Impact of Water-Sediment Regulation on Variations of Amino Acids in the Middle-Lower Yellow River, China

ZHANG Kun, ZOU Li, , DAI Qunying, WANG Jian , JIANG Xueyan,and LIANG Shengkang

1) College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China

2) Key Laboratory of Marine Environment and Ecology, Ministry of Education, Ocean University of China,Qingdao 266100, China

3) SGS-CSTC Standards Technical Services (Qingdao) Co., Ltd., Qingdao 266101, China

4) Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100, China

Abstract In order to examine the impacts of water-sediment regulation on regional carbon cycling, we collected water, particulate and sediment samples from the middle-lower Yellow River in late June and early July, 2015 and analyzed their specific amino acids(AA), DOC, POC, and bacteria abundance. Summarized by 14 specific AA, the total hydrolysable AA (THAA), particulate AA(PAA), and sediment AA (SAA) varied in ranges of 2.29-9.05 μmol L-1, 5.22-22.96 μmol L-1, and 81.7-137.19 μg g-1 dry weight.After the regulation, dissolved free AA (DFAA) decreased by 29% while DCAA increased by 72%. These variations suggested that DFAA were further degraded, while DCAA molecules were further activated. Meanwhile, PAA increased almost 4 times as many as those before regulation, and SAA increased as well. After regulation, the amounts of bioactive amino acids (Asp, Glu and Gly) increased in THAA but decreased in PAA, with little changes in SAA. The ratios of Asp/Gly in different phases increased after regulation, indicating the AA contributions were promoted by calcareous organisms rather than by siliceous organisms. Multiple correlation analysis showed that PAA was primary representatives of AA and organic carbon, followed by DCAA and POC. Moreover, bacterial reproduction played a key role in shaping the AA compositions and properties, followed by the redox condition and acid-base balance.The results of this study provided a clear evidence for the effects of water-sediment regulation on regional biogeochemistry of organic carbon in the middle-lower Yellow River.

Key words the middle-lower Yellow River; water-sediment regulation; amino acids; organic carbon

1 Introduction

Amino acids, as the basic units of biological proteins and polypeptides, were major components of organic nitrogen and carbon in most organisms (Cowie and Hedges,1992). Amino acids in the environment were produced by biological metabolism and decomposition of biological debris (Sharp, 1983; Williams, 1986). As active organic nitrogen, the relative contents of amino acids were closely related to their existing phases. It was reported that,amino acid nitrogen accounted for 42%-72% of total nitrogen in aquatic plankton (Degens and Mooper, 1976), and 40%-65% in water and sediment (Lee and Cronin, 1982).Thus, amino acids were not only the main components of most organisms, but also the key compositions of aquatic organic matter (Cowie and Hedges, 1992).

Generally, the higher the proportion of amino acids in organic matter is, the stronger the bioavailability of organic matter and the lower its degradation degree will be(Davis et al., 2009). Both dissolved free amino acids(DFAA) and dissolved combined amino acids (DCAA)were biogenic organic matters, whose sources and removal processes were positively related to the activities of plankton and bacteria (Burdige, 1991). However,DFAA was easily utilized by both autotrophs (phytoplankton) and heterotrophs (bacteria), and stayed in waters with shorter retention time (Veuger, 2004). PAA was mainly composed of living plankton and its decomposed debris, and was gradually transported from the surface to the deep water and then to the sediment. They were biodegraded and utilized during the transportation (Lee and Cronin, 1982). The compositions and contents of SAA were further regulated by sedimentary environment and diagenesis (Cowie and Hedges, 1992). Amino acids, as protein hydrolysates, can be classified into acidic, neutral,basic, aromatic and sulfuric amino acids according to their functional structures in side chains (Dauwe and Middleburg, 1998), and its structure is related to biological sources and activities. (Jørgensen et al., 1999; Pedersen et al., 2002). For example, components such as Aspartic acid (Asp), Glycine (Gly) and Serine (Ser) originated from active organisms and dominated in PAA, and components of Glutamic acid (Glu) presented more in surface water (Lee and Cronin, 1984). Degradation index of AA was calculated to indicate the degradation status of organic matter (Dauwe and Middelburg, 1998). Therefore,the existence form, composition and distribution of amino acids and organic carbon were linked to demonstrate the biogeochemistry of organic carbon (Whelan, 1977; Cowie et al., 1994).

