Biocarrier selection for the attachment of specific microorganisms in wastewater treatment

doi:10.21203/rs.3.rs-4616296/v1

Download PDF

Research Article

Biocarrier selection for the attachment of specific microorganisms in wastewater treatment

https://doi.org/10.21203/rs.3.rs-4616296/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

With the growing global awareness of sustainable water resource management, biological treatment technologies play a crucial role in the transformation and removal of pollutants in wastewater treatment, water reclamation, and resource recovery. Biofilm technology is particularly promising due to its high efficiency, capacity, and stability. Given the complex sources of wastewater, designing suitable carrier materials is essential to enhance treatment efficiency by supporting various microorganisms growth. In nitrogen-containing wastewater treatment systems, slow-growing autotrophic nitrifying bacteria or anaerobic ammonium-oxidizing bacteria require carriers to extend their retention time, facilitating nitrogen removal.

Recent studies show that the smaller the surface energy difference (γ^bv-γ^sv) between bacteria and materials, the greater the adhesion. Most commercial bio-carriers are made of HDPE, which has a large surface energy difference (11.26 mJ/m²) with nitrifying bacteria, resulting in low adhesion (< 1.3 nN) and slow biofilm growth. This study developed a new biocarrier using polyvinyl alcohol (PVA), known for its bio-affinity. Further modification on PVA reduced the surface energy difference with ammonium-oxidizing bacteria (7.69 mJ/m²) and increased adhesion (6.37 nN), making the target bacteria's adhesion 2.3 times higher than on commercial HDPE carriers.

In continuous biocarrier system tests over 50 days, the effluent of ammonia nitrogen from the treatment system with modified PVA carrier was approximately 3.6–7.8 mg/L, while the effluent of ammonia nitrogen from the treatment system with HDPE carrier system was range from 7.3 to 13.2 mg/L. HDPE carriers failed to meet Taiwan's Environmental Protection Administration's ammonia nitrogen discharge standard for public sewage systems (< 10 mg/L) within 50 days. In addition, the result of real-time PCR quantification showed that the microorganisms on the modified PVA carriers had an ammonium-oxidizing bacteria adhesion amount of 3.73*10⁸ copies/mL, over 60 times higher than those on HDPE carriers.

In summary, the PVA-modified bio-carrier developed in this study enhances the adhesion and growth of ammonium-oxidizing bacteria, leading to rapid biofilm formation, shorter start-up times, and stable water quality. This innovation has broad applicability for high-load secondary biological treatment units, significantly improving current wastewater treatment plant efficiency.

Porous biocarrier

Wastewater treatment

Nitrogen removal

Surface free energy

Chemical modification

Biofilm reactor

The majority (99%) of all microorganisms in the natural environment adhere to surfaces such as stones, suspended particles, sediment, and soil [1]. The adhesion of microorganisms to these surfaces is the first step in biofilm formation, which is critical for various environmental and industrial processes, such as wastewater bioremediation, mineral beneficiation, and microbial fuel cells [2, 3].

In general, biological wastewater treatment methods can be divided into approaches based on suspended or attached growth systems. Attached growth systems are easy to operate and resistant to shock loads but are less versatile than suspended growth systems in terms of control [4]. Many different types of biofilm reactors are used for biological wastewater treatment. Biofilm reactors have been shown to offer advantages such as enhanced biomass retention, higher resistance to shock loads, and improved treatment efficiency compared to traditional suspended growth systems [4]. Generally, biofilm reactors can be divided into fixed bed bioreactors (FBBRs) and moving bed bioreactors (MBBRs) [5].

MBBRs treat wastewater by utilizing biofilms to degrade organic pollutants and nutrients, such as nitrogen and phosphorus [5]. Effective wastewater treatment in MBBR systems is based on small biofilm support media made of various types of plastics, such as polypropylene (PP), polyethylene (PE), and high-density polyethylene (HDPE), which are used to promote the growth of dense and protected biofilms within a well-mixed reactor vessel [6]. However, these plastic biocarriers have low hydrophilicity and poor biological affinity, resulting in slow biofilm growth and susceptibility to detachment [6]. Khan et al found that different biocarrier materials result in different biofilm quantities and nitrification rates; in particular, HDPE yielded a lower attached biomass amount and ammonia removal rate than other materials [7]. Therefore, when using HDPE, a longer startup period is necessary due to the slow biofilm establishment process, and a longer operation period is needed to achieve optimal performance [7].

According to the planetary boundary framework, six of nine boundaries have already been crossed: climate change, biosphere integrity, land system change, freshwater change, biogeochemical flow, and novel entities. It is noteworthy that the changes in nitrogen cycle and biodiversity are estimated to be 100 times greater than they would be without human interference [8]. As Hernández acutely pointed out, nitrogen pollution is being increasingly recognized as a threat to biodiversity; the negative impacts from nitrogen pollution include direct nitrogen toxicity effects, eutrophication, reduced dissolved oxygen levels in water, algal blooms, and direct harm to plant species through competition or to animal species by eliminating their food sources [9].

Marine sponges are among the oldest living organisms on our planet and play a crucial role in nitrogen cycling [10, 11, 12]. Maldonado et al reported that the greatest nitrogen fluxes in sponges are related to nitrification (oxidation of ammonia to nitrite and nitrate by ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea and nitrite-oxidizing bacteria (NOB)), denitrification (conversion of nitrate to N₂ by associated bacteria or archaea), and the conversion of ammonia and nitrite to N₂ by anammox bacteria [13]. These natural systems have inspired the development of biofilm processes aimed at enhancing pollutant removal in wastewater treatment, particularly through the conversion of nitrogen compounds and organic matter.

Ammonia-oxidizing bacteria (AOB), such as Nitrosomonas europaea, play a crucial role in nitrogen removal systems because they initiate the nitrification process. This process is essential for converting toxic ammonia (NH₃) into nitrite (NO₂⁻), which is then further converted to nitrate (NO₃⁻) by nitrite-oxidizing bacteria. This bacteria is a critical step in the nitrogen cycle, enabling the removal of nitrogen from wastewater, thereby preventing environmental pollution and maintaining ecosystem balance [14].

