Effect of light shielding on the photoinhibition of nitrifying bacteria immobilized on a light-shielding hydrogel Open Access

Wastewater generated from human activities contains significant amounts of nitrogen, often in the form of ammonia. Ammonia-rich wastewater is generally converted to nitrogen gas through a combination of nitrification (ammonia oxidation) and denitrification (nitrite reduction). In particular, nitrification requires a significant amount of energy for aeration; thus, there is a need to develop alternative energy-saving methods (Chai et al. 2019). One method involves the coexistence of microalgae and nitrifying bacteria in a single reactor that does not require aeration (Gonçalves et al. 2017; Jiang et al. 2021). Because the oxygen supplied by the blower can be replaced by the oxygen supplied by the photosynthesis of microalgae, no aeration is required. However, a significant challenge in harnessing their full potential is the sensitivity of nitrifying bacteria to photoinhibition, especially under the high-intensity light conditions commonly found in outdoor environments (Merbt et al. 2012; Kang et al. 2018a, b). Lu et al. (2023) reported that ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) are not inhibited by light at intensities within the range of 0–60 μmol photons m⁻² s⁻¹. However, NOB are inhibited at light intensities between 60 and 200  μmol photons m⁻² s⁻¹, and both bacteria experience inhibition at intensities exceeding 200 μmol photons m⁻² s⁻¹ (Lu et al. 2023). To mitigate the photoinhibition of these nitrifying bacteria, Akizuki et al. (2019) used granular sludge with an average particle size of 300 μm to maintain light resistance even under solar irradiation conditions (Akizuki et al. 2019).

Our recent work demonstrated that the use of light-shielding hydrogels, specifically hydrogels incorporating carbon black (CB) and nitrifying bacteria, can effectively mitigate the photoinhibition of nitrifying bacteria and maintain nitrification efficiency even under intense light exposure (Nishi et al. 2020). Despite these advancements, a gap in research remains evident. Previous procedures for light-shielding hydrogel preparation have been largely empirical, meaning the concentration of CB was adjusted to achieve a ‘visually black’ appearance without a detailed investigation of the optimal concentration for effective light shielding. Although this approach is practical, a systematic evaluation to determine the most effective CB concentration to balance optimal light shielding and other functional properties, such as mass transfer, has not yet been conducted. For instance, increasing the hydrogel size to enhance light-shielding performance can restrict mass transfer (Benyahia & Polomarkaki 2005). Because of the trade-off between hydrogel size and mass transfer, the concentration of CB should be considered. Furthermore, although the light transmittance characteristics of composite hydrogels, particularly those containing alginate, have been extensively studied (Yue et al. 2019), there is a dearth of research focusing on the enhancement of the light-shielding properties of poly(vinyl alcohol) (PVA) and sodium alginate (SA) composite hydrogels, especially those containing CB. This presents an opportunity to explore new composite materials that can offer superior light-shielding performance compared with previous procedures.

In this study, we aimed to clarify the effect of light shielding on the photoinhibition of nitrifying bacteria immobilized on a light-shielding hydrogel. Because a spherical hydrogel complicates the evaluation of light transmission in the hydrogel, we prepared a ‘sheet-type light-shielding hydrogel’ for this purpose. This sheet allowed for a more accurate evaluation of the light-shielding properties while maintaining the original composition of the hydrogel under the same preparation conditions. In this study, we conducted a comparative analysis of the light-shielding effect of this sheet-type hydrogel on encapsulated nitrifying bacteria against the results of an actual nitrification experiment. Through this approach, we not only aimed to quantify the light-shielding performance but also to confirm the relationship between theoretical predictions of nitrification and actual observations, thereby contributing to a broader understanding of light-shielding mechanisms in nitrifying bacterial immobilization systems. This study is expected to provide vital insights into the optimization of preparation conditions for light-shielding hydrogels, paving the way for their enhanced application in outdoor wastewater treatment.

