Nutrient dynamics study of overlying water affected by peroxide-treated sediment
© The Author(s) 2017
Received: 18 February 2017
Accepted: 26 July 2017
Published: 31 August 2017
Loading of excess nutrient via bioremediation of polluted sediment to overlying water could trigger anoxia and eutrophication in coastal area. The aim of this research was to understand the changes of overlying water features such as dissolved oxygen (DO); pH; oxidation reduction potential (ORP); chlorophyll-a (Chl-a); and nitrogen nutrients ammonia (N-NH4 +), nitrate (N-NO3 −), and nitrite (N-NO2 −) when the sediment was not treated (control) and treated by calcium peroxide for 5 weeks.
The water samples were analyzed for measuring physical and chemical properties along with the sediment analyzed by polymerase chain reaction (PCR) including denaturing gradient gel electrophoresis (DGGE) for identifying the phylogenetic affiliation of microbial communities.
Results showed that due to the addition of calcium peroxide in sediment, the overlying water exposed the rise of dissolve oxygen, pH, and ORP than control. Among the nitrogen nutrients, ammonia inhibition was higher in calcium peroxide treatment than control but in case of nitrate inhibition, it was reversed than control. Chlorophyll-a was declined in treatment column water by 30% where it was 20% in control column water. Actibacter and Salegentibacter group were detectable in the calcium-peroxide-treated sediment; in contrary, no detectable community ware found in control sediment. Both phylogenetic groups are closely related to marine microflora.
This study emphasizes the importance of calcium peroxide as an oxygen release material. Interaction with peroxide proved to be enhancing the formation of microbial community that are beneficial for biodegradation and spontaneity of nutrient attenuation into overlying water.
KeywordsOxygen release Calcium peroxide Bacteria Anoxia Remediation Nitrification
Marine sediments in coastal sites usually contaminated by pollutants from a wide variety of anthropogenic sources, such as wastewater effluents, industrial sewage, and large amount of aquaculture activities, are posing extensive dangers to the environment (Zhang et al. 2015). Researchers have proven that the overlying water and sediment was the major object of contaminants like excess nutrient loading and stinky odor, where decomposition of organic matter and onsite nitrification might greatly contribute to anoxia of the water body (Wu et al. 2012). However, the depletion of oxygen in the sediment may be contributed to the release of N nutrient from the sediment into the water column. So, increased N nutrient concentration and algal blooms leads to more severe anoxia, and apparently, there is a mutual enhanced effect between these two processes (Liu et al., 2016a, b). However, the remediation of contaminated sediment has gained much attention in the past decade since it has been recognized that substantial improvement in the quality of the overlying water often cannot be achieved without appropriate treatment of contaminated sediment. Thus, great efforts have been made in recent years in exploring the effective remediation approaches for the decontamination of marine sediments (Veetil et al. 2013; Asaoka et al. 2015). Several types of chemicals or biological agents have been applied for in situ sediment dealing through directly injecting into the contaminated sediment for the purposes of odor control, nutrient inactivation, and organic contaminant bioremediation (Nykanen et al. 2012; Perelo 2010). Calcium peroxide (CaO2) is the most used agent injected into the sediment being treated as it has gained great attention to promote contaminant biodegradation (Hanh et al. 2005). Due to the immediate reaction with water, CaO2 has a superior characteristic in improving the oxygen concentration rapidly despite the rise of the pH value of water. In addition, acid volatile sulfide that exists in sediments can be oxidized to sulfate through the autotrophic denitrification process, resulting in sulfide decrease and odor suppression (Liu et al. 2015). Moreover, improved dissolved oxygen can stimulate aerobic respiration of the microorganism in the sediment and reduce the formation of H2S and NH3, etc. and an aerobic membrane is formed to prevent pollutants in the sediment from releasing into the overlying water. So far, few researches have been reported by the method of using oxygen release material to control N nutrient releasing. However, peroxide reacts with water rapidly and the excess released oxygen was also wasted by the demands of the microbes. For above reason, the aerobic condition in the overlying water did not show an immediate increase after the alteration but slowly it increased (Nykanen et al. 2012). Nevertheless, in consideration of avoiding the waste of oxygen, nitrogen nutrient release would be constrained if the oxygen-releasing material were able to release oxygen for several months or years steadily, so that water and sediment restoration could be more effective.
