Molecular Dynamics Simulation of the Aggregation of Extracellular Polymeric Substance

. Extracellular polymeric substance (EPS) play an important role in the transport and transformation of pollutants. At present, some scholars have conducted basic experimental characterization of EPS, but the molecular lev-el interactions among the various components of EPS still need to be fully characterized. Here, we use molecular dynamics (MD) simulation to explore the structural properties of EPS systems in atomic detail. The process and mechanism of the aggregation of different EPS components (polysaccharide, lipid, nucleic acid and protein) were revealed by the simulation results. EPS aggregation consist of a hydrophobic core and an amphiphilic exterior. Lipid tail, as a hydrophobic core, promoted the aggregation of EPS. But strong hy-drophilic nucleic acid and protein components inhibited the aggregation of EPS in water and were located outside the aggregation body. The details of the structure of EPS aggregation are revealed here, which provides a micro molecular mechanism for understanding the interaction between EPS.


Introduction
Natural organic matter (NOM) widely exists in the water environment, and is composed of organic compounds with different molecular weights, components, and isomers [1] . According to different sources of NOM, it can be divided into autochthonous and allochthonous compounds. Autochthonous compound is an organic compound directly released by various microorganisms in water, accounting for a large proportion of organic macromolecules in aquatic ecosystems [2] . The most important component of in situ NOM is the highly viscous EPS secreted by microorganisms into the surrounding environment. EPS is an im-portant component of biofilms, which is a byproduct of metabolism secreted by microorganisms in response to pressure or adaptation to habitats, its secretion greatly improves the survival rate of microorganisms in water [3] . EPS is general-ly composed of four components: high molecular weight polysaccharides, pro-teins, lipids, and nucleic acids [4] . These components not only contain a variety of isomers, but also contain rich chemical groups, such as carboxyl, amino, and carbonyl groups [5] . At the same time, these EPS can be divided into hydrophilic EPS and hydrophobic EPS according to their hydrophilicity and hydrophobicity [6] . The different hydrophilicity and hydrophobicity of EPS will inevitably have different effects on its own aggregation. As a heterogeneous mixture composed of various organic compounds, EPS has received widespread attention in the field of ecological environment in recent years. The complexity, heterogeneity, and variability of EPS increase the difficul-ty of research, making our limited understanding of its interfacial behavior be-tween microbial cells and the aquatic environment. With the continuous devel-opment of molecular characterization and identification technology, the research on the composition of EPS is also deepening. Some studies have confirmed the assembly of humus in water through experiments [7] . However, the self-assembled structure of EPS in water and the molecular mechanism of its formation still need to be studied, and related microscopic issues cannot be verified through experiments, requiring in-depth exploration in combination with MD simulation.

Model construction
The complex EPS was simulated by adding six polysaccharides (Pol), two lipids, one RNA, and one protein to the simulation system. Among these, four Pol components (Pol 1-4) are neutrally charged ( Figure 1A) and two Pol divisions are negatively charged (Pol 5) and positively charged (Pol 6) by deprotonation and protonation of carboxyl and amino groups ( Figure 1B). Lipids consist of 1-palmitoyl-2-oleoyl-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho (POPG). POPG has a negative head group ( Figure 1C). The short RNA possesses the AUCGAUCG sequence ( Figure 1D) and their protein components are represented by Cytochrome type C (Cyt-C) ( Figure 1E), which is secreted by many bacteria to aid in extracellular respiration. The molecular structure of EPS components except Cyt-C was constructed by Materials Studio 2017. These structures are imported into an automatic topology generator to generate interaction parameters for MD simulations. The whole atomic molecular structure of Cyt-C was obtained from the Protein database (PDB:1akk).