Anammox bacteria convert ammonium (NH₄⁺) and nitrite (NO₂⁻) directly into nitrogen gas (N₂) under anoxic conditions. This bypasses the need for complete nitrification and subsequent denitrification, making the nitrogen removal process more efficient [15]. However, AOB and anammox bacteria are slow-growing microorganisms and are reported to be responsible for nitrogen removal. Growth in suspension requires long residence times; hence, biofilm can represent an effective solution for successfully retaining biomass in the reactors [16]. The initial step in bacterial adhesion involves the attraction of bacterial cells to a biotic or abiotic surface [17]. The early stages of biofilm formation are heavily influenced by the properties of the cells and substrate, such as the surface free energy (SFE), ζ potential, roughness, and chemical composition[18, 19]. Among all these influencing factors, the SFEs of microorganisms and solid surfaces provide important information for biofilm formation and adhesion in aqueous environments.

The thermodynamic adhesion energy can be calculated from the surface free energies using Neumann's equation. A negative adhesion energy leads to bacterial adhesion, whereas a positive adhesion energy does not [20]. Despite extensive research efforts, current thermodynamic models that evaluate adhesion energy often struggle to predict bacterial adhesion behavior accurately. Zhang et al discovered that bacterial adhesion is clearly mediated by the SFE difference between bacterial cells and the glass substratum. They found that the smaller the SFE difference, the higher the degree of bacterial adhesion [21]. Thus, the SFE difference was used as a fundamental factor for biocarrier design in this study.

For the biocarrier design, polyvinyl alcohol (PVA) exhibits several desirable properties, including a high content of hydroxyl groups and hydrophilicity. These characteristics endow PVA with excellent water absorption, wear resistance, weather resistance, chemical stability, and biocompatibility [22]. Our objective was to design biocarrier materials by adjusting the SFE to promote the attachment of specific microorganisms during biofilm formation in wastewater treatment. Therefore, considering the difference in SFE between bacteria and substrate, we covalently grafted hydrophobic groups onto the main chain of PVA through esterification and controlled the SFE of biocarrier. Furthermore, we investigated the adhesion of ammonia-oxidizing bacteria (Nitrosomonas europaea) to different biocarrier surfaces by atomic force microscopy (AFM). We evaluated the adhesion force, ammonia nitrogen removal performance, and attached biomass of commercial HDPE and modified PVA biocarriers in terms of the difference in SFE between the bacteria and biocarriers. For the modified PVA, we assessed the microbial community structure and nitrogen metabolic pathway flux by third-generation sequencing (TGS). In this study, we designed biocarrier materials by adjusting the SFE to promote the attachment of ammonia-oxidizing bacteria, resulting in rapid biofilm formation, shorter start-up times, and stable water quality.

2.1 Thermodynamic adhesion energy calculations

Bacterial adhesion can often be qualitatively predicted by thermodynamic principles, particularly the adhesion energy (∆F_adh) [23]. When ΔF_adh is negative, bacterial adhesion is likely to occur, while when ΔFadh is positive, bacterial adhesion is less likely to occur.

$$\varDelta {F}_{adh} = {\gamma }^{bs} - {\gamma }^{bl} -{ \gamma }^{sl}$$

where γ^bs is the bacterium-substrate interfacial tension, γ^bl is the bacterium-liquid interfacial tension, and γ^sl is the substrate-liquid interfacial tension.

The interfacial free energies (γ^bs, γ^bl, and γ^sl) can be calculated from the corresponding surface free energies (SFEs) (γ^bv, γ^sv, and γ^lv) using combining rules [24]. A value of β = 0.0001247 (m²/mJ)² is used in the Neumann equation [25].

$${\gamma }^{bs}={\gamma }^{bv}+{\gamma }^{sv}-2\sqrt{{\gamma }^{bv}{\gamma }^{sv}}{e}^{-\beta {\left({\gamma }^{bv}-{\gamma }^{sv}\right)}^{2}} \left(2\right)$$

$${\gamma }^{bl}={\gamma }^{bv}+{\gamma }^{lv}-2\sqrt{{\gamma }^{bv}{\gamma }^{lv}}{e}^{-\beta {\left({\gamma }^{bv}-{\gamma }^{lv}\right)}^{2}} \left(3\right)$$

$${\gamma }^{sl}={\gamma }^{sv}+{\gamma }^{lv}-2\sqrt{{\gamma }^{sv}{\gamma }^{lv}}{e}^{-\beta {\left({\gamma }^{sv}-{\gamma }^{lv}\right)}^{2}} \left(4\right)$$

Substituting Eqs. 2 − 4 into Eq. 1 yields

$$\varDelta {F}_{adh}=2\sqrt{{\gamma }^{bv}{\gamma }^{lv}}{e}^{-\beta {\left({\gamma }^{bv}-{\gamma }^{lv}\right)}^{2}}+2\sqrt{{\gamma }^{sv}{\gamma }^{lv}}{e}^{-\beta {\left({\gamma }^{sv}-{\gamma }^{lv}\right)}^{2}}-2\sqrt{{\gamma }^{bv}{\gamma }^{sv}}{e}^{-\beta {\left({\gamma }^{bv}-{\gamma }^{sv}\right)}^{2}}-2{\gamma }^{lv} \left(5\right)$$

According to Eq. 5, within the same liquid medium (γ^lv), if the difference in SFE between the bacterial cells (γ^bv) and the solid surface (γ^sv) decreases (i.e., |γ^bv-γ^sv| decreases), the adhesion energy between the bacterial cells and the solid surface will become more negative. Consequently, more bacterial adhesion is expected.

2.2 Biocarrier preparation

To increase the adhesion of AOB, we designed a modified PVA biocarrier based on the difference in SFE between the bacterial cells and the solid surface. Seven and a half grams of PVA (Emperor Chemical Co., Ltd., with a trade number of BP-05) and 50 grams of dimethyl sulfoxide (DMSO) were added to a reaction bottle under a nitrogen atmosphere. Next, the reaction bottle was heated to 80°C to completely dissolve the PVA in the DMSO. After reaction for 30 minutes, 56.3 grams of ε-caprolactone was added to the reaction bottle, and the reaction bottle was heated to 100°C. After reaction for 24 hours, the reaction bottle was cooled to 0°C, and the sample was subjected to reprecipitation. For reprecipitation, the sample was dissolved in 116 grams of acetone and poured into 1160 grams of methanol, and the solid was collected. After repeating this reprecipitation process twice, the obtained product was heated to 80°C under vacuum and dried for 5 hours to obtain modified PVA. The biocarrier preparation was carried out according to United States Patent US-11,795,080-B2.

