Microbial diversity of nitrifying sludge acclimated to high salt concentrations

Durante o processo de extração do petróleo, um elevado volume de água é consumido gerando grandes volumes de efluentes salinos contaminados com compostos tóxicos de difícil degradação. A amônia é um desses compostos, que em concentrações elevadas, pode ser prejudicial para o ambiente. Por isso, antes de ser descartado ou reutilizado, o efluente deve ser tratado. O tratamento biológico por lodos ativados tem sido o mais utilizado devido ao baixo custo e boa qualidade do efluente tratado. O processo biológico de remoção de amônia é denominado nitrificação, onde a amônia é oxidada a nitrito que é oxidado a nitrato. A primeira oxidação pode ser realizada tanto por bactérias quanto por archaeas. O sal é um fator de instabilidade nas estações de tratamento biológico, porque interfere diretamente nos processos de degradação de poluentes como a nitrificação. Então, o uso de microrganismos adaptados ao sal nas estações de tratamento otimizaria o processo. Dessa forma, o objetivo do estudo foi avaliar a diversidade microbiana total e nitrificante no lodo nitrificante ao longo do processo de aclimatação dos microrganismos à altas concentrações salinas, visando um melhor entendimento sobre a ecologia destes microrganismos. Para tal, o lodo foi aclimatado em batelada, com adição semanal de 5 g/l de NaCl ao efluente de alimentação e a taxa de remoção de amônia foi monitorada. O sal foi adicionado até a completa inibição da nitrificação, que ocorreu na concentração de 125 g/l de NaCl. Em cada concentração salina, uma alíquota do lodo foi coletada e fixada. Os pontos com 25, 50, 75, 100, 105, 115 e 125 g/L foram selecionados, e o DNA total foi extraído das amostras. O DNA foi amplificado com primers específicos para a região RNAr 16S de bacteria e Archaea, e a separação dos amplicons foi feita por eletroforese em gel de gradiente desnaturante (DGGE). Após o DGGE, três pontos foram selecionados para o estudo da diversidade através de sequenciamento massivo da região RNAr 16S de bacteria e archaea e quantificação das populações metabolicamente ativas por Flow-FISH. Foram selecionados os pontos com 25 g/l de NaCl, concentração salina normal do efluente, 100 g/l de NaCl, que foi a ultima concentração salina onde a taxa de nitrificação foi de 100 %, e 125 g/l de NaCl, onde a nitrificação chegou a zero. Os resultados mostraram que com o aumento da salinidade a abundancia de bactérias diminuiu e a de Archaea aumentou, e na concentração de 125 g/L de sal, 80 % da população metabolicamente ativa é composta por archaea. Este resultado era esperado, uma vez que muitos microrganismos pertencentes ao domínio Archaea são resistentes à condições extremas como altas salinidades. Os resultados também mostraram que a remoção de amônia nas três concentrações salinas analisadas é feita por um grupo distinto de microrganismos, envolvendo bactérias autotróficas, bactérias heterotróficas e archaeas, evidenciando a ocorrência da nitrificação autotrófica, heterotrófica e processo ANAMMOX.During the process of petroleum extraction, a high volume of water is consumed generating large volumes of saline effluent contaminated with toxic compounds that are difficult to degrade. Ammonia is one of these compounds, which at high concentrations can be harmful to the environment. Therefore, before being discarded or reused, the wastewater must be treated. Biological treatment has been the most widely used due to low cost and good quality of the final effluent. The biological process of removing ammonia is nitrification, where ammonia is oxidized to nitrite which is oxidized to nitrate. The first step may be performed both by bacteria and by archaea. The salt is a factor of instability in biological treatment plants, because it interferes directly on pollutant degradation processes such as nitrification. Then, the use of microorganisms adapted to salt in wastewater treatment station would optimize the process. Thus, the aim of the study was to evaluate the total and nitrifying microbial diversity in nitrifying sludge during the acclimation process of microorganisms to high salt concentrations, seeking a better understanding of the ecology of these microorganisms. For this, the sludge acclimatization was carried out in batch with weekly addition of NaCl to the effluent and the removal rate of ammonia was monitored. The salt was added until complete nitrification inhibition, which occurred in the concentration of 125 g/l NaCl. At each salt concentration, a sludge aliquot was collected and fixed for analysis. The points 25, 50, 75, 100, 105, 115 and 125 g/L were selected, and total DNA was extracted from the samples. The DNA was amplified with specific primers for the 16S rRNA region of bacteria and archaea and the separation of amplicons was performed in denaturing gradient gel electrophoresis (DGGE). After DGGE, three points were selected for the study of diversity by massive sequencing of 16S rRNA region of bacteria and Archaea and quantification of metabolically active population by Flow-FISH. Points were selected with 25 g/l of NaCl, initial salt concentration of the effluent, 100 g/l of NaCl, which was the last salt concentration where nitrification rate was 100 %, and 125 g/l of NaCl, where nitrification reached zero. The results showed an increase in the abundance of archaea and a decrease in the abundance of bacteria over increasing the salt concentration, and in the concentration of 125 g/l of salt, 80 % of metabolically active population is Archaea. This result was expected, since many microorganisms belonging to domain Archaea are resistant to extreme conditions such as high salinity. The results also showed that removal of ammonia in the three salt concentrations analyzed is made by a distinct group of microorganisms, involving autotrophic bacteria, heterotrophic bacteria and archaea, indicating the occurrence of nitrification autotrophic, nitrification heterotrophic and ANAMMOX processes.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Effect of salinity in heterotrophic nitrification/aerobic denitrification performed by acclimated microbiota from oil-produced water biological treatment system

Mixed cultures salt acclimated showed high efficiency in heterotrophic nitrification/aerobic denitrification process in hypersaline conditions. They were able to remove 80% of ammonium in Heterotrophic Nitrification Medium (HNM) with 12 and 14% of salt. Above these salinity, the process still had 40% ammonium removal up to 20% of salt. Chromatography analysis validated the occurrence of the heterotrophic nitrification/aerobic denitrification process in all studied salinities (6%–20% of NaCl). However, with increasing salinity, the N2 production was smaller and took longer than the unsalted control. Microbial diversity analysis of mixed cultures showed that different groups of nitrifying microorganisms were involved in ammonium removal, including heterotrophic nitrifying/aerobic denitrifying genera such as Pseudomonas, Paracoccus, Bacillus, Halomonas, Acinetobacter and Klebsiella. In addition, this analysis also revealed that the acclimation process allowed the adaptation of the microorganisms to high saline conditions and ammonium removal up to 20% of salt. This work showed that heterotrophic nitrification/aerobic denitrification process could occur in high salinity after microbiota acclimation step, and these mixed acclimated cultures have a potential for application in hypersaline effluent treatment