Vi sinh vật hữu ích - Phần 2: Bacillus spp

The probiotic bacteria used in aquaculture are from many phylogenetic lineages; however, most of the probiotics studied belong to two bacterial divisions, the Firmicutes (e.g. Bacillus spp., Lactobacillus spp., Lactococcus spp., Carnobacterium spp. etc.) and the Gammaproteobacteria (e.g. Vibrio spp., Pseudomonas spp., Shewanella spp. etc.), while yeasts remain rarely studied (Gatesoupe 2007).

In the case of crustaceans, the diversity of microorganisms assessed as probiotics is important. The range of microorganisms examined has encompassed Gram-positive bacteria (Bacillus spp., Micrococcus sp., Lactobacillus spp., Lactococcus spp., Pediococcus sp. and Arthrobacter sp.), Gram-negative bacteria (Vibrio spp., Pseudomonas spp., Bdellovibrio spp., Aeromonas spp., Pseudomonas spp., Halomonas sp. and Alteromonas spp.), yeasts (Saccharomyces spp., Phaffia sp.) and microalgae (Tetraselmis sp.).

The main probiotic bacteria documented and used in crustacean farming belong to the Bacillus genus (Moriarty 1998; Rengpipat et al. 1998; 2000; 2003; 2008; Vaseeharan and Ramasamy 2003; Ziaei-Nejad et al. 2006; Decamp et al. 2008; Boonthai et al. 2011). Other Gram-positive bacteria such as Lactobacillus spp. (Villamil et al. 2003; Venkat et al. 2004; Chiu et al. 2007; Viera et al. 2008), Lactococcus lactis (Harzevili et al. 1998), Pediococcus acidilactici (Castex et al. 2008; 2010), Micrococcus sp. (Antony et al. 2011) and Arthrobacter sp. (Li et al. 2006; 2008; Pai et al. 2010) have received some attention and are used in some commercial products (Nimrat and Vuthiphandchai 2011). Autochthonous Gram-negative bacteria strains such as Vibrio spp. (Garriques and Arevalo 1995; Moriarty 1998; Verschuere et al. 2000a; Alavandi et al. 2004; Rodríguez et al. 2007; Krupesha Sharma et al. 2010), Bdellovibrio spp. (Qi et al. 2009), Aeromonas sp. (Verschuere et al. 2000b), Pseudomonas spp. (Chythanya et al. 2002; Alavandi et al. 2004; Vijayan et al. 2006; Hai et al. 2007; 2009; Pai et al. 2010), Halomonas sp. (Zhang et al. 2009) and Alteromonas sp. (Abraham 2001) have also been documented and used in some parts of the world, although the development of commercial products containing such microorganisms would be very limited.

Furthermore, even though the scientific literature available on the use of photosynthetic bacteria is scarce, bacteria such as Thalassobacter utilis (Nogami et al. 1997), Rubrivivax gelatinosa, Rhodobacter capsulata, Rhodobacter spheroides and Phaeospirillum fulvum are extensively used in Chinese aquaculture (Qi et al. 2009), probably being the earliest and most widely used probiotics in China since the 1980s. Finally, yeast probiotic strains belonging to the species Saccharomyces cerevisiae or marine yeasts are also considered, though results to date show that such microorganisms can be efficient probiotics to optimize shrimp cultivation (Scholz et al. 1999; Burgents et al. 2004; Yang et al. 2011).

Bacillus spp.