2.3 Ammonia-oxidizing bacteria preparation

Nitrosomonas europaea (ATCC 19718) was obtained from the Bioresource Collection and Research Center, Hsinchu, Taiwan. The basal mineral medium contained 18.9 mM (NH₄)₂SO₄, 0.40 mM MgSO₄∙7H₂O, 0.03 mM CaCl₂∙2H₂O, 0.17 mM Fe-EDTA, 50.1 mM HEPES, 3.67 mM K₂HPO₄ in ultra-pure water and was adjusted to pH 7.8-8.0 with 5.95 mM NaHCO₃ prior to autoclaving in 120 mL serum bottles sealed with butyl rubber stoppers. Cultures were always protected from the light by shielding with black covers. The shake flask batch cultures (50 ml) of N. europaea were grown at 120 rpm and 28°C for at least 12 days.

2.4 Bacterial adhesion force measurement

A silicon-based tipless cantilever probe (TL-FM, Nanosensors) was used for force modulation for AFM (Bioscope Catalyst, Bruker Scientific Instruments). The spring constant of the cantilever is 2.8 N/m, which is suitable for nano-newton scale force measurement, and all force measurements were performed in a liquid cell. To immobilize bacteria on the AFM cantilevers, a strong positive charge modification process was applied. The AFM cantilevers were cleaned with a UV ozone cleaner (Model 342, Jelight) for 20 min and incubated with 3-APTMS (1%) in ethanol for 1 h. The 3-APTMS-coated tips were washed with ethanol/water several times and then incubated with 1% polyethylenimine in DI water (product number 408727, Sigma‒Aldrich) for 1 h, after which they were ready for N. europaea immobilization. The treated probes were then inserted into growth medium and incubated overnight at 4°C. Washing with DI water was performed before measurement. The adhesion force was determined according to the method described in United States Patent US-10,006,934-B2.

2.5 Surface free energy estimation

A contact angle measurement system (FTA 1000 B, First Ten Ångstroms) was utilized for SFE measurement. The liquids used were pure water, glycerol (Sigma‒Aldrich, product number G5516), and formamide (Sigma‒Aldrich, product number F9037). The sample was fixed on a glass slide, and a measuring liquid was dropped onto the sample. After the sample stabilized, its contact angle was measured. The Zisman critical wetting tension (Zisman plot) was used to calculate the SFE [26].

2.6 Microstructure and surface chemistry analysis

The biocarriers were scanned using a Skyscan 2211 Nano-CT imaging system at 15 µm pixel resolution (Bruker Micro-CT, Kontich, Belgium) in microfocus mode. The voltage was 100 kV, the current was 280 µA with a 5.3 W output power, and a 180-degree scan was used. Reconstructed cross-sections were reoriented, and the region of interest (ROI) was selected by using Dataviewer (Bruker Micro-CT, Kontich, Belgium). Three-dimensional image visualization was performed using CTVox (3.3.0, Bruker Micro-CT). The morphometric indices of the structure and pores were analyzed using CTAn (version 1.19.11.1, Bruker Micro-CT, Kontich, Belgium). Image reconstruction, ring artifact correction and beam-hardening correction were performed using the reconstruction software Instarecon and GPU-Nrecon.

The surface chemistry of biocarriers were analyzed through fourier transform infrared spectroscopy (FTIR) transmission spectra recorded using a PERKIN ELMER Spectrum One FTIR spectrophotometer, the resolution was 0.5 cm ^-1 and the wavenumber accuracy was ± 0.01 cm ^-1. The biocarriers were measured the wave number range of 7700 − 360 cm^-1.

2.7 Reactor setup and experimental conditions

The experiments were conducted in two lab-scale MBBRs (filled with PVA or HDPE biocarriers), with a volume of 6 liters for each reactor, which were operated in continuous mode for 50 days. To evaluate the effect of different biocarrier materials on nitrogen removal, the PVA MBBR was filled with the modified PVA biocarrier prepared as described above, whereas the HDPE MBBR was filled with Mutag BioChip^™ media (Multi Umwelttechnologie AG, Germany). To ensure that the same surface area was available for biofilm development in both reactors, the filling fractions (volume of carriers per volume of reactor, Vcarrier/Vreactor) of PVA and HDPE in the two MBBRs were set to 50% and 55.7%, respectively. Hence, the specific surface area in both reactors was 2644 m²/m³.

Both reactors were inoculated with 3 L of municipal sludge from the Taoyuan North District Water Resources Reclamation Center, Taiwan. To ensure stable substrate concentrations, laboratory-prepared wastewater was used as the influent for the MBBRs. Ammonium chloride (NH₄Cl) and sodium nitrite (NaNO₂) were used as nitrogen sources, and the synthetic influent was prepared with demineralized water and had the following composition: 76.5 ~ 172 mg/L NH₄Cl (corresponding to an influent ammonium content of 20 ~ 45 mg N/L), 143 ~ 271 mg/L NaNO₂ (influent nitrite of 29 ~ 55 mg N/L), 2.5 mg/L KH₂PO₄ (0.55 mg P/L), 30 mg/L MgSO₄*7H₂O, 18 mg/L CaCl₂*2H₂O, and 235 mg/L KHCO₃. The ammonia concentration of the simulated wastewater was approximately 20–45 mg N/L, the flow rate was 6–12 mL/min, and the inflow load of ammonia nitrogen was 0.1–0.2 kg NH₄⁺-N/m³-d. Alkalinity (supplied as KHCO₃) and phosphorus (provided as KH₂PO₄) were also dosed according to the bacterial nutrition requirements during each experimental stage. A trace element solution with a composition described previously was added at a proportion of 1 mL/L medium [27].

Air was introduced into the reactors through porous diffusers placed at the bottom of each tank. The dissolved oxygen (DO) concentration varied within the range of 0–1 mg/L. The ambient temperature was approximately 27 ± 2°C, and the pH was maintained in the range of 7.5 ~ 8.0 by adding either 1 M HCl or 1 M NaOH. The NH₄⁺, NO₂^- and NO₃^- concentrations were determined using IonPac™ CS18 IC and AS4A SC ion exchang columns in ion chromatography (Dionex, Thermo Scientific, USA). The standard process was followed according to Taiwan's National Environmental Research Academy protocols NIEA W461.50B and NIEA W415.54B. All the water samples were filtered through a 0.22 µm filter before analysis.