Because measuring the transmitted light intensity inside a spherical light-shielding hydrogel of approximately 5 mm is difficult, we attempted to prepare sheet-type light-shielding hydrogels of different thicknesses. Nitrifying bacteria obtained from an aerobic sludge tank at the Hokubu Sludge Treatment Center in Kanagawa, Japan were used for nitrification. The immobilizing materials, such as PVA (FUJIFILM Wako Pure Chemical Co., Ltd) and SA (FUJIFILM Wako Pure Chemical Co., Ltd), were dissolved in distilled water at 15 and 2 wt%, respectively, by autoclaving (121 °C, 20 min). The PVA and SA concentrations were customized following the method proposed by Zhang et al. (2007) and Van & Bach (2014). The nitrifying bacteria were concentrated in a centrifuge to 30 g-SS (suspended solids) L⁻¹. The concentrated nitrifying bacteria and polymer solution were mixed in equal volumes (1:1 volume ratio), and CB (Yoneyama Yakuhin Kogyo Co., Ltd) powder, a light-shielding material, was added to the mixture at 0.1 or 0.5 wt%. A sample without CB was prepared using the same procedure. The prepared solution was poured into a plastic petri dish (⌀55 × 17 mm) to a height of 1.0–5.0 mm and frozen at −20 °C. Each resulting sample was thawed at 25 °C and immersed in a solution of 2 wt% calcium chloride and saturated boric acid for 1 h, followed by 2 h in a 0.5 M sodium sulfate solution. Finally, the samples were washed in distilled water for 24 h and refrigerated until light irradiation. The prepared hydrogel sheet samples without nitrifying bacteria and CB were named ‘PVA–SA,’ those with only nitrifying bacteria were named ‘CB0,’ and those with both nitrifying bacteria and CB powder with 0.1 and 0.5 wt% were named ‘CB0.1’ or ‘CB0.5,’ as shown in Table 1. The final concentration of nitrifying bacteria in the prepared hydrogel sheets is 15 g-SS L⁻¹.

Table 1

Sample names and its contents

Sample name .	PVA–SA .	C B0 .	C B0.1 .	C B0.5 .
Bacteria
CB			0.1 wt%	0.5 wt%

Sample name .	PVA–SA .	C B0 .	C B0.1 .	C B0.5 .
Bacteria
CB			0.1 wt%	0.5 wt%

View Large

Light-shielding hydrogel containing 0.5 wt% CB was prepared using the same materials and concentrations as the hydrogel sheets prepared in Section 2.1 using the following procedure. First, the nitrifying bacteria were concentrated in a centrifuge to 30 g-SS L⁻¹. For a polymer solution, PVA and SA were dissolved at 15 and 2.0 wt%, respectively, in distilled water by using an autoclave (121 °C, 20 min). The mixture was prepared by adding an equivalent amount of concentrated nitrifying sludge to the polymer solution. A light-shielding hydrogel was formed immediately after the mixture was dripped into a 2 wt% calcium chloride and saturated boric acid solution through a nozzle with an inner diameter of 2 mm. The sphere light-shielding hydrogel beads were continuously crosslinked for 1 h. The crosslinking solution was replaced with a 0.5 M sodium sulfate solution and kept for 2 h. The prepared light-shielding hydrogels were stored in distilled water until nitrification. Thus, the prepared sphere light-shielding hydrogel was composed of a polymer concentration of 7.5 wt% PVA, 1.0 wt% SA, 15 g-SS L⁻¹ nitrifying sludge, and 0.5 wt% CB concentration. Nitrifying bacteria were used within 10 days of collection from the aerobic sludge tank in the wastewater treatment plant. They were immobilized in hydrogels and cultured by artificial wastewater until use.

Batch nitrification experiments were conducted using the prepared spherical light-shielding hydrogel. To completely seal the reactor, serum bottles with effective volumes of 100 mL were used in this experiment. We used 15 g-SS L⁻¹ of light-shielding hydrogel to achieve a nitrifying bacteria concentration of 0.5 g-SS L⁻¹ in each reactor. The initial NH₄⁺-N concentration in the synthetic wastewater was 50 mg-N L⁻¹. By using a white LED light irradiation device, the nitrification experiment was conducted under the different light intensity conditions of 0, 100, 450, and 1,600 μmol photons m⁻² s⁻¹. Light intensity in the reactor was adjusted to 100, 450, and 1,600 μmol photons m⁻² s⁻¹ by covering the serum bottle using a black sheet with varying thickness. For a light intensity of 0 μmol photons m⁻² s⁻¹, the incident light was completely blocked by covering the serum bottle using aluminum foil. Each bottle was shaken at 180 rpm and kept at a temperature of 25 ± 2 °C for 24 h. Before the experiment, the dissolved oxygen (DO) was saturated by supplying pure oxygen gas for 5 min.

