible benefits can be expected on the immune

and the digestive system of the host (Gatesoupe 2007)

After settlement in fish intestine, the yeasts have to

com-pete with other microorganisms, but it is well known that

some yeasts strains have a strong adhesion potential to fish

intestinal mucus (Va´zquez-Jua´rez et al 1994, 1997; Andlid

et al.1995, 1998)

Even though yeasts might have an impact on the

intesti-nal ecology and nutrition of the fish, the topic merits

fur-ther investigations Mandal & Ghosh (2010) described

tannase activity of Pichia spp and Candida spp isolated

from the GI tract of fresh water fish Tovar-Ramı´rez et al

(2002) compared the effects of two yeasts (Debaryomyces

hansenii HF1 and S cerevisiae X2180) on European sea

bass larvae fed compound diets Debaryomyces hansenii

improved survival and vertebral conformation of the

lar-vae, possibly due to the observed acceleration of the

matu-ration of the digestive system On the other hand, these

effects were not observed using S cerevisiae Conversely,

Wache´ et al (2006) observed that the maturation of the

digestive system took place before day 20 poststart feeding

in rainbow trout fry, and the colonization by D hansenii

was too late to accelerate the onset In such conditions,

since start feeding, dietary supplementation of S cerevisiae

var boulardii CNCM I-1079 stimulated the activity of three

enzymes (alkaline phosphatase, c-glutamyl-transpeptidase

and leucine – amino-peptidase N) in the brush border

membrane of the enterocytes at day 10, but without any

effect on growth

Several studies have characterized and identified

enzyme-producing bacteria from homogenates of the intestinal

digesta by culture-based techniques and selective media,followed by conventional morphological and biochemicalassays Ghosh et al (2002) identified the enzyme-producinggut bacteria, B circulans Lr 1.1, B pumilus Lr 1.2 and

B cereus Lr 2.2, based on morphological, physiologicaland biochemical characteristics Kar et al (2008) identifiedgut isolates as B subtilis and B cereus, while Saha et al.(2006) isolated bacilli from the alimentary tracts of Chinesegrass carp and tilapia and identified them as B megaterium(CI3) and B circulans (TM1), respectively More recently,attempts have been made to identify enzyme-producing gutbacteria by 16S rRNA gene sequencing and subsequentcomparison with data available in NCBI GenBank or RDPdatabases Ray et al (2010) identified the 10 most promis-ing enzyme-producing strains isolated from the threeIndian major carps by 16S rRNA gene sequence analysis ofwhich five belonged to the genus Bacillus Mondal et al.(2010) identified B licheniformis (BF2) and B subtilis(BH4) from the gut of bata on the basis of phenotypiccharacteristics as well as 16S rDNA sequence analysis.Lately, Ghosh et al (2010) used SEM evaluation and cul-ture-based analysis to confirm the presence of autochtho-nous bacteria in the GI tract of rohu and that theseautochthonous bacteria possess enzymatic activity Theauthors altogether isolated 59 adherent bacterial strainsfrom the GI tract of rohu and identified 16 of them by 16SrRNA gene sequencing, of which 11 strains belonged tobacilli, two strains to Pseudomonas, one strain to Aeromo-nas, one strain was most closely related to Enterobacterwhile one strain was treated as unknown because of<97%16S rRNA sequence similarity in BLAST program Askari-

an et al (2012a) evaluated the effect of chitin (5% mentation) on the adherent aerobic/facultative anaerobicintestinal microbiota of Atlantic salmon (Salmo salar L.).The authors identified 64 of 139 autochthonous gut bacte-ria by 16S rRNA gene sequencing and tested for protease,amylase, cellulase, phytase, lipase and chitinase activities.This study indicated that dietary chitin modulates the gutmicrobiota, but not the portion of enzyme-producing gutbacteria The most promising gut bacteria isolated withrespect to enzyme production and in vitro growth inhibitionshowed high similarity to Bacillus thuringiensis by 16SrRNA gene sequencing In a subsequent study, Askarian

supple-et al (2012b) isolated 79 autochthonous gut bacteria fromAtlantic cod (Gadus morhua L.) fed fishmeal, soybean meal

or bioprocessed soybean meal and tested the bacteria forprotease, amylase, cellulase, phytase, lipase and chitinaseactivities The most promising enzyme-producing gutbacteria (48 isolates) were identified by 16S rRNA gene

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Aquaculture Nutrition 18; 465–492 ª 2012 Blackwell Publishing Ltd

sequencing and further tested for in vitro growth inhibition

of four important fish pathogens The most promising

bac-terias with respect to enzyme production and in vitro

growth inhibition belonged to Brochothrix sp and

Brocho-thrix thermosphacta

It is generally accepted that conventional culture-based

techniques are time consuming, lack accuracy (Asfie et al

2003) and do not represent a correct picture of the bacterial

diversity in fish gut, even if several different media are used

(Ray et al 2010) Therefore, to present more reliable

infor-mation on the gut microbiota of fish, nowadays, several

cul-ture-independent molecular technologies methods such as

polymerase chain reaction (PCR), random amplified

poly-morphic DNA, denaturing gradient gel electrophoresis

(DGGE), fluorescence in situ hybridization (FISH), confocal

microscopy and EM have been used to identify and detect

the microbial community in the GI tracts of fish (e.g

Spanggaard et al 2000; Walter et al 2001; Holben et al

2002; Ringø et al 2003; Temmerman et al 2004; Pond et al

2006; Hovda et al 2007; Kim et al 2007; Li et al 2008a,b;

Peter & Sommaruga 2008; Navarrete et al 2009; Ferguson

et al.2010; He et al 2010; Zhou et al 2011) However, one

has to bear in mind that the presence of any

microorgan-ism within the GI tract does not necessarily signify its

functional role The uses of culture-independent methods

are therefore an important supplement in gathering

infor-mation on the microbial community in the GI tract of

fish with respect to enzyme production However,

charac-terization and identification of the intestinal microbiota

designated with its functional role, conventional methods

should be used in combination with molecular methods

like 16S rRNA/26S rDNA sequence analysis (in case of

bacteria and yeasts, respectively) as suggested in some

recent studies (Ghosh et al 2010; Mondal et al 2010;

Ray et al 2010)

The term ‘probiotics’ is constructed from the Latin word

pro (for) and the Greek word bios (life) (Zivkovic 1999)

and was created by Kollath (1953) The definition of a

pro-biotic used in aquaculture differs greatly depending on the

source (Gram et al 2005; Merrifield et al 2010a), but

gen-erally, probiotics offer potential alternatives by providing

benefits to the host primarily via the direct or indirect

mod-ulation of the intestinal microbiota, enhanced immune

sys-tem and growth, stimulate enzyme activity and improved

disease resistance However, only few studies carried out

on fish have focused on contribution of the gut microbiotarelated to nutrition Even though earlier investigations havesuggested that gut bacteria have a beneficial effect on thedigestive processes of fish (for review, see Ringø et al

1995; Austin 2006; Nayak 2010a), the topic merits furtherinvestigations and especially related to the probioticapproach

An extensive range of enzymes (for review, see Tables 1–

6) produced by GI bacteria could be a contributing source

to digestive enzymes in fish For example, the presence of ahigh concentration of Aeromonas in the GI tract can play

an important role in digestion as Aeromonas species secreteseveral proteases and chitinase (Pemberton et al 1997;