2.8 Real-time PCR analysis

Modified PVA biocarrier and HDPE biocarrier samples (three pieces) were taken from the laboratory-scale MBBRs for analysis. An UltraCleanTM Soil DNA Isolation Kit (Mo Bio, USA) was used to extract the DNA from the biosamples following the manufacturer’s protocol. A total of 1 µL of purified cDNA was added to 0.5 µL of forward primer, 0.5 µL of reverse primer, and 24 µL of PCR mix. The primers and reaction conditions are shown in Table 1 and Table 2 and were consistent with the standard operating method for the ABI PRIAM 7000 Sequence Detection System (Applied Biosystems, USA).

Table 1

Real-time PCR primers
Primer	Specificity		Reference
hzsA 526f	Anammox bacteria	WTYGGKTATCARTATGTAG	[28]
hzsA 1857r	Anammox bacteria	AAABGGYGAATCATARTGGC	[28]
CTO 189f A/B	16S rRNA AOB	GGAGRAAAGCAGGGGATCG	[29]
CTO 189f C	16S rRNA AOB	GGAGGAAAGTAGGGGATCG	[29]
CTO 654r	16S rRNA AOB	CTAGCYTTGTAGTTTCAAACGC	[29]

Table 2

Real-time PCR conditions
Function	Program	Number of cycles
Denaturation	96 ℃, 3 min	1
Annealing	96 ℃, 30 sec; 55 ℃, 30 sec; 72 ℃, 30 sec	40
Extension	72 ℃, 5 min	1

2.9 Microbial analysis and bioinformatic analysis

Biocarrier samples, including the initial seeding phase (PVA0) and after 50 days of operation (PVA50), were taken from the laboratory-scale MBBRs for further analysis. DNA was extracted from the biosamples using a DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol and used for shotgun metagenomics sequencing. Library preparation of the extracted genomic DNA was performed by using an Oxford Nanopore Technologies (ONT) Ligation Sequencing Kit according to the manufacturer’s protocol. The libraries were sequenced with the ONT PromethION platform using R9.4.1 flow cells. For sequence assembly, adapters were removed from the reads using Porechop v0.2.4. High-quality reads (Phred score > Q7) were selected using longQC v1.2.0b. Reads shorter than 1K bps were filtered using Filtlong v0.2.0 and assembled into metagenome-assembled genomes (MAGs) using Flye v2.8.3. The MAGs were polished twice with Racon v1.4.21 to correct simple sequencing errors. The remaining systematic errors were then removed by Medaka v1.2.5 and Homopolish v0.0.2. For taxonomic classification and quantification, the quality-controlled reads were aligned against the NCBI RefSeq database using minimap2 and mash dist to select candidate references and were then assigned to taxa using the average nucleotide identity (ANI). The microbial proportion, which represents the relative abundance of each species in the microbial community (with dominant and sub-dominant microbial groups defined by relative abundances of > 1% and 0.01%-1%, respectively), was calculated based on the read count and corrected by the genome size of each species [30]. The analysis of the microbial community on the biocarrier focused on changes in these dominant groups.

2.10 Nitrogen metabolic pathway flux analysis

The nitrogen metabolic pathway flux was determined to assess the relative abundance of microorganisms associated with various nitrogen metabolic pathways in the environment. In this study, the Kegg Orthology (KO) numbers of the MAGs were obtained and compared to the nitrogen metabolism KO numbers in the Kyoto Encyclopedia of Genes and Genomes (KEGG) for identification. The relative abundance of a MAG was considered in subsequent calculations when the MAG contained at least one copy of a given pathway gene. The gene statistics and data visualization were performed using the Python (v3.10.5) environment. Duplicates were removed and relative abundances summed using pandas (v1.5.0) and numpy (v1.23.0). The nitrogen metabolic pathway flux was visualized with matplotlib (v3.6.2), utilizing the matplotlib.pyplot, matplotlib.patches, and ScalarMappable modules.

3.1 Estimation of the SFE of bacteria (γ^bv) from the contact angle

The aim of this study is to design and evaluate biocarrier materials that enhance the attachment and growth of specific microorganisms, particularly ammonia-oxidizing bacteria, in wastewater treatment systems. The SFE of bacteria is one of the critical factors governing the mechanism of bacterial adhesion. According to Daffonchio [25], bacteria are considered hydrophobic when they have a contact angle greater than 45° and hydrophilic when they have a contact angle less than 45°. Previously published data indicated that many aerobes and facultative anaerobes are hydrophilic (contact angle less than 45°), Bacillus cereus, Lactobacillus strains, Staphylococcus strains exhibited low contact angles [21, 23]. On the other hand, the acetogenic strain and methanogenic strains exhibited high contact angles [31].

It was observed in previous studies that acetogens and methanogens appear to be more hydrophobic than acidogens and aerobes. Aerobes and facultative anaerobes are involved in the initial breakdown of oxygen and organic matter. Acetogens and methanogens then act as intermediate and ultimate degraders, respectively, further metabolizing the products of earlier degradation stages and ultimately producing simpler end products such as acetate and methane.

In the surface thermodynamics approach [23], steric and electrostatic forces are neglected, and γ^bv is used to distinguish between hydrophilic and hydrophobic cells. Therefore, Fig. 1 presents data compiled from multiple studies [20, 21, 23, 25, 31], showing the relationship between contact angles and γ^bv. The relationship (a linear relationship with an R² value of 0.9985) between the SFE and contact angles with different bacterial strains, the smaller contact angles, the higher SFE of bacterial strains. This relationship can then be used to estimate γ^bv from the contact angle of a given bacterial strain. A previous study showed that the contact angle of N. europaea was 56 ± 3 degrees [32], and the SFE of N. europaea was estimated to be 48.26 mJ/m².

This study aims to design biocarrier materials that improve the attachment and growth of specific microorganisms. By adjusting the SFE of the biocarriers to match the SFE of the target bacteria (N. europaea), this study ensures that the biocarriers have properties conducive to rapid biofilm formation and stable wastewater treatment performance.

3.2 Designing Biocarriers for Nitrogen Removal Bacteria

According to Eq. 1, the adhesion energy can be correlated with the difference in SFE (|γ^bv − γ^sv|) between bacterial cells and a solid substrate. Zhang found that the smaller the SFE difference, the higher bacterial adhesion [21]. Nakanishi [33] reported that hydrophobic bacteria, or bacteria with a low SFE, preferentially attached to hydrophobic surfaces, or surfaces with a low SFE. However, bacteria with hydrophilic properties (high SFE) preferentially adhered to hydrophilic surfaces, or surfaces with a high SFE. Thus, the absolute SFE difference can serve as a quantitative indicator for the design of biocarriers.

