Aquaculture 290 (2009) 290295

Contents lists available at ScienceDirect

Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Transformation of trichothecene mycotoxins by microorganisms from sh digesta

Shu Guan a,b, Jianwei He b, J. Christopher Young b, Honghui Zhu b, Xiu-Zhen Li b, Cheng Ji a, Ting Zhou b,
a b

National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100193, China Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph, Canada N1G 5C9

a r t i c l e

i n f o

a b s t r a c t
Trichothecene mycotoxins are commonly found in cereals worldwide and bring signicant threats to the food industry and animal production. The aim of this research was to search for microbes from sh guts capable of transforming trichothecenes into less toxic compounds. Digesta of 62 shes from nine species were screened for their ability to transform 4-deoxynivalenol (DON). Liquid chromatography-mass spectrometry was used to determine the reduction of DON concentrations and structures of DON-transformation products. The microbial community from one catsh Ameiurus nebulosus, namely microbial culture C133, completely transformed DON to deepoxy DON (dE-DON) at 15 C in full medium after 96 h incubation. Various media and culture conditions were tested to evaluate their effect on DON transformation. Microbial culture C133 maintained high transformation ability over a broad range of temperatures from 4 to 25 C and pH values from 4.5 to 10.4. The transformation of DON to dE-DON was enhanced in a rich medium such as full medium, nutrient broth and corn meal broth. Microbial culture C133 was then tested for its ability to transform other trichothecene mycotoxins; most of the toxins were transformed to deacetyl and/or deepoxy products. This is the rst report on trichothecene transformation by microbes from the intestinal tract of sh. Crown Copyright 2009 Published by Elsevier B.V. All rights reserved.

Article history: Received 22 December 2008 Received in revised form 16 February 2009 Accepted 17 February 2009 Keywords: Ameriurus nebulosus Catsh Microbial culture Trichothecene Mycotoxin Transformation

1. Introduction Trichothecenes are a group of mycotoxins commonly found in wheat, barley and corn primarily infected with Fusarium spp. They have been shown to reduce performance, affect reproductive systems, impair immune function in livestock and aquatic species, and pose a severe threat to humans consuming contaminated cereal products (Woodward et al., 1983; Arukwe et al., 1999; Eriksen et al., 2004; Diaz, 2005; Morgavi and Riley, 2007; Zhou et al., 2008). Mycotoxins pose a potential threat to aquaculture since the plant feedstuffs that have been increasingly used in sh and shrimp diets over the past decade may be contaminated with these deleterious substances (Spring and Fegan, 2005; Fegan and Spring, 2007). Studies in sh have shown that trichothecene mycotoxins cause performance reduction, immune impairment and organ lesions (Arukwe et al.,1999; Manning, 2004; Manning et al., 2003), which are similar to clinical signs of livestock such as cattle, poultry, swine and sheep when they consumed feed contaminated with mycotoxins (Desjardins, 2006). Transformation of trichothecene mycotoxins to different compounds by naturally occurring microorganisms has been demonstrated since the

Abbreviations: 3ADON, 3-acetyldeoxynivalenol; amu, atomic mass units; APCI, atmospheric pressure chemical ionization; dA, deacetyl; dE, deepoxy; dV, deisovaleryl; DAS, diacetoxyscirpenol; DON, 4-deoxynivalenol; FUS, fusarenon X; LC, liquid chromatography; MS, mass spectrometry; NEO, neosolaniol; NIV, deacetyl fusarenon X (Nivalenol); UV, Ultraviolet; VER, verrucarol. Corresponding author. Tel.: +1 519 780 8036; fax: +1 519 829 2600. E-mail address: Ting.Zhou@agr.gc.ca (T. Zhou).

