The Bioremediation For Nitrogen-polluted Marine Aquaculture Water By Marine Purple Sulfur Bacterium Marichromatium Gracile YL28

For many years,ammonia?NH₄⁺?,nitrite?NO₂^-?and nitrate?NO₃^-?,widely present in the area of offshore aquaculture sea,have been ranked first in many pollutants,which not only restricted the development of aquaculture,but also caused potential threat to the coastal or offshore ecological environment.Microbial remediation employing microbial metabolisms is an effective way for removal of harmful pollutants,such as ammonia and nitrite,and the effective regulation of mariculture ecosystems in aquaculture water.However,currently,the most studied and applied microorganisms are originated mainly from the soil or freshwater environment,while less reports for species from the oceans.There is no clear conclusion as to whether these terrestrial or freshwater microorganisms can survive and function in complex mariculture water or high-salt environments in the oceans.Present studies have showed that microorganisms generally have ammonification,which could convert organic nitride into ammonia and then release into the environment.However,at present,there are few strains have been reported in this area and the majority microorganisms showed reduced capacity,even completely inhibited in the high level of organic nitrogen environment.In addition,the sediment-water interface?SWI?of the mariculture water is a nutrient-rich,microbial metabolism active ecological environment,but with insufficient dissolved oxygen?DO?and light,resulting the organics cannot be completely degraded into a variety of soluble small molecular substances and released into mariculture water?such as inorganic nitrogen,amino acids,peptides,organic acids,alcohols,sugars and the like?,which were considered as the main source of eutrophication or deterioration of mariculture water.Therefore,it is of great significance to develop a new type of high efficiency nitrogen removal microbial preparation for the healthy and sustainable development of aquaculture and marine environment that can adapt into marine aquaculture water,especially SWI environment with low-light,hypoxic or even anaerobic and nutrient-rich.In the early studies of our lab,a strain of purple sulfur bacterium YL28 was obtained,which could grow by using nitrite as the only nitrogen source and efficiently remove high concentration of inorganic nitrogen?ammonia,nitrite and nitrate?from water,then identified as Marichromatium gracile.It is one of the widely present anoxygenic phototrophic bacteria?APB?strains that with the highest removal capacity of nitrous nitrate to date.The basic law of growth and nitrogen removal characteristics of the strain and the molecular mechanism of high efficiency nitrogen removal were preliminarily elucidated,but the removal ability of ammonia the strain was greatly affected in organic nitrogen environment.Based on this,this study selected the YL28as the research object,through the method of rejuvenation and screening,the strain with high efficiency ammonia removal ability was selected out in the presence of peptone and urea.Then the culturing conditions of the strain were optimized.Then the influence factors,such as light,oxygen and a series of environmental possible carbon and nitrogen sources in the evaluation system of high concentration inorganic nitrogen,were used to investigatthe adaptability of the strain to the environment,especially the SWI of mariculture water.The effect and the regulatory effects of nitrogen metabolism related functional genes?nifH,amoA,narG and nos Z?of the strain on the bacterial community structure of sediments in the mariculture system were studied by using the method of high throughput sequencing of 16S rRNA sequences?V3-V4?and qPCR with the physical and chemical indexes of mariculture water,respectively.The main results are as follows:1)In view of the limitation of the ability of organic nitrogen to remove inorganic nitrogen from microorganisms,the method of rejuvenation and screen was used to obtain a strain for the high efficiency removal of ammonia under the conditions of organic nitrides.Compared with the starting strain,the removal rate was increased more than 90%.Because of the strain in the culture and storage process,easy to precipitate stratification,the formula of optimized medium was obtained by method of single factor optimization on the basis of the original medium.Compared with before,the biomass(OD₆₆₀)was increased by 54.20%,BChl and Car increased by 50.73%and52.69%respectively.Liquid preparation was saved 810 days in the dark static conditions,subsidence ratio still reached more than 55%,at the same time,ammonia removal rate reached more than 80%.2)Considering the complexity of mariculture water,17 kinds of organic carbides and their compounds,2 kinds of organic nitrogen and baits were selected in the inorganic nitrogen systems to investigate the adaptability of YL28 to environmental factors.The results showed that small molecule organic carbon?acetic acid and pyruvate,etc.?were good carbon sources for the growth and nitrogen removal of this strain.Alga-oligosaccharides is not a good carbon source for growth,but it could significantly promote the growth and nitrogen removal activity in the presence of good carbon sources.The