In this study, we focused on nitrogen removal bacteria, such as AOB and anammox bacteria. Therefore, to promote AOB attachment, we designed materials with a small difference in SFE (the SFE of N. europaea is 48.26 mJ/m²).

According to the results of surface free energy estimation, the SFE of the unmodified PVA biocarrier was 58.07 mJ/m². Therefore, the difference in SFE between N. europaea and the unmodified PVA biocarrier was 9.83 mJ/m². To increase AOB attachment, this SFE difference was decreased by covalently grafting hydrophobic groups to the main chain of PVA by esterification. FTIR analysis of functional groups revealed that the peak corresponding to the OH region (3273.34 cm^− 1) of PVA disappeared after the esterification reaction (Fig. 2). There was a strong C = O peak at 1720 cm^− 1, corresponding to the formation of ester bonds, and the peak at 1639 cm^− 1 was attributed to the asymmetric stretching vibration of C = O bonds in the poly(ε-caprolactone) (PCL) side chain; these results indicate that the hydrophobic segment was successfully grafted to PVA.

The difference and adhesion force in SFE between N. europaea and the modified PVA biocarrier were 7.69 mJ/m² and 6.37 nN, respectively. On the other hand, the difference and adhesion force in SFE between N. europaea and the HDPE biocarrier were 11.26 mJ/m² and 1.29 nN, respectively (Table 3). By modifying N. europaea on the AFM probe, the adhesion force of N. europaea on various biocarriers can be measured. The greater the adhesion force, the more difficult it is to detach from the biocarrier. The difference in the SFE of modified PVA (relative to the SFE of N. europaea) was considerably smaller than those of unmodified PVA and HDPE, and the adhesion force of N. europaea to modified PVA was four times greater than those to unmodified PVA and HDPE. This result indicates that a smaller SFE difference between N. europaea and the biocarrier material is associated with an increased microbial adhesion force. This observation suggests that microorganisms have difficulty detaching from the modified PVA surface, achieving secure positioning and reducing the probability of detachment.

Table 3

Different biocarrier material of bacterial adhesion force and the SFE difference between *N. europaea*
	PVA	HDPE	Modified PVA
Biocarrier material surface energy (mJ/m²)	58.07	37.0	40.57
N. europaea SFE (mJ/m²)	48.26	48.26	48.26
SFE difference between N. europaea and the biocarrier material (mJ/m²)	9.83	11.26	7.69
Adhesion force (nN)	1.31	1.29	6.37

3.3 Biocarrier and reactor performance

3.3.1 Biocarrier physical characteristics

The pore size, porosity, and specific surface area of the biocarriers were analyzed by micro-CT, and the associated characteristics of the biocarriers are listed in Table 4. The average pore sizes of the modified PVA and HDPE biocarriers were 302 and 427 µm, respectively. On the other hand, the porosities of the two biocarriers were 34.4% and 47.2%, respectively. The modified PVA and HDPE biocarriers had specific surface areas of 5288 and 4743 m²/m³, respectively. To ensure that the same surface area (2644 m²/m³) was available for biofilm development in both reactors, the filling fractions of PVA and HDPE in the two MBBRs were set to 50% and 55.7%, respectively.

3.3.2 Ammonium removal

The two reactors with different biocarriers (modified PVA and commercial HDPE) were operated simultaneously. As shown in Fig. 3, the removal rate of ammonia in the PVA system was approximately 75–95%, and the concentration of ammonia nitrogen in the effluent was 3.6–7.8 mg/L, which met the Taiwan's Environmental Protection Administration's ammonia nitrogen discharge standard (< 10 mg/L). In contrast, the effluent quality of the HDPE system was unstable, and the ammonia concentration increased over time. The concentration of ammonia nitrogen in the effluent was 7.3–13.2 mg/L, which fails to fully comply with the Taiwan's Environmental Protection Administration's ammonia nitrogen discharge requirement (< 10 mg/L).

3.3.3 Bacterial abundance

To quantify the abundances of AOB and anammox bacteria on the HDPE and modified PVA biocarriers, we analyzed samples (collected after 30 days of operation) with real-time PCR (Table 5). For the modified PVA biocarrier the copy numbers of AOB and anammox bacteria genes were 3.73∙10⁸ copies/mL and 1.39∙10⁶ copies/mL, respectively. However, for the HDPE biocarrier, the copy number of AOB genes was 5.54∙10⁶ copies/mL, but the copy number of anammox bacteria genes was below the detection limit (2.88∙10⁵ copies/mL). The copy numbers of AOB and anammox bacteria genes were more than 10 times greater for the modified PVA biocarrier than the HDPE biocarrier. These results indicated that AOB and anammox bacteria can attach and grow more rapidly on the surface of modified PVA than HDPE.

Table 4

Characteristics of the modified PVA and HDPE biocarriers
	Modified PVA	HDPE
Shape	Round	Round
Diameter (mm)	22	30
Thickness (mm)	0.9–1.3	0.8–1.2
Pore size (µm)	302	427
Porosity (%)	34.4	47.2
Specific surface area (m²/m³)	5288	4743

Table 5

Abundances of AOB and anammox bacteria on the different biocarriers
	Modified PVA	HDPE
Copy number of AOB genes (copies/mL)	3.73∙10⁸	5.54∙10⁶
Copy number of anammox bacteria genes (copies/mL)	1.39∙10⁶	N.D.*
*Below the limit of detection (2.88∙10⁵ copies/mL)

3.4 Evolution of the microbial community

To further understand the process of microbial development, metagenomics was utilized to analyze the evolution of the microbial community on the modified PVA biocarrier (Fig. 4). Sequencing of samples from the initial seeding phase (PVA0) revealed the dominant microbial taxa (the relative abundance ≧ 1%) at the genus level, included Longilinea (1.4%), Levilinea (1.3%), Caldilinea (1.2%), Bellilinea (1.2%), and Anaerolinea (1.1%). In the PVA50 samples, the dominant microbial taxa at the genus level included Longilinea (1.9%), Anaerolinea (1.8%), Nitrospira (1.4%), Ca. Brocadia (1.3%), Levilinea (1.3%), and Nitrosomonas (1.2%). Longilinea, Anaerolinea, and Levilinea were identified as common dominant genera in both PVA0 and PVA50. All three genera belong to the class Anaerolineae, members of which have a filamentous structure and play a crucial role in promoting cell aggregation and maintaining the compactness of sludge. Moreover, these three microbial groups have been recognized for their significant contributions to anammox granulation [28–31]. Notably, the relative abundance of Longilinea and Anaerolinea, which contribute to anammox granulation, increased in PVA50 compared to PVA0. This observation suggests that the modified PVA biocarrier supports the growth of anammox co-cultivating bacteria, promoting the growth of anammox community.