early 1960s (Horvath and Varga, 1961) and mixed cultures of microorganisms from various sources such as animal guts, soil and plants have been found to possess the ability to transform a variety of toxins (Yoshizawa et al.,1983; Kiessling et al., 1984; Swanson et al., 1988; Beeton and Bull, 1989; He et al.,1992; Kollarczik et al., 1994; Matsushima et al., 1996; Volkl et al., 2004; Zhou et al., 2005). However, such microorganisms have not been reported from sh gut. Mycotoxin-transforming microorganisms have been investigated with some success for their potential applications in detoxifying mycotoxins in contaminated food and feed (Karlovsky, 1999; Volkl et al., 2004; Zhou et al., 2008). Typically, the microorganisms obtained from different sources are only active under specic conditions that are similar to where they have been isolated. These conditions often include nutrition, presence or absence of oxygen (aerobic vs. anaerobic), pH and temperature. Microorganisms originating from aquatic species should have signicant advantages when used to detoxify mycotoxins in aquaculture industry, as compared with the use of microorganisms obtained from other sources. Microbial populations in sh digestive tracts have been well studied (Trust and Sparrow, 1974; Lindsay and Harris, 1980; Lesel et al., 1986). These populations grow mainly upon the food consumed by the host animal and digestive secretions (Lesel, 1993). The sh gut bacterial ora represents a diversied enzymatic potential, and it is logical to propose that the bacteria themselves or the enzymatic metabolites might interfere with the feedstuff consumed by the host animal. Therefore, there is a high probability of discovering trichothecene-transforming microorganisms from sh intestinal systems. The purpose of this study was to search for microorganisms from various types of sh gut that could transform trichothecenes. Fishes

0044-8486/$ see front matter. Crown Copyright 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2009.02.037

S. Guan et al. / Aquaculture 290 (2009) 290295

291

belonging to different species from local ponds or rivers were collected and their digesta were tested for mycotoxin-transformation activities. One microbial community, originating from catsh (Ameriurus nebulosus) gut and designated microbial culture C133, showed an effective ability to transform DON to dE-DON (also reported in the literature as DOM-1). The ability of the culture C133 to transform other trichothecene mycotoxins has also been examined. Additionally, we studied mycotoxin transformation efcacy and conditions of the microbial culture, providing new information for an understanding of microbial detoxication of mycotoxins. 2. Materials and methods 2.1. Fishes and their digesta Table 1 presents the feeding habits, average body length and number of the 62 adult freshwater sh from 9 species with different feeding habits. The sh were sampled from several ponds and rivers around Guelph, Ontario, August to November 2007 and April to June 2008. Water temperatures varied between 10 and 18 C during the sampling period. The gut contents were collected from alimentary tracts within 10 h of the sh being caught. The gut contents were diluted with sterile distilled water and then used for the DON transformation test. 2.2. Mycotoxins and antibiotics The mycotoxins 3-acetyldeoxynivalenol (3ADON), diacetoxyscirpenol (DAS), 4-deoxynivalenol (DON), fusarenon X (FUS), deacetyl fusarenon (NIV, Nivalenol), neosolaniol (NEO), and verrucarol (VER) were obtained from Sigma-Aldrich (Oakville, ON). Standard solutions were prepared in methanol or water. They were diluted with methanol or water to make stock solutions of 500 g/mL concentration. The antibiotics, streptomycin sulfate and penicillin G, were obtained from Sigma-Aldrich (Oakville, ON). Stock solutions (10,000 g/mL) were prepared in methanol. Structures of the various mycotoxins are shown in Fig. 1. 2.3. Culture media The following media were used for screening microbial cultures for DON transformation ability: nutrient broth (NB), tryptone soya broth (TSB), lauria bertani (LB), corn meal broth (CMB), minimal medium (MM), full medium (FM) and sole nitrogen media (SN). NB, TSB and LB were obtained from Sigma-Aldrich (Oakville, ON). Corn meal broth (CMB): 40 g corn meal soaked in 1 L water at 58 C for 4 h, allowed to stand for 2 h, and then ltered through Whatman No. 1 lter paper (Whatman; Maidstone, Kent, UK). To 1 L of the ltrate was added 3.0 g (NH4)2SO4, 1.0 g K2HPO4, 0.5 g MgSO4, 0.5 g K2SO4, 0.01 g FeSO4,
Table 1 Fish samples tested for ability to transform trichothecene mycotoxins. Species Ameiurus nebulosus Esox lucius Common name Feeding habits Average Sample length (cm) numbers 28 45 40 30 15 20 22 26 36 39 1 13 1 3 1 2 1 1

Fig. 1. Structures of the various mycotoxins concerned. This graph was drawn using ChemDraw ActiveX/Plugin Viewer 11.0, CambridgeSoft Corporation.

