The enhancement effect of polyethylene (PE) surfaces modified with poly(lactic acid) (PLA) on formation of nitrifying biofilms in biological aerated filter (BAF) was investigated in this study. X-ray photoelectron spectroscopy, scanning electron microscope, and protein absorption analysis revealed that modified PE surfaces produced active groups, including carboxyl (O═C─O), hydroxyl (C─OH), and carbonyl (C═O), increased surface roughness, and enhanced the adsorption of both the bovine serum albumin and fibrinogen. During the startup period of 33 days, the average removal rates of ammonia nitrogen (NH3-H) were 68 and 72% before and after modification which were 36 and 43% for total nitrogen, 47 and 45% for total organic carbon. The results of denaturing gradient gel electrophoresis experiments demonstrated that modified carriers adsorbed and gathered more species of bacteria on surfaces. Thus, surface modification of PE suspended carrier by PLA improved the efficiency of reactor startup for drinking water treatment.
Nitrification is a two-step sensitive process containing nitrozation and nitration, oxidized by Nitrosomonas species and Nitrobacteria species, respectively; in particular, the first step is more sensitive and normally rate-limiting (Lackner et al. 2009). The responsible nitrifying bacteria keeps a low growth level and forms strong biofilm with difficulty because of long generation period and poor extracellular polymeric substance (EPS) production (Bassin et al. 2012), which can potentially lead to a longer startup period of the nitrifying biofilm reactor for drinking water treatment.
In addition, EPSs play key roles in promoting superficial adhesion and developing the formation of biofilm (Xue et al. 2012). It is generally appreciated that EPSs contain polysaccharides, proteins, phospholipids, humic acid, and other polymeric compounds (Sheng et al. 2010). Sometimes, proteins among the components are more dominant (McSwain et al. 2005). Thus, the higher ability of adsorbing proteins on the surfaces of carriers could enhance bacterial adhesion.
Carrier surface modification by poly(lactic acid) (PLA) can be one choice to improve the start efficiency of biofilm reactors. PLA has been widely applied in various areas due to its biodegradability, biocompatibility, and accessibility from renewable resources (Chen et al. 2003). Therefore, it will be safe and feasible to make use of this green-friendly material. However, to our knowledge, studies on enhancing the formation of nitrifying biofilms and subsequently shortening the startup period of reactors for drinking water treatment by PLA loaded on surfaces of suspended carriers are lacking.
To shorten the period of startup in nitrifying biofilm reactor, common polyethylene (PE) suspended carriers were modified by green materials PLA, and its surface property and adhesion characteristics were improved to achieve the end goal that target pollutants were fast removed in the raw water.
This report shows that PLA deposited on the suspended carrier (PE) surface could increase surface roughness and protein adsorption, facilitating the formation of nitrifying biofilm and shortening the period of biological aerated filter (BAF) startup. Subsequently, it improved the removal rate of ammonia nitrogen (NH3-H) and total nitrogen (TN).
MATERIALS AND METHODS
Preparation of PLA coated PE
PE fluidized filler was chosen for the carrier in this assay because of its wide application in biofilm reactors and modified by loading PLA which was purchased from Sigma Co. Ltd., St Louis, MO, USA, possessing biocompatibility and active groups through the following procedures. One gram of PLA and 2 g sodium chloride were dissolved in 100 ml dichloromethane solution at 60 °C for 1 h. Then the solution was poured into a beaker with suspended PE carriers and stirred evenly. The carriers were separated and dried in a draught cupboard for 24 h to remove dichloromethane absolutely at room temperature (Saito et al. 2006).
X-ray photoelectron spectroscopy analysis
The surface of the modified and unmodified carriers was characterized on an X-ray photoelectron spectrometer (XPS; PHI 5700 ESCA System, Waltham, MA, USA). C1s peaks were used as an inner reference calibration peak at 284.6 eV. Curve fitting was performed with a computer program XPS peak provided by the equipment manufacturer, and the software allows manipulation of the peak positions (Ruan & Feng 2003).