The results of the microbial structure analysis showed that ammonia-oxidizing microorganisms (AOM), nitrite-oxidizing bacteria (NOB) and anammox bacteria were dominant in PVA50. Among the nitrifying microbial community, AOM such as Nitrosomonas, Nitrosospira, Ca. Nitrosotenuis, Nitrosopumilus, and Nitrosovibrio were identified. Nitrosomonas europaea emerged as the dominant species, exhibiting a substantial increase in relative abundance from 0.01–1.2% from PVA0 to PVA50. This result indicates that the modified PVA biocarrier has a high affinity for Nitrosomonas. The NOB community comprised Nitrospira, Nitrospina, Nitrobacter, Nitrolancea, and Nitrospirillum. Notably, Ca. Nitrospira inopinata, Ca. Nitrospira nitricans, and Ca. Nitrospira nitrosa, which belong to Nitrospira and are capable of complete ammonia oxidation (comammox) [32, 33], were observed, and the relative abundance of Ca. Nitrospira inopinata increased from 0.08–0.12% after 50 days of operation. The results indicate that the modified PVA enhances the redundancy of nitrifying communities, which helps maintain the nitrogen metabolism function of the system.

Known anammox bacteria are primarily classified into five genera: Kuenenia, Brocadia, Anammoxoglobus, Jettenia, and Scalindua. The relative abundance of the anammox community increased from 0.29–1.26% from day 0 to day 50 of reactor operation. To assess the affinity of the modified PVA for different anammox bacteria, we compared the changes in their relative abundances.

The of Ca. Brocadia sinica, Ca. Brocadia sapporoensis, and Ca. Kuenenia stuttgartiensis increased from 0.27–0.93%, 0.001–0.23%, and 0.03–0.09%, respectively. This phenomenon implies that the newly developed carrier exhibits a greater affinity for Ca. Brocadia than Ca. Kuenenia.

3.5 Nitrogen metabolic pathway flux

The metagenomics data were compared with the KO numbers of nitrogen metabolism pathways in the KEGG database to reveal the potential nitrogen metabolism functions in the microbial community. The nitrogen metabolism KO numbers were extracted from the metagenomics data of samples PVA0 and PVA50 and combined with the proportions of MAGs to visualize the nitrogen metabolic pathway flux.

Nitrogen metabolic pathway flux analysis revealed that the dissimilatory nitrate reduction to ammonium (DNRA) pathway, involving nitrite reduction to ammonia, was the predominant pathway in both the PVA0 and PVA50 samples. The relative abundance of this pathway was 13.1% in PVA0 and 11.6% in PVA50 (Fig. 5 and Fig. 6). Upon comparing the abundance of the biological community between PVA0 and PVA50, it was noted that the abundances of microorganisms associated with nitrite reductase, Nor, Nxr, nitrate reductase, and Nir decreased. Conversely, the abundances of microorganisms related to Hao, Nos, Hzs, Hzo, and Amo increased (Fig. 7).

Wastewater treatment relies on two main pathways for nitrogen removal: traditional denitrification, facilitated by the Nor gene pathway, and the anammox process, driven by the Hzo gene [35, 36, 37, 38]. The initial relative abundances of microorganisms with the NorBC gene and Hzo gene were 9.8% and 1.2%, respectively (Fig. 5). This observation showed that denitrification predominantly occurred through the conventional nitrification-denitrification pathway in the initial feed sludge. After the system was operated for 50 days, the proportions of microorganisms with the NorBC gene and Hzo gene changed to 5.0% and 3.1%, respectively (Fig. 6). This change in proportions revealed that the nitrogen removal pathway might transform from traditional denitrification at the start of an operational period to partial nitrification and annamox reactions as time passes.

During 50 days of reactor operation, the population of microorganisms carrying the Amo gene, known as AOM, increased in abundance from 0.35–0.65%. Additionally, the relative abundance of microorganisms with the Hao gene that is associated with hydroxylamine to nitrite reaction, increased from 4.1–7.3%. However, the relative abundance of NOB, which carry the Nxr gene associated with the nitrite oxidation reaction, decreased from 8.9–7.1%. These changes indicated that in the PVA system, partial nitrification became dominant after 50 days of operation. In summary, carrier modification enables the selection of anammox and AOB microbial communities, thereby enhancing the occurrence of partial nitrification and anammox processes.

This study introduces a novel method for designing biocarriers based on thermodynamic considerations, representing a significant advancement in the field. Our findings demonstrate that the difference in surface free energy (SFE) between bacteria and substrates serves as a simple and effective criterion for predicting bacterial adhesion on both hydrophobic and hydrophilic surfaces, and this offers a clear, quantifiable approach to optimizing biocarrier materials. The modified PVA biocarrier showed substantially higher abundances of ammonia-oxidizing bacteria (AOB) and anammox bacteria in the attached biofilm compared to the HDPE biocarrier. This breakthrough suggests that custom-designed biocarriers can selectively enhance the adhesion of specific microorganisms, significantly improving the degradation of particular pollutants and thereby greatly enhancing wastewater treatment efficiency. The nitrogen removal performance of the modified PVA bioreactor was notably superior to that of the HDPE bioreactor. The PVA system achieved an ammonia removal rate of approximately 75–95%, with effluent ammonia nitrogen concentrations of 3.6–7.8 mg/L, consistently meeting Taiwan's Environmental Protection Administration's discharge standards. In contrast, the HDPE system showed unstable performance, with effluent ammonia nitrogen concentrations ranging from 7.3 to 13.2 mg/L, failing to meet the required standards consistently. From an engineering perspective, these innovations hold significant potential for advancing wastewater treatment technologies and contributing to environmental sustainability. Custom-designed biocarriers, based on the principles established in this study, can lead to more efficient and effective treatment solutions, ultimately supporting the goal of sustainable water resource management.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Availability of data and materials

All data generated during this study are available from the corresponding author ([email protected]) upon request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by the Semiconductor Low-carbon Emission Manufacturing Process and Electronic Material Innovation Technology (113-EC-17-A-27-1903/2024), the Ministry of Economic Affairs and the National Science and Technology Council, Taiwan (109-2221-E-007-058, 110-2221-E-007-054 and 110-2731-M-007-001).

