Effects Of Hydroponics Peppermint Beds And Combined Fillers On Bacterioplankton Communities In Fish Production Systems

Recently,the wild capture fisheries report a decline as the industry tries to meet the global per capita consumption.The freshwater and marine fisheries project collapse in a few decades if the current trend continues.With worldwide consideration of freshwater aquaculture and mariculture enhancement,these will overcome the detrimental effects.Improvement of global aquaculture production methods through modern farming techniques including;cage culture,aqua tanks,integrated pond,and re-circulatory aquaculture systems(RAS)are focused upon to sustain the fish production,food security,improvement and modification of the applied technologies.These technologies apply with minimal or limited recycled water through mechanical and biological treatments.Further,aquaculture systems usually encounter severe nitrogenous waste production under intensive production and temperature rises(global warming)during summer production.This dissertation suggests two conventional methods of bio-augmentation involving hydroponic peppermint bed and bio-filter(combined filler)technologies alongside microbial enhancements as biological and more cost-effective methods for subsistence and commercial application.Several studies designed to determine the effects of the hydroponic peppermint beds and combined filler technologies on the water quality,bacterioplankton communities especially the ammonia-oxidizing nitrifiers,fish safety,and the effluent dynamics.1.Effects of combined fillers and hydroponic peppermint bed technologies on bacterioplankton communities in intensive Grass(Ctenopharyngodon idella)and silver(Hypophthalmichthys molitrix)carp pond cultures.This study proposed further in-depth knowledge through comparison of the roles and mechanisms of biodegradation of the organic matter of specific bacterioplanktons using either combined filler or hydroponic peppermint bed technologies alongside the natural oxidizing bacterial communities to enhance a better nitrogen removal process.The effects of the combined fillers and hydroponic peppermint bed technologies on the bacterioplanktons under intensive grass and silver carp production investigated in a breeding facility in southern China.Bacterioplankton community profiles were generated using Illumina high-throughput pyrosequencing to assess the expression of the 16S rRNA genes followed by the DNA sequence analysis.Forty-four phyla identified dominated by Proteobacteria(37.82%),Actinobacteria(14.14%),Un-classified bacteria(11.35%),Bacteroidetes(8.34%),and Verrucomicrobia(6.57%).Furthermore,Beta(31.55%),Alpha(23.90%),Delta(23.14%)and Gamma(16.11%)Proteobacteria subclasses displayed the majority sequences in the total identified microbes.Distinctive microbial phyla analyzed revealed phyla BRC1,Diapherotrites,Elusimicrobia,Euryarchaeota,Spirochaetes,and Synergistetes survived in the Control;Aminicenantes,Cyanobacteria,Deinococcus-Thermus,and Ignavibacteriae(combined filler);while Microgenomates,Thaumarchaeota,and Thermotogae existed in hydroponic peppermint technology-operating ponds.Identification of various genera in phyla Acidobacteria,Actinobacteria,Bacteroidetes,Chloroflexi,Planctomycetes,Proteobacteria,and Verrucomicrobia suggested a vivid understanding of the mechanisms,roles,and responsibilities of the different bacterioplanktons in the nitrogenous waste breakdown.The non-parametric tests revealed spatial and temporal differences between the different pond technologies,with higher richness,evenness,diversity,and coverage registered within floating bed ponds,as compared to the bio-filter ponds.Physiochemical parameter assessed to determine environmental characteristics on the microbial communities revealed combined filler ponds had cleaner and healthier water quality.The NO3-,NO2-,and COD among other studied parameters alongside alkalinity were indicative of heterotrophic denitrification.Temperatures influenced nutrient removal within the specific fish species ponds significantly eliminating harmful metabolic wastes.Correlation analyses by Non-multidimensional scaling(NMDS),Analysis of Similarities(ANOSIM),and linear effect size(LEfSe),Co-occurrence networks revealed linkages from shared niches,segregations,co-operation and relationships between and among microbial species of the member communities.2.Effects of combined fillers,Pre-,and Probiotics on bacterioplankton communities within tanks growing tilapia(Oreochromis niloticus)Following the remarks in our earlier study,this study proposed a higher metagenomic study as we consolidated the effects of bio-filter technologies.Bacterioplanktons especially ammonia nitrifiers within bio-filters regulating nitrogenous waste effects in aquaculture tanks.In this study,we obtained an in-depth knowledge of the microbial communities involved in this process through characterization and identification of those microbes using Illumina high-throughput pyrosequencing through 16S rRNA gene clone libraries.Bacterioplankton communities