Poly-b-hydroxybutyrate-accumulating bacteria protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii Dirk Halet1, Tom Defoirdt1,2, Petra Van Damme1, Han Vervaeren1, Ilse Forrez1, Tom Van de Wiele1, Nico Boon1, Patrick Sorgeloos2, Peter Bossier2 & Willy Verstraete1 1
Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Ghent, Belgium; and 2Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Ghent, Belgium
Correspondence: Willy Verstraete, Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Ghent, Belgium. Tel.: 1 32 0 9 264 59 76; fax: 132 0 9 264 62 48; e-mail:
[email protected] Received 14 September 2006; revised 16 January 2007; accepted 18 January 2007. First published online 28 March 2007. DOI:10.1111/j.1574-6941.2007.00305.x Editor: Riks Laanbroek
Abstract A poly-b-hydroxybutyrate (PHB)-accumulating enrichment culture was obtained using activated sludge from a polyphosphate-accumulating reactor as inoculum. PHB accumulated by the enrichment culture significantly enhanced the survival of Artemia nauplii, infected with the virulent pathogen Vibrio campbellii LMG 21363. A strain was isolated from the enrichment culture, based on its ability to accumulate PHB, and 16S rRNA gene sequencing of the isolate revealed 99% sequence similarity to Brachymonas denitrificans AS-P1. The isolate, named PHB2, showed good PHB-accumulating activity (up to 32% of the cell dry weight). PHB accumulated by isolate PHB2 was able to protect Artemia completely from the V. campbellii strain. Our data indicate that PHB-accumulating bacteria, such as B. denitrificans PHB2, could be used as an an effective and economically interesting alternative strategy to control infections in aquaculture.
Keywords brine shrimp; PHB; luminescent vibriosis; Brachymonas denitrificans .
Introduction Aquaculture is a rapidly expanding industry worldwide. However, disease outbreaks are considered to be a significant constraint to the development of the aquaculture sector (Subasinghe et al., 2001). Chronic disease can affect the growth rate and feed efficiency of the cultured animals and mortalities contribute directly to a loss of investment in time, labour and feed. Thus far, conventional antibiotics have had only limited success in the treatment of aquatic diseases (Subasinghe et al., 2001). Moreover, their frequent use is leading to the rapid development of (multiple) resistance (Schmidt et al., 2000; Teo et al., 2000, 2002; Molina-Aja et al., 2002; Vivekanandhan et al., 2002). Therefore, there is an urgent need for alternative control techniques. Short-chain fatty acids are well known to inhibit the growth of enterobacteria (Cherrington et al., 1991). We recently showed that this type of compound also inhibits the growth of pathogenic Vibrio campbellii LMG 21363 (Defoirdt et al., 2006). Moreover, the addition of fatty acids to the culture water of Artemia nauplii infected with the pathogen significantly increased the survival of the shrimp. In another report, we showed that the addition of the wellFEMS Microbiol Ecol 60 (2007) 363–369
known bacterial storage compound poly-b-hydroxybutyrate (PHB), a polymer of the short-chain fatty acid b-hydroxybutyrate, also protected Artemia nauplii from the virulent V. campbellii strain (Defoirdt et al., 2007). A wide variety of microorganisms are known to accumulate PHB as an intracellular energy and carbon storage compound, usually when an essential nutrient (such as nitrogen) is limited in the presence of excess of carbon source (Lee, 1996). PHB is produced, for instance, in polyphosphate-accumulating organisms under controlled conditions of nutrients such as nitrogen, oxygen and/or minerals (Guisasola et al., 2004). The biosynthesis and degradation of PHB is a cyclical mechanism (Senior & Dawes, 1973). When submitted to consecutive periods of external substrate accessibility (‘feast’) and unavailability (‘famine’), bacteria show so-called unbalanced growth (Serafim et al., 2004). During the excess of external carbon substrate, carbon uptake is mainly driven to PHB storage and, to a lesser extent, to biomass growth. After substrate exhaustion, PHB degradation starts with the depolymerization to b-hydroxybutyrate monomers, which can then further be used as an energy and carbon source (Kadouri et al., 2005). 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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The aim of the present research was to select an enrichment culture, to isolate pure cultures of PHB-accumulating bacteria, and to examine their efficiency to protect Artemia from pathogenic V. campbellii.