0.007 g MnSO4, and 5.0 g yeast extract, pH 7.2. FM contained 5.0 g yeast extract, 10.0 g nutrient broth (Sigma), 1.0 g glucose, 1.75 g K2HPO4 and 0.75 g KH2PO4/L, pH 7.2 (Volkl et al., 2004). SN medium was composed of 10.0 g nitrogen source, 6.0 g glucose, 1.75 g K2HPO4, 0.75 g KH2PO4 and 5.0 g NaCl/L. The following nitrogen sources were used, respectively: tryptone, peptone, yeast extract, (NH4)2HPO4 and NH4NO3. Media were solidied with agar when required. 2.4. Transformation of trichothecene mycotoxins by sh gut microbes Transformation tests of DON by sh gut microbes were carried out in liquid medium. In the tests, DON was adjusted to a nal concentration of 50 g/mL. Five mL of sterilized water was added to 5 mL of sh digesta and mixed by vortex. The mixtures were used as microbial inocula. An aliquot of 100 L of the individual inocula suspension was mixed with 100 L of 500 g/mL aqueous DON solution and 800 L of FM medium. The inoculated mixtures were incubated in the dark at 15 C without shaking for 96 h before they were extracted and then analyzed by liquid chromatography-mass spectrometry (LC-MS) according to the methods in the following section. A sh digesta diluted with sterile water without DON served as a blank control; the autoclaved digesta dilution with addition of DON served as a physical absorption control; and the digesta dilution passed through a 0.22 m lter plus DON served as a chemical reaction control. Transformation tests of other trichothecene mycotoxins were carried out in the same way. For 3ADON and FUS, the 96 h reaction mixtures were transferred to fresh FM medium and incubated for another 7 days. 2.5. Effects of media, incubation length, temperature, pH and antibiotic on transformation of DON by microbial culture C133 To nd appropriate media for DON transformation, microbial culture C133 was tested in NB, TSB, LB, CMB, MM, FM and SN media.

Brown bullhead Fish, insects and plants catsh Northern pike Fish, water voles and ducklings Micropterus Largemouth Fish, frogs and mammals salmoides bass Catostomus White sucker Small invertebrates and commersonii plant Perca Yellow perch Minnows,maggots avescens and craysh Lepomis Bluegill sunsh Small invertebrates macrochirus and sh Pomoxis White crappie Insects and crustaceans annularis Salmo trutta Brown trout Drifting invertebrates Oncorhynchus Pink salmon Small crustaceans gorbuscha and sh

292

S. Guan et al. / Aquaculture 290 (2009) 290295

Fig. 2. Transformation of DON by microbial culture C133 in FM medium at 15 C. Starting concentration of DON was 50 g/mL.

Fig. 4. Transformation of DON by microbial culture C133 at different temperatures. The culture was incubated with 50 g/mL DON for 96 h at each designed temperature.

The tests were carried out at 15 C for 72 h, respectively. To determine optimal incubation time, DON was incubated with culture C133 in FM medium at 15 C for 48, 72, 96, 120 and 168 h, respectively. A wide range of temperatures was tested by incubation of DON in FM medium with culture C133 for 96 h at 4, 7.5, 15, 20, 25 and 37 C, respectively. The optimal initial pH value was determined by adjusting the pH of the culture C133 inoculated FM medium with DON to 2.0, 3.8, 4.5, 5.0, 7.2, 7.6, 8.2, 8.9, 9.4, 9.9, 10.4, respectively. The tests were carried out at 15 C for 96 h. FM medium with DON but without culture C133 adjusted to the same pH values were set as controls. The antibiotic solutions were added to the FM inoculated with the culture C133 to the nal concentration of 200 g/mL. DON was added to the mixtures for a nal concentration of 50 g/mL 24 h after addition of the antibiotics. The test was conducted at 15 C for 8 days. 2.6. Analysis of mycotoxins and transformation products The analysis of mycotoxins and their transformation products was done according to the method of Young et al. (2006). To each 500 L subsample of suspension cultures, 500 L methanol was added. The mixture was allowed to stand for 2 h and centrifuged at 6000 g for 10 min before being analyzed by LC-MS. Identication and quantication of tested mycotoxins and their transformation products were achieved using a Finnigan LCQ DECA ion trap LC-MS instrument (ThermoFinnigan, San Jose, CA, USA). The HPLC system was equipped with a Finnigan SpectraSystem UV6000LP ultraviolet (UV) detector and an Agilent Zorbax Eclipse XDB C18 column (4.6 150 mm, 3.5 m). The binary mobile phase consisted of solvent A (methanol) and solvent B (water). The system was run with a gradient program: 25% A at 0 min, 2541% A for 5 min, isocratically at 41% A for 2 min and 4125% A for 1 min. There was a 3 min post-run at its starting condition to allow for the equilibration of the column. The ow-rate was controlled at 1.0 mL/min. UV absorbance was measured at 218 nm. Atmospheric pressure chemical ionization (APCI) positive ion mode was used for MS detection. DON standard was used to tune the instrument to its maximum response. The system was operated as follows: shear gas