This research was to test a method for increasing the oxygen level of sediments treated by calcium peroxide (CaO2) as a compound for the slow release of oxygen and to stimulate the aerobic microbial population to degrade organic matter in the sediment. The objectives of this research were to (1) investigate the effect of calcium peroxide treatment (inside sediment) on the physical parameter flux (dissolved oxygen (DO), pH, oxidation reduction potential (ORP)) in different levels of water column (2) measure vertical distribution of N nutrients (N-NH4 +, N-NO3 −, and N-NO2 −) and Chl-a in the system, and (3) identify the bacterium classes growth in the sediment.
Sample collection and study
Physiochemical characteristics of the sediment and water
N-NO2 − (mg/L)
N-NO3 − (mg/L)
Column packing and incubations
The chemical oxygen demand (COD) was analyzed by the reducing agent of potassium permanganate followed by the iodometric titration method, and the acid-volatile sulfide (AVS) was measured by the sulfide detection tube (Detector tube No. 201H; GASTEC, Kanagawa, Japan) as follows: 2 g of a sample were mixed with 2-mL concentrated sulfuric acid, which was continuously pumped into the tube, until the color of the tube changed. Water samples were filtered through a glass microfiber filter (GF/C, Whatman, Brentford, UK). The concentration of nitrogen ammonium (N-NH4 +) was determined by indophenol blue method, and nitrate (N-NO3 −) and nitrite (N-NO2 −) concentration was measured by N-(1-naphthyl)-ethylenediamine adsorption spectrophotometry and Cd–Cu reduction N-(1-naphthyl)-ethylenediamine adsorption spectrophotometry, respectively. All spectroscopic analyses were done by UV Mini-1240 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Chlorophyll-a have been measured as extractable chl-a from water samples and analyzed by the spectrofluoro-meter (Shimadzu Mod., RF 1501) methods described in Antonietta et al. (2009). Analytical grade chemicals were used in all experiments. For all samples, sediment and overlying water were analyzed in triplicates and all data are reported as the average of the three subsamples.
Microbial community analysis
The microbial community was analyzed via FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) following the methods described by Cho et al. (2014). First, 16S rDNA out of the extracted DNA samples were identified using primers 27f and 1492r. The second touch-down polymerase chain reaction (PCR) was run through the 16S rDNA V3 region and re-amplified with primer GC-341F equipped with 40-bp GC-clamps (Bioneer Inc., Daejeon, Korea). In PCR, a sample was heated at 95 °C for 5 min and was denatured for 30 s. Then, it was annealed starting at 65 °C decreasing by 0.5 °C per cycle, 15 more cycles at 55 °C, and 45 s of elongation at 72 °C within 10 min. The products of PCR were identified in 1% of agarose gel. DNA fragments were cut from bands on the denaturing gradient gel and were washed with highly purified distilled water. After being added with TE buffer (25 uL), the DNA samples were centrifuged at 13,500g for 1 min. The collection was frozen (−70 °C) and thawed (45 °C) three times for 15 min each. After centrifugation, the supernatant was collected for further analysis. The finalized collections were amplified again for an NCBI BLAST (Basic Local Alignment Search Tool) search designated for the most probable phlemonic similarity.
Data were analyzed using SPSS 18.0.1 statistical packages. The relationships between different parameters were tested statistically using general correlation coefficient (Pearson) procedures in SPSS.
Results and discussion
Changes of dissolve oxygen, pH, and oxidation reduction potential in overlying water
pH increases in the sediment and overlying water of the treatment column which is caused by the hydrolysis of calcium. Oxides are converted to hydroxides over time (as oxygen is released), and even larger pH differences are observed because Ca(OH)2 helps in increasing the pH. But the bacterial density were not affected negatively and stayed elevated (Nykanen et al. 2012). Hence, the advantages of CaO2 are low disproportionation and long-lasting effectiveness for in situ remediation (Liu et al., 2016a, b).