unserviceable carbon sources?carrageenan,sodium alginate and cyclodextrin,etc.?were combined with good carbon sources,the growth and nitrogen removal activity of the strain had not been significantly affected.The combination of organic nitrogen and good carbon sources did not affect the ability of the strain to remove inorganic nitrogen,but significantly affected the growth characteristics.3)Light and oxygen are important factors affecting the growth and metabolism of APB.In this study,in the only nitrogen source and inorganic nitrogen coexistence system,the relationship and law of the effect of light and oxygen on the growth and nitrogen removal of the strain were elucidated by single factor test and response surface optimization,the optimum DO?expressed by sample loading quantity,SLQ?,illuminance and photoperiod condition the range of light and oxygen parameters feasible in practical application is obtained by using the expected analysis.In this study,the relationship between light,oxygen and YL28 growth and nitrogen removal was clarified through single factor test and response surface optimization analysis,and the optimal conditions of dissolved oxygen?represented by sample loading quantity?,light intensity and photoperiod were obtained in theory.We found that YL28 can well grown and nitrogen removal of the strain in the light and anaerobic,the growth activity decreases in the light and aerobic,but the activity of nitrogen removal did not change significantly.YL28 can maintain good nitrogen removal ability under dark and anaerobic conditions,the conversion rate of inorganic nitrogen reached 81.41%.The analysis of response surface optimization shows that biomass(OD₆₆₀)increased by21.28%,and ammonia removal rate could reach 93.79%on the basis of optimizing the medium.4)The change of microbial community characteristics and directional regulation of mariculture ecosystem is one of the important indexes to reflect the health level of mariculture water.In this study,the high-throughput sequencing technique with 16S rRNA?V3-V4?was used to analyze the effect of the strain on the change of bacterial community structure and its diversity in Penaeus vannamei mariculture water.The results showed that the strain could significantly improve the mariculture water quality,achieve the Class?quality requirements of Fisheries Water Quality Standards?GB11607-89?,and could also be implanted in mariculture system to regulate bacterial community structure.Addition of this strain can significantly improve the bacterial community diversity of SWI,but also significantly improve the species abundance of the Proteobacteria,Gammaproteobacteria,Chromatiales,Chromatiaceae and Marichromatium,as well as does not alter its position as a dominant species at the classification level.We also found Marivita and OM27 can be act as a potential useful indicator of the health status of aquatic ecosystem and a symbiotic relationship between OM27 and Marichromatium.In addition,the strain can effectively reduce the abundance and variety of Vibrio and the inhibition rate was more than 62%.5)In order to reveal the relationship between the YL28 and other microorganisms relating nitrogen metabolisms,we analyzed the changes of the abundance about16S rRNA?AOB and AOA?and functional genes associated with nitrogen metabolisms,such as nitrogen fixation?nifH?,nitrification?amo A?,denitrification?narG and nos Z?and so on,by method of qPCR.It was speculated that YL28 had no effect on change of AOA,but increased the abundance of AOB.Adding lower dose of YL28 could effectively increase the abundance of AOB by compared with higher dose of YL28.Results showed the greatly positive relationship of abundance between YL28 and nif H.High dose of YL28 adding into culture could increase the abundance of amoA,but not the narG and nosZ.Finally,on the basis of the above findings,5 d?low dose?or10-12 d?high dose?may be the best usage period for YL28 in the mariculture water which can effectively increase the growth of Penaeus vannamei.Collectively,this study selected a strain?YL28?,which can effectively remove inorganic nitrogen in the organic nitrogen environment with wide microbial ammonia effect that seriously influence microbial nitrogen removal activity for released ammonia.Then,we optimized the culturing conditions for this strain and the systematically investigated a number of influencing factors?such as carbon and nitrogen source,light and oxygen etc?on the growth and nitrogen removal activity.Whereafter,we investigated the influence of the strain on the bacterial community structure in the SWI,as well as expounded the adaptive law and characteristics in the mariculture water.We found that alga-oligosaccharides could significantly promote the growth and nitrogen removal activity,and remarkably improve the subsidence ratio in the process of bacterial culture and storage,which put forward a novel way for the application.YL28 could keep growth-well and nitrogen removal ability in the dark and anaerobic conditions and also be implanted in the SWI and adapt well,as well as effectively control the release of harmful substances such as ammonia and nitrite from the pollutants in the mariculture water.This study provides a theoretical basis for the development and application of APB water quality regulator.