Sugita et al 1999) Similarly, the p-nitrophenyl-glucosaminide-, chitin-, cellulose- and collagen-degradingability of gut bacteria may indicate their involvement in thenutrition of fish Characterization of the microbial popula-tions in the intestinal microenvironment of fish and under-standing the physiological interactions between theindigenous microbiota and the host might have importantimplications (Silva et al 2005) The major biochemicalactivity of the heterotrophic bacteria is the dissimilation oforganic matter Enzymes produced by intestinal fish micro-biota might have a significant role in digestion, especiallyfor substrates such as cellulose, which few animals candigest, and also for other substrates (Smith & Halver1989) Luczkovich & Stellwag (1993) opined that the GImicrobiota of pinfish (L rhomboides) might contribute tothe breakdown of plant material Recent observations havedocumented that fish harbour proteolytic, amylolytic andcellulolytic bacteria in their digestive tracts (Bairagi et al

b-N-acetyl-2002a; Ghosh et al 2002; Saha et al 2006; Roy et al

2009) Kar et al (2008) indicated that the ing gut bacteria are able to utilize carbohydrates, such asmannose, xylose, raffinose, cellobiose and cellulose Thesesubstances are mainly found in plant feedstuffs Therefore,cellulase and amylase activities by the gut bacteria mayindicate their ability to aid in digestion of plant feedstuffs

enzyme-produc-The use of such beneficial bacteria as probiotics has a longtradition in animal husbandry (Stavric & Kornegay 1995)

Beneficial bacteria could be introduced in commercialaquaculture by incorporating them into formulated fishdiets, or in the form of bacteria biofilm to achieve coloniza-tion in the fish GI tract at a higher degree (Bairagi et al

2002b, 2004; Ghosh et al 2002, 2003; Ramachandran et al

2005; Ramachandran & Ray 2007; Askarian et al 2011;

Saha & Ray 2011)

Probiotics could be beneficial in various ways Thesemight include: inhibition of a pathogen via production of

.

antagonistic compounds, competition for attachment sites,

competition for nutrients, alteration of enzymatic activity

of pathogens, immunostimulatory functions and nutritional

benefits such as improving feed digestibility and utilization

and by breakdown of indigestible components (Fuller 1989;

Fooks et al 1999; Irianto & Austin 2001; Bomba et al

2002; Austin 2006) Most often, the probiotic issue is

pre-sented that the bacteria must adhere and colonize the

mucosal surfaces of the GI tract, replicate to high numbers,

produce antimicrobial substances and withstand the acidic

environment of the GI tract (Ziemer & Gibson 1998;

Dun-ne et al 1999; Gismondo et al 1999; Mombelli &

Gis-mondo 2000) However, these descriptions are suggested

misleading as probiotic need only to possess one mode of

action (Kesarcodi-Watson et al 2008) Previous beliefs are

based on the understanding that a probiotic must become

a permanent member of the intestinal microbiota as

pre-sented in terrestrial animals Therefore, much of the

probi-otic research focuses on the adherence capacity of bacteria

On the other hand, it has been demonstrated that transient

bacteria can also exert beneficial effects (Isolauri et al

2004) However, multistrain and multispecies probiotics

might be developed to cover a wide angel of beneficial

aspects as indicated by Temmerman et al (2004)

Gut microbiota in many freshwater teleosts are fairly

dominated by Bacillus spp (e.g Ghosh et al 2002; Kar

et al 2008; Ray et al 2010; Mondal et al 2010, Ghosh

et al 2010), and Bacillus spp has been shown to possess

adhesion abilities, provide immunostimulation and produce

bacteriocins (Cherif et al 2001; Cladera-Olivera et al 2004;

Duc et al 2004; Barbosa et al 2005) Commercial products

containing such bacilli have been demonstrated to improve

shrimp production to a level similar to that observed when

antibiotics are used (Decamp & Moriarty 2006) Bacillus

spp hold added interest in probiotics as they can be kept

in the spore form and therefore stored indefinitely on the

shelf (Hong et al 2005) Although enzyme-producing

abil-ity may lead to designate a gut microorganism as

probiot-ics, it should be mentioned that the antimicrobial/

immunostimulatory potential of the enzyme-producing

Bacillus spp isolated from fish gut has not been evaluated

These issues together with challenge studies should be given

high priority to explore their full potential in commercial

aquaculture A proposed scheme of work has been

pre-sented to address this issue in future works (Fig 1)

According to Conway et al (1996), a microorganism is

able to colonize the GI tract when it can persist there for a

long time, by possessing a multiplication rate higher than

the expulsion rate Nikoskelainen et al (2001) suggested

that mucosal adhesion is one of the five important criteriafor selection of probiotics in fish However, in their recentreview devoted to probiotic and prebiotic applications forsalmonids, Merrifield et al (2010a) proposed an extendedlist of eleven essential and favourable criteria for potentialprobionts, and the authors proposed that probiotic coloni-zation of intestinal epithelial surface is favourable criteria.Whereas some authors suggested that probiotic lactic acidbacteria colonization of intestinal mucus involves hostspecificity (Lin & Savage 1984; Fuller 1986; Askarian et al.2011), other authors reported the absence of specificitywhen binding host intestinal epithelial surface (Gildberg &Mikkelsen 1998; Ringø 1999; Rinkinen et al 2003; Salinas

et al 2008) However, to the authors’ knowledge, suchinformation is not available with respect to gut bacteriawith high enzymatic activities, and this topic merits furtherresearch

Various mechanisms have been proposed to explain thebeneficial effects of probiotics such as (i) antagonismtowards pathogens, (ii) competitions for adhesion sites, (iii)competition for nutrients, (iv) improvement of water qual-ity, stimulation of host immune responses and (v) enzy-matic contribution to digestion Several studies havedocumented nutritional effect of algae, probiotic bacteriaand Saccharomyces on the digestive enzymes of fish andshellfish larvae (Cahu et al 1998; Tovar-Ramirez et al.2004; Tovar-Ramı´rez et al 2002; Wache´ et al 2006; Wang

& Xu 2006; Ghosh et al 2008; Suzer et al 2008; Iehata

et al 2009; Saenz de Rodriganez et al 2009; Askarian

et al.2011) Tovar-Ramirez et al (2004) reported improvedactivity of the digestive enzyme; trypsin, amylase andlipase in European sea bass larva by adding live yeast(D hansenii) to the diet Wache´ et al (2006) tested twostrains of Saccharomyces cerevisiae as probiotics for rain-bow trout fry After 10 and 20 days poststart feeding,higher activities of three brush border membrane enzymes(alkaline phosphatize, c-glutamyl-transpeptidase and leu-cine-amino-peptidase) were noticed when the fry were fed

S cerevisiae var boulardii compared with fish fed either

S cerevisiae strain NCYC Sc or the control fish Based ontheir findings, the authors suggested an earlier maturation

of the digestive system Furthermore, Wang & Xu (2006)showed significant difference (P< 0.05) of digestiveenzymes activity; protease, amylase and lipase in commoncarp by using Bacillus sp as probiotics In a later study,Ghosh et al (2008) tested four different inclusion levels of

.

Aquaculture Nutrition 18; 465–492 ª 2012 Blackwell Publishing Ltd

B subtilis isolated from C mrigala on survival, feed

con-version ratio, specific growth rate, proximate composition,

intestinal amylase and protease activity and gut microbiota

of four live-bearing ornamental fishes Length, weight,

sur-vival, body ash and protein content and gut enzyme

activ-ity were significantly improved by including bacilli in the

diets Furthermore, the population level of gut bacteria

belonging to motile aeromonads, presumptive Pseudomonas

and total coliforms was significantly reduced by probiotic

feeding Moreover, improved disease resistance against

A hydrophilainfection was noticed in fish fed bacilli Theauthors recommend that probiotic concentrations of 106–

108 bacilli g 1 are adequate for use in live-bearing mental fishes Suzer et al (2008) reported improved activity

orna-of the intestinal enzymes; alkaline phosphatase and peptidase and pancreatic trypsin, amylase and lipase byusing the Lactobacillus spp as probiotic in gilthead seabream larvae Iehata et al (2009) used a Lactobacillus plan-tarum originally isolated from rice bran and an Enterococ-cus mundtii originally isolated from horse manure in their

Screening for extracellular enzyme production

Screening for antagonism against pathogenic bacteria

Protease Amylase Cellulase Lipase Phytase Tannase Chitinase Xylanase Glucanase etc.