Authors' contributions

All the authors conceptualized the work. TTC designed the experiments, analyzed the data, and conceptualized and wrote the manuscript. YHL analyzed the data and designed and performed the experiments. YJJ performed the microbial community and nitrogen metabolic pathway flux analyses. SYC, WKC, KFC, WYS and PYC supervised the work. All the authors have read, revised, and approved the final manuscript.

Acknowledgments

The authors wish to thank the Ministry of Economic Affairs and the National Science and Technology Council for their support within the scope of 113-EC-17-A-27-1903/2024, MOST 109-2221-E-007-058, 110-2221-E-007-054 and 110-2731-M-007-001.

Sperling M von. Basic principles of wastewater treatment. vol. 45. 2008. https://doi.org/10.5860/choice.45-2632.
Dunne WM. Bacterial adhesion: Seen any good biofilms lately? Clin Microbiol Rev 2002;15:155–66. https://doi.org/10.1128/CMR.15.2.155-166.2002.
Logan BE, Regan JM. Fuel Cells Fuel cells. Fuel Cells Technol Fuel Process 2006:1–32.
Musa MA, Idrus S. Physical and biological treatment technologies of slaughterhouse wastewater: A review. Sustain 2021;13:1–20. https://doi.org/10.3390/su13094656.
Choi HJ, Lee AH, Lee SM. Comparison between a moving bed bioreactor and a fixed bed bioreactor for biological phosphate removal and denitrification. Water Sci Technol 2012;65:1834–8. https://doi.org/10.2166/wst.2012.847.
Chen S, Cheng X, Zhang X, Sun D. Influence of surface modification of polyethylene biocarriers on biofilm properties and wastewater treatment efficiency in moving-bed biofilm reactors. Water Sci Technol 2012;65:1021–6. https://doi.org/10.2166/wst.2012.915.
Khan MMT, Chapman T, Cochran K, Schuler AJ. Attachment surface energy effects on nitrification and estrogen removal rates by biofilms for improved wastewater treatment. Water Res 2013;47:2190–8. https://doi.org/10.1016/j.watres.2013.01.036.
Rockström J, Steffen W, Noone K, Persson Å, Stuart Chapin, F. Lambin E. “A Safe Operating Space for Humanity” (2009). Futur Nat 2023;461:491–505. https://doi.org/10.2307/j.ctt5vm5bn.53.
Hernández DL, Vallano DM, Zavaleta ES, Tzankova Z, Pasari JR, Weiss S, et al. Nitrogen Pollution Is Linked to US Listed Species Declines. Bioscience 2016;66:213–22. https://doi.org/10.1093/biosci/biw003.
Brain CKB, Prave AR, Hoffmann KH, Fallick AE, Botha A, Herd DA, et al. The first animals: Ca. 760-million-year-old sponge-like fossils from Namibia. S Afr J Sci 2012;108:1–8. https://doi.org/10.4102/sajs.v108i1/2.658.
Hoffmann F, Radax R, Woebken D, Holtappels M, Lavik G, Rapp HT, et al. Complex nitrogen cycling in the sponge Geodia barretti. Environ Microbiol 2009;11:2228–43. https://doi.org/10.1111/j.1462-2920.2009.01944.x.
Martins E, Rapp HT, Xavier JR, Diogo GS, Reis RL, Silva TH. Macro and Microstructural Characteristics of North Atlantic Deep-Sea Sponges as Bioinspired Models for Tissue Engineering Scaffolding. Front Mar Sci 2021;7:1–18. https://doi.org/10.3389/fmars.2020.613647.
Maldonado M, Ribes M, van Duy lFC. Nutrient Fluxes Through Sponges. Biology, Budgets, and Ecological Implications. vol. 62. 2012. https://doi.org/10.1016/B978-0-12-394283-8.00003-5.
Shrestha NK, Hadano S, Kamachi T, Okura I. Dinitrogen production from ammonia by Nitrosomonas europaea. Appl Catal A Gen 2002;237:33–9. https://doi.org/10.1016/S0926-860X(02)00279-X.
Kartal B, Maalcke WJ, De Almeida NM, Cirpus I, Gloerich J, Geerts W, et al. Molecular mechanism of anaerobic ammonium oxidation. Nature 2011;479:127–30. https://doi.org/10.1038/nature10453.
Chaali M, Naghdi M, Brar SK, Avalos-Ramirez A. A review on the advances in nitrifying biofilm reactors and their removal rates in wastewater treatment. J Chem Technol Biotechnol 2018;93:3113–24. https://doi.org/10.1002/jctb.5692.
Katsikogianni M, Missirlis YF, Harris L, Douglas J. Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimating bacteria-material interactions. Eur Cells Mater 2004;8:37–57. https://doi.org/10.22203/eCM.v008a05.
Teughels W, VanAssche N, Sliepen I, Quirynen M. Effect of material characteristics and/or surface topography on biofilm development. Clin Oral Implants Res 2006;17:68–81. https://doi.org/10.1111/j.1600-0501.2006.01353.x.
Briandet R, Meylheuc T, Maher C, Bellon-Fontaine MN. Listeria monocytogenes Scott A: Cell surface charge, hydrophobicity, and electron donor and acceptor characteristics under different environmental growth conditions. Appl Environ Microbiol 1999;65:5328–33. https://doi.org/10.1128/aem.65.12.5328-5333.1999.
Busscher HJ, Weerkamp AH, van Der Mei HC, van Pelt AW, de Jong HP, Arends J. Measurement of the surface free energy of bacterial cell surfaces and its relevance for adhesion. Appl Environ Microbiol 1984;48:980–3. https://doi.org/10.1128/aem.48.5.980-983.1984.
Zhang X, Zhang Q, Yan T, Jiang Z, Zhang X, Zuo YY. Quantitatively predicting bacterial adhesion using surface free energy determined with a spectrophotometric method. Environ Sci Technol 2015;49:6164–71. https://doi.org/10.1021/es5050425.
Kamarul T, Krishnamurithy G, Salih ND, Ibrahim NS, Raghavendran HRB, Suhaeb AR, et al. Biocompatibility and Toxicity of Poly(vinyl alcohol)/N,O-Carboxymethyl Chitosan Scaffold. Sci World J 2014;2014. https://doi.org/10.1155/2014/905103.
Absolom DR, Lamberti FV., Policova Z, Zingg W, van Oss CJ, Neumann AW. Surface thermodynamics of bacterial adhesion. Appl Environ Microbiol 1983;46:90–7. https://doi.org/10.1128/aem.46.1.90-97.1983.
Sharma PK, Hanumantha Rao K. Analysis of different approaches for evaluation of surface energy of microbial cells by contact angle goniometry. Adv Colloid Interface Sci 2002;98:341–463. https://doi.org/10.1016/S0001-8686(02)00004-0.
Daffonchio D, Thaveesri J, Verstraete W. Contact angle measurement and cell hydrophobicity of granular sludge from upflow anaerobic sludge bed reactors. Appl Environ Microbiol 1995;61:3676–80. https://doi.org/10.1128/aem.61.10.3676-3680.1995.
Zisman WA. Relation of the Equilibrium Contact Angle to Liquid and Solid Constitution 1964:1–51. https://doi.org/10.1021/ba-1964-0043.ch001.
Van De Graaf AA, De Bruijn P, Robertson LA, Jetten M S M, Gijs Kuenen J. Autotrophic growth of anaerobic ammonium-oxidizing microorganisms in a fluidized bed reactor. Microbiology 1996;142:187–9.
Harhangi HR, Le Roy M, van Alen T, Hu B lan, Groen J, Kartal B, et al. Hydrazine synthase, a unique phylomarker with which to study the presence and biodiversity of anammox bacteria. Appl Environ Microbiol 2012;78:752–8. https://doi.org/10.1128/AEM.07113-11.
Kowalchuk GA, Stephen JR, De Boer W, Prosser JI, Embley TM, Woldendorp JW. Analysis of ammonia-oxidizing bacteria of the β subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments. Appl Environ Microbiol 1997;63:1489–97. https://doi.org/10.1128/aem.63.4.1489-1497.1997.
Guo F, Zhang T. Profiling bulking and foaming bacteria in activated sludge by high throughput sequencing. Water Res 2012;46:2772–82. https://doi.org/10.1016/j.watres.2012.02.039.
Grotenhuis JTC, Plugge CM, Stams AJM, Zehnder AJB. Hydrophobicities and electrophoretic mobilities of anaerobic bacterial isolates from methanogenic granular sludge. Appl Environ Microbiol 1992;58:1054–6. https://doi.org/10.1128/aem.58.3.1054-1056.1992.
Khan MMT, Ista LK, Lopez GP, Schuler AJ. Experimental and theoretical examination of surface energy and adhesion of nitrifying and heterotrophic bacteria using self-assembled monolayers. Environ Sci Technol 2011;45:1055–60. https://doi.org/10.1021/es101389u.
Nakanishi EY, Palacios JH, Godbout S, Fournel S. Interaction between biofilm formation, surface material and cleanability considering different materials used in pig facilities—an overview. Sustain 2021;13. https://doi.org/10.3390/su13115836.
Liu Y, Niu Q, Wang S, Ji J, Zhang Y, Yang M, et al. Upgrading of the symbiosis of Nitrosomanas and anammox bacteria in a novel single-stage partial nitritation–anammox system: Nitrogen removal potential and Microbial characterization. Bioresour Technol 2017;244:463–72. https://doi.org/10.1016/j.biortech.2017.07.156.
Ribeiro H, Wijaya IMW, Soares-Santos V, Soedjono ES, Slamet A, Teixeira C, et al. Microbial community composition, dynamics, and biogeochemistry during the start-up of a partial nitritation-anammox pathway in an upflow reactor. Sustain Environ Res 2022;32:1–11. https://doi.org/10.1186/s42834-022-00130-1.
Daims H, Lebedeva EV., Pjevac P, Han P, Herbold C, Albertsen M, et al. Complete nitrification by Nitrospira bacteria. Nature 2015;528:504–9. https://doi.org/10.1038/nature16461.
Van Kessel MAHJ, Speth DR, Albertsen M, Nielsen PH, Op Den Camp HJM, Kartal B, et al. Complete nitrification by a single microorganism. Nature 2015;528:555–9. https://doi.org/10.1038/nature16459.
Zumft WG. Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 1997;61:533–616. https://doi.org/10.1128/mmbr.61.4.533-616.1997.