Protein absorption study
Films prepared with PLA modified and unmodified PE carriers were first washed three times with phosphate buffer solution. Then the samples were immersed in bovine serum albumin (BSA) and fibrinogen solutions (1 mg/ml, Sigma Co. Ltd, USA) and incubated at 37 °C in 4 ml centrifuge tubes for 2 h. After that, these samples were washed three times with phosphate buffered saline (PBS) to remove the loosely attached protein. Consequently, films were immersed in 1.5% SDS solution (w/v) at 37 °C for 1 h. After washing with PBS again, micro-bicinchoninic acid protein assay was used to determine the amount of protein (Eon BioTek) at a wavelength of 562 nm (Shan et al. 2009).
Scanning electron microscope analysis
The biofilm samples obtained from each reactor in the startup period were first fixed in a 0.1 mol/l phosphate buffer solution (pH 7.2) containing 2.5% (v/v) glutaraldehyde at 4 °C for 1.5 h. Then the samples were washed three times with phosphate buffer solution (0.1 mol/l, pH 7.2). Consequently, these chemically stabilized biofilm samples were dehydrated using ethanol in series: 50% (v/v); 70% (v/v); 90% (v/v); 100% (v/v); 100% (v/v); 100% (v/v) for 10 min, respectively. After that, the biofilm was air dried overnight at room temperature. The samples were attached with conductive tape and coated with 15 nm thick gold by a precision etching coating system (Gatan Model 682). The samples were finally examined with a scanning electron microscope (SEM) (Quanta 200F) operated at 5 kV.
The two laboratory scale reactors used in this experimental study were cylinders made of Perspex with dimensions of 550 mm high and 55 mm internal diameter and a conic bottom. Working volume was 1 l for each column. The system was operated at ambient room temperature (22–28 °C but typically at 25 °C). The reactors were filled up to 33.3% of their active volume with a floating PE and PLA modified particles with average diameter of 10 mm.
Raw water was supplied from Songhua River in Harbin City, China. The essential component for biomass synthesis, namely nitrogen, was added in the form of NH4Cl in order to keep NH4+-N at 1.5–2.0 mg/l. Sodium acetate was also added to make total organic carbon (TOC) at 40 mg/l. The hydraulic retention time was 1 h. pH, dissolved oxygen, and TN were 7.2–7.5, 5–7 mg/l, and 2–3 mg/l, respectively.
The samples of influent and effluent of BAF were analyzed on NH4+-N, TOC, and TN. During the startup and steady phase, the operational parameters were measured continuously every 3–5 days. The samples withdrawn from BAF were analyzed for determination of NH4+-N with the procedure described by Standard Methods (APHA, 2005). TOC and TN were determined by the TOC analyzer (TOC-VCPH, Shimadzu, Japan). The samples were pre-filtered by 0.45 μm membrane.
Polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE) technology facilitates the research on bacterial community and microbial diversity due to separation nucleotide sequences without isolation and cultivation of microorganism (Merlino et al. 2013).
Biofilm samples were collected from PE and PLA modified PE carriers of BAF and centrifuged to provide 0.25 g total biomass removing excessive water. Then DNA extraction was conducted with a DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA). The V3 region of 16S rRNA for extracted DNA was amplified by PCR using universal bacterial primers (343F, 5′-ACTCCTACGGGAGGCAGCAG-3′ and 534R, 5′-ATTACCGCGGCTGCTGG-3′ with a GC clamp 5′-CGCCCGCCGCGCGCGGC GGGCGGGGCGGGGGCCCGGGGG-3′). The 50 μl PCR reaction mixture comprised 0.25 μl Taq DNA polymerase (Takara, Dalian, China), 5 μl PCR buffer (Mg2+ Plus), 4 μl dNTP mixture (2.5 mmol/l), 2.5 ng DNA template, 1 μl 343F primer (20 μmol/l), 1 μl 534R primer (20 μmol/l), and 35.75 μl Milli-Q water. The PCR conducted according to the procedure of 10 min preheating at 94 °C, 30 cycles of 1 min denaturation at 94 °C, 30 s annealing at 55 °C, 90 s elongation at 72 °C, followed by final extension at 72 °C for 10 min and kept at 4 °C. After preparation with 1% agarose gel electrophoresis and 0.1 (μl/ml) ethidium bromide, DGGE analyses of PCR products were performed using the Dcode™ universal mutation detection system (Biorad Laboratories, Hercules, CA, USA) as the method below. Denaturing gradient gels (30–60% denaturant gradient, 100% denaturant comprises 7 mol/l urea and 40% deionized formamide) were loaded by adding PCR amplification products 10 μl and 6 × loading buffer 2 μl in all, and then electrophoresed at 150 V and 60 °C for 7 h; finally gels were scanned by a transmission scanner.