Pathogenicity test towards target organism

Use as bacteria Bio-film

Assessments for fish growth, carcass composition, enzyme production and disease resistance Use for

processing

of feed ingredients

Figure 1 Scheme for designating enzyme-producing fish gut bacteria as probiotics.

.

study of Abalone Both bacteria increased volatile

short-chain fatty acids in the gut With respect to increase gut

enzyme activity, L plantarum increased amylase activity,

while E mundtii elevated protease activity Askarian et al

(2011) displayed enhanced specific growth rate, survival

and an increase in digestive enzyme (amylase, protease and

lipase) activity in beluga (Huso huso) and Persian sturgeon

(Acipenser persicus) fed two lactic acid bacteria

(Lactococ-cus curvatus and Leuconostoc mesenteroides) The authors

suggested that the enhanced digestive enzyme activities

observed in some of the treatment groups of sturgeon

might be attributed to improved gut maturation as

previ-ously suggested by Tovar-Ramı´rez et al (2002) in a study

using D hansenii originally isolated from the gut of

rain-bow trout (O mykiss) In addition to this direct effect,

some authors have suggested that the main modes of action

and beneficial effects of probiotics are prevention of

intesti-nal disorders and predigestion of antinutrient factors

pres-ent in the ingredipres-ents (Thompson et al 1999; Verschuere

et al 2000; Suzer et al 2008) To clarify the mechanisms

involved, further studies have to be carried out

Complementary enzymes produced by the symbiotic

bacte-ria contribute to digestion and assimilation of plant

food-stuffs in endothermic animals as well as fish (McBee 1971;

Ghosh et al 2002; Esakkiraj et al 2009) However,

symbi-oses are well studied in terrestrial animals compared with

aquatic animals A number of experiments have been

con-ducted with terrestrial animals (especially ruminants)

regarding contribution of microbes to digestive functions

In an in vitro digestion study, Hino & Russell (1987)

incu-bated various protein sources with mixed ruminal

microor-ganisms (protozoa and bacteria) from a cow fed timothy

hay and commercial concentrate feed (50:50) to determine

deamination under enzyme-limiting substrate-excess

condi-tions Their results suggested that (i) soluble proteins are

primarily degraded by bacteria, (ii) protozoa could

contrib-ute to the degradation of insoluble, particulate proteins,

(iii) protozoans possess limited ability to assimilate peptides

(or amino acids), and (iv) low molecular weight products

could be fermented more readily by bacteria Lee et al

(2000) assessed the relative contribution of bacteria,

proto-zoa and fungi in rumen fluids to overall process of

degra-dation of orchard grass cell walls in the artificial rumen

ecosystem The protozoal fraction was reported to inhibit

cellulolysis of cell wall material by both the bacterial and

fungal fractions, while in the co-culture between the

bacte-rial fraction and the fungal fraction, a synergistic tion was detected In an in vitro digestibility study of wheatstraw by rumen microorganisms of water buffalo of Khuze-stan in Iran and Holstein cow, Jabbari et al (2011)observed that the dry matter (DM) digestibility of wheatstraw by rumen microbial population of Khuzestani buf-falo was higher than that by rumen microorganisms ofHolstein cow Under the same condition, the DM and neu-tral detergent fibre (NDF) digestibility by rumen microor-ganisms of Khazestani buffalo was 1.22- and 1.51-foldshigher than the Holstein cow, respectively The researchefforts towards establishment of in vitro digestion model infish would be pertinent not only to evaluate the effective-ness of the gut microbiota, but also to adopt strategies forprocessing of feed ingredients utilizing autochthonous gutmicrobiota as the organism itself and their metabolites pos-sibly would not cause harm to the fish providing the basisfor mutual relationship

interac-Although the relative numbers and type of bacteria ated with the GI tract of healthy fish are interesting, it isthe role of the gut microbiota that is of importance Dur-ing the last two decades, several comprehensive reviewpapers have been published focusing on the use of probiot-ics related to growth, improvement in immune activity byimproving barrier properties of mucosa, modulating pro-duction of cytokines, modulating the gut microbiota,improvement of fish diseases, competition between the

associ-‘good’ and pathogenic bacteria in the fish gut and tion of antimicrobial compounds (e.g Ringø & Birkbeck1999; Verschuere et al 2000; Irianto & Austin 2001; Gram

produc-et al 2005; Ringø et al 2005, 2010; Gatesoupe 2007;Panigrahi & Azad 2007; Magnadottir 2010; Merrifield et al.2010a; Nayak 2010b) as well as enzyme-producing bacteriaisolated from the GI tract of fish (Ringø et al 1995; Austin2006; Nayak 2010a; the present review) and aquatic inver-tebrates (Harris 1993) Moreover, among the microbialpopulation in the fish gut, beneficial bacteria (enzyme-pro-ducing) are continuously competing with pathogensthrough competitive exclusion, and this topic should beaddressed in in vitro, ex vivo and in vivo studies

From our point of view, it is of high importance to seethe enzyme-producing bacteria in a greater context There-fore, we recommend the use of synbiotics, a mixture ofprobiotics (enzyme-producing bacteria with antagonisticactivity against fish pathogens) and prebiotics, to evaluategrowth, feed conversion rate, gut enzyme activity, gut

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Aquaculture Nutrition 18; 465–492 ª 2012 Blackwell Publishing Ltd

maturation, gut immunology, modulation of the gut

micro-biota and disease resistance

When it comes to the hypothesis of whether or not the

gut microbiota can contribute to nutrition, our conclusion

is the gut microbiota may to some extent contribute to

nutrition but further investigations related to the fact that

gut microbiota utilizing different substrates in vitro do not

necessary have any in vivo effect merits further

investiga-tions On the other hand, for example, information is

avail-able concerning microbial cellulase production in the GI

tract of fish One of these species investigated is grass carp

a fish that feeds only on hydrophytes in natural waters

Therefore, there must be some kind of mechanism for them

to use plant resources effectively, which suggests that

cellu-lase might play an important role in their digestive system

Because grass carp cannot produce cellulase by itself,

cellu-lase-producing microbes in the grass carp’s intestine

proba-bly contribute to nutrition Cellobiose, the repeating

disaccharide unit of cellulose, has ab(1?4) glycosidic

link-age, and cellobiose utilization is often used to differentiate

members of Vibrionaceae As bacteria belonging to bacilli

seem to be the main contributors to cellulose utilization in

the digestive tract of the fish species investigated, we

rec-ommend gene clusters evaluation of cellobiose utilization

by gut bacilli One should also bear in mind that diet

for-mulation should be taken into account when evaluating the

enzyme-producing bacteria From our point of view, it is a

paradox that most studies on enzyme-producing gut

bacte-ria isolated from fish are carried on different carp species

while few studies are carried out on salmonids and Atlantic

cod (Askarian et al 2012a,b) Therefore, we recommend

that the topic enzyme-producing gut bacteria isolated from

salmonids merits further investigations, especially related to

chitinase activity as chitin is one of the most renewable

bio-polymers on earth and might be useful as a constitutive

material in formulated fish feed in the future Even though

dietary chitin modulates the intestinal microbiota,

influ-ences disease resistance, susceptibility and innate immune

parameters, these topics are not fully understood and merit

further studies (Ringø et al 2012)

Although several studies during the last two decades

have demonstrated that fish gut bacteria produce vitamins

(e.g., vitamin B12, which may be of value to the host)

(Kashiwada & Teshima 1966; Kashiwada et al 1970, 1971;

Sugita et al 1991) and polyunsaturated fatty acids

(Yaza-wa et al 1988; Jøstensen et al 1990; Ringø et al 1992a,b;