and auxiliary ow-rates were set at 95 and 43 (arbitrary units); voltages on the capillary, tube lens offset, multipole 1 offset, multipole 2 offset, lens, and entrance lens were set at 30.00, 17.00, 3.70, 8.50, 35.00, and 42.00 V, respectively; capillary and vaporizer temperatures were set at 200 and 450 C, respectively; and the discharge needle current was set at 7 A. Identication of mycotoxins was achieved by comparing their retention time, UV spectra and mass spectrum with those of their standards. Quantication of DON and products was based on their UV and MS calibration curves. All tests were run in triplicate and means of the results were calculated. 2.7. Statistical analysis The results were means of triplicate incubations and were expressed as mean standard error. Data were analyzed as a completely randomized single factor design by ANOVA using the general linear model procedure in SAS. Signicant F tests at the 0.05 levels of probability are reported. When a signicant F-value was detected, Duncan's Multiple Range Test was used to determine signicant differences among means. 3. Results 3.1. Screening for DON-transforming microorganisms from sh digesta Among digesta of the 62 sh screened, DON reduction was detected in only one of the samples, which was from digesta of a brown bullhead catsh (Ameiurus nebulosus). The microbial community derived from the sample, named culture C133, completely transformed DON to deepoxy DON (dE-DON, Fig. 1) when it was incubated in FM medium with DON at 15 C for 96 h. No reduction of DON levels was detected for other sh gut samples screened under the same or different conditions. 3.2. Transformation of DON by microbial culture C133 DON concentration in FM medium with microbial culture C133 decreased by 43.8 and 100% after incubation for 72 and 96 h,

Fig. 3. Transformation of DON by microbial culture C133 in different media. The culture was incubated in each medium with 50 g/mL DON at 15 C for 72 h. MM: minimal medium (MM); LB: lauria bertani; TSB: tryptone soya broth; NB: nutrient broth; CMB: corn meal broth; FM: full medium.

Fig. 5. Transformation of DON by microbial culture C133 in FM media at different pH values. The DON (50 g/mL) was incubated in the media at 15 C for 96 h.

S. Guan et al. / Aquaculture 290 (2009) 290295

293

respectively (Fig. 2). The incubation was extended to 168 h and only dE-DON was detected in the mixture. When culture C133 was tested in different media with 50 g/mL DON, the most efcient medium was FM medium, in which the concentration of DON was reduced to 21.9 g/mL (i.e. 56.2% of DON transformed) when incubated for 72 h (Fig. 3). No transformation was observed in MM and LB media during the same periods. DON transformation was comparatively low in media with solo nitrogen such as media containing only tryptone, peptone, yeast extract, (NH4)2HPO4 or NH4NO3. Transformation of DON by culture C133 prefers lower temperature; it was observed at temperatures as low as 4 C. The optimum temperature for the transformation was 15 C. No transformation was observed at 37 C (Fig. 4). The effect of initial pH level of culture medium on DON transformation is shown in Fig. 5. Culture C133 was able to transform DON to dE-DON under a wide range of pH values from 4.5 to 10.4; no transformation occurred at pH levels below 4.5. The highest percentages of DON to dE-DON transformation were detected at pH levels of 7.2 to 8.9. pH 7.2 was chosen for further DON transformation tests. The presence of microbial cells was necessary for DON to dE-DON transformation. No transformation was observed when the culture C133 was either autoclaved or ltered through a 0.22 m membrane lter (data not shown). Although culture C133 treated with penicillin G (200 g/mL) was still able to completely transform DON to dE-DON, streptomycin completely eliminated the transformation ability. 3.3. Transformation of other trichothecene mycotoxins by microbial culture C133 Microbial culture C133 was also examined for its ability to transform other trichothecene mycotoxins. Deacetylation and deepoxidation were the major transformation reactions. Table 2 summarizes the results. The three non-acetylated trichothecenes DON, NIV and VER were essentially completely transformed to their corresponding deepoxy metabolites (Fig. 1). The acetylated trichothecenes reacted differently. For monoacetyl 3ADON, only 40.9% transformation was observed; after the acetyl group was removed (36.7%), there was only 4% subsequent deepoxidation. No transformation occurred with monoacetyl FUS. However, when the incubation was extended, there was very little unreacted material and
Table 2 Product ratios for treatment of selected trichothecene mycotoxins with the digesta from catsh gut. Mycotoxina Alcohol DON NIV VER MonoAcetyl 3ADON 3ADONi FUS FUSi DiAcetyl DAS NEO
a b c d e f g h i

