Application of nitrogen isotope in amino acids for marine biogeochemistry and ecology

Abstract

Understanding food web structure as a route of energy flow from primary production to the higher organisms is important in marine ecology. Stable isotope analysis has been applied in the food web studies for decades, but has reached various limitations. Recently compound specific isotope analysis (CSIA) of nitrogen within amino acids has been developed as an alternative for isotope analysis of bulk tissue in trophic ecology. However, CSIA of amino acid is still required improvement to increase availability in various approaches. This thesis attempted the alternative applications from traditional approaches using CSIA technic in marine ecology. Nitrogen isotope ratios of individual amino acids were analyzed using GC/C/IRMS, via N-pivaloyl/isopropyl (Pv/iPr) derivatization, after hydrolyzation of approximately 5mg of dried biota samples. Nitrogen isotope ratio of glutamic acid and phenylalanine were used as representative of trophic amino acid and source amino acid, respectively. CSIA of nitrogen within amino acids significantly reduce the uncertainty in the estimated trophic position (TP) of organisms based on the isotopic difference between glutamic acid and phenylalanine within a single organism. However, because the initial offset (ß) between glutamic acid and phenylalanine differs between aquatic algae (ca. +3.4‰) and vascular plants (ca. −8.4‰) in food webs that rely on both resources, ß should be replaced by a value adapted to the admixture of primary producers for each specimen. This thesis established a new method involving the ß value (ßmix) of each consumer specimen determined based on its bulk tissue δ13C value and successfully obtained realistic TPs (TPmix) for organisms in a complex seagrass meadow food web. Remarkable differences between the TPmix and traditional TP values were found in deposit feeders due to the large contribution of seagrass to their basal resources. The estimated TPs of organisms increased by up to 1.5 units (from traditional TP to TPmix), in terms of trophic transfer, when their diets included substantial seagrass-derived contributions. Thus, combinatorial analysis of the amino acid δ15N and specimen-specific ßmix values provides better understanding of the trophic interactions in food webs, even in complex seagrass meadow ecosystems. CSIA of nitrogen in source amino acid also is applied to determine the basal nitrogen isotope ratio (δ15Nbaseline) in the food web, due to the negligible isotopic difference between diet and consumer (eg. δ 15N of phenylalanine, δ15Nphe). However, different isotopic turnover time between bulk tissue and single amino acid in an organism and nitrogen isotopic offset between bulk tissue and single amino acid in primary producer should be considered. δ15N values of primary producer (δ15NPP), as time integrated δ15Nbaseline, were determined from δ15Nbulk and trophic enrichment factor of the mussel with trophic position based on nitrogen isotope of glutamic acid and phenylalanine in the coastal region. The differences between estimated δ15NPP and δ15Nphe values were ranged –1.5‰ to 1.6‰, showing strong correlation between both isotope values. In addition, δ15NPP was determined from δ15Nphe of zooplankton in the western North Pacific Ocean. Although 1.3‰ offset of nitrogen isotope ratio between both δ15NPP estimations were found, similar spatial variation indicates both new δ15Nbaseline estimations are applicable to determine δ15Nbaseline. CSIA of nitrogen in amino acid was applied to understand the influence of oyster culture in the coastal environment. Size dependent selective consumption of particulate organic matter (POM) in oyster is well known. However, nitrogen isotope ratio between estimated diet of oyster and oyster preferred size of POM (larger than 20 µm) showed distinct difference in longline oyster culture system at high density. Additionally, POC/chl-a ratio in water column of oyster farm showed remarkably high value. Meanwhile, trophic position estimated by nitrogen isotope ratios within amino acids of cultured oyster was close to 2, suggesting its herbivorous feeding behavior. Accordingly, extremely high POC/chl-a ratio showed in the water column of long line oyster farm can be interpreted as a consequence of rapid consumption by oysters cultured at high density. Moreover, the irregularly enriched nitrogen isotope ratio of large POM can be interpreted as result from dominance of heterotrophic organism in the water column. The influence of long line cultured oyster at high density to the marine ecosystem was figured out by the trophic position information. To the best of our knowledge, the CSIA approach in the aquaculture system is never been accessed. However, this case study showed that CSIA can be extensively applicable to interpret alteration of nature by culturing system. This thesis suggests improvement for CSIA of nitrogen isotope ratio within amino acid. The alternated estimation of trophic position using “ßmix” will enhance the application to the mixed food web. Also estimation of δ15Nbaseline is highly valued not only in ecology but also in biogeochemistry in marine science. The case study showed successfully compensate the defect of stable isotope of bulk tissue approach using trophic position estimated by CSIA of nitrogen isotope within amino acid. Above all things, this thesis has great value as the first report for CSIA of nitrogen isotope within amino acid in Korean marine science society.