Yano et al 1994), it is not clearly known whether these

bacteria make a significant contribution to fish nutrition

This topic merits further investigations and especially

related to the early developmental stages where such bution can be of vital importance to distinguish betweensuccess and failure

contri-There are published some studies using terrestrial gutsystems which suggest contributions (or drains) to overallenergy availability by gut microbiota Vervaeke et al

(1979) suggested that 6% of the net energy in the pig diet

is lost to the microbiota On the other hand, Von hardt et al (1985) suggested that the gut microbiota ofruminants could contribute up to 80% of maintenanceenergy and that up to 20% of maintenance energy could beprovided by the gut of carnivorous terrestrial species How-ever, to the authors’ knowledge, there is no informationavailable on this topic on fish

Engel-Another aspect that merits further research is the gutmicrobiota in fast and slow growing individuals In anearly study by Ringø et al (1997), it was demonstrated thetotal culturable population level in proximal intestine ofdominant individuals of Arctic charr was higher compared

to subordinate individuals, while the population level indistal intestine was similar In a more recent study, by Sun

et al (2009), the population level of gut bacteria in fastand slow growing grouper (Epinephelus coioides) was simi-lar However, in both studies, the gut microbiota differs

Whether these modulations in gut microbiota can ute to nutrition merits further investigations, but it is worthnoticing that bacilli (Bacillus spp.) were one of the domi-nant bacterial genera isolated from the GI tract of fastgrowing grouper Furthermore, we recommend using cul-ture-independent techniques such as denaturing gradientgel electrophoresis, fluorescence in situ hybridization, tem-poral temperature gradient gel electrophoresis or clonelibraries in combination with traditional cultivation for thescreening of the gut microbiota in fast and slow growingfish and wood-eating fish species

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National Renderers Association, Causeway Bay, Hong Kong, China

An 8-week growth trial was conducted using a 2· 3 factorial

design to evaluate the effect of substitution of fishmeal (FM)

by rendered animal protein blend [APB, comprised of

400 g kg)1 poultry by-product meal, 350 g kg)1 meat and

bone meal, 200 g kg)1hydrolysed feather meal (HFM) and

50 g kg)1spray-dried blood meal] in diets of Siberian

stur-geon, Acipenser baerii Brandt Two isoenergetic control diets

were formulated to contain two different protein levels

[high-protein control (400 g kg)1), with 483 g kg)1 of FM] and

[low-protein control (360 g kg)1), with 400 g kg)1 of FM]

At each protein level, dietary FM protein was replaced by

APB at 75% and 100% levels and supplemented with

crys-tallized essential amino acid under ideal protein concept The

six diets were named as HC, HAPB75, HAPB100, LC,

LAPB75 and LAPB100, respectively No significant

differ-ences were found in weight gain rate (WGR) and specific

growth rate (SGR), but fish fed with the low-protein diets

showed higher feed intake and feed conversion ratio Plasma

growth hormone and insulin-like growth factors I of each

group were not significantly different (P > 0.05) The

whole-body composition and liver composition were not affected by

dietary protein levels, replacement or their interaction

Muscle protein and lipid contents of fish fed with diet

LAPB100 were significantly lower than those of HC group

Digestibility of nitrogen (N) and phosphorus (P) were

reduced with higher APB inclusion levels, but productive N

and P values of all groups were not different Lower N and P

intake induced lower nutrients losses (P < 0.05) The results

suggested that dietary protein level could be reduced to

360 g kg)1from 400 g kg)1without affecting WGR or SGR

and significantly reduced nutrients lose Furthermore, dietary

FM protein can be totally replaced by APB in feed lation either at 400 g kg)1or at 360 g kg)1protein level

formu-key words: fishmeal, nitrogen, phosphorus, protein levels,rendered animal protein blend, Siberian sturgeon

Received 27 May 2011, accepted 18 August 2011 Correspondence: Y Qin, National Aquafeed Safety Assessment Station, Feed Research Institute, The Chinese Academy of Agricultural Sciences, Beijing 100081, China E-mail:yuchangqin1963@gmail.com

Fishmeal (FM) is the most important and one of mostexpensive protein source in fish feed However, high demandsand limited supply lead to high price for FM in present andfuture Using low-cost, reasonable plant or terrestrial animalprotein ingredients to replace FM can reduce feed cost ofaquaculture Rendered animal protein ingredients, forexample poultry by-product meal (PBM), meat and bonemeal (MBM), spray-dried blood meal (SDBM) and HFM aregenerally economical protein sources These ingredients havebeen investigated in a wide range of carnivorous fish species,such as rainbow trout (Oncorhynchus mykiss) (Bureau et al.2000), hybrid striped bass (Morone saxatilis· M chrysops)(Gaylord & Rawles 2005), cuneate drum (Nibea miichthio-ides) (Wang et al 2006) and Malabar grouper (Epinephelusmalabricus) (Wang et al 2008)

Formulation of the practical diet at optimal protein leveland an ideal essential amino acid (EAA) profile is a pre-requisite for fish growth and nitrogen retention (Luo et al.2006; Peres & Oliva-Teles 2009) Contents of the EAA,especially lysine (Lys), methionine (Met) and threonine

(Thr), are generally limiting amino acids in economical

alternative protein sources The deficiency of an EAA will

lead to poor utilization of the dietary protein (Wilson 2002)

To improve nitrogen utilization, it is important to reduce

dietary protein level based on optimal EAA profile, and it had

been practiced well not only in poultry and livestock diets

(Dari et al 2005; Libao-Mercado et al 2006), but also in

some fish species (Yamamoto et al 2005; Gaylord & Barrows

2009; Peres & Oliva-Teles 2009; Rawles et al 2011)

All sturgeon species and their caviar are listed in

Con-vention on International Trade in Endangered Species

(CITES), and Siberian sturgeon is widely farmed not only for

caviar, but also for meat in China In sturgeon farming,

commercial rainbow trout diets and marine fish diets are

often used, and their results are acceptable in terms of growth

and survival However, these diets are considered to be

suboptimal because prolonged feeding with these may result

in malformations and physiological disorders (Sicuro et al

2011) There have been a few reports studied feeding and

nutrition of the Siberian sturgeon (Acipenser baerii Brandt)

(Me´dale et al 1995; Liu et al 2009) Kaushik et al (1989)

reported that protein requirement for optimal growth of

Siberian sturgeon (90–400 g) would be around 360–

420 g kg)1in the practical diets Besides, we had successfully

replaced 50% of FM by an animal protein blend (APB,

including PBM, MBM, SDBM and HFM) with

supplemen-tary of crystallized amino acids (CAAs) in the study by Zhu

et al (2011) The objectives of the present study were to

investigate the effects of partial (75%) or total replacement

FM with APB at two protein levels on the growth

perfor-mance, body compositions, input and output of nitrogen (N)

and phosphorus (P) in Siberian sturgeon

Poultry by-product meal (pet-food grade), MBM and HFM

were supplied by National Renderers Association, Ltd.,

Hong Kong, China FM was produced in Peru and supplied

by International FM and Fish Oil Organization, St Albans,

Hertfordshire, UK Other ingredients were obtained from

local market A blend of animal protein source was

com-posed of PBM, MBM, SDBM and HFM at the ratio of

40 : 35 : 20 : 5 to formulate at same protein level as FM, in

which, Lys, Met and Thr were the limiting amino acids

compared with FM

The diet treatments were designed following a 2· 3

fac-torial layout Two isoenergetic control diets were formulated

to contain two different protein levels [high-protein control(400 g kg)1), HC with 483 g kg)1 of FM] and [low-proteincontrol (360 g kg)1), LC with 400 g kg)1 of FM] FM wasdesigned as the primary protein source in control diets Ateach protein level, dietary FM protein was replaced by APB

at 75% and 100% levels and supplemented with crystallizedEAA under ideal protein concept [(digestible EAA/digestibleprotein (DP)] of Siberian sturgeon (Zhu et al 2011) The sixdiets were named as HC, HAPB75, HAPB100, LC, LAPB75and LAPB100, respectively Crystallized Lys-H2SO4 (65%),