about 96% the products from both 3ADON and FUS resulted in sequential deacetylation and then deepoxidation. The diacetyl trichothecenes DAS and NEO both showed unreacted material (42.0 and 71.5%, respectively). Monodeacetylation (28.5%) was the only transformation step observed in NEO. However, in DAS treatment, the major product (54.2%) resulted from sequential deacetylation followed by deepoxidation. There was no evidence for dideacetyated products. 4. Discussions This study found the presence of microorganisms capable of transforming trichothecene mycotoxins from the digesta of brown bullhead catsh. To our knowledge this is the rst report on mycotoxin-transforming microorganisms in sh gut (digesta), although a number of studies have reported transformation of trichothecenes by microorganisms from other animal intestinal systems, such as rumen uid, chicken, rat, and swine digesta (Kiessling et al., 1984; King et al., 1984; Prelusky et al., 1988; Swanson et al., 1988; Beeton and Bull, 1989; Young et al., 2007). With the increased use of plant feedstuffs in feed for aquatic animals, the deleterious effects of mycotoxins are becoming a threat to the aquatic animal industry. The risk arises not only from the introduction of toxins into feeds during manufacture but also from increased toxin production during storage (Spring and Fegan, 2005). The discovery of the mycotoxin-transforming microorganisms may provide new opportunities for managing mycotoxin problems in aquaculture. It has been found that sh harbour a large number of microbial populations in their digestive tracts. Many studies have characterized functional metabolites by gut microbes from a variety of sh species over the past few years (David et al., 1994; Erasmus et al., 1997; Bairagi et al., 2002; Saha et al., 2006; Rae et al., 2007; Ray et al., 2007). The identication of the active microbial culture C133 has clearly demonstrated that sh gut microorganisms can be resources for mycotoxin-transforming microorganisms although more research is needed to explain why only one sample showed signicant activity in reducing DON levels in the media among more than 60 shes of nine species that were screened using various media and cultural conditions. Mycotoxin-transforming microorganisms may be present in sh with frequent exposure to mycotoxins, thus, the quality of the water, habits of the sh, or even the sampling time may be signicant determinant factors for their existence. Manning (2004) has reported that channel catsh are more tolerant to dietary DON up to a level of 10 g/mL compared to other shes; this plus the habits of bottom feeding and plants as food sources may partially explain the identication of mycotoxin-transforming microorganisms from a brown bullhead catsh instead of other types of shes. Transformation of trichothecene mycotoxins by microbial culture C133 occurred mainly via deacetylation and deepoxidation pathways. At the outset, deepoxidation was presumed to be the primary metabolic reaction by microbial culture C133. This proved to be the case for the three non-acylated trichothecenes: DON (in agreement with Young et al., 2007), NIV (Hedman and Pettersson, 1997; Eriksen et al., 2002) and VER, where essentially complete transformations to the deepoxy metabolites were observed. The 12, 13-epoxide group has been demonstrated as being essential for the toxicity of trichothecenes although the number and position of acetyl groups are also known to inuence the toxicity (Ehrlich and Daigle, 1987; Rotter et al., 1996). Many studies have shown that the toxicity of dE-DON is much lower compared with DON (Eriksen et al., 2004). Under the same conditions that DON was completely deepoxidized, microbial treatment of the acylated trichothecenes producted different products. For 3ADON, both deacetylation and de-epoxidation were detected. Similar trends were found in microbial treatment by pig faeces (Eriksen et al., 2002) and chicken intestinal microbes (Young et al., 2007). Interestingly, FUS, another monoacetylated trichothecene, failed to react under these conditions. However,

UnRxb 0 0 0

dEc 100 100 92.7 2.7h

dAd nag na na

dAdEde na na na

Unkf

7.3 2.7

59.1 8.7 0 100 4.6 2.5

0 0 0 0

36.7 5.5 3.5 1.7 0 0

4.3 3.4 96.5 2.5 0 95.4 2.5

42.0 16.0 71.5 14.8

0 0

3.7 1.3 28.5 14.8

54.2 14.7 0

The structures of the mycotoxins are shown in Fig. 1. Unreacted material. Deepoxidation. Deacetylation. Deepoxidation deacetylation. Unknown. Not applicable. Mean standard error for triplicate determinations. Extended incubation.

