Bacterial communities and predicted nitrogen metabolism of heterotrophic‐ and probiotic‐based biofilms used for super‐intensive indoor shrimp culture

Biofilm‐based aquaculture systems constitute a promising alternative for intensive shrimp rearing. Microorganisms forming biofilms can recycle the nitrogen compounds the production units improving the water quality while using zero or limited water exchange. This study aimed to compare the taxonomic profiles and the predicted functions related to the transformation of nitrogenous compounds between a heterotrophic‐ (HtB) and a probiotic biofilm (PrB), and the effect of these on the water quality and the productive response of cultured shrimp. Libraries of the 16S‐rRNA gene (V3‐V4 region) were prepared and sequenced to be used as a taxonomic biomarker. Analysis of metagenomic datasets revelated that genera Halomonas, Planctomycetes and Rhodopirellula were the most abundant genera in HtB; meanwhile, Bacillus, Halobacillus and Flavobacterium dominated in PrB. Regarding nitrogen metabolism, the proportion of genes encoding enzymes catalyzing the six pathways shaping the nitrogen metabolism showed differences between biofilms, which could also explain the difference in water quality between treatments. Concerning the productive response of shrimp, no significant differences were detected except for survival, which was higher in PrB. Finally, the results suggest that biofilms harbour functions for nitrogen metabolism, including dissimilatory nitrate reduction, assimilatory nitrate reduction, denitrification, nitrification, nitrogen fixation and anammox; however, the balance of these functional capabilities seems to be relevant to maintain water quality.     

Biofilm consumption shapes the intestinal microbiota of shrimp (Penaeus vannamei)

The effect using biofilm as complementary feed for shrimp (Penaeus vannamei) on the taxonomic and functional profile of its intestinal microbiota was evaluated. Shrimp were cultured for 30 days in two similar low‐water exchange systems, one using commercial feed and the other a combination of commercial feed with biofilm as complementary feed source. Six bacteria phyla were detected in the intestine of shrimp from both treatments (Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, Fusobacteria and Proteobacteria), being Proteobacteria the most abundant (81%–92%). However, this later phylum was significantly less dominant in shrimp fed with biofilm; this drop was associated with a decrease in Vibrio abundance. Differences in the taxonomic profile of bacteria between treatments were also observed through principal component analysis where the diet was the main influencing factor (PC1 72.4%). Regarding functional inference, similarities were detected between treatments. Carbohydrate and energy metabolism were the most abundant functions regardless of time or diet, followed by metabolism of cofactors and vitamins, translation processes, nucleotide metabolism and others. Results suggest that biofilm consumption influenced the taxonomic profile bacteria colonizing the shrimp intestine; however, a functional redundancy was observed despite the use of biofilm as complementary feed. Finally, shrimp fed with biofilm showed a better productive response.     

Effects of biological water purification grid on microbial community of culture environment and intestine of the shrimp Litopenaeus vannamei

Biological water purification grid (BWPG), a type of biofilm, could increase aquaculture production by improving the growth of shrimp and culture environment. In this study, the microbial community composition and diversity of water, sediment and shrimp intestine were analysed using the Illumina Miseq high‐throughput sequencing technique. Installation of BWPG did not only increase the dissolved oxygen, pH and decreased inorganic nutrients, but also increased shrimp body length by 5.39% and weight by 16.35%. Furthermore, it was found that the microbial community diversity of water and shrimp intestine in test pond with BWPG was higher than that of control pond without BWPG. Cyanobacteria, Bacillus and Lactococcus were enriched in the test pond with BWPG. However, Rhodobacter was mainly identified in the test pond. It thus seems to suggest that the application of BWPG in shrimp culture pond enhanced the microbial species richness, types of species and proportion of beneficial bacteria in culture environment as well as shrimp intestine. The results from this study will therefore provide some scientific basis for the improvement and development of shrimp aquaculture.     