Materials and methods Enrichment of PHB-accumulating bacteria PHB-accumulating bacteria were enriched in a sequencing batch reactor inoculated with activated sludge from a laboratory-scale polyphosphate-accumulating reactor as described by Serafim et al. (2004). Briefly, the sequencing batch reactor, with a working volume of 2 L, was operated with consecutive periods of external substrate accessibility and unavailability. Each reactor cycle consisted of 10.5 h of aerobiosis, 1 h of settling (agitation and air bubbling switched off), and 0.5 h withdrawing half of the volume, which was replaced with fresh medium during the first 5 min at the beginning of the next cycle. The total cycle duration was 12 h and the hydraulic retention time was 24 h. Every day, a defined volume (200 mL) was removed before settling to keep a sludge retention time of 10 days. Oxygen was supplied by an air compressor. The temperature was kept at 28 1C and the stirring rate at 250 r.p.m. Acetate was monitored as described by Nollet et al. (1997) with a Di200 gas chromatograph (GC; Shimadzu, ’s-Hertogenbosch, the Netherlands). The GC was equipped with a capillary-free fatty-acid-packed column [EC-1000 Econo-Cap column (Alltech, Laarne, Belgium), 25 m 0.53 mm; film thickness 1.2 mm], a flame ionization detector and a Delsi Nermag 31 integrator (Thermo Separation Products, Wilrijk, Belgium). Nitrogen was used as the carrier gas at a flow rate of 20 mL min1. The column temperature was set at 130 1C and the temperature of the injector and detector was set at 195 1C. The mineral salts medium used in the sequencing batch reactor contained (per litre of distilled water) 2.4 g sodium acetate, 600 mg MgSO4 7H2O, 160 mg NH4Cl, 100 mg EDTA, 92 mg K2HPO4, 45 mg KH2PO4, 70 mg CaCl2 2H2O and 2 mL l 1 of trace solution (Serafim et al., 2004). The trace solution consisted of (per litre of distilled water) 1500 mg FeCl3 6H2O, 150 mg H3BO3, 150 mg CoCl2 6H2O, 120 mg MnCl2 4H2O, 120 mg ZnSO4 7H2O, 60 mg Na2MoO4 2H2O and 30 mg CuSO4 5H2O. Allylthiourea (10 mg L 1) was added to inhibit nitrification. The initial pH was set at 7.2.
Isolation of pure cultures of PHB-accumulating bacteria Microbiological isolation of PHB-accumulating bacteria was carried out by the spread-plate method described by Spiekermann et al. (1999), with slight modifications. To prepare solid medium for isolation, 15 g of Technical Agar (Difco, 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Detroit, MI) was added to 1 L of the mineral salts medium used in the sequencing batch reactor. After autoclavation, 0.002% (v/v) of a solution of 0.25 mg Nile blue A (Sigma, St Louis, MO) per millilitre dimethylsulfoxide (DMSO) was added to the medium to give a final concentration of 0.5 mg dye (mL medium) 1. The agar plates were exposed to UV light (312 nm) after cultivation to detect PHB accumulation in the grown colonies. The isolates were grown for 24 h in LB medium at 28 1C with shaking. After incubation, the cells were centrifuged (8 min, 2000 g) and resuspended in the mineral salts medium used in the sequencing batch reactor. The cultures were aerated and samples were taken every hour in order to determine the PHB content.
Determination of the PHB content PHB concentrations were measured with a Di200 GC (Shimadzu) following the procedure described by Oehmen et al. (2005). The GC was equipped with a capillary-free fatty-acid-packed column [EC-1000 Econo-Cap column (Alltech), 25 m 0.53 mm; film thickness 1.2 mm], a flame ionization detector and a Delsi Nermag 31 integrator (Thermo Separation Products). Nitrogen was used as the carrier gas at a flow rate of 3 mL min 1.