The most well studied probiotics for crustacean applications belong to the genus Bacillus (Farzanfar et al. 2006). Generally, Bacillus species tested in shrimp culture have been selected according to their antimicrobial activities toward pathogenic Vibrio spp. based on in vitro antagonism assays (Rengpipat et al. 1998; Decamp et al. 2008). Bacillus spp. are saprophytic Gram-positive spore forming bacteria naturally present in a wide range of environmental conditions (in air, water, dust, soil and sediment). It is reported that Bacillus species are ubiquitous and even dominate the marine microbiota (Priest 1993). Present in the water column as well as the sediment, Bacillus spp., especially Bacillus subtilis, Bacillus licheniformis and Bacillus coagulans, are considered autochthonous members of the crustacean’s environment and a component of the intestinal microbiota entering the gut via the food or water. However, it is interesting to highlight that the most recent studies, which used molecular techniques to characterize the gut microbiota of crustaceans, rarely report the presence of Bacillus species within the gastrointestinal (GI) tract (Li P. et al. 2007; Liu H. et al. 2011a), suggesting either a minor component of the microbiota belongs to this genus or it constitutes a transient population.

Moriarty (1998) emphasized several reasons for the use of Bacillus, rather than antibiotics, to improve shrimp culture success: Bacillus strains naturally produce many different antibiotic compounds, secrete many enzymes, compete for nutrients and surface adhesion sites, and hence are able to inhibit other bacteria. Furthermore, the robustness of these spore forming bacteria is often viewed as a great advantage as it may enable passage across the gastric barrier and offer better stability during feed processing and shelf life/storage. The large interest in the Bacillus genus in shrimp aquaculture can indeed be explained by these characteristics, but it is also for historical reasons as the first successful studies of probiotic in shrimps were conducted with Bacillus species (Rengpipat et al. 1998; Moriarty 1998).

Among Bacillus spp., B. subtilis is the most recurrent species being tested and used in crustaceans either alone (Rengpipat et al. 1998; 2000; 2003; Moriarty 1998; Dalmin et al. 2001; Meunpol et al. 2003; Vaseeharan and Ramasamy 2003; Marques et al. 2006a; Balcázar et al. 2007; Yu et al. 2008; Liu et al. 2009; Tseng et al. 2009; Liu K.-F. et al. 2011b; Shen et al. 2010) or in combination with other Bacillus spp. (Decamp et al. 2008; Boonthai et al. 2011), other probiotic species (Wang et al. 2005; Wang 2007) or prebiotic supplements (Li et al. 2009; Daniels et al. 2010; Zhang et al. 2011). The other Bacillus species documented so far as probiotics for crustacean are B. licheniformis (Li K. et al. 2007), B. coagulans (Zhou et al. 2009) and B. pumilus (Hill et al. 2009).

Interestingly B. subtilisis also widely used in terrestrial animals and in humans, with claims of bacteriotherapy and bacterioprophylaxis of GI disorders (Cutting 2011). In shrimp, Vaseeharan and Ramasamy (2003) reported the beneficial effect of B. subtilis, isolated from shrimp culture ponds, on the survival of black tiger shrimp (Penaeus monodon) juveniles challenged with Vibrio harveyi, when the probiotic was delivered in the culture water at a density of 10⁶ –10⁸ CFU ml–1. Furthermore, these authors demonstrated under in vitro and in vivo conditions that the probiotic was able to control the growth of V. harveyi. Balcázar and Rojas-Lunas (2007) also evaluated the dietary supplementation of a B. subtilis strain (UTM 126) on the survival of juvenile Pacific white shrimp Litopenaeus vannamei following an immersion challenge with Vibrio parahaemolyticus.

The strain was initially isolated from the digestive tract of adult L. vannamei according to its antimicrobial activity against V. harveyi, Vibrio vulnificus, and Vibrio fluvialis (Balcázar et al. 2007). The results indicated that the probiotic strain exhibited in vitro antagonism toward V. parahaemolyticus and could induce a significant increase in the survival rate and performance of the host (weight gain and feed conversion ratio, FCR) when fed for 28 days to juveniles at 10⁵ CFU g–1 of diet. Rengpipat et al. (1998) also isolated a Bacillus strain (strain S11) from the gut of healthy Penaeus monodon with antimicrobial properties against V. harveyi and V. parahaemolyticus.