Breeding and life cycle of the ornamental freshwater shrimp Neocaridina davidi in a biofilm‐based culture system

Neocaridina davidi is a popular shrimp in the aquarium industry; however, information regarding its husbandry is scarce. In this study, we investigated the contribution of biofilm to its life cycle, comprising three successive phases: (1) the evaluation of biofilm growth on plastic nets (PN), plastic sheets (PS) and agrovelo (AV); (2) the reproduction of adult shrimp to get juveniles (JI); and (3) the effects of biofilm on the survival and growth performance of JI. Trials were performed in aquaria with zero water exchange and natural environmental conditions. Biofilm was composed mainly of microalgae, diatoms, cyanobacteria and ciliates and used as the sole diet. Survival, biomass and biochemical reserves of JI reared in this culture system were significantly higher in the presence of PN and AV substrates. The occurrence of ovarian maturation and egg incubation of female shrimp in these treatments indicate that biofilm supplied the energy required for somatic growth and fecundity. Harvested females also displayed the size and the red pigmentation associated with premium pricing. Based on these results, it is concluded that N. davidi can complete the life cycle and display characteristic life history traits in a low‐cost biofilm technology system without losing economic value as ornamental species.     

Characterization by whole-cell hybridization of bacterial populations associated with shrimp hatchery biofilms

The impact of shrimp larvae development, as well as water and food inputs upon the increase of bacterial populations within the bacterial community of hatchery tank biofilms, was studied. For this study, a total of 68 biofilm samples were collected from concrete tanks at three larvae production times in a commercial shrimp hatchery. Seventeen samples were taken at each larval development stage (Zoea I, Mysis I, postlarvae 1 and postlarvae 16), as well as 37 samples from water, shrimp nauplii and food, introduced into the shrimp hatchery tanks. Culturable and direct bacterial counts were performed and 16S‐rRNA‐targeted oligonucleotide probes were used to quantify the presence of specific bacterial groups. An average of 27–70% of DAPI total cell counts were detected with the EUB338 probe, while the GAM42a probe signal ranged from 1% to 11%. Vibrio‐like bacteria (VLB) counts in TCBS agar ranged from <10 to 10¹ VLB/cm⁻², with a tendency to increase at the last postlarvae stage. The most significant external source of bacteria registered with GAM42a probe and TCBS agar were found in live Artemia nauplii, used as food; nevertheless, biofilms remain with low counts of these groups.     

Effects of a probiotic (Bacillus subtilis FY99‐01) on the bacterial community structure and composition of shrimp (Litopenaeus vannamei,Boone) culture water assessed by denaturing gradient gel electrophoresis and high‐throughput sequencing

In China, many commercial probiotics are added as water additives into shrimp culture ponds with no sediment. In these ponds, probiotics should have great effects on the bacterial community of culture water at different culture stages. However, the specific effects have not been clarified yet. In this study, the effects of applying the probiotic Bacillus subtilis FY99‐01 on the bacterial community of shrimp (Litopenaeus vannamei) culture water at different culture stages were determined by denaturing gradient gel electrophoresis and high‐throughput sequencing. The water quality parameters, shrimp production, survival rate and feed conversion ratio (FCR) were also analysed. The results showed that application of probiotics improved the water quality by reducing the levels of pH, nitrite and soluble reactive phosphorus, whereas there was no significant effect on shrimp production, survival rate and FCR. Application of probiotics brought no obvious effect on bacterial α diversity while had effect on bacterial β diversity. In addition, probiotics brought different effects on the bacterial community structure at different culture stages, and the effects are stronger in the early and middle phase than in the late phase. Sequencing results demonstrated that probiotics could increase the abundance of Flavobacteria, whereas decrease the abundance of α‐Proteobacteria in the late phase of culture. Besides, principle component analysis results indicated that using probiotics also could increase the abundance of beneficial microalgae (Bacillariophyta and Chlorophyta) and decrease the abundance of Vibrionaceae.     