Phylogenetic identification of pure cultures DNA extraction from the pure cultures and PCR were performed as described previously (Boon et al., 2000). During the PCR step, a 1315-bp fragment of the 16S rRNA gene of the bacteria was amplified using the bacterial primer set P63F and R1378r (Øvre˚as et al., 1997). Subsequently, the PCR product was cleaned with the QIAquicks PCR Purification Kit (Qiagen, Westburg, the Netherlands) and the size of the PCR product was verified on a 1% (w/v) agarose gel. DNA sequencing of the 1315-bp PCR products was carried out at IIT Biotech (Bielefeld, Germany). Analysis of DNA sequences was completed with the BLAST server of the National Center for Biotechnology Information using the BLAST algorithm and the BLASTN program for comparison of a nucleotide query sequence against a nucleotide sequence database. Nucleotide sequences for the 1315-bp fragments have been deposited in the GenBank database under accession numbers DQ648571 and DQ648572. A phylogenetic tree was constructed using the RDP PHYLIP 3.5c Interface (Maidak et al., 2001). Distance matrix analyses were carried out with the Jukes–Cantor correction (Jukes & Cantor, 1969) and tree construction was via the neighbourjoining method (Saitou & Nei, 1987).
Axenic hatching of Artemia franciscana All challenge tests were performed with highquality hatching cysts of Artemia franciscana (EGs Type, batch 6940, INVE FEMS Microbiol Ecol 60 (2007) 363–369
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Vibrio campbellii LMG 21363 (isolated from the lymphoid organ of diseased shrimp) was stored in 40% glycerol at 80 1C. Ten microlitres of this stored culture was inoculated into fresh Marine Broth (Difco Laboratories) and incubated for 24 h at 28 1C under constant agitation (100 min 1). The grown cultures of V. campbellii were washed in filtered and autoclaved artificial seawater and added to the Artemia culture water at c. 105 CFU mL 1. Aeromonas hydrophila LVS3 (Verschuere et al., 1999) was used as feed for the nauplii (Defoirdt et al., 2005). The strain was grown overnight on Marine Agar (Difco Laboratories), suspended in sterile artificial seawater, autoclaved and added to the Artemia culture water at c. 107 cells mL 1.
In vivo challenge tests Challenge tests were performed as described by Defoirdt et al. (2005), with slight modifications. Briefly, after hatching, groups of 20 nauplii were transferred to new sterile 50mL tubes that contained 20 mL of filtered and autoclaved artificial seawater. The tubes were inoculated with V. campbellii LMG 21363 (except for the control, where no pathogen was added) and fed with LVS3. PHB-accumulating bacteria were added to the Artemia culture water at c. 107 CFU mL 1. After feeding and the addition of the appropriate bacteria, the falcon tubes were put back on the rotor and kept at 28 1C. Survival of Artemia was scored 2 days after the addition of the pathogen. All manipulations were done under a laminar flow hood in order to maintain sterility of the cysts and nauplii. Each treatment was done in triplicate.
Results Enrichment of PHB-accumulating bacteria A sequencing batch reactor inoculated with activated sludge from a laboratory-scale polyphosphate-accumulating reactor was subjected to cyclical carbon supply, using acetate as carbon source under N-limiting conditions. After several weeks, a PHB-accumulating enrichment culture with stable performance was growing in the reactor. PHB concentrations of 25 5% of the VSS were obtained. Figure 1 shows a typical concentration profile during a reactor operation cycle. Acetate was quickly taken up and partially used for growth (increase of VSS) and partially stored as PHB (sharp increase of PHB concentration). After substrate exhaustion, the PHB concentration decreased back to the initial level.
The effect of the PHB-accumulating enrichment culture on the survival of Artemia nauplii infected with V. campbellii LMG 21363 In a first in vivo challenge test, the effect of the PHBaccumulating enrichment culture on the survival of Artemia nauplii infected with the pathogenic isolate V. campbellii LMG 21363 was investigated. The enrichment culture was growing as aggregates in the sequencing batch reactor and many aggregates were too large to be ingested by Artemia. Consequently, the culture was subjected to different treatments that aimed at making the PHB more available for Artemia by decreasing the size of the aggregates. If it was subjected to three cycles of freezing and thawing prior to addition to the culture water, the addition of the enrichment culture (containing 15% PHB on VSS or more) significantly enhanced the survival of the infected nauplii (Table 1). Adding the culture untreated or after pasteurization 4
30 Acetate VSS PHB
3
25 20
2
15 10
1 5 0
Statistics For each experiment, mean survival of Artemia subjected to different treatments was compared by independent samples FEMS Microbiol Ecol 60 (2007) 363–369
PHB content (% of VSS)
Preparation of the inocula for in vivo challenge tests
t-tests, using the SPSS software, version 12.0. Differences were considered significant at P o 0.05.
