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Poly-β-hydroxybutyrate as a microbial agent in aquaculture

(2010)
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Abstract
Aquaculture is generally accepted as being the main sector in which mankind puts its hope to comply with future global food requirements. Although the popular belief is that the production of low-cost protein-rich food by aquaculture is performed in a sustainable way and relieves pressure from ocean stocks, these statements are only partly valid. Aquaculture practices have too long been performed unsustainably. Firstly, the prophylactic and massive (mis-)use of antibiotics to control pathogenic infections has lead to resistance in pathogens and trace amount of antibiotics in the environment and harvested biomass. Secondly, the discharge of waste nutrients and carbon has put a burden on the environment. And thirdly, by its dependence on pelagic aquatic species for fish meal and fish oil as feed ingredients, aquaculture puts pressure on natural stocks and indirectly on its own existence. The aquaculture scene is aware of these problems and little by little solutions and alternative approaches are investigated and developed to render aquaculture a sustainable industry that is integrated into our future and in the environment. To control the massive mortalities caused by pathogenic infections, more and more focus is being put on biological strategies. These imply either that the natural resistance mechanisms counteracting detrimental pathogenic activity are enhanced or that anti-pathogenic treatments are used that cause no negative effects on the environment. The two most famous terms in this respect are probiotics and prebiotics. The former relates to living microbial actors that are added to the culture water or the feed to improve the health of the cultured aquatic animals. The activity of probiotics can be situated either in the surrounding water or the intestinal tract of the animals. Prebiotics relate to non-digestable compounds that are added to the feed to stimulate health-promoting microbial actors in the intestinal tract. Recently, a bio-control strategy was suggested in the form of poly-β-hydroxybutyrate (PHB). This is a compound that is accumulated by a large variety of micro-organisms as an internal carbon and energy reserve. It was found that this compound could protect gnotobiotic Artemia franciscana against vibriosis. The release of the PHB monomer β-hydroxybutyric acid was suggested to inhibit the growth and/or the activity of the pathogens, although the complete mode of action is not yet known. In this work, for the first time, PHB was applied as a feed supplement to aquaculture animals (Chapter II). No adverse effects were observed in juvenile European sea bass, even when 10% of the normal feed was replaced with PHB. Moreover, at levels of 2% and 5% in the diet, PHB seemed to significantly enhance the growth performance of the fish with a factor 2.4 and 2.7, respectively. Decreasing pH values at higher PHB supplementation levels indicated for the release of acidic compounds. Remarkably, the richness and genetic diversity of the intestinal microbial community in terms of the range-weighted richness seemed to be closely correlated with the growth performance of the fish. This indicated for a host-microbial interaction that was steered by the presence of PHB. It remains to be elucidated whether the condition of the intestinal microbiota was causal to the increased growth performance or whether it should merely be considered an indicator of the health status of the host. At the earliest life stages, opportunistic rather than specific obligate pathogens cause mortalities when the culture animals experience stress. The composition of the “pathogenic” microbiota in the intestine is largely determined by the colonization at mouth opening and first feeding. Also, the way the micro-organisms are organized in the community relative to each other can determine the outcome of the host. It can thus be important to control the colonization of the intestine by selecting for beneficial species or to steer towards a beneficial microbial community structure. The supplementation of PHB had a steering effect in juvenile European sea bass resulting in converging microbial community similarities between treated fish (Chapter III). A core-community of micro-organisms that comprised up to 60% of the total bacterial diversity was sustained at the highest PHB level of 10%. PHB induced more equal abundances between the different bacterial species; higher PHB levels resulted in a higher degree of evenness. Based on literature reports, it is hypothesized that this contributes to an increased resistance against pathogenic infections. Additional experimenting, including PHB feeding experiments in combination with following challenge tests will have to be performed to verify whether or not this is the case. The feeding of aquatic animals with PHB can be considered as an in vivo procedure for the enrichment in micro-organisms able to degrade PHB. The isolation and application of such specialized strains in combination with PHB may considerably increase the efficiency of PHB treatments. PHB-degrading bacteria were isolated from the intestinal environment of juvenile sea bass, sturgeon and giant river prawn that had been fed with PHB (Chapter IV). They were combined with PHB in - what we call - a synbiotic strategy, although the latter does not completely fulfill the requirements of a prebiotic. This approach increased the survival of Artemia franciscana nauplii with a factor 2 to 3 when challenged with a pathogenic Vibrio strain compared to the PHB or the isolates alone. It was verified that the not a feed effect but the increased degradation of PHB by the activity of the PHB