The Yellow River ranked at the 20th of the world largest rivers, carrying the water flux of 49×109m3yr-1, but ranked at the 2nd with a very high sediment flux 1100×106tyr-1(Milliman and Meade, 1983; McKee et al., 2004).The Yellow River had an important impact to the material cycling and climate change in both regional and global scales (Chen et al. 2008; Wang et al., 2010). In order to remove deposited sediment in the reservoirs and channels of the Yellow River, the Ministry of Water Resources of China started to regulate water and sediment deliberately since 2002. The water and sediment fluxes accounted for 14%-56% and 33%-93% of the whole year during the period of water-sediment regulation in 2002, 2003 and 2004 (Wang, 2005), and these values became 22.8% and 34.6% in 2016 (Li et al., 2015). These high fluxes in short time led to a concentration effect. Water flux was increased by two times and groundwater flux by more than six times in one single water-sediment regulation (Xia et al.,2016), while nutrient flux was increased by 8-30 times(Liu, 2015). The regulation impacted on the particulate organic carbon (POC), dissolved organic carbon (DOC)(Zhang et al., 2015; Xue et al., 2017). POC fluxes in 2009 and 2015 were only 6.4% and 1% of that in 1987 (Zhang et al., 1992; Wang et al., 2012; Xue et al., 2017). Compounds from terrestrial vascular plants were increased after the regulation (Tao et al., 2018), including the characteristic n-alkanes (Tao et al., 2015). It was stated that the artificial water-sediment regulation altered the composition and transportation of organic particles temporally and spatially in the middle-lower Yellow River.

This study was designed to examine the compositions of amino acids in the phases of water, particulate, and sediment before and after water-sediment regulation, hoping to reveal the sources, transportation, and degradation of amino acids in the middle-lower Yellow River. The ultimate goal is to clarify the impacting mechanism of regulation on the alteration of amino acids. The results would help to further understand the effects of watersediment regulation on the biogeochemistry of organic matter in the middle-lower Yellow River and the longterm influence on climate change in regional and global scales.

2 Methods

2.1 Study Sites and Sample Collection

The study area was located in the middle-lower Yellow River (Fig.1), and the sampling sites were set up from the start place of water-sediment regulation to the estuary area, including Xiaolangdi (XL), Huayuankou (HY),Gaocun (GC), Dongda (DD), and Lijin (LJ). The field work was carried out in two periods: before (from 29 June to 03 July) and after (from 4 to 8 July) the regulation.The water samples were collected and filtered through Whatman GF/F (pre-combusted at 450℃ for 4 h),and the filtered water samples were sterilized with saturated HgCl2and then frozen in dark for DOC and THAA analysis. Suspended particles retained on the filters were kept frozen for POC and PAA analysis. Part of the suspended particles were kept in room temperature for grain size analysis. The water samples for accounting bacterial abundances were collected aseptically, added with formaldehyde solution to a final concentration of 2%, and stored in dark and under 4 ℃ . Surface sediment sa mples (0-3 cm) were collected by grab bucket or flat shovel, packaged in sterilized plastic bags and kept frozen for analyses of organic carbon, amino acids and bacteria abundance.

2.2 Analytical Methods

2.2.1 Grain size

Approximately 0.5 g dried sediment sample was weighted and added into 2% sodium hexameta phosphate solution (10 mL) to separate sand from silt-clay. After fully dispersed, sediment particle size was measured by laser particle size analyzer (Rise-2002, Ocean University of China, China), referenced on the national standard of China (GB/T 12763.8-2007), and the measurement deviation was less than 0.71%.

2.2.2 DOC

Water sample was first treated with drops of HCl solution (1 mol L-1) to remove inorganic carbon, and then was measured by TOC-VCPH analyzer (Shimadzu, Ocean University of China, China). The detection limit was 4 μg L-1and the relative deviation was less than 3%.

2.2.3 POC

POC sample was first treated with HCl (1 mol L-1) to remove inorganic carbon, and then was dried, grinded and wrapped with tin cup for instrumental analysis. Prepared POC sample was measured with an element analyzer(PE2400 II, Ocean University of China, China), and the relative deviation was less than 0.03%.

2.2.4 Amino acids

The filtered water samples were directly analyzed for DFAA on the instrument. The samples for DCAA analysis were hydrolyzed by HCl (Henrichs, 1991). After cooled,filtered, concentrated and volume-fixed, they were measured with the same method as DFAA.