RESULTS AND DISCUSSION
Surface characteristics of suspended PE carriers
Various types of suspended carriers have found widespread applications in different water treatment technologies by reason of low density, holder free, homogeneous fluidization, low-energy consumption, such as PE, polypropylene, polyvinyl chloride, etc. (Gapes & Keller 2009). However, it is necessary to modify the surface of the carrier because the smooth surface acts against initial adhesion of microorganism.
Polymer surface modification includes a series of techniques to improve surface characteristics, a such as wet chemical and ultraviolet irradiation (Goddard & Hotchkiss 2007). Surface characteristics containing surface morphology, surface roughness, biocompatibility, and porosity could be improved and promoted by surface modification (Hou et al. 2013). A solvent evaporation method was used to introduce PLA coating on the PE fluidized filler surface in this research. Also, the modification method in this research is universally applicable for the kinds of above-mentioned suspended carriers or others with smooth surfaces. Normally, there is a general consensus about application of micrographic and spectral methods including SEM for analysis and XPS (Buonomenna et al. 2007).
The supporting materials of the biofilm and its surface roughness could affect the performance of protein adsorption and bacterial adhesion (Janjaroen et al. 2013); therefore, it is necessary to observe the differences between the modified and unmodified PE surfaces. SEM images were taken to demonstrate the morphology of modified and unmodified suspended PE carrier surfaces. As compared to surfaces of unmodified carriers (Figure 1(a)), surfaces of carriers modified with PLA were rougher and more uneven (Figure 1(b)) and consequently provided more living environments for autotrophic and heterotrophic bacteria, which might relate to dissolution of saturated sodium chloride and the process of separation from surfaces of modified suspended PE carriers. In reactors operated for a period of time, diverse species of bacteria adhered to the surfaces of suspended PE carriers, as illustrated in Figures 1(c) and 1(d). Besides Bacillus, which was in a dominant position (Figure 1(c)), various species of cocci appeared and coexisted with other Bacillus sp. (Figure 1(d)).
The chemical surface composition of the sample was identified by using XPS. Figures 2(a) and 2(b) show the XPS C1s region of the suspended PE unmodified and modified carriers, respectively. It could be seen that the shape of XPS spectra of the unmodified and modified surfaces was almost unidentical. Curve fitting by the PC-ACCESS software gave the percentage area for the feature of PLA modified PE surface. The peak area of number 2–4 (binding energy 287.85, 288.9, and 285.55 eV, respectively) possessed 12.76, 11.07, and 55.12% corresponding to carbonyl (C=O), carboxyl (O=C–CO), and hydroxyl (C–OH), which implied that modification by PLA introduced C–O functional groups on modified surfaces and therefore increased the probability of combining and adsorbing more bacteria. Previous observation also demonstrated that the introduction of carboxyl by surface modification could greatly enhance bacterial adhesion to carriers (Eichler et al. 2011). In addition, formation of new chemical bonds contributed to binding of carrier and EPS (Fang et al. 2012); thus, it should be reasonable to predict that PLA modified PE surfaces could lead to better biocompatibility.
Accompanied by surface modification of carriers, the level of protein adsorption altered (Pei et al. 2011). High protein adsorption capacity of carriers could indicate high EPS adsorption on the carrier surface (Bhatia et al. 2013), which subsequently promotes and strengthens the formation of biofilm (Ras et al. 2011). Figure 3 illustrates the adsorptions of BSA and fibrinogen to the unmodified and modified surfaces of suspended PE carriers. The BSA and fibrinogen that were used in this study which have smaller and larger molecular weights had different performances in amount of adsorption. Compared to unmodified surfaces (adsorption 6.24 μg/cm2), a minor increase of BSA adsorption was 17% for the modified surfaces (adsorption 7.31 μg/cm2), while a major increase of fibrinogen adsorption was 27% for the modified surfaces (adsorption 25.25 μg/cm2) as compare to unmodified surfaces (adsorption 19.88 μg/cm2). The results revealed that modification remarkably enhanced protein adsorption capacity.
