Investigating the Effect of Bacilli and Lactic Acid Bacteria on Water Quality, Growth, Survival, Immune Response, and Intestinal Microbiota of Cultured Litopenaeus vannamei.

Shrimp is one of the most important aquaculture industries. Therefore, we determined the effect of nitrifying-probiotic bacteria on water quality, growth, survival, immune response, and intestinal microbiota of Litopenaeus vannamei cultured without water exchange. In vitro, only Bacillus licheniformis used total ammonia nitrogen (TAN), nitrites, and nitrates since nitrogen bubbles were produced. TAN decreased significantly in the treatments with B. licheniformis and Pediococcus pentosaceus and Leuconostoc mesenteroides, but no differences were observed in nitrites. Nitrates were significantly higher in the treatments with bacteria. The final weight was higher only with bacilli and bacilli and LAB treatments. The survival of shrimp in the bacterial treatments increased significantly, and superoxide anion increased significantly only in lactic acid bacteria (LAB) treatment. The activity of phenoloxidase decreased significantly in the treatments with bacteria compared to the control. Shrimp treated with bacilli in the water showed lower species richness. The gut bacterial community after treatments was significantly different from that of the control. Linoleic acid metabolism was positively correlated with final weight and superoxide anion, whereas quorum sensing was correlated with survival. Thus, bacilli and LAB in the water of hyperintensive culture systems act as heterotrophic nitrifers, modulate the intestinal microbiota and immune response, and improve the growth and survival of shrimp. This is the first report on P. pentosaceus and L. mesenteroides identified as nitrifying bacteria.

In-depth assessment of microbial communities in the full-scale vertical flow treatment wetlands fed with raw domestic wastewater.

A multiphase study was proposed to examine microbial communities linked to the nitrogen cycle in the first stage of four full-scale French vertical flow treatment systems. To this end, denaturing gradient gel electrophoresis (DGGE) was performed for structural assessment and quantitative PCR (qPCR) to enumerate the abundance of ammonia-oxidizing (AOB). 16S rRNA sequencing was used to assess the taxonomic profile followed by putative assessment of functional genes. The samples were collected under different conditions, such as operational time (presence/absence of sludge layer on the surface of the filters), season (winter and summer), sampling depth (0, 15 and 30 cm) and operation cycle (rest and feed periods). A structural disparity was noted in the upper layers, whereas higher similarity at 30 cm was observed highlighting the effect of organic matter on bacterial diversity. The 7th rest day was highlighted by an apparent decline in the microbial community abundance. Additionally, qPCR indicated that the largest amount of AOB was found at 30 cm depth and during the feeding period. From the taxonomic profile, Mycobacterium, Acinetobacter, Flavobacterium, and Nitrospira were the most abundant genre found in all systems. The functional prediction results showed predicted genes linked to the denitrification process. The results suggested that operating time and season were responsible for the pattern of the microbial community behavior. This study allowed us to further understand the bacterial dynamics and to advance the idea of design modifications made in the first stage of the classical French system to improve nitrogen removal are promising.

Evaluation of nitrogen removal and the microbial community in a submerged aerated biological filter (SABF), secondary decanters (SD), and horizontal subsurface flow constructed wetlands (HSSF-CW) for the treatment of kennel effluent.