Development of a Model to Simulate Nitrogen Dynamics in an Integrated Shrimp–Macroalgae Culture System with Zero Water Exchange

The effluents of traditional shrimp monoculture cause pollution and promote eutrophication and hypernutrification of the receiving coastal ecosystems. Integrated aquaculture and a recirculating aquaculture system (RAS) have been proposed as an alternative to address these problems. In this study, we developed a dynamic model to simulate the concentration of total ammonia nitrogen (TAN), nitrite, and nitrate in an integrated culture of whiteleg shrimp, Litopenaeus vannamei, and seaweed, Gracilaria vermiculophylla, in a recirculating and zero water exchange system, and the effect of nitrifying and heterotrophic bacteria was also included. The experiments demonstrated that a dynamic model can explain the concentrations of dissolved inorganic nitrogen and variations in these concentrations over time in the integrated culture. The results also suggest that nitrifying and heterotrophic bacteria play an important role in the transformation of dissolved nitrogenous compounds; therefore, these bacteria should be considered within the dynamics of nitrogen in integrated systems with low water exchange.     

Abundance of sulphur‐oxidizing bacteria in coastal aquaculture using soxB gene analyses

Molecular techniques based on sequencing of metagenomic clone libraries provide an insight into the diversity of microbial populations. Using nucleic acid‐based methods, the diversity of soxB genes was examined to detect and characterize sulphur‐oxidizing bacteria in Indian coastal aquaculture environments. Gene‐specific degenerate primers were used to amplify various fragments (710, 753, 483–503, 280 and 239 bp) of soxB genes. Metagenomic clone libraries were constructed for 753, 483–503 and 239 bp fragments of soxB genes. The abundance of soxB revealed the presence of sulphur‐oxidizing organisms. Amino acids in parts of the soxB‐encoded proteins were aligned to known conserved amino acid residues. The level of conservation ranged from 23% to 30%. A phylogenetic tree constructed from aligned amino acid sequences of SoxB revealed different clusters associated with the branches of phototrophic α‐ and γ‐proteobacteria. In general, soxB is widespread among the various phylogenetic groups, although this does not necessarily mean that the organism can use sulphur compounds. Our results suggest that the chemolithoautotrophy based on sulphur oxidation in coastal aquaculture is primarily sustained by the presence of sulphur oxidizers, which involve the soxB gene. This study aids identification of the phylogenetic characteristics related to sulphur bioremediation in poorly characterized coastal aquaculture environments.     

Characterization of bacterial community in intestinal and rearing water of Penaeus monodon differing growth performances in outdoor and indoor ponds

Penaeus monodon provides a high‐quality protein source for humanity, and pond cultured shrimp often presents asynchronous growth. Microbial communities are important for the digestion and immunity of shrimp. Therefore, in this study, we investigated the bacterial characteristics of the intestine and rearing water of asynchronously growing P. monodon that were cultured in outdoor and indoor pond respectively. The results showed that the bacterial community of the rearing water was more complex than that of the intestine; the fast‐growing shrimp in the indoor pond had higher intestinal bacterial diversity. Besides, the dominant bacterial composition of the water was more complex than that of the intestinal. Specifically, the abundance of Proteobacteria in the intestine was consistent with the growth performance of shrimp in the outdoor pond, which was exactly the opposite in the indoor pond. At the genus level, two cyanobacteria, Limnothrix and Cyanobium PCC‐6307, were dominant in the indoor and outdoor water respectively. In the outdoor pond, Bacillus was dominant in the slow‐growing shrimp intestines, while Vibrio was dominant in the fast‐growing shrimp. The intestinal microbes of the fast‐growing shrimp had higher proteasome metabolic capacity. These results can provide new insights into microbiome characteristics involved in the asynchronous growth of shrimp.     