294

S. Guan et al. / Aquaculture 290 (2009) 290295

when the FUS reaction mixtures were transferred to fresh FM medium and incubated for another 7 days, 95.4% of the FUS was transformed and the major product resulted from deacetylation and de-epoxidation, presumably in that order. The diacetyl trichothecenes DAS and NEO both showed unreacted material. Monodeacetylation was observed in both cases. A minor product (3.7%) dA-DAS had congruent LC retention times and mass spectra with 15-monoacetoxyscirpenol (15-MAS), which strongly suggests preferential loss of the C-3 acetyl. The relative stability of the C-15 acetyl can be explained by steric hindrance at that position as noted by Young et al. (2007). Presumably the sh microbes then quite readily deepoxidized dA-DAS (MAS) to dAdE-DAS (de-MAS), the major product (54.2%). For NEO, dA-NEO is the only product with no evidence for dENEO or dAdE-NEO. Given the consistent stability of the C-15 acetyl, it is reasonable to propose that NEO preferentially loses the C-4 acetyl. Swanson et al. (1987, 1988) demonstrated that the products obtained from treatment of DAS were dependent on the microbial source. Microbes from cattle rumen uid and rat intestine, as well as faecal material from cattle, rat, and swine produced deepoxidation and deacetylation products. Faecal microbes from chickens, horses and dogs yielded only deacetylation products. The same pattern was also found by rumen protozoa (Kiessling et al., 1984) and 14 strains of rumen bacteria (Matsushima et al., 1996). Test conditions, such as nutrition, temperature, and pH, can signicantly affect the effectiveness of mycotoxin-transforming microorganisms. Microorganisms originated from different sources often require different conditions, which may limit their applications in mycotoxin detoxications. Medium is an important factor in trichothecene transformation. Microbial culture C133 requires rich nutrients such as FM, CMB and NB for trichothecene transformation. Since sh gut is a complex matrix, it is reasonable to propose that gut microbes favor rich medium. Several studies have reported transformation of trichothecene mycotoxins by microorganisms from animal gut contents, rumen uid and faeces. However, most of these microorganisms showed activity only under relative high temperatures over 30 C (Westlake et al., 1987; Swanson et al., 1988; Kollarczik et al., 1994). King et al. (1984) observed DON transformation by rumen microorganisms under 40 C. Transformation of DON to 3-keto-DON by a bacterial isolate from soil was achieved at 30 C (Shima et al.,1997). Relatively high temperature of over 30 C was also required for transformation of trichothecenes by bacterial isolates from chicken intestines (Young et al., 2007). In the current study, microbial culture C133 from catsh digesta completely transformed DON to dE-DON within 96 h of incubation at 15 C. Interestingly, culture C133 maintained a DON transformation ability at low temperatures (4 and 7.5 C) whereas this ability was lost at 37 C. Since aquatic animal production is mainly under relative low temperatures, the culture C133 should be more suitable for application in aquaculture. Most of the reported microbial transformations of trichothecene mycotoxins were conducted in neutral medium (Ueno et al., 1983; Beeton and Bull, 1989; Swanson et al., 1988; Shima et al., 1997). He et al. (1992) observed that the optimal pH for DON transformation function by chicken gut bacteria was in the pH range of 5.72 and 6.91; low pH 5.2 completely inhibited DON transformation activity. In the current study, microbial culture C133 maintained high transformation activity over a wide pH range from 4.5 to 10.4, indicating a broader range of possible incubation conditions and thus fewer limitations in its potential application. 5. Conclusions Microbial culture C133 from catsh digesta has shown the ability to transform DON to dE-DON, a product much less toxic than DON. The optimum conditions required for this transformation have been determined; the culture C133 is able completely to transform DON in FM medium at 15 C over 96 h. Culture C133 is also capable of

