Bioremediation Of Eutrophic Sediment And Response Of Microbial Communities To Bioturbation By Benthic Macrofauna

The concentration of nitrogen and phosphrous in seawater was increasing dramatically, due to the increasing input of effluents which is rich in nutrients. Subsequently, the seawater became eutrophic then sediments became eutrophic too since the deposition of nutrients in sediments. So far, the coastal sediments in south of Jiangsu province became eutrophic duo to input of nutrients, in turn the biodiversity decreased in sediment. Nowadays, lots of researchers focused on the eutrophication, explored the bioremediation for the eutrophic sediment. In this study, we combined the bioturbation, physical disturbance, microrganisms agent to remove the nutrients in sediment exploying response surface methodology（RSM） and orthogonal method in laboratory and in situ intertidal flats respectively, exploring the combined effects on nutrients removal from sediment. Additionally, we studied the influence of bioturbation by clam（Meretrix meretrix）, polychaetes（Perinereis aibuhitensis）, mixed macrofauna（clam plus polychaetes） on parterns of nutrients and chacteristics of microbial communties in sediment, comparing the difference between experimental plots. Finally, we studied the relationship of microbial communities between intestinal tract of Meretrix Meretrix and sediment on its shell surface. The results of stuies are expected to provide a reference for bioremediation of eutrophic sediment and an understanding of the mechnisms of bioturbation of clam Meretrix meretrix. 1. Isolation and testing removal abilities of microrganisms for bioremediation from sedimentIn this study, we isolated several strains of nitrifying-aerobic denitrifying bacteria called MD5 and MD8 from intertidal sediments at Jiangsu coastal, identified their biology taxonomy, and tested the ability of removal of nitrogen. The morphology, 16 S r RNA sequencing and functional gene（nir S） results showed that MD5 and MD8 were identified as Halomonas and Alcaligenesx respectively with Gen Bank sequence number of KM362826 and KM406394. In the experiment with ammonium as solo nitrogen resource, MD5 and MD8 reached 81% and 88.3% removal rate respectively. While in the experiment with nitrate as solo nitrogen resource, they reached 85.3% and 92.1% removal rate respectively. In experiment with combination of ammonium and nitrate, MD5 and MD8 reached 72% and 76.8% total removal rate respectively. The p H, salinity and C: N ratio influenced the nitrogen removal ability of MD5 and MD8. For instance, the removal abilities of MD5 and MD8 were significantly inhibited（p<0.05）, then the optimum p H was 7.5⁸.5; MD5 increased its nitrogen removal ability with the increasing of salinity within salinity of 10³0, whereas MD8 reached its high removal ability within salinity of 20²5; MD5 and MD8 were significantly inhibited under C:N ratio of 6（p<0.05）, nevertheless, the C: N ratio ranges from 8 to 10 which made MD5 reach its highest ability. The above results revealed that MD5 and MD8 were two strains of nitrifying-aerobic denitrifying bacteria, which could be applied for bioremediation of aquaculture effluents. 2. Optimization of hard clams, polychaetes, physical disturbance and denitrifying bacteria of removing nutrients in marine sedimentMarine organisms are known to play important roles in transforming nutrients in sediments, however, guidelines to optimize sediment restoration are not available. We conducted a laboratory mesocosm experiment to investigate the role of hard clams, polychaetes, the degree of physical disturbance and denitrifying bacterial concentrations in removing total nitrogen（TN）, total phosphorus（TP）, and total organic carbon（TOC） in marine sediments. Response surface methodology was employed to analyze the results of initial experiments and in a subsequent experiment identified optimal combinations of parameters. Balancing the TN, TP, TOC removal efficiency, our model predicted 39% TN removal, 33% TP removal, and 42% TOC removal for a 14-day laboratory bioremediation trial using hard clams biomass of 1.2 kg m^-2, physical disturbance depth of 16.4 cm, bacterial density of 0.18 L m^-2, and polychaetes biomass of 0.16 kg m^-2, respectively. These results emphasize the value of combining different species in field-based bioremediation.3. Manipulate macrofauna, physical disturbance and bacteria to remove total nitrogen and total phosphorus from sediments in situ field bioremediationCoastal marine sediments play a key role in the transformation, transport and burial of nutrients. Many factors influence these ecosystem processes and often they interact, making it difficult to develop clear guidelines on how to use ecological processes to remediate nutrient contaminated sediments. Therefore we manipulated hard clam biomass, physical disturbance, denitrifying bacteria additions and polychaete biomass to examine the effects of the interaction of removal the total nitrogen（TN）, total phosphorus（TP） from sediments. The orthogonal experiment combined three levels of those factors, and the results showed that there were different combinations maximizing TN and TP removal efficiency during the different sampling periods. At the last sampling period（3.5 months）, the seventh treatment plot combined hard clam biomass of 0.8 kg m^-2, physical disturbance depth of 25 cm, denitrifying bacteria additions of 0.3 L m^-2 and polychaete biomass of 0.2 kg m^-2, reaching the highest TN（18.46%） and TP removal efficiency（18.88%）. Pore-water nutrient profiles in this treatment plot were also strongly influenced by the combination, suggesting that hard clams, denitrifying bacteria, and polychaetes influenced the pore-water characteristics dramatically. The interactions between denitrifying bacteria additions and hard clam biomass and polychaete biomass seemed to be positively correlated. However, the interaction between hard clam biomass and polychaete biomass seemed to be constrained by each other. Our results highlight the importance of interactions between macrofaunal bioturbation, sediment stability and nitrogen cycling bacteria for the nutrient transport in sediments. This experiment illustrates that manipulation of biomass of hard clams, physical disturbance, denitrifying bacteria and polychaetes could effectively eliminate TN and TP removal efficiency and restore the eutrophic sediments. 4. The influence of bioturbation on physicochemical characteristic and microbial communities in sedimentIn this study, we conduct an experiment on intertidal flat which contained the clam plots, polychaetes plots, mixed plots（clam plus polychaetes） to study the influence of bioturbation on physicochemical characteristic and microbial communities in sediment. The results showed that physicochemical characteristic was changed significantly by the bioturbation of clam, polychaetes and mixed macrofauna（P<0.05）, which showed the TN, TP, TOC were decreased significantly in sediment（P<0.05）. The microbial communities in bioturbated sediment changed in some degree, however, it were not altered significantly between control plots and bioturbated plots（P>0.05）, between bioturbating plots（P>0.05）. Additionally, the number of amo A with Real-time PCR suggested that the number of AOB was increased significantly by bioturbation rather than AOA. 5. The relationship of microbial communities between the intestinal tract of clam Meretrix meretrix and sediment on surface of its shellIn this study, we investigated the difference of microbial communities between the intestinal tract of clam Meretrix meretrix and sediment on surface of its shell. The high-throughput sequencing results showed that there were different microbial communities between intestinal tract of clam Meretrix meretrix and sediment on surface of its shell. However, there was no significant different between them. Additionally, the nitrogen cycling functional gene of arch-amo A and amo A was amplified and quantified, indicating that the nitrifying and denitrifying bacteria inhabited in the intestinal tract of clam Meretrix Meretrix. Our study suggested that the microbial communities in the intestinal tract of clam Meretrix Meretrix had direct contribution to nitrogen cycling process, besides significantly changing the phical structure, biochemical and microbial process in sediment, indicating the interaction between bioturbation of clam Meretrix meretrix and sediment.