Acetate or VSS (g L )
Aquaculture, Baasrode, Belgium). Two hundred milligrams of cysts were hydrated in 18 mL of tap water for 1 h. Sterile cysts and nauplii were obtained via decapsulation, adapted from the protocol described by Marques et al. (2004). Briefly, 660 mL of NaOH (32%) and 10 mL of NaOCl (50%) were added to the hydrated cyst suspension. The decapsulation was stopped after 2 min by adding 14 mL of Na2S2O3 (10 g L 1). During the reaction, 0.22- mm filtered aeration was provided. The decapsulated cysts were washed with filtered (0.22 mm) and autoclaved artificial seawater containing 35 g L 1 of Instant Ocean synthetic sea salt (Aquarium Systems Inc., Sarrebourg, France). The cysts were resuspended in a 50-mL tube containing 30 mL of filtered and autoclaved artificial seawater and hatched for 30 h on a rotor (4 min 1) at 28 1C with constant illumination (c. 2000 lux).
0 0
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Time (h)
Fig. 1. Acetate, VSS and PHB concentrations as a function of time during a PHB enrichment cycle of the mixed culture.
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Table 1. Percentage survival of Artemia nauplii (mean SE of three replicates) after 2 days challenge with Vibrio campbellii LMG 21363 Treatment
Survival (%)
Control LMG 21363 LMG 21363 1 culture (2% PHB, freezing and thawing) LMG 21363 1 culture (15% PHB, freezing and thawing) LMG 21363 1 culture (25% PHB, freezing and thawing) LMG 21363 1 culture (25% PHB, untreated) LMG 21363 1 culture (25% PHB, pasteurized)
83 2 10 3 72 43 2 67 7 12 4 18 3
The PHB-accumulating enrichment culture was added to the Artemia culture water at the start of the experiment, either untreated or after pasteurization or freezing and thawing. The enrichment culture was sampled at three time points and contained 2%, 15% or 25% PHB on VSS. Survival significantly different from the treatment with pathogen and without PHB-accumulating bacteria (P o 0.01).
(30 min, 60 1C) had no effect on survival of the infected Artemia. The enrichment culture was sampled after 2 and 6 h during PHB enrichment and after 1 day of starvation. By so doing, the same enrichment culture was obtained with different PHB concentrations (25%, 15% and 2% of the VSS, respectively). Importantly, the concentration of VSS was the same (c. 3 g L 1) in all three cases. Survival of the infected nauplii was clearly proportional to the PHB content of the enrichment culture (Table 1).
reactor. Maximal PHB contents were obtained after 2 h of incubation and were 2% and 32% of the VSS for isolate PHB1 and PHB2, respectively. We decided to perform further experiments only with isolate PHB2 as isolate PHB1 showed only minor PHB-accumulating capacity.
The effect of isolate PHB2 on the survival of Artemia nauplii infected with V. campbellii LMG 21363 A further in vivo experiment aimed at testing whether isolate PHB2, enriched with PHB (32% of the VSS), could protect Artemia nauplii from the pathogenic Vibrio campbellii. Isolate PHB2 was added to the Artemia culture water either untreated or after drying (5 h at 40 1C), pasteurization (30 min, 60 1C) or three cycles of freezing and thawing. If added together with the pathogen, isolate PHB2 (either untreated or subjected to the different treatments) significantly enhanced the survival of infected Artemia (Table 2). The protection offered by the isolate was complete as no significant mortality occurred in infected nauplii treated with PHB2 (as compared with uninfected nauplii; P 4 0.25). The addition of starved PHB2 (containing 2% PHB on VSS) did not result in increased survival. If added 1 day after the start of the challenge, PHB2 also significantly increased the survival of infected Artemia. However, in this case the protection was not complete and there was still significant mortality in infected nauplii treated with PHB2 (P o 0.01).