transforming other trichothecene mycotoxins either through deepoxidation or deacetylation pathways. The discovery of this active culture and knowledge obtained in this research provide a basis for the identication of the pure bacterial isolates responsible for the microbial transformations and for future applications to microbial mycotoxin detoxication in aquaculture and livestock production. Acknowledgement Financial support by Agriculture and Agri-Food Canada and the National Natural Science Foundation of China (Contract number: 30571353) is gratefully acknowledged. References
Arukwe, A., Grotmol, T., Haugen, T.B., Knudsen, F.R., Goksyr, A., 1999. A sh model for assessing the in vivo estrogenic potency of the mycotoxin zearalenone and its metabolites. Sci. Total Environ. 236, 153161. Bairagi, A., Ghosh, K.S., Sen, S.K., Ray, A.K., 2002. Enzyme producing bacterial ora isolated from sh digestive tracts. Aquac. Int. 10, 109121. Beeton, S., Bull, A., 1989. Biotransformation and detoxication of T-2 toxin by soil and fresh water. Appl. Environ. Microbiol. 55, 190197. David, W., Brown, C., McCoy, P., George, E.R., 1994. Experimental feeding of Fusarium moniliforme culture material containing fumonisin B1 to channel catsh, Ictalurus punctatus. J. Vet. Diagn. Invest. 6, 123124. Desjardins, A.E., 2006. Fusarium Mycotoxins: Chemistry, Genetics, and Biology. American Phytopathological Society, St. Paul, MN. Diaz, D.E., 2005. The Mycotoxin Blue Book. Nottingham University Press, Nottingham, UK, pp. 145146. Ehrlich, K.C., Daigle, K.W., 1987. Protein synthesis inhibition by 8-oxo-12,13-epoxytrichothecenes. Biochim. Biophys. Acta 923, 206213. Erasmus, J.H., Cook, P.A., Coyne, V.E., 1997. The role of bacteria in the digestion of seaweed by the abalone Haliotis midae. Aquaculture 155, 377386. Eriksen, G.S., Pettersson, H., Johnsen, K., Lindberg, J.E., 2002. Transformation of trichothecenes in ileal digesta and faeces from pigs. Arch. Anim. Nutr. 56, 263274. Eriksen, G.S., Pettersson, H., Lundh, T., 2004. Comparative cytotoxicity of deoxynivalenol, nivalenol, their acetylated derivatives and de-epoxy metabolites. Food Chem. Toxicol. 42, 619624. Fegan, D.F., Spring, P., 2007. Recognizing the reality of the aquaculture mycotoxin problem: searching for a common and effective solution. Nutritional biotechnology in the feed and food industries. Proceedings of Alltech's 23rd Annual Symposium, Lexington, Kentucky, USA, pp. 343354. He, P., Young, L.G., Forsberg, C., 1992. Microbial transformation of deoxynivalenol (vomitoxin). Appl. Environ. Microbiol. 58, 38573863. Hedman, R., Pettersson, H., 1997. Transformation of nivalenol by gastrointestinal microbes. Arch. Anim. Nutr. 50, 321329. Horvath, I., Varga, M., 1961. Enzymatic inactivation of trichothecin and crotocin. Nature 192, 88. Karlovsky, P., 1999. Biological detoxication of fungal toxins and its use in plant breeding, feed and food production. Nat. Toxins 7, 123. Kiessling, K.H., Pettersson, H., Sandholm, K., Olsen, M., 1984. Metabolism of aatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen uid, rumen protozoa, and rumen bacteria. Appl. Environ. Microbiol. 47, 10701073. King, R.R., McQueen, R.E., Levesque, D., Greenhalgh, R., 1984. Transformation of deoxynivalenol (vomitoxin) by rumen microorganisms. J. Agric. Food Chem. 32, 11811183. Kollarczik, B., Gareis, M., Hanelt, M., 1994. In vitro transformation of the Fusarium mycotoxins deoxynivalenol and zearalenone by the normal gut microora of pigs. Nat. Toxins 2, 105110. Lesel, R., 1993. Does a digestive active bacterial ora exist in sh? In: Kaushik, S.J., Luquet, P. (Eds.), Fish Nutrition in Practice, pp. 655664. Biarritz, France. Lesel, R., Fromageot, C., Lesel, M., 1986. Cellulose digestibility in grass carp, Ctenopharyngodon idella and in goldsh, Carassius auratus. Aquaculture 54, 1117. Lindsay, G.J.H., Harris, J.E., 1980. Carboxymethylcellulase activity in the digestive tracts of sh. J. Fish Biol. 16, 219233. Manning, B., 2004. Mycotoxin problems in aquaculture. Information presented at Alltech's 2nd Aquaculture Workshop. Dunboyne, Ireland. Manning, B.B., Li, M.H., Robinson, E.H., Gaunt, P.S., Camus, A.L., Rottinghaus, G.E., 2003. Response of channel catsh Ictalurus punctatus to diets containing T-2 toxin. J. Aquat. Anim. Health 15, 230239. Matsushima, T., Okamoto, E., Miyagawa, E., Matsui, Y., Shimizu, H., Asano, K., 1996. Deacetylation of diacetoxyscirpenol to 15-acetoxyscirpenol by rumen bacteria. J. Gen. Appl. Microbiol. 42, 225234. Morgavi, D.P., Riley, R.T., 2007. Fusarium and their toxins: mycology, occurrence, toxicity, control and economic impact. Anim. Feed Sci. Technol. 137, 199200. Prelusky, D.B., Hartin, K.E., Trenholm, H.L., Miller, J.D., 1988. Pharmokinetic fate of 14Clabelled deoxynivalenol in swine. Fundam. Appl. Toxicol. 10, 276286. Rae, T.B., Heather, M.S., Robert, J.L., Carys, L.M., 2007. Debromination of polybrominated diphenyl ether-99 (BDE-99) in carp (Cyprinus carpio) microora and microsomes. Chemosphere 69, 987993. Ray, A.K., Bairagi, A., Ghosh, K.S., Sen, S.K., 2007. Optimization of fermentation conditions for cellulase production by Bacillus subtilis CY5 and Bacillus circulans TP3 isolated from sh gut. Acta Ichthyol. Piscat. 37, 4753.