To ensure microbial activity and a reaction equilibrium with efficiency and energy saving, it is important to know the factors that influence microbiological nitrogen removal in wastewater. Thus, it was investigated the microorganisms and their products involved in the treatment of kennel effluents operated with different aeration times, phase 1 (7 h of continuous daily aeration), phase 2 (5 h of continuous daily aeration), and phase 3 (intermittent aeration every 2 h), monitoring chemical and physical parameters weekly, monthly microbiological, and qualitative and quantitative microbiological analyzes at the end of each applied aeration phase. The results showed a higher mean growth of nitrifying bacteria (NB) (106) and denitrifying bacteria (DB) (1022) in phase with intermittent aeration, in which better total nitrogen (TN) removal performance, with 33%, was achieved, against 21% in phase 1 and 17% in phase 2, due to the longer aeration time and lower carbon/nitrogen ratio (15.7), compared with the other phases. The presence of ammonia-oxidizing bacteria (AOB), the genus Nitrobacter nitrite-oxidizing bacteria (NOB), and DB were detected by PCR with specific primers at all phases. The analysis performed by 16S-rRNA DGGE revealed the genres Thauera at all phases; Betaproteobacteria and Acidovorax in phase 3; Azoarcus in phases 2 and 3; Clostridium, Bacillus, Lactobacillus, Turicibacter, Rhodopseudomonas, and Saccharibacteria in phase 1, which are related to the nitrogen removal, most of them by denitrifying. It is concluded that, with the characterization of the microbial community and the analysis of nitrogen compounds, it was determined, consistently, that the studied treatment system has microbiological capacity to remove TN, with the phase 3 aeration strategy, by simultaneous nitrification and denitrification (SND). Due to the high density of DB, most of the nitrification occurred by heterotrophic nitrification-aerobic. And denitrification occurred by heterotrophic and autotrophic forms, since the higher rate of oxygen application did not harm the DB. Therefore, the aeration and carbon conditions in phase 3 favored the activity of the microorganisms involved in these different routes. It is considered that, in order to increase autotrophic nitrification-aerobic, it is necessary to exhaust the volume of sludge in the secondary settlers (SD), further reducing the carbon/nitrogen ratio, through more frequent cleaning, whose periodicity should be the object of further studies. Graphical abstract.

Tetracyclines lead to ammonium accumulation during nitrification process.

The effect of tetracyclines used for swine food-production (tetracycline and oxytetracycline) on enriched nitrifying bacteria cultures over time was investigated in this study. Short-term exposure assays were performed in different concentrations of each antibiotic, using ammonia oxidizing bacteria (AOB) culture and nitrifying bacteria. The results pointed out a higher inhibitory effect of tetracycline on both bacterial communities. The AOB was more sensitive to antibiotic exposure when compared to the nitrifying culture. Although high antibiotic concentrations were applied, the half maximal inhibitory concentration (IC50) was achieved only for the AOB culture exposed to tetracycline at a concentration of 273 mg L-1. Nonetheless, the long-term exposure assay demonstrated a reduction of the tetracycline inhibition effect against AOB. The exposure to 100 mg L-1 of tetracycline (TC) did not show relevant influence over ammonium conversion efficiency; however, at 128 mg L-1 of TC, the efficiency decreased from 94% to 72%. Further investigation revealed that TC reduced the final effluent quality due to the development of a resistance mechanism by AOB culture against this antibiotic. This mechanism involves increasing the excretion of extracellular polymeric substances (EPS) and soluble microbial products (SMP), which probably increases BOD, and reduces ammonia consumption by the bacterial culture.

Assessment of the nitrification process in a culture of pacific white shrimp, using artificial substrate and bacterial inoculum in a biofloc technology system (BFT) / Avaliação do processo de nitrificação no cultivo do camarão branco do pacífico Litopenaeus vannamei com uso de substrato artificial e inóculo de bactérias em sistema de bioflocos (BFT)

Litopenaeus vannamei is the most cultured marine shrimp in all types of systems including the Bioflocs Technology System (BFT). Bioflocs are formed by microorganisms, among these, autotrophic bacteria are responsible for the nitrification process. This study aimed to identify and promote the development of nitrifying bacteria by adding artificial substrates and biofloc inoculum in L. vannamei culture in a BFT system. The experiment consisted of four treatments with three replics (4x3) as follows: (1) Control: clear water in which bioflocs were formed; (2) IN (10%): clear water with biofloc inoculum (10%); (3) IB: clear water with substrate (immature bioballs); and (4) MB: clear water with mature bioballs inoculum from a recirculation system. Treatments were stocked with shrimp juveniles (4.92±0.45 g) in 12 tanks with 200 L working volume at a stocking density of 200 shrimp/m³. Shrimps were fed twice a day with a commercial feed (38% CP) following a feeding table, and daily observations intake were made over the four weeks of the experiment. Biofloc and bioballs samples were collected to detect the growth of the population of nitrifying and heterotrophic bacteria by FISH. There was no significant difference between treatments (P>0.05) for survival, obtaining mean values greater than 88%. The IN (10%) treatment had lower concentrations of ammonia and nitrite, and nitrate concentration increased, while MB had a higher weight and biomass final, productivity, weekly weight gain and lower conversion of apparent feed for production performance results.(AU)