Isolation of pure cultures of PHB-accumulating bacteria Plating of the PHB-accumulating enrichment culture on medium with the PHB-staining fluorescent dye Nile blue A revealed that there were two dominant PHB-accumulating strains present in the culture, tentatively named PHB1 and PHB2. Isolate PHB1 grew faster and produced colonies with a diameter of 5 mm after 24 h of cultivation at 28 1C. Isolate PHB2 grew more slowly, yielding colonies with a diameter of 1 mm on the same medium after 48 h of incubation. However, the fluorescence intensity of PHB2 colonies was clearly higher than that of PHB1 colonies and PHB2 was present in much higher numbers than PHB1. Determination of the nearest phylogenetic neighbour sequences for the 16S rRNA gene sequences of isolates PHB1 and PHB2 by the BLAST search program showed that the 16S rRNA gene of isolate PHB1 had 99% sequence similarity with Comamonas testosteroni CNB-1 (Wu et al., 2005). The 16S rRNA gene of isolate PHB2 had 99% sequence identity with Brachymonas denitrificans AS-P1 (Hiraishi et al., 1995). The isolates were enriched with PHB in batch culture using the same medium as was used in the sequencing batch 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Table 2. Percentage survival of Artemia nauplii (mean SE of three replicates) after 2 days challenge with Vibrio campbellii LMG 21363 Treatment
Survival (%)
Control LMG 21363 LMG 21363 1 PHB2 (2% PHB, freezing and thawing, added at start) LMG 21363 1 PHB2 (32% PHB, freezing and thawing, added at start) LMG 21363 1 PHB2 (32% PHB, untreated, added at start) LMG 21363 1 PHB2 (32% PHB, dried, added at start) LMG 21363 1 PHB2 (32% PHB, pasteurized, added at start) LMG 21363 1 PHB2 (32% PHB, freezing and thawing, added after 1 day)
87 3 17 2 15 3 82 2 82 2 87 3 90 3 45 3
The PHB-accumulating strain PHB2 was added to the culture water at the start of the experiment or 1 day after addition of the pathogen, either untreated or after pasteurization, drying or freezing and thawing. Survival significantly different from the treatment with pathogen and without PHB-accumulating bacteria (P o 0.01).
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Discussion Our previous research (Defoirdt et al., 2007) showed that the addition of PHB particles to the Artemia culture water protected the shrimp from a virulent V. campbellii strain. In this study, we investigated the use of PHB-accumulating bacteria as a new biocontrol strategy for aquaculture, using our gnotobiotic Artemia model system. Initially, a sequencing batch reactor was inoculated with activated sludge from a laboratory-scale phosphate-removing reactor and subjected to transient carbon supply, in which a short time of excess of substrate was altered with a long period of lack of substrate. These conditions are known to select for bacteria with a high PHB storage capacity (Beccari et al., 1998; Beun et al., 2002; Serafim et al., 2004). The PHB-accumulating enrichment culture growing in the sequencing batch reactor had a PHB storage capacity of 25 5% on VSS. This is comparable with the results of Serafim et al. (2004), where a sequencing batch reactor was running under the same conditions and where a PHB storage capacity of 35 5% on VSS was obtained. In a first challenge test, we aimed at investigating whether the PHB-accumulating enrichment culture could be used to protect Artemia from the pathogenic V. campbellii strain LMG 21363. The enrichment culture was growing as aggregates in the sequencing batch reactor and was subjected to different treatments in order to make the PHB more available for Artemia. The culture protected the nauplii from the pathogen only if it had a PHB content of at least 15% of the VSS and if it had been subjected to freezing and thawing. Adding the culture untreated or after pasteurization had no effect on the survival of infected nauplii. As observed by microscopy, the freezing and thawing treatment had resulted in a higher concentration of aggregates smaller than 50 mm (data not shown). Only these smaller aggregates can be ingested by Artemia (Sorgeloos et al., 1986). Freezing and thawing is known to transform aggregates into a more compact form (Vesilind et al., 1991) and the above-mentioned observations indicate that this was also the case for the aggregates of the enrichment culture. The protective effect of the enrichment culture (subjected to freezing and thawing) was clearly related to the PHB