DL-Met (98%) andL-Thr (98%) were supplemented to meetthe EAA requirements of Siberian sturgeon in diets of APBused (Zhu et al 2011) In addition, 1.0 g kg)1yttrium oxide(Y2O3) was used as an inert marker in each diet for deter-mining digestibility and discharging of N and P Diets for-mulation and proximate compositions are shown in Table 1,and EAA profiles are shown in Table 2

All ingredients were ground into fine powder through a246-lm mesh before extrusion The diets were made into drypellets (diameter: 2 mm) under the extrusion condition asfeeding section (90C per 5 s), compression section (130 Cper 3 s) and metering section (60C per 4 s) using a twin-screwed extruder (TSE65; Yanggong Machine, Beijing,China), air-dried for about 36 h at 20C and kept in freezer

at)20 C

Siberian sturgeon was obtained from Sturgeon farm ofBeijing Fisheries Institute (Beijing, China) Fish were accli-mated to the recirculation system and fed with the HC dietfor 2 weeks before the trials Siberian sturgeon [initial bodyweight (IBW) = 39.0 ± 0.2 g] were randomly distributedinto 24 tanks with flat bottom (diameter: 80 cm; volume:

0.25 m3) Water flow rate in each tank was maintained at1.2 L min)1, and water was drained through bio-filters todecrease microorganism, reduce ammonia concentration andremove solid substances in the system Four replicates tankswere randomly assigned to each diet group, and 20 fish werebatch weighed and stocked in each tank During the 8-weekfeeding period, fish were fed with the experimental diets toapparent satiation three times daily at 9:00, 15:00 and 21:00,respectively Any uneaten feed was collected 1 h after eachmeal, dried to constant weight at 70C and reweighed

Leaching loss in the uneaten diet was estimated by leavingfive samples of each diet in tanks without fish for 1 h,recovering, drying and reweighing Before the experiment, 20fish from the same population were randomly selected for thedetermination of initial whole-body proximate composition

.

At the end of the experiment, in each tank, three fish of

similar weights as the groups were sampled for whole-body

composition analysis, and other five fish from each tank were

sampled for blood sampling, muscle and liver composition

analysis Blood samples were taken from the caudal vein

using heparinised syringes to obtain plasma samples after

centrifugation (4000 g for 10 min) at 4C Individual body

weight, body length, liver weight and visceral weight were

recorded to calculate condition factor, hepatosomatic index

(HSI) and viscerosomatic index (VSI) All samples werestored at)80 C until determination

During the 8-week feeding period, water temperature wascontrolled at 20–22C, pH = 7.5–8.0, ammonia was lowerthan 0.4 mg L)1, nitrite was lower than 0.1 mg L)1 anddissolved oxygen was higher than 6.0 mg L)1 Aeration wassupplied to each tank 24 h day)1, photoperiod was12D : 12L and light intensity was 7 1· Faeces were collected

1 h after each meal, and faeces collection method was same

as described by Liu et al (2009)

Dry matter, crude protein, crude lipid, ash, energy and totalphosphorus were analysed for all ingredients, experimentaldiets, faeces and fish samples (AOAC 1995) Amino acidswere analysed for ingredients and diets Dry matter wasanalysed by drying the samples to constant weight at 105C.Crude protein was determined by measuring nitrogen(N· 6.25) using the Kjeldahl method (Kjeltec 2300 ProteinAnalyzer; Foss, Hillerød, Denmark) Crude lipid was mea-sured by acid hydrolysis with a Sotex System Hotplate 2022Hydrolyzing Unit (Foss), followed by Soxhlet extractionusing a Sotex system 2050 (Foss) Ash was examined bycombustion in a muffle furnace at 550C for 16 h Grossenergy was determined by Parr 1281 Automatic Bomb Cal-orimeter (Parr, Moline, IL, USA) The amino acids ofingredients and diets were determined by amino acid analyzer(Hitachi 8900, Hitachi, Tokyo, Japan) after hydrolysis in 6 NHCl for 22–24 h at 110C For sulphur amino acids deter-mination, an oxydrolysis in performic acid for 30 min at

55C was conducted before hydrolysis by 6 N HCl Faeceswere collected twice per day until sufficient dry faeces pre-pared for analysis Pooled faeces from each group of fishwere freeze-dried prior to the analysis for yttrium oxide,moisture, crude protein and phosphorus content Yttrium

Table 1 Formulation (g kg ), proximate compositions (g kg )

and energy content (MJ kg)1) of the experimental diets in the growth

trial for 8 weeks

Ingredients HC

HAPB 75

HAPB

100 LC

LAPB 75

LAPB 100

DP, digestible protein; HFM, hydrolysed feather meal; MBM, meat

and bone meal; PBM, poultry by-product meal; SDBM, spray-dried

blood meal; CP, crude protein; CL, crude lipid; TL, total phosphorus;

APB, animal protein blend; NFE, nitrogen-free extracts.

1 Fishmeal (FM): steam-dried FM, (COPENCA Group., Lima, Peru),

4 NFEs of diets were calculated: NFE = 1000 ) (Crude protein +

Crude lipid + Crude ash).

5 Ideal protein concept was according to the study by Zhu et al.

HAPB

LAPB 75

LAPB 100 EEAs

oxide (Y2O3) and phosphorus in the diet and faeces were

determined by inductively coupled plasma-atomic emission

spectrophotometer (ICP-OES, Vista-Mpx; Varian, Hansen

Way, Palo Alto, CA, USA) after perchloric acid digestion

Plasma growth hormone (GH) and insulin-like growth factor

I (IGF-I) were measured by using a commercially available

125

I radioimmunoassay (RIA) kit (Jiuding Biotech

Corpo-ration, Shenzhen, China) Duplicate analyses were conducted

for each sample

A 2· 3 factorial trial was designed to evaluate effects of

dietary protein levels, replacement levels and their

interac-tion Significant differences (P < 0.05) of each variable were

firstly detected in one-way ANOVA test, and then DuncanÕs

multiple range test was used to rank the group All data were

subjected to two-way analysis of variance, followed by

DuncanÕs multiple-range test by STATISTICA 8.0 (Statsoft.,

Tulsa, OK, USA) Differences were regarded as significant

when P < 0.05 All data are expressed as mean ± SE

Results of growth performance and morphometric

para-meters are shown in Table 3, and plasma GH, IGF-I results

are shown in Fig 1 During the whole growth trial, survivalrate of all groups was 100% No significant differences werefound in WGR, SGR, condition factor (CF), HSI and VSIamong dietary treatments (P > 0.05) Feed intake (FI) andfeed conversion ratio (FCR) were not affected by FMreplacing level, but fish fed with low-protein diets showedhigher FI and FCR than those of fish fed with the high-protein diets (P < 0.05) Plasma GH (0.72–0.79 ng mL)1)and IGF-I (11.22–15.65 ng mL)1) after 24 h starvation ofeach group were not significantly different (P > 0.05)

The whole-body, muscle and liver compositions of Siberiansturgeon fed with the experimental diets are presented inTable 4 The whole-body and liver compositions were notaffected by dietary protein level, replacement or their inter-action The muscle moisture and lipid contents were affected

by dietary replacement level Muscle protein and lipid tents of fish fed with diet LAPB100 were significantly lowerthan those of HC group

con-The results of input and output of dietary N and P are shown

in Table 5, Figs 2 & 3, which include intake, digestion,retention and discharging of N and P Apparent digestibilitycoefficient of N and P was not affected by dietary protein

Table 3 Growth performance and morphometric parameters of Siberian sturgeon fed with the experimental diets for 8-week (IBW 39 g,

means ± SE, n = 4)

Two-way ANOVA (P-values)

PL RL PL · RL Final body weight (g) 143.3 ± 4.1 144.8 ± 5.3 138.5 ± 4.9 143.8 ± 4.5 130.9 ± 6.2 137.2 ± 4.0 0.230 0.416 0.295

PL, protein levels; HSI, hepatosomatic index; IBW, initial body weight; PL · RL, protein levels · replacement levels.