Oxidation reduction potential
Changes of N-NH4 +, N-NO3 − and N-NO2 − concentrations in overlying water
Coefficients of correlation between environmental parameters and nutrients
The correlations between environmental parameters and nitrogen compounds at the sediment-water interface of the control
Effect of pH and DO on ammonia and nitrate dynamics in overlying water
Previous researches have revealed that nitrification has a significant effect on oxygen consumption in various aquatic systems (Hsiao et al. 2014). Our results showed that N-NO3 − was positively correlated with DO. During the early period of the experiment, DO showed an increase trend in control, and as a consequence, the vitality of nitrifying microorganisms was stored and the concentration of nitrate increased to a certain extent. Once started, nitrification costed additional oxygen and might result in an anoxic condition. This would be an important reason for the decreasing trend of both DO and N-NO3 − after 2 weeks of the experiment. Without supplement of oxygen, DO levels were limited in control; thus, nitrification was less intensive than that in the CaO2-treated column because nitrification cannot proceed under low DO condition. On treatment column, nitrification remained in the first stage until week 3, indicating the periodic burst of oxygen consumption associated with nitrification and organic matter degradation process which constituted a significant impact factor to oxygen depletion. After week 3, N-NO3 − began to increase in treatment, explaining the simultaneous increase of DO by CaO2 but finally, some of the resulting nitrate being lost from the system indicates the denitrification process. Denitrification is the main process for nitrate attenuation (Jing et al. 2013). pH is another most important factor of nutrient release. Comparably, under high DO condition, the N-NH4 + concentration were apparently higher at low pH than that at high pH. This phenomenon could be due to the sufficient H+ under acid conditions (pH <7) that would inhibit the activity of nitrifying bacteria at the water-sediment interface, resulting in slower rate of conversion from ammonium to nitrate. Thereafter, ammonium accumulated abundantly in the surface sediment and is further released, leading to higher ammonium concentration in overlying water. Enough molecular oxygen can also inhibit the activity of denitrifying enzymes, especially that of nitrite reductase enzyme (Jing et al. 2013). Concurrently, in overlying water, denitrification rates tended to be higher in regions of low pH low oxygen concentration (Zhang et al. 2014). But in treatment column, under an aerobic and alkaline condition, abundant OH− was present, which reacts with the ammonium released from the sediment into overlying water. Hence, the ammonium would be converted directly to N-NO3 − and escaped from the water body, which was reflected in the lower ammonium concentration in overlying water.
DGGE band analysis and community’s phylogenetic affiliation
Base sequence of 16S rDNA revealed from DGGE bands
NCBI accession no.
Uncultured marine bacterium
Uncultured Actibacter sp.
Salegentibacter flavus strain
The control and agent-treated sediment columns have not showed bacterial identity at beginning of the experiment. Although this form was unchanged for control sediment, agent-treated sediment showed bacterial identity after 5 weeks of experiment. It could be associated that the agent has an effect to the growth bacterial species.
The water-sediment system is usually disturbed by biodegradation process of organic and inorganic matter inside sediment. This study observed the system parameters changes during 5 weeks of experiment especially parameters of overlying water. Most of the study focuses on sediment remediation; although, the parameter changes in the overlying water could massively affect the ecology. Sediment and water exposed significant and visual changes by the internal parameter changes due to application of calcium peroxide. Parameters of overlying water were found greatly altered in calcium-peroxide-treated sediment column than in non-treated sediment column. These could be due to chemical composition changes in sediment and nutrient dynamics from sediment to water layer. The DO in the control column water was found unchanged until 2 weeks when it was 1 week for agent-treated column. After the pointed time, DO was changed by decline in control column overlying water and was inclined in treatment column overlying water until 5 weeks. By way of pH, result has pointed that the agent treatment in sediment suppresses excess nutrient loading in overlying water than what occurred in the control for a long lasting period. The ORP at the beginning of 3 weeks was not distinguishable between the control and treatment columns’ overlying water over the 5 weeks of study. ORP in overlying water of treatment column were found to be increased by 2% from the start to the end of the study. Agent showed 1.5% efficacy than natural condition by changes in ORP of overlying water. Oxygen-releasing agent calcium peroxide was affecting nutrient dynamics of N-NH4 + suppression by 63% than the overlying water of the control column when it was reverse by 7% for N-NO3 −. But changes of N-NO2 − were found inconsistent and negligible for the overlying water in both columns. Photosynthesis pigment chlorophyll-a concentrations were found in a declining pattern for the overlying water in both columns. But this decrease was higher in the agent-treated column’s overlying water by 10% in compare with control column overlying water. The microbial community residing in the sediment was found to be species closely related to the genus Actibacter and Salegentibacter group. Both phylogenetic groups of natural marine microflora were observed in treated sediment at the end of the experiment whereas no detectable species were observed in the control sediment; although, they have showed physical color change from transparent to black in the column during the study.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science ICT & Future Planning (2017R1A2B4008720) and BK21 plus program in the Republic of Korea.
The funding was provided by the Ministry of Science ICT & Future Planning (2017R1A2B4008720) and BK21 plus program, South Korea.
Availability of data and materials
Data and materials are available to all.
MNH participated in the research work and wrote down the manuscript. SHK conceived the idea and directed the research work. Both authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
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