DFAA was measured by high-performance liquid chromatography (Agilent 1260 HPLC) using o-phthalaldehyde (OPA) pre-column derivatization method (Kaiser and Benner, 2005). The measurement conditions are listed as follows: excitation at 330 nm and emission at 450 nm;Echipse AAA analytical column (150 mm×4.6 mm, 5 μm)with protective column; a mixture of methanol and acetonitrile (v:v = 2:1) for gradient elution mobile- phase A and a solution of 40 nmol L-1potassium dihydrogen phosphate (pH=7.20) for mobile-phase B.

2.2.5 Bacterial abundance

Water samples were stained with 4’,6-diamidino-2-phenylindole (DAPI), filtered to black filter membrane(Whatman) and counted via fluorescence microscope(Kepner and Pratt, 1994).

3 Results and Discussion

3.1 Physical, Chemical and Biological Factors Before and After Water-Sediment Regulation

The physical, chemical and biological factors before and after water-sediment regulation at the study sites are shown in Table 1. The particulate particles were primarily composed of silty sand and partially clay. The median diameter (MD) of particulate particles varied obviously after the regulation. The temperature (T), pH and salinity(S) of river water at the study sites generally changed little after the regulation, while DO decreased slightly from HY to the downstream stations. Except for HY station, bacterial abundance (BA) decreased by 4%-57%after the regulation at each station.

Table 1 Parameters of MD, T, pH, DO, S and BA before and after water-sediment regulation (from 29 June to 08 July)at study sites in the middle-lower Yellow River

Fig.2 Concentrations of POC, DOC and organic carbon in sediments before and after water-sediment regulation (from 29 June to 08 July) at study sites in the middle-lower Yellow River (before the regulation; □ after the regulation).

3.2 Variations of Organic Carbon Before and After Water-Sediment Regulation

Contents of POC, DOC and organic carbon in sediments are shown in Fig.2. POC, DOC and organic carbon in sediments ranged at 0.22%-0.44%, 117-158 μmol L-1and 0.019%-0.067% correspondingly before the regulation. The regulation led to the increases of DOC by 29%-121% and organic carbon in sediments by 4%-463%, but the decreases of POC by 15%-62%.

3.3 Compositions of Amino Acids Before and After Water-Sediment Regulation

Fig.3 The concentrations of amino acids in different phases before and after water-sediment regulation (from 29 June to 08 July) at study sites in the middle-lower Yellow River (before the regulation; □ after the regulation).

Fig.4 The mole% of various amino acids in different phases before and after water-sediment regulation (from 29 June to 08 July) at study sites in the middle-lower Yellow River. b, before the regulation; a, after the regulation.

A number of 14 amino acids was detected at study sites,THAA, PAA and SAA ranged at 2.29-9.04 μmol L-1,5.22-22.96 μmol L-1and 81.72-137.19 μg g-1, respectively. Including acidic amino acids (Asp and Glu), neutral amino acid (Ser, Gly, Threonine (Thr), Alanine (Ala),Valine (Val), Leucine (Leu), and Isoleucine (Ile)), basic amino acid (Histidine (His) and Arginine (Arg)), aromatic amino acids (Tyrosine (Tyr) and Phenylalanine (Phe)),and sulfuric amino acid (Methionine (Met)). Concentrations of total amino acids and molecular compositions of amino acids in different phases before and after water-sediment regulation are shown in Figs.3, 4. The average THAA in the study sites was slightly higher than that in the Amazon River (0.2-10.6 μmol L-1) (Aufdenkampe,2001), and much higher than that in the Yangtze River(0.2-1.0 μmol L-1) (Zhang et al., 2015). Meanwhile,DCAA generally dominated for 71%-98% of THAA at study sites. Concentrations of DFAA decreased significantly by 46%-59% at XL and DD sites after the regulation, while those at other stations increased by 19%-41%.Except for the 45% decrease at LJ, DCAA showed an increasing trend. After the regulation, the highest increase of concentrations of DCAA was 4.7 times at GC site, and the increases at other sites ranged at 37%-128%. Compare to other rivers, the average concentrations of PAA and SAA at the study sites were relatively higher: For example, PAA was 7 times higher than that in the Yangtze River (Wu et al., 2007) and 5 times higher than that in the Pearl River (Chen et al., 2004). Except decreases by 20%and 27% at DD and LJ, content of PAA increased by 2.0-7.5 times after the regulation, and SAA increased by 54%-156% spontaneously.

The compositions of amino acids varied significantly in different regions and phases due to the differences in biological species and activities (Siezen and Mague, 1978).Comparatively, the relative composition of amino acids varied little in different phases at the study area, but the absolute contents of amino acids varied significantly at the same time.