Intestinal microbiota of white shrimp,Litopenaeus vannamei,fed diets containing Bacillus subtilis E20‐fermented soybean meal (FSBM) or an antimicrobial peptide derived from B. subtilis E20‐FSBM

The purpose of the present study is to profile the microfloral structure in the intestines of the white shrimp, Litopenaeus vannamei, after being fed a control diet, in comparison with the diets containing Bacillus subtilis E20‐fermented soybean meal (FSBM) or an antimicrobial peptide (AMP) isolated from B. subtilis E20‐FSBM (diets designated FSBMD and AMPD, respectively) for 60 days. Metagenomic data showed detection of eight phyla, 20 classes, 40 orders, 68 families and 96 genera. Despite no statistically significant difference, an evaluation of microbial diversity recorded higher species richness, Shannon–Weaver diversity index and evenness in the AMPD and FSBMD groups, compared to the control diet group. Venn diagrams showed that 58 of the operational taxonomic units (OTUs) were shared among the control, FSBMD and AMPD groups, but additional beneficial microbes were only found in the FSBMD and AMPD groups. In addition to the augmentation of beneficial bacteria in the FSBMD and AMPD groups, the abundance of potential pathogens, Vibrio and Flavobacterium, was lower in the gut of shrimp fed the FSBMD and AMPD. The results clearly suggest that the FSBMD and AMPD have the capability to change the microfloral structure of shrimp intestines and could be used for disease prevention in shrimp aquaculture.     

长毛对虾海水养殖环境以及虾肠道微生物群落结构研究

为了研究长毛对虾养殖环境以及对虾肠道微生物种群结构的特征,实验分别采集养殖区进水口水体、养殖池底泥、养殖池水体以及长毛对虾肠道样品,采用构建16S rRNA基因克隆文库的方法对不同样品间的微生物群落组成进行了研究。结果表明,4组样品中共获得621条序列,操作分类单元(OTU)总数达212个,表明养殖环境微生物群落结构具有高度的多样性。从遗传进化树分析发现,进水口水体中细菌优势种群为蓝细菌(53.97%)、α-变形杆菌(13.76%)和γ-变形杆菌(10.58%);养殖池水体细菌优势种群为蓝细菌(33.55%)、γ-变形杆菌(14.84%)、厚壁菌(14.19%)、拟杆菌(12.26%)和α-变形杆菌(9.68%);养殖池底泥细菌优势种群大部分属于厚壁细菌(79.12%);对虾肠道细菌优势种群为厚壁细菌(75.79%)、梭杆菌(13.68%)和γ-变形杆菌(10.53%)。在目分类水平上,养殖池底泥、养殖池水体和对虾肠道中芽孢杆菌占有较高的比例,分别占克隆数的69.78%、13.55%和72.63%;进水口水体和养殖池水体中红细菌的比例较高,分别占克隆数的10.05%和9.68%。本研究分析了养殖环境以及对虾肠道微生物的群落结构,揭示微生物从水源到对虾肠道内的演替规律。总体上,本养殖系统微生物群落结构良好,但在养殖池水体和对虾肠道中也检测到黄杆菌类群和少量的弧菌。本研究有助于了解养殖环境对于对虾肠道微生物组成的影响,并为长毛对虾病害的预防提供参考。     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Short‐term effect of the inoculation of probiotics in mature bioflocs: Water quality parameters and abundance of heterotrophic and ammonia‐oxidizing bacteria

The aim of this study was to evaluate the short‐term effect of probiotic inoculation on the abundance of heterotrophic and ammonia‐oxidizing bacteria in mature biofloc, as well as on total suspended solids (TSS), chlorophyll‐a and nitrogenous compounds in water. A completely randomized design consisting of five treatments (three commercial probiotics, one native consortia and one control) was performed. At the beginning of the experiment (day 1), each treatment was inoculated with the respective probiotic: PondPlus^® (PP), Efinol^® PT (EF) and Epicin^® ponds (EP), native consortia UE, whereas the control was not inoculated. Water parameters and bacterial abundance were evaluated at 0, 2, 4, 6 and 8 days. The addition of probiotics, either native or commercial, did not show any significant effect on the TSS, Chl‐a and colony‐forming unit (CFU) of heterotrophic bacteria when they were added to the systems containing mature biofloc. A significant increase in ammonium oxidizing bacteria was registered with the probiotics PP and EP, although the levels of total ammonia nitrogen, NO₃‐N and NO₂‐N were statistically similar among all treatments. Modifications on most of the parameters measured were associated with the factor of time, rather than the inclusion of probiotics. Results suggest that the bacterial conglomerates in mature stage contain well‐established bacterial communities that are difficult to be affected by the addition of probiotics.     