Values in the same row with different superscripts are significantly different (P < 0.05).

1 Weight gain rate (WGR) (%) = 100 · [(final body weight ) initial body weight)/initial body weight].

2 Specific growth rate (SGR) (% per day) = 100 · [ln (final body weight) ) ln (initial body weight)]/days.

3

Feed intake (FI) (% per day) = 100 · total amount of the feed consumed/[days · (initial body weight + final body weight)/2].

4 Feed conversion ratio (FCR) = total amount of the feed consumed/(final body weight ) initial body weight).

5 Condition factor (CF) = 100 · fish whole-body weight/(body length) 3

6 HSI (%) = 100 · (liver weigh/body weight).

7 Viscerosomatic index (VSI) (%) = 100 · (visceral weight/body weight).

.

levels or interaction of the two factors but decreased when

FM was replaced by APB No significant differences wereobserved in the retention of N and P [productive nitrogenvalue (PNV) and productive phosphorus value (PPV)] amongdietary treatments N (P) intake, digestible N (P) intake,faecal N (P) loss, and total N (P) loss were significantlyaffected by both of dietary protein levels and FM replace-ment (P < 0.05) Faecal N loss was increased when FM wasreplaced by APB Fish fed with low-protein diets showedlower N and P intake, which significantly induced lowerfaecal and total nutrients loss for Siberian sturgeon N and Pintake of fish showed closely linear relationship with total

N loss (y = 0.6538x + 0.6417, R2

= 0.9241) (Fig 2) andtotal P loss (y = 0.8666–0.189, R2= 0.9127) (Fig 3),respectively

It has been noted that the muscle or whole-body EAA profile

is well related to the ideal protein model of feed in fish (Ng &Hung 1994; Mambrini & Kaushik 1995; Rollin et al 2003;Peres & Oliva-Teles 2009; Zhu et al 2011) Under intensivefish farming conditions, FM and fish oil are the most com-mon ingredients supplying the essential nutrients of farmedfish PBM has been widely studied as an alternative proteinsource for FM in fish feed and seemed to be a good proteinsource, which hold similar amino acid profile to FM Inrecent years, tremendous improvement has been achievedthat PBM could be able to replace FM at a very high level or

up to 100% on a balanced amino acid basis (Gaylord &

00.20.40.60.811.2

–1)

0510152025

Figure 1 Plasma growth hormone (GH) (a) and insulin-like growth

factors I (IGF-I) (b) levels for each diet group Data of the six groups

are presented as mean ± SE (n = 20).

Table 4 Proximate composition (g kg)1) and energy content (MJ kg)1) in whole-body, muscle, and liver of Siberian sturgeon fed with the experimental diets for 8-week (in wet basis; means ± SE, n = 8)

Two-way ANOVA (P-values)

Muscle composition

Moisture 740 ± 11 a 748 ± 9 a 766 ± 8 ab 750 ± 6 ab 751 ± 5 ab 774 ± 7 b 0.272 0.014 0.899 Crude protein 165 ± 1 b 163 ± 1 ab 163 ± 2 ab 162 ± 2 ab 163 ± 2 ab 157 ± 3 a 0.127 0.182 0.452 Crude lipid 86 ± 12 b 82 ± 8 ab 64 ± 9 ab 76 ± 5 ab 76 ± 6 ab 58 ± 5 a 0.243 0.033 0.944 Liver composition

Moisture 536 ± 27 573 ± 23 559 ± 17 557 ± 17 538 ± 15 583 ± 22 0.849 0.495 0.283 Crude lipid 315 ± 27 280 ± 17 301 ± 17 285 ± 20 321 ± 20 276 ± 27 0.801 0.817 0.216 Values in the same row with different superscripts are significantly different (P < 0.05).

.

Aquaculture Nutrition 18; 493–501  2012 Blackwell Publishing Ltd

Rawles 2005; Rawles et al 2010) However, compared to

FM, MBM and HFM are deficient in Lys and Met HFM

holds high content of isoleucine (Ile) and conditional EAA,

such as cystine, proline and hydroxylproline (Li et al 2011)

In addition, blood meal is rich in Lys, but lacks Met and Ile

Utilization of blend protein sources to replace FM was a

more efficient way than using a single alternative protein

source (Milliamena 2002; Wang et al 2006; El-Haroun et al

2009) Therefore, it can be used to balance dietary EAAcontent when PBM, MBM, HFM and SDBM are used incombination to replace dietary FM substitutes (Wang et al

2008; El-Haroun et al 2009) Zhu et al (2011) recentlyreported that 50% of FM (240 g kg)1) replaced by APB didnot show any negative effect on growth of the Siberian

y = 0.866x – 0.189

R2 = 0.912

11.11.21.31.41.51.61.71.8

–1)

P intake (g fish–1)Figure 3 Correlation of phosphorus (P) intake with total P loss in Siberian sturgeon fed the experimental diets.

Siberian sturgeon fed the experimental diets.

Table 5 Intake (g), digestion (g), retention (%), and discharging (g) of nitrogen and phosphorus in Siberian sturgeon fed with the experimental

diets for 8-week (means ± SE, n = 4)

Two-way ANOVA (P-values)

Values in the same row with different superscripts are significantly different (P < 0.05).

1 N (P) intake (g per tank) = feed consumption (g per tank) · N (P) content of diet/100.

2

Apparent digestibility coefficient (ADC) of N (P) (%) = [1 ) (dietary Y2O3/faecal Y2O3) · (faecal N (P)/dietary N(P))] · 100.

3 Digestible N (P) intake (g per tank) = N (P) intake (g per tank) · ADC of N (ADC of P) of diet.

4 Productive nitrogen value (PNV) (%) = 100 · (whole-body nitrogen gain/nitrogen consumption).

5

Faecal N (P) loss (g per tank) = N (P) intake (g per tank) · [1 ) ADC of N (ADC of P) of diet].

6 Total N (P) loss (g per tank) = N (P) intake (g per tank) ) [final whole-body N (P) content (g per tank) ) initial N (P) whole-body content

(g per tank)].

7 Productive phosphorus value (PPV) (%) = 100 · (whole-body phosphorus gain/phosphorus consumption).

.

sturgeon when dietary Lys and Thr were balanced by CAAs.

In the present study, the ability of Siberian sturgeon to utilize

rendered animal protein was further certified Fish fed with

diet HAPB100 or LAPB100, with 100% APB replacement

and balanced with crystallized Lys, Met and Thr under ideal

protein concept, showed as high growth performance as that

of control group It progressively confirmed that Siberian

sturgeon hold similar characteristic to other carnivorous fish

species, such as rainbow trout, turbot (Psetta maxima),

European seabass (Dicentrarchus labrax) and Japanese

seabass (Lateolabrax japonicus), which can utilize CAAs

efficiently (Fournier et al 2004; Peres & Oliva-Teles 2006;

Nang Thu et al 2007; Hu et al 2010)

