Mining cultured and uncultured microbes from shrimp pond water for improving sustainable shrimp aquaculture in Vietnam
Creators: name: TRAN, Thanh
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310509
subject: Genomics

description: Shrimp aquaculture is the most highly profitable economic sector and shrimp exports account for the largest part of the total seafood export in Vietnam. However, shrimp aquaculture faces problems with nitrogen pollution, which is the accumulation of ammonia and nitrite in the water column. During shrimp cultivation, ammonia and nitrite are produced, which cause lower growth rates of shrimp and can lead to large-scale mortalities on farms. In the past, treatments such as ion transfer and adsorption to reduce ammonia and nitrite levels have caused significant environmental problems and impacted on shrimp quality. Therefore, to improve sustainable shrimp aquaculture in Vietnam, bacteria with nitrifying activity, which could lead to a reduction in shrimp pond nitrification, have become a topic of major interest.
In Chapter 1, I review relevant literature for this thesis and a provide an overview about the development of shrimp farming in Vietnam related to seafood exports and the impact of environmental issues on the shrimp cultivation due to the accumulation of ammonia and nitrite caused nitrogen pollution. The involvement of two bacterial groups (ammonia-oxidizing bacteria and nitrite-oxidizing bacteria) involved in the nitrification process to solved nitrogen pollution in nature was shown. The benefit of metabarcoding and metagenomics sequencing technologies were revealed to understand the fundamental information about the composition and diversity of bacteria in shrimp pond water and predicted functions of bacterial groups

In Chapter 2, I generate a total of 6,295,650 high-quality sequences (bacteria of 99.93%, archaea of 0.065% and unclassified sequences of 0.005%) from four samples (three Litopenaeus vannamei ponds and one Penaeus monodon pond) in Soc Trang province, Vietnam to understand the diversity and dominance of bacterial communities in Litopenaeus vannamei ponds. I found that the distribution of the top ten phyla in the three L. vannamei communities was similar. The top three largest dominant phyla in Litopenaeus vannamei ponds were Proteobacteria, Bacteroidetes and Actinobacteria. The distribution of the top ten genera among the three L. vannamei communities was different from the distribution of top ten phyla. I selected the largest dominant group, Pseudomonas, to further analysis the relationship between environmental factors and the relative abundance of bacterial groups. I found the correlation between Pseudomonas and the reduction of ammonia in shrimp ponds.

In Chapter 3, I selected two more similar composition and abundance of bacteria which were ST1 and ST4 (Litopenaeus vannamei ponds) to do whole-genome shotgun sequencing. I obtained the data about the presence of genes in all members in two samples. I found that the composition and abundance of the bacterial group at the phyla and genus level were consistent with the results in Chapter 2 based on 16S gene sequences analysis. Especially, the dominant of Pseudomonas in these two samples were confirmed. The data on sequences of all genes were annotated and screened against Clusters of Orthologous Groups (COG) database, Carbohydrate active enzyme database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database. I found that the main roles of the bacterial groups in the two samples were the degradation of biomass and the synthesis of natural products.

In Chapter 4, in order to focus on cultured microbes, I collected fifteen water samples from shrimp ponds in Quang Ninh province (northern Vietnam), Thanh Hoa province (mid-Vietnam), and Soc Trang, Bac Lieu province (southern Vietnam). All samples were enriched in a culture medium for raising the relative abundance of AOB and NOB (known as oxidizing nitrate bacteria or nitrifying bacteria) and testing their nitrifying activities. Higher ammonia-oxidizing activity of bacterial groups was indicated in the third month of enrichment. When bacterial group activities were at a peak and stable level, I found the presence of ammonia- oxidizing bacteria and the absence of nitrite-oxidizing bacteria based on the ammonia, nitrite, nitrate concentration measurement. Pseudomonas as a member of ammonia-oxidizing bacteria was found based on quantitative PCR analysis.

In Chapter 5, I successfully isolated six Pseudomonas strains from fifteen ponds (Chapter 4). The six potential colonies showed their heterotrophic nitrification activities by completely convert ammonia concentration of 200 mg/L after cultivation at 96 h. I found the involvement of carbamoyl phosphate synthetase I in the conversion of ammonium in Pseudomonas isolates based on the protein assays. I selected TT321 and TT322 based on higher heterotrophic nitrification activity to do whole-genome analysis. Nitrogen metabolic pathways are suggested based on genome analysis of two Pseudomonas strains in comparison with 31 other genomes of Pseudomonas. In particular, the ammonia conversion in TT321 and TT322 was predicted following three pathways comprising L-Glutamine and L- Glutamate metabolism, arginine and proline metabolism and glycine and serine and threonine metabolism.

Finally, in Chapter 6, I summarise the findings comprising the relationship between environmental factors and the relative abundance of bacteria group, the detection of a benefit bacteria group as Pseudomonas will be a suggestion to stimulate this group in nature and the benefit of the metagenomics approach to reveal genes source and ammonia conversion pathways in TT321 and TT322 strains. Moreover, I pointed out several possibilities for future research such as a metatranscriptomics analysis to confirm the regulation and expression of genes involved in heterotrophic nitrification activity, labelling nitrogen input by using 15N and thin- layer chromatography and high performance liquid chromatography (HPLC) analysis to understand the mechanisms of ammonia conversion in Pseudomonas.