Characterization of virulence,cell surface characteristics and biofilm‐forming ability of Aeromonas spp. isolates from fish and sea water

Members of the genus Aeromonas are emerging human pathogens, causing a variety of extra‐intestinal, systemic and gastrointestinal infections in both immunocompetent and immunocompromised persons. Aeromonas virulence is multifaceted and involves surface‐associated molecules, motility, biologically active extracellular products and biofilm formation. Aeromonads, isolated from diverse freshwater fish species as well as sea water, were screened for biofilm formation, with varying physicochemical parameters including temperature, agitation and nutrient availability. Motility, cell surface characteristics (auto‐aggregation, hydrophobicity and S layer), and extracellular virulence factor production (haemolysis, proteolysis, DNase production) were also assessed to identify potential associations with the biofilm phenotype. Biofilm formation was influenced by environmental conditions, with isolates preferentially forming biofilms in nutrient‐rich media at 30 °C, although strong biofilm formation also occurred at 37 °C. Strong biofilm formation was observed for Aeromonas culicicola isolates following exposure to nutrient‐rich conditions, while Aeromonas allosaccharophila isolates preferred nutrient‐poor conditions for biofilm formation. Source‐/species‐specific correlations, ranging from weak to strong, were observed between biofilm formation and motility, cell surface characteristics and/or extracellular virulence factor production. Understanding the specific mechanisms by which Aeromonas species adhere to abiotic surfaces may aid in preventing and/or treating disease outbreaks in aquaculture systems and could lead to effective eradication of these fish pathogens.     

Biofilm feeding by postlarvae of the pink shrimp Farfantepenaeus brasiliensis (Decapoda,Penaidae)

The effect of biofilm was assayed for Farfantepenaeus brasiliensis postlarvae fed with commercial pellets. Indoor tanks in a zero water exchange system were used, considering: shrimp fed with biofilm and commercial feed (B+F), and shrimp fed only with commercial feed (F); both receiving polyethylene sheets as artificial substrates. For B+F, sheets were placed 15 days before the trial into a heterotrophic medium (containing diatom Thalassiosira weissflogii, commercial feed, molasses and wheat bran in a 20 C:1 N ratio) to promote biofilm development. For F, clean sheets were used and daily replaced to avoid biofilm formation. Biofilm chlorophyll a concentration, micro‐organisms density and water quality were determined weekly. Also, a stomach content analysis was made. An increase in chlorophyll a concentration was observed in biofilm during the experiment, characterized mainly by pennate diatoms, filamentous cyanobacteria, flagellates, ciliates, nematodes and rotifers. Most of these items were found in the stomach of shrimp; however, no significant differences in growth were detected between treatments. Survival was significantly higher and nitrite concentrations were significantly lower when biofilm was present. The results indicate that the use of biofilm could be considered a good tool during F. brasiliensis nursery phase, mainly by improving survival through the maintenance of water quality.     

Phytoplankton community structure and diversity in the indoor industrial aquaculture system for Litopenaeus vannamei revealed by high‐throughput sequencing and morphological identification

The explosive multiplication of phytoplankton caused by water eutrophication often occur in the intensive shrimp aquaculture. To comprehensively assess the diversity and community structure of phytoplankton in the waters of typical indoor industrial aquaculture system for Litopenaeus vannamei, a combination of high‐throughput sequencing and morphological identification methods were used in the present study. A total of 41 genera belong to nine phyla were detected by both methods. Chlorophyta, Cyanophyta and Bacillariophyta were found to be three dominant phyla. The high‐throughput sequencing revealed that green algae and cyanobacteria were the most dominant phytoplankton; however, diatoms were the first dominant phytoplankton by using the morphological identification. At the genus level, Picochlorum and Synechococcus were dominant, accounting for 20.94%–97.19% and 0.01%–52.81% of total phytoplankton, revealed by the high‐throughput sequencing. Therefore, more attention should be paid to their ecological impacts on the surrounding sea areas or potential toxicity to shrimp. Cyclotella was the most dominant genus revealed by the morphological identification. High‐throughput sequencing revealed a high diversity and small‐sized phytoplankton which were undetected by microscopy. Both methods provide similar information on the environmental drivers of phytoplankton community. NO₃^?, NH₄⁺, DIP, DSi, DON and DOP concentrations were the main factors influencing the phytoplankton community structure and diversity.     