obtained from the water samples in twelve tanks i.e.,Control,Lactic acid bacteria,and Pseudomonas strain enrichments,alongside the environmental bacteria tanks were determined.Bacterial 16S rRNA gene sequences from this study revealed broad phylogenetic diversities exhibiting 31 phyla,dominated by Beta,Alpha,Gamma and Delta-Proteobacteria(45.36%),Firmicutes(13.62%),Bacteriodetes(9.6%),Actinobacteria(7.28%).Acidobacteria(3.87%),Planctomycetes(2.94%),Verrucomicrobia(2.79%),and Chloroflexi(2.63%).Illumina profiles and the real-time quantitative PCR analyses revealed similar patterns of bacterial diversity among the different tanks,and spatial distribution variances existed in the different tank enrichments.Statistical analysis showed that bacterial species distribution strongly correlated with water quality variables,including TN(2.69-20.43);COD(9.34-31.47);NH4+-N(0.44-11.78);NO2--N(0.00-3.67);NO3--N(0.05-1.82),mg/L and DO(1.47-10.31 μg/L)in varying ranges,suggesting that microbial community structure and composition significantly influenced the nitrogen nutrients in the aqua tanks.Temperature rises to over 40℃ affected the environmental variables.This study had important implications for the comparison with probiotic microbes that contributed to the improvement of effluent dynamics,eutrophication and nitrate leaching as determining factors that structure bacterial communities in freshwater ecosystems.3.The effects of hydroponic peppermint beds on the spatial-temporal distribution for the ammonia-oxidizing microbial community in pond cultures.In this study,we noted that intensive production and high temperature rises in aquaculture generate detrimental nitrogenous components.The study proposed using peppermint(Mentha haplocalyx Briq)on hydroponic floating beds alongside the natural oxidizing bacterial communities enhancing nitrogen removal.The influence of bioremediation on ammonia oxidizers within Silver(Hypophthalmichthys molitrix)and Grass carps(Ctenopharyngodon idella)production investigated in Suzhou city;China.Ammonia oxidizer profiles were generated using denaturing gradient gel electrophoresis to assess the expression of the 16S rRNA genes followed by the DNA sequence analysis.Next-generation sequencing of equimolar mixtures of degenerate primer pairs CTO189f-GC and CTO654r,together with 338F and 518r used for analysis and estimation of bacterial biodiversity and temporal community changes.Nitrifier microbes including genera Brevundimonas,Pseudarthrobacter,Methylobacillus,Methylophilus,and Ralstonia were among the dominant community microbes identified.Real-time qPCR analysis revealed higher ammonia-oxidizing bacteria(AOB)gene abundances and expression within the control ponds than the treatment ponds.Physiochemical parameters including:COD(2.76mg/L);TN(1.315mg/L);TP(0.0325mg/L);NH4+(0.516 mg/L)and Chl.a(91.905mg/m3)suggested water quality improvement within the treatment ponds compared to the control ponds.Temperature rises over 40℃ significantly affected the water quality especially TN and PO43-content and the NO3-and NO2-content declined under the same conditions resulting in a direct or indirect effect on the bacterial communities.Finally,an alternative method to understand the ammonia oxidizer communities recommended for the degenerate CTO primers.4.Effects of combined fillers on methanogenic ammonia-oxidizing nitrifiers in tank cultures.The discovery of aerobic and anammox bacteria capable of generating methane in bio-filters in freshwater aquaculture systems generated interest in studies to understand the activity,diversity,distribution,and roles of these environmental microbes.In this study,we used microbial enrichment alongside combined fillers to assess their effect on water quality Profiles of ammonia-oxidizing bacterial communities generated were determined using nested PCR and fingerprint(DGGE)methods to assess the expression of 16S rRNA genes through DNA sequencing.Five dominant ammonia-oxidizing bacterial strains-clones;KB.13,KB.15,KB.16,KB.17,and KB.18-were isolated and identified by phylogenetic analysis as environmental samples closely related to genera Methylobacillus,St.anieria,Nitrosomonas,and Heliorestis.Further,the methyl ammonia-oxidizing microbes thereby found suggested a biochemical pathway involving electron donors and carbon sources,and all strains were functional in freshwater aquaculture systems.Environmental parameters assessed varied in the ranges in the different tanks.Principal component analysis revealed that these water quality parameters significantly influenced the ammonia-oxidizing microbial community composition.Temperature rises to about 40℃ significantly affected environmental characteristics-especially DO,TN,and NH4+-N-and directly or indirectly affected the microbial communities.Although the nested PCR design was preferred due to its high sensitivity for amplifying specific DNA regions,a more concise method recommended,as an equimolar mixture of degenerate PCR primer pairs,CTO189f-GC and CTO654r,never amplified only 16S rRNA of ammonia-oxidizing bacteria.