accumulated in the bacteria as the survival of infected nauplii treated with the culture was proportional to its PHB content. Two PHB-accumulating strains were isolated from the mixed enrichment culture. The determination of the nearest phylogenetic neighbours for the 16S rRNA gene sequences of the isolates showed that the first isolate, PHB1, was found to be closely related to C. testosteroni. Comamonas testosteroni has been reported before to accumulate PHB (Thakor et al., 2003). The second isolate, PHB2, was shown to be closely related to B. denitrificans. Brachymonas denitrificans is a representative species of activated sludge microbial FEMS Microbiol Ecol 60 (2007) 363–369
communities (Hiraishi et al., 1995), and has been identified as an iron-reducing (Ivanov et al., 2005) and an efficiently denitrifying bacterium (Leta et al., 2005). However, as far as we are aware, this is the first report mentioning PHB accumulation in this species. Isolates PHB1 and PHB2 were enriched with PHB in batch culture and isolate PHB2 was found to accumulate much more PHB (32% of the VSS) than isolate PHB1 (2% of the VSS). However, the PHB content of isolate PHB2 was still lower than values achieved with pure cultures of other species (between 80% and 90% of the VSS; Reddy et al., 2003). However, optimalization of the PHB production by isolate PHB2 was not the focus of this study and will be part of future research. In a second challenge test, the capacity of isolate PHB2 enriched with PHB (32% of the VSS) to protect Artemia from the pathogenic Vibrio was investigated. The isolate was added to the Artemia culture water either untreated or after drying, pasteurization or freezing and thawing and completely protected the infected Artemia from the pathogenic V. campbellii in all cases (no significant difference in survival with uninfected naupllii). This was in contrast to the challenge test with the enrichment culture, which only had an effect after freezing and thawing. Probably, owing to the absence of aggregates in the pure culture of isolate PHB2, all PHB-enriched cells could be ingested by Artemia in all treatments. This could explain why the isolate was more effective than the mixed culture although the PHB content was not much higher. In addition to the preventive mode of action of PHB-enriched PHB2, this strain was also shown to have curative properties. The same effects as we obtained with PHB2 were seen when using PHB-biopolymer particles (average diameter 30 mm) at a dosage of 1 g PHB L 1 (Defoirdt et al., 2007). In this study, the dosage of PHB added in the form of PHB-enriched PHB2 was c. 10 mg L 1, which is 100 times lower than for the PHB-biopolymer particles. The difference in efficiency is probably due to the fact that the PHB particles (added as PHB2 cells) were obviously much smaller than 30 mm, resulting in a more efficient release of bhydroxybutyrate. Indeed, Forni et al. (1999) also reported a much higher digestibility in sheep of smaller poly-(bhydroxybutyrate-co-b-hydroxyvalerate) when compared with larger particles of the same polymer. Polyhydroxyalkanoates (such as PHB) are already industrially synthesized by bacteria for the production of bioplastics. The production price of these bioplastics is around $16 kg 1 (Lee, 1996). However, as mentioned by Salehizadeh & Van Loosdrecht (2004), process scaling-up is believed to half the production costs. Moreover, laboratory-scale fedbatch tests using Alcaligenes latus indicated that it would be even possible to lower the costs to $2.6 kg 1 (Lee & Choi, 1998). Hence, PHB-accumulating bacteria might not only be an effective, but also an economically interesting alternative biocontrol strategy. 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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In conclusion, the results obtained in this study showed that the application of PHB-accumulating bacteria constitutes an effective alternative method for the abatement of pathogenic bacteria in aquaculture. This alternative method could lead to greater ecological and economic sustainability of the aquaculture industry, minimizing the amount of antibiotics entering the environment.
Acknowledgements This work was supported by the ‘Fonds voor Wetenschappelijk Onderzoek’ (project no. G0230.02N) and the ‘Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen’ (IWT Grant no. 33205).
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