Variability of the nitrifying bacteria in the biofilm and water column of a recirculating aquaculture system for tilapia (Oreochromis niloticus) production

The aim of this study was to evaluate variability of nitrifying bacterial community in the biofilm and in the water of a recirculating aquaculture systems (RAS) in a tilapia farming in order to determine if nitrification process is dependent, or not, of nitrifying bacteria abundance. Biofilm and water samples were collected periodically for 30 days and analysed with the fluorescent in situ hybridization (FISH) technique, used to quantify ammonia‐oxidizing bacteria (AOB) and nitrite‐oxidizing bacteria (NOB). Ammonia presented the peak in the first week, while the nitrite's maximum was recorded in the second week. Nitrate increased steadily, indicating nitrification activity. Total bacterial abundance in biofilm increased continuously, while in water, it did not change significantly. In the biofilm, number of AOB was high at beginning, decreased after few days and increased again following augment of ammonia. Number of NOB also showed an increase in abundance in biofilm following the increment of nitrite and nitrate. In water, AOB and NOB did not show major variability. Relative abundance of nitrifying bacteria represented more than 30% of total bacteria in biofilm at beginning of the experiment. Their contribution decreased to >3% in last days. It indicates that nitrifying bacteria are biofilm colonizers, and that their activity seems to be directly related to the concentration of nitrogen compounds. However, contribution of nitrifying bacteria did not vary much along the time. We may conclude that the biofilm‐nitrifying bacteria plays major role in nitrification process in RAS and that the activity of these organisms is dependent of their abundance in response to the concentration of nitrogen compounds.     

Evaluation of Agave lechuguilla by‐product crude extract as a feed additive for juvenile shrimp Litopenaeus vannamei

Agave lechuguilla is a succulent plant species, mainly distributed in the northeast of Mexico and south of the United States of America. The main use for this plant is the fibre´s extraction (known as Tampico fibres), resulting in 15% of fibres and 85% of a by‐product waste named guishe. The lechuguilla collectors, normally incinerate the guishe, thus causing environmental contamination. Interestingly, recent studies showed that guishe contains molecules with nutritional properties, such as saponins, flavonoids and sugars. Therefore, in this work, we evaluated the effect of the crude extract of guishe as a feed additive in whiteleg shrimp diets. According to that, MS‐HPLC analysis of the extract showed the presence of saponins such as diosgenin, smilagenin, hecogenin, manogenin, tigogenin hexose, yucagenin, chlorogenin, diosgenin diglucoside and the flavonol, quercetin. After chemical analysis, the crude extract was included into an experimental diet in four levels; 0% (L0%), 0.1% (L0.1%), 0.3% (L0.3%) and 0.6% (L0.6%). Dietary incorporation of the extract was evaluated by zootechnical performance, haemolymph biochemistry, histomorphology and digestive enzyme activity of shrimps. After 5‐week feeding, the L0.3% diet showed significantly higher growth and better feed utilization among treatments. A significant increase in tubule epithelium height and tubule coverage area from hepatopancreas in shrimp under L0.3% diet compared with the control diet suggest an improvement of the health and nutritional status of the shrimp. Inclusion of L0.3% and L0.6% of the crude extract resulted in a reduction in amylase activity, without effect in glucose levels in the haemolymph. Thus, we suggest that lechuguilla guishe crude extract contains nutritional molecules that may be used as a feed additive to promote shrimp productivity.