Samples were collected from the top of the bottle using a syringe needle, as shown in Figure S1. Prior to analysis, all collected samples were filtered through a 0.45 μm glass filter (GC-50, Advantec, Taiwan). The SS concentration was measured and calculated after drying at 105 °C. Nitrogen compounds such as and were analyzed using high-performance liquid chromatography (Shimadzu, Japan) with an anion column IC NI-424 (Shodex, Japan). For HPLC analysis, the mobile buffer of 8 mM 4-hydroxybenzoic acid + 2.8 mM Bis-Tris + 2 mM phenylboronic acid + 0.005 mM CyDTA aq. was used. -N and -N peaks were detected during a 15-min retention time in a column oven at 40 °C. The analysis was detected by the conductivity detector.

In this experiment, the synthetic substrate used contained the following concentrations: 2.4 g L⁻¹ (NH₄)₂SO₄, 1.9 g L⁻¹ NH₄Cl, 2.8 g L⁻¹ KH₂PO₄, 2.0 g L⁻¹ MgSO₄, 2.0 g L⁻¹ NaCl, 17.5 g L⁻¹ NaHCO₃, 1.28 g L⁻¹ CaCl₂·2H₂O, and 3 mL L⁻¹ trace metal solution. The trace metal solution comprised 1.0 g L⁻¹ Na₂EDTA·2H₂O, 200 mg L⁻¹ FeCl₃·6H₂O, 36 mg L⁻¹ MnCl₂·4H₂O, 10.4 mg L⁻¹ ZnCl₂, 4.0 mg L⁻¹ CoCl₂·6H₂O, and 2.5 mg L⁻¹ NaMoO₄·2H₂O.

The light intensity transmitted through the hydrogel sheet was measured by the photon flux density values in the visible light region of 380–780 nm using a light analyzer directly below the sheet. For the light analyzer, the exposure time and wavelength interval were set to 1,000 ms and 1 nm, respectively.

Here, I₀ is the incident light intensity, I is the transmitted light intensity, A is the absorbance, is the absorption coefficient, L is the thickness of the hydrogel sheet, and c is the concentration of CB.

The results show that the nitrification efficiency is in good agreement between the experimental and predicted values in the light intensity range of 900–1,600 μmol photons m⁻² s⁻¹. At 1,600 μmol photons m⁻² s⁻¹, the experimental result (plot) showed 36.5%, while the predicted value (dashed line) was 37.9%. While discrepancy is observed for the light intensity range less than 500 μmol photons m⁻² s⁻¹. This is because nitrification activity in the actual experiment suffers from photoinhibition even under weak light irradiation of less than 500 μmol photons m⁻² s⁻¹.

Based on these findings, it is possible to theoretically determine the distance from the outer surface at which light attenuates to 500 μmol photons m⁻² s⁻¹ by calculating the absorption coefficient. To further improve the light-shielding effect, either increasing the bead size of the light-shielding hydrogel or increasing the CB concentration are potential solutions. However, in the former case, increasing the bead size could potentially hinder the diffusion of oxygen necessary for nitrification to adequately reach the interior and limit mass transfer to other components, such as ammonia or NO_x (Benyahia & Polomarkaki 2005). Therefore, the best option is to increase the CB concentration to enhance the light-shielding effect. In this study, the mass transfer inside the hydrogel was not examined. However, an increase in nitrifying bacteria and CB concentrations increased diffusion resistance, especially for large molecular ions. Therefore, in future work, the mitigation performance of the photoinhibition of nitrifying bacteria and the mass transfer for the light-shielding hydrogel will be investigated.

In this study, light transmission in the sheet-type light-shielding hydrogel was evaluated to clarify the effect of light shielding on the photoinhibition of nitrifying bacteria immobilized on a spherical light-shielding hydrogel. The results showed that the light absorption coefficient increased with increasing CB concentration, and the presence of bacteria and CB improved light attenuation in the light-shielding hydrogel. Light transmission through CB0.5, 1 mm thick, was 2.5%, indicating that the light-shielding hydrogel is expected to provide an effective light-shielding effect for the photoinhibition of nitrifying bacteria. In nitrification experiments with spherical light-shielding hydrogels, nitrifying activity decreased with increasing light intensity, e.g., to 36.5% of the original at 1,600 μmol photons m⁻² s⁻¹. The theoretical prediction agreed well with the obtained nitrification efficiency decrease with increasing light intensity in the range of 900–1,600 μmol photons m⁻² s⁻¹. These findings indicate that the proposed light-shielding hydrogel and its analytical methods represent a novel approach to cultivating photoinhibitory microorganisms.

This work was supported by JSPS KAKENHI (Grant Numbers JP20KK0249 and JP23K20026). We thank the Yokohama Hokubu Treatment Sludge Center for providing nitrifying bacteria.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.