probiotics was the cause. In a second line of this work, it was focussed on sustainable effluent treatment as water quality is an important factor in the level of stress aquatic animals experience. Most of the currently available treatments systems score rather limited regarding sustainability as no focus is being put on recovery of resources except for the treated water. The recently developed bio-flocs technology (BFT) performs much better in this respect as it combines water treatment with the internal recovery of nutrients in the form of bio-flocs (Chapter V). These bio-flocs can be reused by the cultured animals as additional sources of feed. It could be calculated that the feed costs kg-1 fish produced can be lowered with 10 - 20% by the application of this technology. Moreover, the specific elements required for the water treatment can be estimed to be twice as expensive in terms of capex + opex for conventional nitrogen removal (nitrification) than for BFT. The performance of bio-flocs technology requires either the dosage of carbon substrate directly to the pond or the use of feed with a higher carbohydrate content. In both cases, these are supplemented at regular time intervals in the pond resulting in a feast and famine regime for the bio-flocs. Such a carbon regime triggers micro-organisms to accumulate PHB into their biomass. PHB can be considered the third added value associated with BFT, next to water treatment and nutrient recycling by in situ feed production. It was shown that the accumulation of PHB in bio-flocs easily reaches up to 16% (w/w) and higher values can be expected upon process optimization (Chapter VI). This value represented a PHB level of about 1% on the total feed supply (normal feed + bio-flocs) of a 50 tons ha-1 yr-1 aquaculture farm. The accumulation of PHB in the bioflocs represented a net positive balance of about 8000 € ha-1 yr-1 compared to the use of normal feed supplemented with PHB (Chapter VII). In conclusion, this study elaborated on the application of PHB in aquaculture. This is a biological compound that can bring added value to aquaculture by its ability to fight pathogenic infections and steer the intestinal microbial community in aquatic animals. By integrating the accumulation of PHB in bio-flocs, this technique can potentially reduce the degree of mortality during larval and juvenile stages of aquaculture animals and can thus become even more cost effective. Overall, PHB can be considered a microbial agent with a large potential to be applied in aquaculture.
Keywords
molecular techniques, microbial ecology, aquaculture, bio-flocs technology, poly-β-hydroxybutyrate

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Citation

Please use this url to cite or link to this publication:

Chicago
De Schryver, Peter. 2010. “Poly-β-hydroxybutyrate as a Microbial Agent in Aquaculture”. Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
APA
De Schryver, P. (2010). Poly-β-hydroxybutyrate as a microbial agent in aquaculture. Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Vancouver
1.
De Schryver P. Poly-β-hydroxybutyrate as a microbial agent in aquaculture. [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2010.
MLA
De Schryver, Peter. “Poly-β-hydroxybutyrate as a Microbial Agent in Aquaculture.” 2010 : n. pag. Print.
@phdthesis{1057637,
  abstract     = {Aquaculture is generally accepted as being the main sector in which mankind puts its hope to comply with future global food requirements. Although the popular belief is that the production of low-cost protein-rich food by aquaculture is performed in a sustainable way and relieves pressure from ocean stocks, these statements are only partly valid. Aquaculture practices have too long been performed unsustainably. Firstly, the prophylactic and massive (mis-)use of antibiotics to control pathogenic infections has lead to resistance in pathogens and trace amount of antibiotics in the environment and harvested biomass. Secondly, the discharge of waste nutrients and carbon has put a burden on the environment. And thirdly, by its dependence on pelagic aquatic species for fish meal and fish oil as feed ingredients, aquaculture puts pressure on natural stocks and indirectly on its own existence. The aquaculture scene is aware of these problems and little by little solutions and alternative approaches are investigated and developed to render aquaculture a sustainable industry that is integrated into our future and in the environment.
To control the massive mortalities caused by pathogenic infections, more and more focus is being put on biological strategies. These imply either that the natural resistance mechanisms counteracting detrimental pathogenic activity are enhanced or that anti-pathogenic treatments are used that cause no negative effects on the environment. The two most famous terms in this respect are probiotics and prebiotics. The former relates to living microbial actors that are added to the culture water or the feed to improve the health of the cultured aquatic animals. The activity of probiotics can be situated either in the surrounding water or the intestinal tract of the animals. Prebiotics relate to non-digestable compounds that are added to the feed to stimulate health-promoting microbial actors in the intestinal tract. 
Recently, a bio-control strategy was suggested in the form of poly-\ensuremath{\beta}-hydroxybutyrate (PHB). This is a compound that is accumulated by a large variety of micro-organisms as an internal carbon and energy reserve. It was found that this compound could protect gnotobiotic Artemia franciscana against vibriosis. The release of the PHB monomer \ensuremath{\beta}-hydroxybutyric acid was suggested to inhibit the growth and/or the activity of the pathogens, although the complete mode of action is not yet known.