S. Guan et al. / Aquaculture 290 (2009) 290295 Rotter, B.A., Prelusky, D.B., Pestka, J.J., 1996. Toxicology of deoxynivalenol (vomitoxin). J. Toxicol. Environ. Health 48, 134. Saha, S., Roy, R.N., Sen, S.K., Ray, A.K., 2006. Characterization of cellulase-producing bacteria from the digestive tract of tilapia, Oreochromis mossambica (Peters) and grass carp, Ctenopharyngodon idella (Valenciennes). Aquac. Res. 37, 380388. Shima, J., Takase, S., Takahashi, Y., Iwai, Y., Fujimoto, H., Yamazaki, M., Ochi, K., 1997. Novel detoxication of the trichothecene mycotoxin deoxynivalenol by a soil bacterium isolated by enrichment culture. Appl. Environ. Microbiol. 38253830. Spring, P., Fegan, D.F., 2005. Mycotoxins a rising threat to aquaculture. Proceedings of Alltech's 21st Annual Symposium, Lexington, Kentucky, USA. Swanson, S.P., Nicolrtti, J., Rood, H.D., Buck, W.B., Cote, L.M., 1987. Metabolism of three trichothecene mycotoxins, T-2 toxin, diacetoxyscirpenol and deoxynivalenol, by bovne rumen microorganisms. J. Chromatog. 414, 335342. Swanson, S.P., Helaszek, C., Buck, W.B., Rood, J.H.D., Haschek, W.M., 1988. The role of intestinal microora in the metabolism of trichothecene mycotoxins. Food Chem. Toxicol. 26, 823829. Trust, T.J., Sparrow, R.A.H., 1974. The bacterial ora in the alimentary tract of freshwater salmonid shes. Can. J. Microbiol. 20, 12191228. Ueno, Y., Nakayama, K., Ishii, K., Tashiro, F., Minoda, Y., Omori, T., Komagata, K., 1983. Metabolism of T-2 toxin in Curtobactenum sp. strain 144-2. Appl. Environ. Microbiol. 46, 120127.

295

Volkl, A., Vogler, B., Schollenberger, M., Karlovsky, P., 2004. Microbial detoxication of mycotoxin deoxynivalenol. J. Basic Microbiol. 44, 147156. Westlake, K., Mackie, R., Dutton, M.F., 1987. T-2 toxin metabolism by ruminal bacteria and its effect on their growth. Appl. Environ. Microbiol. 53, 587592. Woodward, B., Young, L.G., Lun, A.K., 1983. Vomitoxin in diets for rainbow trout (Salmo gairdneri). Aquaculture 35, 93101. Yoshizawa, T., Takeda, H., Ohi, T.,1983. Structure of a novel metabolite from deoxynivalenol, a trichothecene mycotoxin, in animals. Agric. Biol. Chem. 47, 21332135. Young, J.C., Zhu, H.H., Zhou, T., 2006. Degradation of trichothecene mycotoxins by aqueous ozone. Food Chem. Toxicol. 44, 417424. Young, J.C., Zhou, T., Yu, H., Zhu, H.H., Gong, J.H., 2007. Degradation of trichothecene mycotoxins by chicken intestinal microbes. Food Chem. Toxicol. 45, 136143. Zhou, T., Gong, J., Young, J.C., Yu, H., Zhu, H., Su, X.J., Li, X.Z., Yang, R., de Lange, C.F.M., Du, W., Cao, R., 2005. Detoxication of vomitoxin with biotransforming microorganisms. Proceeding of the 23rd Annual Centralia Swine Research Update, Kirkton, Ont., 2005, pp. 2330. Zhou, T., He, J., Gong, J., 2008. Microbial transformation of trichothecene mycotoxins. World Mycotoxin J. 1, 2330.