Generally, neutral amino acids dominated for 43%-72% in three phases, followed by acidic amino acids accounting for 14%-24%, basic amino acids for 9%-20%,and then aromatic and sulfuric amino acids for 4%-13%and 1%-6% respectively. The relative contributions of acidic amino acids increased by 77% from dissolved to particulate phases, and then decreased by 22% to sediment phase, while those of neutral amino acids decreased by 25% from dissolved to particulate phases and then 20%to sediment phase, and those of basic, aromatic and sulfuric amino acids increased by 41%, 59% and 92% from dissolved to particulate phases and then 50%, 93% and 188% to sediment phase, correspondingly. Thr and Gly,accounted for 29% and 17% of the content in DFAA and DCAA, which were significantly higher than other amino acids. Meanwhile, Tyr and Leu contributed only 1.3% and 1.2%, which were lower than other amino acids. Ser and Asp contributed the most important relative fractions,both for 12% in PAA and SAA phases, while Ile and Val contributed the lowest relative fractions for 3% and 2%.

3.4 Changes in Source of Amino Acids Before and After Water-Sediment Regulation

The monomer amino acids could be used to track the source, transportation and transformation of biogenic materials (Andersson et al., 2000). For example, plankton particles contained relative high abundance of Asp, Gly,Glu, Ala and Lys, while diatom enriched in Thr, Ser and Gly (Hecky et al., 1973; Andersson et al., 2000). Moreover, the sum of Asp, Gly and Glu could exceed 30% of THAA, but acidic, sulfuric and aromatic amino acids were detected in small percentages (Cowie and Hedges,1992; Braven et al., 1995). The sum of fractions of Asp,Glu and Gly (SAGG) in this study was 19%-36%, 32%-43% and 30%-33% in the THAA, PAA and SAA before regulation. However, after the regulation, the SAGG increased by 42% in the THAA, but decreased by 18% and 7% in the PAA and SAA. This result indicated that the water-sediment regulation promoted the releasing of amino acids from PAA and SAA to the dissolved phase by 4%-17%.

Asp was observed to dominate in calcareous organisms,while Gly dominated in siliceous organisms (Ittekkot et al.,1984). Thus, the ratio of Asp to Gly could indicate the predominant biological sources of amino acids and proteins. In this study, Asp/Gly ratios in the THAA, PAA and SAA increased from 0.28, 0.93 and 0.80 before the regulation to 0.29, 1.11 and 1.26 after the regulation, respectively. The values of Asp/Gly indicated that the siliceous organisms were dominant sources of THAA, while the calcareous organisms were dominant sources of PAA and SAA. The increases of Asp/Gly ratios (0.21-1.26) after regulation implied that the calcareous amino acids or proteins in three phases were less affected by the regulation compared to siliceous amino acids or proteins, although the more alterations of amino acids occurred from dissolved to particulate and sediment phases. It could be inferred that the amino acids or proteins originated from calcareous organisms would be relatively well preserved in the study area.

3.5 Degradation Characteristics of Amino Acids Before and After Water-Sediment Regulation

Degradation index (DI) was calculated to indicate the degradation status of amino acid compounds (Dauwe and Middelburg, 1998). DI was calculated as follows,

where varirepresents mole% of certain amino acids to total amino acids, AVG variis the average value of this amino acid, STD variis the standard deviation of this amino acid, and fac.coef.iis the factor coefficient of this amino acid. The lower the DI is, the higher the degradation degree will be (Dauwe and Middelburg, 1998). Principal component analysis (PCA) was also applied to examine the relationship between the mole% of amino acids and the degradation degree of organic matter.

The results of amino acid DI before and after watersediment regulation in this study were shown in Fig.5.Compared with the Pearl River and the Jiaozhou Bay, DI of PAA and SAA were relatively high in study areas(Zhang and Ran, 2014; Zhou et al., 2018). Overall, the average DI of DFAA was higher than that of DCAA. DI of DFAA decreased while the DI of DCAA increased after water-sediment regulation. These results showed that DCAA was degraded more significantly than DFAA, indicating that DFAA was deactivated and DCAA was activated in dissolved phase after the regulation. Also because of the regulation, the DI of PAA increased and freshly-produced amino acids were imported into PAA.