Litopenaeus vannamei é o camarão marinho mais cultivado em todos os tipos de sistemas, incluindo o Sistema de Tecnologia de Bioflocos (BFT). Os bioflocos são formados por microorganismos, entre estes, bactérias autotróficas que são responsáveis pelo processo de nitrificação. Este estudo teve como objetivo identificar e promover o desenvolvimento de bactérias nitrificantes pela adição de substratos artificiais e inóculo de bioflocos no cultivo de L. vannamei em sistema BFT. O experimento consistiu de quatro tratamentos com três repetições (4x3), sendo: (1) Controle: água limpa, na qual foram formados os bioflocos; (2) IN (10%): água limpa com inóculo de bioflocos (10%); (3) BI: água limpa com substrato (bioballs imaturos); e (4) BM: água limpa com inóculo de bioballs maduros de um sistema de recirculação. Para tanto, os tratamentos foram estocados com juvenis de camarão (4,92±0,45 g) em 12 tanques com 200 L de volume útil com densidade de 200 camarões/m³. Os camarões foram alimentados duas vezes ao dia com ração comercial (38% PB) seguindo uma tabela de alimentação, e observações diárias foram feitas ao longo das quatro semanas de experimento. Amostras de bioflocos e bioballs foram coletadas para detectar o crescimento da população de bactérias nitrificantes e heterotróficas por FISH. Não houve diferença significativa entre os tratamentos (P>0,05) para sobrevivência, obtendo-se valores médios superiores a 88%. O tratamento com IN (10%) apresentou menores concentrações de amônia e nitrito, e a concentração de nitrato aumentou, enquanto o BM apresentou maior peso e biomassa final, produtividade, ganho de peso semanal e menor conversão alimentar aparente para resultados de desempenho zootécnico.(AU)

Assessment of the nitrification process in a culture of pacific white shrimp, using artificial substrate and bacterial inoculum in a biofloc technology system (BFT) / Avaliação do processo de nitrificação no cultivo do camarão branco do pacífico Litopenaeus vannamei com uso de substrato artificial e inóculo de bactérias em sistema de bioflocos (BFT)

ABSTRACT: Litopenaeus vannamei is the most cultured marine shrimp in all types of systems including the Bioflocs Technology System (BFT). Bioflocs are formed by microorganisms, among these, autotrophic bacteria are responsible for the nitrification process. This study aimed to identify and promote the development of nitrifying bacteria by adding artificial substrates and biofloc inoculum in L. vannamei culture in a BFT system. The experiment consisted of four treatments with three replics (4x3) as follows: (1) Control: clear water in which bioflocs were formed; (2) IN (10%): clear water with biofloc inoculum (10%); (3) IB: clear water with substrate (immature "bioballs"); and (4) MB: clear water with mature "bioballs" inoculum from a recirculation system. Treatments were stocked with shrimp juveniles (4.92±0.45 g) in 12 tanks with 200 L working volume at a stocking density of 200 shrimp/m³. Shrimps were fed twice a day with a commercial feed (38% CP) following a feeding table, and daily observations intake were made over the four weeks of the experiment. Biofloc and "bioballs" samples were collected to detect the growth of the population of nitrifying and heterotrophic bacteria by FISH. There was no significant difference between treatments (P>0.05) for survival, obtaining mean values greater than 88%. The IN (10%) treatment had lower concentrations of ammonia and nitrite, and nitrate concentration increased, while MB had a higher weight and biomass final, productivity, weekly weight gain and lower conversion of apparent feed for production performance results.