Performance of modified and unmodified carriers for reactor startup
Removal efficiencies of impurities including NH3-N, TN, and TOC in BAF with unmodified and modified carriers are exhibited in Figure 4. NH3-N removal through nitrifying biofilms on modified suspended PE carriers was relatively good, as more than 78% of influent NH3-N was removed on average, surpassing 90% in the stable operation phase. Compared to the unmodified suspended PE carriers with which 71% average removal rate was achieved, higher removal rate with modified carriers indicated that surface modification could be propitious to formation of biofilms (Figure 4(a)). The reason for the relatively low performance was due to the inert, single, level surfaces of unmodified carriers that were incapable of attracting enough microorganism for the initial adhesion. Differences of removal efficiencies between unmodified and modified suspended carriers also revealed reduction of TN in BAF. During the 33 days of the startup period, parallel-running reactors, respectively, reduced TN levels by an average of 36 and 42.9% with unmodified and modified PE suspended carriers, suggesting that surface modification not only improves the process of nitrification but also promotes the process of denitrification (Figure 4(b)). However, the removal characteristics of TOC show different tendencies, as illustrated in Figure 4(c). TOC removal rates with unmodified and modified suspended PE carriers were 47.8 and 48% on average, respectively. The removal rates of ammonia nitrogen and TN in this test were superior to previous observation, especially in the stable operation period (Guo et al. 2013).
Diversity of microbial community on surfaces of suspended carriers
DGGE, which is regarded as a trusty experimental technique to explore the microbial community (Araujo & Schneider 2008) was applied in this study to observe the diversities of bacterial species on the surfaces of unmodified and modified PE suspended carriers listed in Table 1. Aggregately 10 DGGE bands were identified in the unmodified and modified carrier biofilm samples (modified 1–6 DGGE bands, unmodified 7–10 DGGE bands). Nitrosococcus halophilus (Lipponen et al. 2004) and Nitrosospira multiformis (Choi et al. 2010) were autotrophic bacteria responsible for oxidizing ammonia to nitrite; in addition, Pseudomonas sp. and Pseudomonas nitroreducens oxidized biochemical and biodegradable organic substances (Wang et al. 2009). The conclusion has been arrived at that distinct microbial communities were distributed on the surfaces of modified and unmodified carriers and much abundant bands obtained through modification.
|Band .||Accession .||Blast alignment results .||Identity (%) .|
|1||KC449292||Uncultured alpha proteobacterium||95|
|5||NR074585||Alicycliphilus denitrificans BC||94|
|Band .||Accession .||Blast alignment results .||Identity (%) .|
|1||KC449292||Uncultured alpha proteobacterium||95|
|5||NR074585||Alicycliphilus denitrificans BC||94|
For modified surfaces of suspended carriers, Nitrosococcus halophilus, Alpha proteobacterium, and Alicycliphilus denitrificans BC became parts of the biofilm with the functions of nitrification and denitrification, and also Nitrobacter which belongs to Alphaproteobacteria could oxidize nitrite to nitrate, avoiding the accumulation of spare nitrite and promoting the process of nitrozation in turn, which were the reasons for its higher removal efficiencies of ammonia nitrogen (NH3-H) and TN. Meanwhile, the competition between autotrophic and heterotrophic bacteria decreased the removal rate of TOC for modified carriers, consequently declaring the cause of removal efficiency in this research from the microscopic view.
Based on the obtained results, it was the first time to demonstrate the improved efficiency of reactor startup in BAF by PLA. Modified suspended PE carriers introduced active groups, increased surface roughness, and enhanced the protein adsorption. As compared to PE carriers, modified suspended carriers achieved higher removal efficiencies of NH3-H and TN in BAF. In addition, surfaces of modified suspended PE could accumulate more kinds of bacteria on surfaces.
This work was supported by the project on the study on control techniques of nitrogen and nitrite nitrogen in urban drinking water, which was from Guangdong Provincial Key Laboratory of Urban Water Cycle and Water Quality Security Technology (NHD201208).