Fig.5 Amino acid degradation index (DI) in THAA, PAA and SAA before and after water-sediment regulation (from 29 June to 08 July) in the middle-lower Yellow River (BDFAA and ADFAA-DFAA before and after the regulation; BDCAA and ADCAA-DCAA before and after the regulation; BHTAA and ATHAA-THAA before and after the regulation; BPAA and APAA-PAA before and after the regulation; BSAA and ASAA-SAA before and after the regulation).

On the other hand, the DI of SAA decreased and the amino acids were further transformed in SAA.

3.6 Effects of Regulation on the Compositions of Amino Acids

DFAA, DCAA and PAA were used to characterize the composition of amino acids in water, POC and DOC were used to characterize the total organic carbon in water, MD,pH, T, S, DO and bacterial abundance were used to characterize the potential influencing factors to organic carbon. Multiple correlation analysis was applied to evaluate the effect of water-sediment regulation on the compositions of amino acids. It was discovered (Fig.6) that the characteristics of amino acids were primarily represented by DFAA, DCAA and PAA. The environmental factors of MD, pH, T, S, DO and BA were related closely with the composition and behavior of amino acids before and after water-sediment regulation. Before the regulation,DCAA and PAA dominated in the compositions and characteristics of amino acids and organic carbon, followed by DFAA and POC. BA was the most important factor regulating the compositions and characteristics of amino acids and organic carbon, followed by DO, pH and T, while MD was the least relating factor. DFAA and PAA played a key role in changing the composition and characteristics of amino acids and organic carbon after the regulation, followed by DCAA and POC. PAA, which might indicate the primary production in situ, kept as the most important factor affecting the characteristics of amino acids and organic carbon before and after the regulation. DFAA instead of DCAA was more labile and its change implied existence of more active organic carbon after the regulation. The whole DOC pool presented more conservative than the specific amino acids. BA was the most important factor regulating the composition and characteristics of organic carbon and specific amino acids,followed by DO, S and T, while MD and pH worked in a minor mode. BA represented the biological reworking on organic carbon, and DO implied the redox conditions.The MCA results suggested that microbial transformation played a critical role on the composition of amino acids and organic carbon, and the redox reactions presented a great influence as well.

Fig.6 Multiple correlation analysis on the organic carbon and environmental factors before and after water-sediment regulation (from 29 June to 08 July) at study sites in the middle-lower Yellow River. The shape size represents the weight of each composition, and the solid line and dotted line stand for positive and negative correlations.

4 Conclusions

The water-sediment regulation in the middle-lower Yellow River in 2015 changed not only the temporal and spatial carbon flux, but also the compositions and transformation process of organic matter in this region. The major conclusions of this study were listed as followed:

1) The regulation led to the increases of DOC (29%-121%) and organic carbon in sediments (4%-463%), but decreases of POC (15%-62%).

2) 14 specific amino acids were detected in the study sites. THAA, PAA and SAA ranged at 2.29-9.04 μmol L-1,5.22-22.96 μmol L-1and 81.72-137.19 μg g-1, respectively. DCAA dominated for 71%-98% of the THAA. Neutral amino acids were the main components in THAA,PAA and SAA, followed by acidic and basic amino acids,whereas sulfuric and aromatic amino acid were minor.The compositions of amino acids in different phases changed differently after the regulation. Content of neutral amino acids decreased from THAA to PAA and then to SAA, but content of basic and aromatic amino acids increased correspondingly.

3) The regulation increased the content of bioactive amino acids (Asp, Glu and Gly) in THAA but decreased those in PAA and changed little of those in SAA. Asp/Gly in different phases indicated that, siliceous organisms contributed more to THAA, while calcareous organisms contributed more to PAA and SAA. The regulation promoted the relative contributions of amino acids by calcareous organisms rather than by siliceous organisms.

4) DI values of DFAA were much higher than those of DCAA before the water-sediment regulation. But after the regulation, DI values of DFAA decreased while DI values of DCAA increased. The results showed DCAA were more readily degraded than DFAA, suggesting that the regulation activated DCAA and PAA but deactivated DFAA and SAA.

5) Multiple correlation analysis showed that PAA was the primary component representing the properties of amino acids and organic carbon in this study area, and bacterial reproduction played a key role in affecting the compositions and properties of amino acids, followed by redox condition.

Acknowledgements

This study is supported by the National Key Research and Development Program of China (No. 2018YFC1407 601) and the National Natural Science Foundation of China (No. 41176064). Thanks for the Qingdao Dairy Compound Detection Expert Workstation, hosted by SGS-CSTC Standards Technical Services (Qingdao) Co.,Ltd.