Download PDF

Reviewers agreed at journal
30 Jul, 2024
Reviewers invited by journal
30 Jul, 2024
Editor assigned by journal
25 Jun, 2024
First submitted to journal
21 Jun, 2024

You are reading this latest preprint version

Biocarrier selection for the attachment of specific microorganisms in wastewater treatment

Status:

Version 1

Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Thermodynamic adhesion energy calculations

2.2 Biocarrier preparation

2.3 Ammonia-oxidizing bacteria preparation

2.4 Bacterial adhesion force measurement

2.5 Surface free energy estimation

2.6 Microstructure and surface chemistry analysis

2.7 Reactor setup and experimental conditions

2.8 Real-time PCR analysis

2.9 Microbial analysis and bioinformatic analysis

2.10 Nitrogen metabolic pathway flux analysis

3 Results and Discussion

3.1 Estimation of the SFE of bacteria (γ^bv) from the contact angle

3.2 Designing Biocarriers for Nitrogen Removal Bacteria

3.3 Biocarrier and reactor performance

3.3.1 Biocarrier physical characteristics

3.3.2 Ammonium removal

3.3.3 Bacterial abundance

3.4 Evolution of the microbial community

3.5 Nitrogen metabolic pathway flux

4 Conclusions

Declarations

References

Status:

Version 1

Biocarrier selection for the attachment of specific microorganisms in wastewater treatment

Status:

Version 1

Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Thermodynamic adhesion energy calculations

2.2 Biocarrier preparation

2.3 Ammonia-oxidizing bacteria preparation

2.4 Bacterial adhesion force measurement

2.5 Surface free energy estimation

2.6 Microstructure and surface chemistry analysis

2.7 Reactor setup and experimental conditions

2.8 Real-time PCR analysis

2.9 Microbial analysis and bioinformatic analysis

2.10 Nitrogen metabolic pathway flux analysis

3 Results and Discussion

3.1 Estimation of the SFE of bacteria (γbv) from the contact angle

3.2 Designing Biocarriers for Nitrogen Removal Bacteria

3.3 Biocarrier and reactor performance

3.3.1 Biocarrier physical characteristics

3.3.2 Ammonium removal

3.3.3 Bacterial abundance

3.4 Evolution of the microbial community

3.5 Nitrogen metabolic pathway flux

4 Conclusions

Declarations

References

Status:

Version 1

3.1 Estimation of the SFE of bacteria (γ^bv) from the contact angle