Simulation system
Placing EPS single molecules at 10 nm × 10 nm × 10 nm in a cubic box and solvated, 10 ns equilibrium was performed to obtain a small molecule after equilibrium.
To study EPS assembly, the components of polysaccharides, lipids, nucleic acids, and proteins were sequentially mixed at 15 nm × 15nm × 15 nm in a cube box, solvate with water and add Na+ and Cl-to neutralize the system charge to prepare four increasingly complex EPS systems. The simulation time was 200 ns.
(1) EPS1 consists of only polysaccharides, with 8 molecules added to each molecule; (2) In the EPS2 system, 8 POPCs and 8 POPGs were added to the EPS1 system to explore the effect of lipid components on aggregation; (3) In the EPS3 system, 8 nucleic acids were added to the EPS2 system to explore the effect of nucleic acid components on aggregation; (4) In the EPS4 system, eight proteins are added to the EPS3 system to explore the effect of protein components on aggregation.

Simulation detail
All MD simulations were performed using the GROMACS software package version 4.6.7 [8] , the Gromos 54a7 force field and TIP3P water model [9] . The assembly was simulated using a NPT ensemble. Using an isotropic Berendsen constant pressure gauge (P = 1 bar), the coupling constant in all three directions was 0.2 ps, and the compressibility coefficient was 4.5 × 10 -5 bar -1 using a V-rescale thermostat with a coupling constant of 0.2 ps to maintain the temperature at T = 300 K. The truncation radius of both the van der Waals interaction and the Coulomb interaction is 1.2 nm. The Lennard-Jones (LJ) and Coul potentials smoothly shift to zero between 0 nm and 1.2 nm. The particle grid Ewald summation method is used to deal with long-range electrostatic interactions. The time step for all simulations is 1 fs, and the neighbor list is updated every 10 steps. Periodic boundary conditions are considered in all three directions. The molecular conformation is presented through VMD software [10] .

Characterization
The Hydrophobicity of EPS components was estimated by solvation free energy (Figure 2). The smaller the free energy of solvation, the stronger the hydrophilicity, and the larger the free energy of solvation, the stronger the hydrophobicity. Because the protein molecule has a hydrophobic core and a hydrophilic surface, the default protein component is the most hydrophilic. The results showed that the order of hydrophobicity of all components was lipid > Pol 1-4 > RNA > Pol 5-6 > Cyt-C.

Effects of each component on EPS aggregation
In order to elucidate the effects of each component on EPS assembly, full-atom MD simulations of EPS1, EPS2, EPS3 and EPS4 were carried out. In EPS1, 48 polysaccharides in the system formed two aggregations ( Figure 3A), indicating a weak interaction between polysaccharides. In the EPS2 system with added lipids, all molecules in the system formed an aggregation ( Figure  3B), which proved that lipid components promoted the aggregation of EPS in water. In the EPS3 system with the addition of nucleic acid, although all molecules in the system formed an aggregation ( Figure 3B), the aggregation was more irregular than that of EPS2, indicating that in the EPS3 system, lipid components still promoted the aggregation of EPS in water, but RNA inhibited the formation of aggregation ( Figure 3C). When eight protein molecules were added to the system (EPS4), the entire system did not aggregation, and other components formed several smaller clusters and were connected to each other by Cyt-C ( Figure 3D). The protein component inhibited EPS aggregation.

Surface properties of EPS aggregation
EPS aggregation is affected by the hydrophobicity of components, and the driving force of aggregation is hydrophobic interaction. Hydrophobicity components promoted EPS aggregation, while hydrophilic components inhibited EPS aggregation. The hydrophobicity component promotes aggregation and is located at the core of the aggregation, while the hydrophilic component is located at the surface of the aggregation. In addition, Pol 5-6 is not only hydrophilic, but also has positive or negative charges, so there are charged groups on the surface of the aggregations. As shown in Figure 3, components with strong hydrophilicity are all on the surface of aggregations.

Conclusion
This study mainly studied the related mechanism of EPS aggregation in water. As a hydrophobic core, lipid components promoted EPS aggregation through hydrophobic action, while nucleic acid and protein components were highly hydrophilic and inhibited EPS aggregation. After the aggregation of EPS, the surface of the aggregation is hydrophilic and has charge.