In several P. monodon studies, and under various experimental conditions (Rengpipat et al. 1998; 2000; 2003; 2008), it has been reported that this probiotic strain, when administered as fresh cells at a concentration of 10⁹ to 10¹⁰ CFU g–1 of diet, could (1) increase growth and survival in pond and tank culture, (2) increase resistance to pathogenic V. harveyi strains, (3) activate both cellular and humoral immune defences, and (4) provide competitive exclusion in the shrimp gut.

Meunpol et al. (2003) confirmed the efficiency of this strain and reported, in association with an ozone treatment, the beneficial effect conferred by Bacillus S11 on the survival of P. monodon post larvae, concomitantly with a reduction in V. harveyi concentration in the culture water. More recently, evaluations of Bacillus spp. probiotics were conducted on other crustacean species. Daniels et al. (2010) investigated the effect of a combination of dietary Bacillus sp. and yeast mannan oligosaccharides (MOS) on European lobster (Homarus gammarus L.) larvae. Beyond the classic effects on performance and survival, this study highlights for the first time the effect of a Bacillus spp. mixture on various parameters of lobster larvae such as modification in the gut microbial communities and improvements in intestinal morphology.

As described later in this chapter, one of the mechanisms of action by which Bacillus spp., especially B. subtilis, provide host benefits is by modulation of the immune response of the host. Such effects have been well documented in humans, terrestrial animals (Green et al. 1999; Cutting et al. 2011), fish (Avella et al. 2010) and also in crustaceans (Tseng et al. 2009; Liu et al. 2011b). The effect of Bacillus spp. on digestion and their putative contribution to nutritional processes have also been evaluated in crustaceans.

It is important to add here that Bacillus strains are also being studied, and extensively used, as bioremediation agents in shrimp farming (Moriarty 1999). The potential role and benefits of such microorganisms on the nutrient cycle and microbial processes in aquaculture ponds have been described by Moriarty (1997) and readers with a specific interest in this topic are referred to that review article. For instance, the author compared results from different farms in the Philippines and concluded that the addition of several Bacillus spp. to the pond water, at a concentration of 10⁴ to 10⁵ CFU/ml, allowed the culturing of shrimps over 160 days without an outbreak of luminescent Vibrio disease, while control ponds were affected by luminescent Vibrio disease before 80 days of culture (Moriarty 1998; 1999). The author demonstrated that the probiotic treatment modified the bacterial species composition of the pond water and sediment with a particular reduction in the number of luminous Vibrio spp.

Apart from pathogenic bacteria, another main concern in intensive shrimp farming is organic enrichment and nitrogenous waste, including ammonium and ammonia in the culture pond. In relation to this, probiotics such as selected Bacillus spp. (Moriarty 1998; Song et al. 2011), eventually coupled with other bacterial strains (Devaraja et al. 2002; Matias et al. 2002; Wang and He 2011), have been added to pond water with the aim of boosting mineralization of the organic matter and increasing water quality. Thus, bacterial species belonging to the Bacillus, Pseudomonas, Acinetobacter, Cellulomonas, Rhodopseudomonas, Aerobacter, Nitrosomonas and Nitrobacter genera are known to promote mineralization of organic wastes (Thomas et al. 1992). For instance, Nitrosomonasspp. will help in the oxidation of ammonia while Nitrobacterspp. will oxidize nitrites and this process can help to prevent the build-up of toxic ammonia.

Recently, a B. coagulans strain isolated from highly intensive shrimp ponds in China was also reported to show interesting aerobic denitrification characteristics for nitrite removal in shrimp ponds (Song et al. 2011). Some of the benefits of using such bacterial products include the reduction of blue-green algal populations and of nitrate, nitrite, ammonia and phosphate levels, the increase of dissolved oxygen concentrations and the promotion of organic matter decomposition (Boyd 1995). Unfortunately well designed studies on the benefits of applying such bacterial products in aquaculture rearing systems are still scarce (Shariff et al. 2001; Devaraja et al. 2002).

Sources: Mathieu Castex, Carly Daniels and Liet Chim - Probiotic Applications in Crustaceans - Aquaculture Nutrition, pages 293 - 295.