Protein is the most expensive component in aquaculture

diets The least cost formulation should be to feed less dietary

protein and to support a desirable performance (Webster

et al 1997) Some advantages of low-protein feed are to

maximally use EAA for protein synthesis but not as energy

source, which would reduce feed cost and decrease nutrients

excretion to environment (Webster et al 1997; Rawles et al

2011) Recent result in sunshine bass (Morone

chrys-ops· M saxatilis) suggested that reduction of 40 g kg)1 of

dietary protein level was possible on ideal protein basis

(Rawles et al 2011) Gaylord & Barrows (2009) found that

dietary protein content of plant-based diets for rainbow trout

could be significantly decreased by supplementing Lys, Met

and Thr without reduction in growth and even with an

improvement in protein retention efficiency Similarly, in the

present study, dietary protein level could be reduced from

400 to 360 g kg)1 with supplementation of limiting amino

acids without affecting growth rate, but higher FCR for

low-protein groups was shown (Table 3) In contrast, it was

reported that reducing dietary 20–40 g kg)1 crude protein

levels would lead to significantly decreased growth

perfor-mance for Japanese seabass (Hu et al 2010) even balanced

amino acids profile on digestible basis These reminded that

except for amino acids profile, dietary palatability and

availability of non-protein energy sources should be

consid-ered when FI was decreased (Nankervis et al 2000; Morais

et al.2001; Ai et al 2004) The GH/IGF-I system plays an

important role not only in mammalian growth (Jones &

Clemmons 1995), but also for fish (Perez-Sanchez & Le Bail

1999; Dyer et al 2004) It has been suggested that the

pitu-itary/hepatic (GH/IGF-I) system was defined as an ÔaxisÕ with

IGF-I mediating the physiological action of GH (Butler & Le

Roith 2001) It is well documented that plasma IGF-I was

positively correlated to growth in Atlantic salmon (Salmo

salar) (Dyer et al 2004), channel catfish (Ictalurus punctatus)

(Li et al 2006), and Nile tilapia (Oreochromis niloticus) (Vera

Cruz et al 2006) Nutritional status affects production ofhepatic and plasma IGF-I in several species of fish, such asgilthead sea bream (Sparus aurata) (Perez-Sanchez et al.1995), coho salmon (Oncorhynchus kisutch) (Larsen et al.2001) and Nile tilapia (Uchida et al 2003) Perez-Sanchez

et al.(1995) reported a positive correlation between plasmaIGF-I hormone and growth rates in gilthead sea bream byregulating nutrition using different feeding and dietary pro-tein levels Go´mez-Requeni et al (2004) reported that total

FM replacement by plant protein in gilthead sea bream nificantly decreased plasma IGF-I, hepatic IGF-I mRNAexpression and growth performance Therefore, GH/IGF-Iaxis could be used as a marker of growth performance andnutritional status in cultured fish (Perez-Sanchez & Le Bail1999; Vera Cruz et al 2006) In the present study, there were

sig-no significant differences in plasma GH (0.72–0.79 ng mL)1)and IGF-I (11.22–15.65 ng mL)1) levels among all treat-ments, which were in accordance with the result of growthperformance At overnight fasting, gilthead sea bream fedwith fish meal-based diet show lower GH levels than that offish fed with plant-based diet (5–15 ng mL)1), but plasmaIGF-I levels decreased with the increase of fish mealreplacement (50–70 ng mL)1) (Go´mez-Requeni et al 2004).The different plasma GH and IGF-I levels might be attrib-uted to different fish species used in the trials

Nutrients (N and P) discharge in the environment isincreasingly concerned in aquaculture production as they arethe most important pollution sources Nutrients discharge isaffected by several factors including diet digestibility, FI,dietary nutrients contents and protein sources (Talbot &Hole 1994; Cho & Bureau 1997; Cai et al 2005) Dietarytotal N excretion increased linearly and significantly with thedietary protein levels as it increased for European seabass(Ballestrazzi et al 1994) and juvenile white shrimp, Litope-naeus vannamei(Gonzalez-Felix et al 2007) Similarly, in thepresent study, fish fed with the high-protein diets showedhigher total N (P) losses than those of fish fed with the low-protein diets, which were associated with the increased N (P)intakes (Table 5, Figs 2 & 3) Except MBM, digestibility of

N and P of PBM, HFM and SDBM was similar to that of

FM for Siberian sturgeon (Liu et al 2009) Phosphorus isone of the most important pollution sources from aquacul-ture P is a component of different chemical compounds infeeds, including bone-P, phytate-P, organic P, calcium (Ca)monobasic supplement, etc Bone-P is the main form for both

of FM and terrestrial animal protein (PBM and MBM) Hua

& Bureau (2006) established mathematical models toestimate the P digestibility of ingredients for salmonids.They reported the negative interaction of bone-P and Ca

.

Aquaculture Nutrition 18; 493–501  2012 Blackwell Publishing Ltd

monobasic Pi supplement In the present study, 6 g kg)1 of

calcium phosphate monobasic was supplied for all

experi-mental diets, and bone-P was decreased with higher APB

inclusion levels The lower digestible P for APB diets should

be owing to the lower P digestibility of MBM (Hua & Bureau

2006; Liu et al 2009)

In conclusion, results of the present study showed that

(i) dietary protein level could be reduced to 360 g kg)1from

400 g kg)1without affecting WGR or SGR and significantly

reduced N and P losses, (ii) furthermore, dietary FM protein

can be totally replaced by APB in feed formulation either at

400 g kg)1or at 360 g kg)1 protein level, without negative

effects on growth performance of the Siberian sturgeon

Financial support was provided by National Natural Science

Foundation of China Project No 31072220, the Special

Fund for Agro-Scientific Research in the Public Interest

(201203015)

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1 1 1 2 1

1

Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Shimoarata, Kagoshima, Japan;

2

Fisheries Science on Resources and Environment, The United Graduate School of Agricultural Science, Kagoshima

University, Korimoto, Kagoshima, Japan

A 2· 3 factorial design with triplicates examined the

interaction between dietary inorganic phosphorus (IP) and

phytase on growth, mineral utilization and phosphorus (P)

mineralization in juvenile red sea bream The treatments

were three levels of dietary IP supplementation at 0, 2.5 and

5 g kg)1, either without or with phytase supplementation

[2000 FTU kg)1; phytase unit is defined as the amount of

enzyme activity which liberates 1 micromol of inorganic

phosphorus per minute at pH 5.5 and 37C at a substrate

concentration (sodium phytate) of 5.1 mmol L)1] Juvenile

red sea bream (IBW = 1.3 g ± 0.1) were stocked twelve

fish per tank and fed for 50 days Growth and feed

efficiency were significantly (P < 0.05) enhanced by both

dietary P and phytase supplementation Feed intake and

survival rate were not significantly affected by the dietary

treatments Both dietary IP and phytase supplementation

significantly increased plasma IP and Mg levels

Concen-tration of vertebral mineral and scale P was significantly

increased by both dietary treatments A skeletal

malfor-mation syndrome of scoliosis occurred in fish fed both

non-IP and non-phytase supplemented diet Interaction between

main dietary effects was detected for vertebral Zn, scale P

and whole-body ash and Mg content With regard to

growth and other examined productivity traits, phosphorus

requirement of juvenile red sea bream can be met if

sup-plemented with 2000 FTU phytase kg)1or in the absence of

phytase, by dietary inclusion of 2.5–5 g kg)1 of IP

key words: digestibility, feed intake, phosphorus, phytase,

red sea bream, scoliosis

Received 29 January 2011, accepted 18 August 2011 Correspondence: Asda Laining, Research Institute for Coastal Aquacul- ture, Ministry of Marine Affairs and Fisheries, Jl Makmur Dg Sitakka

No 129, Maros, South Sulawesi, Indonesia E-mail: asdalaining@

yahoo.com

Phosphorus (P) is an essential element in the diet of all tebrate animals including fish Its requirement for growth,bone mineralization, synthesis of nucleic acids, reproductionand energy metabolism has been well documented (Lovell1989; Lall 2002; Sugiura et al 2004) Thus, deficiency of Phas implications not only for hard tissues but also for dis-turbances of intermediary metabolism leading to impairment

ver-of growth To fulfil the P requirement, fish must obtain Pfrom dietary source (NRC 1993) because freshwater and seawater are low in P concentration ranging from 0.005 to0.07 mg L)1(Boyd 1971) Various studies have been reportedregarding the P requirements for fish species (NRC 1993) andother animals (NRC 1994, 1998) However, the lack ofaccurate data on P requirements of fish was of minorimportance in practical diet formulations that were mainlyfishmeal-based as the amount of P in these diets considerablyexceeded the estimated requirements (Rodehutscord &