RESUMO: Litopenaeus vannamei é o camarão marinho mais cultivado em todos os tipos de sistemas, incluindo o Sistema de Tecnologia de Bioflocos (BFT). Os bioflocos são formados por microorganismos, entre estes, bactérias autotróficas que são responsáveis pelo processo de nitrificação. Este estudo teve como objetivo identificar e promover o desenvolvimento de bactérias nitrificantes pela adição de substratos artificiais e inóculo de bioflocos no cultivo de L. vannamei em sistema BFT. O experimento consistiu de quatro tratamentos com três repetições (4x3), sendo: (1) Controle: água limpa, na qual foram formados os bioflocos; (2) IN (10%): água limpa com inóculo de bioflocos (10%); (3) BI: água limpa com substrato ("bioballs" imaturos); e (4) BM: água limpa com inóculo de "bioballs" maduros de um sistema de recirculação. Para tanto, os tratamentos foram estocados com juvenis de camarão (4,92±0,45 g) em 12 tanques com 200 L de volume útil com densidade de 200 camarões/m³. Os camarões foram alimentados duas vezes ao dia com ração comercial (38% PB) seguindo uma tabela de alimentação, e observações diárias foram feitas ao longo das quatro semanas de experimento. Amostras de bioflocos e "bioballs" foram coletadas para detectar o crescimento da população de bactérias nitrificantes e heterotróficas por FISH. Não houve diferença significativa entre os tratamentos (P>0,05) para sobrevivência, obtendo-se valores médios superiores a 88%. O tratamento com IN (10%) apresentou menores concentrações de amônia e nitrito, e a concentração de nitrato aumentou, enquanto o BM apresentou maior peso e biomassa final, produtividade, ganho de peso semanal e menor conversão alimentar aparente para resultados de desempenho zootécnico.

Variación espacial y estacional de grupos funcionales de bacterias cultivables del suelo de un bosque tropical seco en México / Spatial and seasonal variation of soil culturable-bacterial functional groups in a Mexican tropical dry forest

Microbial biomass and activity in soils are frequently studied in tropical dry forests, but scarce information is available about the relationships between functional bacterial groups and soil fertility, where relief interacts with rainfall seasonality. The culturable-bacterial groups and nutrients were studied during two consecutive years in soils from two topographic areas of different relief (hilltop vs hillslope) in a tropical dry forest from Chamela Jalisco, Mexico. We expected that seasonal and spatial variation in soil resources availability affects the abundance of functional culturable-bacterial groups. To evaluate this, fifteen soil cores (1kg), 0-5cm depth, were taken in the dry, early rainy and rainy seasons, from each of the ten replicate plots in hilltop and hillslope areas located in three microbasins. We found that hilltop soils were more organic and had higher concentration of labile C and total nutrient forms than hillslope soils, for which these soils had higher counts of colony-forming units (CFU) of total heterotrophic and P solubilizing bacteria. In both hilltop and hillslope soils, C and nutrient concentrations, as well as the counts of CFU of heterotrophic and P solubilizing bacteria generally decreased from the dry to the rainy season during the two study years. In contrast, the counts of CFU nitrifying and cellulolytic bacteria were higher at the hillslope than at the hilltop soils. The seasonal pattern of both groups was opposite to that of heterotrophic bacteria, presumably associated with a decrease in soil labile C and organic matter quality. In conclusion, our study suggests that available C appears to be the main factor that controls the structure of soil bacterial groups and soil fertility, where relief, rainfall seasonality and intraand inter-annual variations are critical factors that interactively modify bacterial dynamics related to soil C availability in the tropical dry forest.

Los bosques tropicales secos tienen una amplia heterogeneidad ambiental y por lo tanto se podría esperar una variación temporal y espacial en la abundancia y tipo de grupos bacterianos activos. Se evaluó, durante dos años consecutivos, el efecto de la estacionalidad de la lluvia sobre algunos grupos bacterianos cultivables y nutrimentos del suelo en dos posiciones topográficas (crestas vs laderas) en el bosque tropical seco, Chamela Jalisco, México. La hipótesis fue que la variación temporal y espacial de la disponibilidad de recursos afectaría la abundancia de los grupos bacterianos funcionales (heterótrofas totales, celulolíticas, solubilizadores de fosfatos y nitrificantes) del suelo. En las crestas, el suelo fue más orgánico y con mayor concentración de nutrimentos totales y C orgánico lábil que en la ladera, registrando más bacterias heterótrofas y solubilizadoras de fosfato. En ambas posiciones topográficas la concentración de C y nutrimentos, así como el número de bacterias heterótrofas y solubilizadoras de P, decrecieron de la estación seca a la lluviosa en ambos años. En contraste, las bacterias nitrificantes fueron mayores en el suelo de la ladera, y al igual que las celulolíticas, tuvieron un patrón estacional opuesto a las bacterias heterótrofas, asociado a la disminución del C lábil y de la calidad de la materia orgánica. En conclusión, se sugiere que el relieve, la estacionalidad y la variación inter-anual de la lluvia regulan interactivamente la relación entre el C disponible y la estructura de las comunidades bacterianas del suelo en el bosque tropical seco de Chamela.