In this work, for the first time, PHB was applied as a feed supplement to aquaculture animals (Chapter II). No adverse effects were observed in juvenile European sea bass, even when 10\% of the normal feed was replaced with PHB. Moreover, at levels of 2\% and 5\% in the diet, PHB seemed to significantly enhance the growth performance of the fish with a factor 2.4 and 2.7, respectively. Decreasing pH values at higher PHB supplementation levels indicated for the release of acidic compounds. Remarkably, the richness and genetic diversity of the intestinal microbial community in terms of the range-weighted richness seemed to be closely correlated with the growth performance of the fish. This indicated for a host-microbial interaction that was steered by the presence of PHB. It remains to be elucidated whether the condition of the intestinal microbiota was causal to the increased growth performance or whether it should merely be considered an indicator of the health status of the host.
At the earliest life stages, opportunistic rather than specific obligate pathogens cause mortalities when the culture animals experience stress. The composition of the {\textquotedblleft}pathogenic{\textquotedblright} microbiota in the intestine is largely determined by the colonization at mouth opening and first feeding. Also, the way the micro-organisms are organized in the community relative to each other can determine the outcome of the host. It can thus be important to control the colonization of the intestine by selecting for beneficial species or to steer towards a beneficial microbial community structure. The supplementation of PHB had a steering effect in juvenile European sea bass resulting in converging microbial community similarities between treated fish (Chapter III). A core-community of micro-organisms that comprised up to 60\% of the total bacterial diversity was sustained at the highest PHB level of 10\%. PHB induced more equal abundances between the different bacterial species; higher PHB levels resulted in a higher degree of evenness. Based on literature reports, it is hypothesized that this contributes to an increased resistance against pathogenic infections. Additional experimenting, including PHB feeding experiments in combination with following challenge tests will have to be performed to verify whether or not this is the case. 
The feeding of aquatic animals with PHB can be considered as an in vivo procedure for the enrichment in micro-organisms able to degrade PHB. The isolation and application of such specialized strains in combination with PHB may considerably increase the efficiency of PHB treatments. PHB-degrading bacteria were isolated from the intestinal environment of juvenile sea bass, sturgeon and giant river prawn that had been fed with PHB (Chapter IV). They were combined with PHB in - what we call - a synbiotic strategy, although the latter does not completely fulfill the requirements of a prebiotic. This approach increased the survival of Artemia franciscana nauplii with a factor 2 to 3 when challenged with a pathogenic Vibrio strain compared to the PHB or the isolates alone. It was verified that the not a feed effect but the increased degradation of PHB by the activity of the PHB probiotics was the cause.
In a second line of this work, it was focussed on sustainable effluent treatment as water quality is an important factor in the level of stress aquatic animals experience. Most of the currently available treatments systems score rather limited regarding sustainability as no focus is being put on recovery of resources except for the treated water. The recently developed bio-flocs technology (BFT) performs much better in this respect as it combines water treatment with the internal recovery of nutrients in the form of bio-flocs (Chapter V). These bio-flocs can be reused by the cultured animals as additional sources of feed. It could be calculated that the feed costs kg-1 fish produced can be lowered with 10 - 20\% by the application of this technology. Moreover, the specific elements required for the water treatment can be estimed to be twice as expensive in terms of capex + opex for conventional nitrogen removal (nitrification) than for BFT.
The performance of bio-flocs technology requires either the dosage of carbon substrate directly to the pond or the use of feed with a higher carbohydrate content. In both cases, these are supplemented at regular time intervals in the pond resulting in a feast and famine regime for the bio-flocs. Such a carbon regime triggers micro-organisms to accumulate PHB into their biomass. PHB can be considered the third added value associated with BFT, next to water treatment and nutrient recycling by in situ feed production. It was shown that the accumulation of PHB in bio-flocs easily reaches up to 16\% (w/w) and higher values can be expected upon process optimization (Chapter VI). This value represented a PHB level of about 1\% on the total feed supply (normal feed + bio-flocs) of a 50 tons ha-1 yr-1 aquaculture farm. The accumulation of PHB in the bioflocs represented a net positive balance of about 8000 {\texteuro} ha-1 yr-1 compared to the use of normal feed supplemented with PHB (Chapter VII).
In conclusion, this study elaborated on the application of PHB in aquaculture. This is a biological compound that can bring added value to aquaculture by its ability to fight pathogenic infections and steer the intestinal microbial community in aquatic animals. By integrating the accumulation of PHB in bio-flocs, this technique can potentially reduce the degree of mortality during larval and juvenile stages of aquaculture animals and can thus become even more cost effective. Overall, PHB can be considered a microbial agent with a large potential to be applied in aquaculture.},
  author       = {De Schryver, Peter},
  isbn         = {9789059894020},
  keyword      = {molecular techniques,microbial ecology,aquaculture,bio-flocs technology,poly-\ensuremath{\beta}-hydroxybutyrate},
  language     = {eng},
  pages        = {VIII, 237},
  publisher    = {Ghent University. Faculty of Bioscience Engineering},
  school       = {Ghent University},
  title        = {Poly-\ensuremath{\beta}-hydroxybutyrate as a microbial agent in aquaculture},
  year         = {2010},
}