Pfeffer 1995) Excess dietary P is excreted by fish, which mayhave an undesirable environmental impact because ofeutrophication effect of the discharged P

In recent years, there has been a trend towards using plantprotein sources as partial or total replacements of the fishmeal

in aquaculture diets because they are comparatively sive and readily available However, the presence of antinu-tritional factors (ANFs) such as trypsin inhibitor and phytic

acid in plant protein ingredients has been a significant

impediment to their use in fish diet (Francis et al 2001) Phytic

acid, or myo-inositol hexakisphosphates (IP6), is the main

storage form of both phosphate and inositol in plant seeds and

grains Even though IP6 can constitute up to approximately

70% of the total P in plant seeds per grains, fish are not able to

effectively utilize the P bound to IP6 owing to the negligible

amounts of the endogenous phytase in their digestive tract

Moreover, IP6 is a polyanionic molecule with the potential to

chelate positively charged nutrients particularly cations such

as Ca, Mg and Zn This is almost certainly a major factor

contributing to the antinutritive properties of IP6 Poor P

availability and retention (Storebakken et al 1998; Laining

et al.2010a; respectively) and impaired mineral utilization, in

particular Ca (Papatryphon et al 1999; Fredlund et al 2006),

Mg (Pallauf & Pietsch 1998; Denstadli et al 2006) and Zn

(Denstadli et al 2006), have been documented as negative

properties of IP6 in fish

Phytase (myo-inositol hexakisphosphate

phosphohydro-lases) has been studied recent years because of the interest in

applying this enzyme in diets to make available the phosphate

group of IP6 as fish lack the endogenous phytase By

increasing the availability of P from plant ingredients in the

diet, less of the plant P will be discharged to environment

(Vielma et al 2002; Dalsgaard et al 2009) In addition,

inclusion of exogenous phytase may help to improve the

uptake of certain trace minerals and reduce the amount of

inorganic P that otherwise might be needed to be included in

the diet, thereby lowering feed costs (Selle & Ravindran 2007)

Some of the most critical factors affecting phytase efficacy

in vivo are pH stability, (Kemme et al 2006), source of

phytase (Liebert & Portz 2007) and composition of minerals

in diet (Applegate et al 2003; Tamim et al 2004) Low

die-tary levels of Ca and P and narrow Ca/P ratio appear to

facilitate the activity of exogenous phytases in poultry (Qian

et al.1997; Manangi & Coon 2007) Similar studies on fish

species so far are still very limited In our previous study with

tiger puffer, Takifugu rubripes, (Laining et al 2010b) an

interaction effect between Ca/P ratio (achieved by

supple-menting different levels of Ca at constant level of P) and

dietary phytase on several traits including specific growth

rate (SGR), feed intake (FI), whole-body Zn, digestibility of

P and Zn as well as vertebral P and Zn was observed It was

concluded that based on growth performances, dietary Ca/P

ratio of 0.5 (without Ca supplementation) with 2000 FTU

phytase kg)1might be the optimum dietary supplementation

for tiger puffer

In the present experiment, diet containing different levels

of dietary inorganic phosphorus (IP) with constant level of

Ca was formulated either without or with dietary phytase at

2000 FTU phytase kg)1and fed to juvenile red sea bream todetermine the main and interactive effects on growth, mineralutilization and P deficiency sign in the fish

This experiment was arranged according to a 2· 3 factorial(two levels of dietary phytase and three levels of dietary P)with triplicates Six diets were formulated as detailed inTable 1 The three levels of dietary P were achieved by sup-plementing IP at 0, 2.5 and 5 g kg)1 Sodium monophos-phate (NaH2PO4) was used as the IP source Two levels ofphytase were obtained by adding commercial phytase(Ronozyme P5000 (CT); DSM Nutritional Product Ltd,Basel, Switzerland) at 0 and 2000 FTU kg)1diet Concen-tration of phytase applied in this experiment was based onour previous experiment (Laining et al 2006) This phytase isproduced from Peniophora lycii by submerged fermentation

of a genetically modified Aspergillus oryzae strain withactivity of 5000 FTU g)1 Fishmeal, krill meal and soybeanprotein isolated were used as protein sources Pollack liver oiland highly unsaturated fatty acid served as lipid sources Thechemical composition of test diets is shown in Table 2.All dry ingredients were weighed and mixed, and then,the lipid source was added and mixed again until homoge-nized Deionized water was added (300 mL kg)1dry ingre-dients mixture) and mixed again The wet dough waspelletized using meat chopper and then dried with oven at

40C until moisture content around 10% Dried pellet wasstored at)20 C until required to feed to the fish

Three hundred juvenile red sea bream, Pagrus major, werepurchased from a private fish hatchery (Ogata Suisan Co,Kumamoto, Japan) The red sea bream were pooled into asingle population and maintained on a commercial diet(Higashimaru, Kagoshima, Japan) for acclimatization Atthe start of the feeding trial, fish with average initial bodyweight 1.3 g were randomly distributed into 18 tanks of

100 L capacity Each tank was stocked with 12 fish, and threereplicate tanks were used for each dietary treatment Filteredsea water was supplied to each tank with a flow-throughsystem (1.5 L min)1) and aerated using air–stone diffusers.Fish were fed test diets to apparent satiation twice a day

at 8:00 and 16:00 for 50 days Fish were weighed every

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Aquaculture Nutrition 18; 502–511  2011 Blackwell Publishing Ltd

10 days Water temperature and salinity ranged between

22 and 28C and between 33 and 34 g L)1, respectively

At the end of the 50-day feeding period, three fish from each

tank representative of the mean body weight of fish in the

tank were euthanized, freeze-dried, blended and stored at

)20 C until analysed for chemical composition A bloodsample was taken from another three fish in each tankfollowing anaesthetization in a water bath containing2-phenoxyethanol (Wako Pure Chemical Industries, Ltd,Tokyo, Japan) at 200 mg L)1 Blood was drawn from thecaudal vein using heparinized (1500 IU mL)1) syringe(1 mL, needle size 25G· 1; Terumo Co., Tokyo, Japan) andpooled into a 1.5-mL microtube Blood samples were

Table 1 Formulation of experimental diet (g kg )

Powesh A; Oriental Yeast Co, Ltd, Tokyo, Japan.

3 Vitamin mixture (g kg)1diet): b-carotene 0.0256; vitamin D3 0.0253; menadione 0.1223; a-tocopherol acetate 1.0267; thiamine nitrate

0.153; riboflavin 0.513; pyridoxine–HCl 0.1227; cyanocobalamin 0.00024; d-biotin 0.0153; inositol 10.264; nicotinic acid 2.052;

Ca–panto-thenate 0.7188; folic acid 0.0384; choline chloride 20.984; q-aminobenzoic acid 1.022; cellulose 3.798.

4 L-ascorbyl-Na/Ca (DSM Nutritional Product Ltd).

5

Calcium/phosphorus-free mineral mixture (g kg)1diet): KCl 1.856; MgSO4Æ5H2O 5.067; Fe Citrate 1.098; Al(OH)3 0.0069; ZnSO4Æ7H2O 0.132;

CuSO4 0.0037; MnSO4Æ5H2O 0.029; K(IO3)2 0.006; CoSO4Æ7H2O 0.037; Cellulose 31.75.

6 Wako Pure Chemical Industries, Ltd.

7 Ronozyme P5000; DSM Nutritional Product Ltd, Basel, Switzerland (declared activity = 5000 FTU g)1product).

8

Attractant (g kg)1diet): betaine 2.0.

Table 2 Proximate composition and mineral content of diet containing different amount of dietary inorganic P (IP) and microbial phytase