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In this work, we present a luminous-exothermic hollow optical element (LEHOE) that performs spectral beam splitting in the visible spectral range for the enhancement of biofilm growth and activity. The LEHOE is composed of a four-layer structure with a fiber core (air), cladding (SiO2), coating I (LaB6 film), and coating II (SiO2-Agarose-Medium film). To clarify the physical, optical and photothermal conversion properties of the LEHOE, we determined the surface morphology and composition of the coating materials, and examined the luminous intensity and heating rate at the LEHOE surface. The biofilm activity on the biocompatible LEHOE is far greater than that of commercial fibers, and the biofilm weight on the LEHOE is 4.5 × that of the uncoated hollow optical element.
© 2017 Optical Society of America
1. Introduction
A bacterial biofilm is an organized community of microorganisms attached to a support material under submersion or in contact with water. The presence of biofilm in an industrial system can be detrimental or beneficial. For example, in the water industry, the optimized reuse of water in closed loop water circuits can be contaminated by circuit fouling due to biofilms, deposits, and scale formations [1,2]. Conversely, effective bioremediation and bioenergy require high-quality biofilms [3,4]. In the bioenergy field in particular, biofilm attachment is considered a highly efficient technology for hydrogen production using photosynthetic bacteria (PSB, see Tab. 1 for list of abbreviations) and biodiesel production by microalgae. Biofilms, therefore, have attracted intense interest for their potential advantages including high conversion yield, avoidance of biomass–liquid separation, and dual functionality in wastewater biodegradation and bioenergy production [5,6]. Although promising, the bioenergy production performance still faces two critical problems: low biofilm development and activity because of the limits based on light and temperature [7,8].
Tab. 1. List of Abbreviations
There are many studies demonstrating that cell adhesion, biofilm growth and activity are significantly affected by photo-conditions including light intensity and spectral composition [9,10], as ATP synthesis, electron transfer and carbon source transport properties are light-dependent. To obtain appropriate light conditions, solid optical fiber (SOF) was recently developed to transport light from external sources into a fiber core and luminescence light at a fiber surface [11,12]. Although biomaterials based on SOF can act as light sources for cell adhesion, the cell immobilization efficiency and immobilized cell activity are not significantly improved through this use. The SOF surface is rather smooth and without chemoattractants, and the luminous intensity and penetration depth at the SOF surface (which are dependent on the evanescent field) leave much to be desired. To overcome these limitations, we developed a biomaterial which is a GeO2-SiO2-chitosan-medium (GSCM-)-coated hollow optical element (HOE) for PSB immobilization [5,7,13]. We found that the GSCM-coated HOE exhibits a high luminous intensity and uniform light distribution along the fibers because of the increase in refractive index when moving from fiber core to fiber coating. These favorable luminescence properties enhanced the cell adhesion capacity, increased the biofilm biomass, and improved the hydrogen production performance of the biofilm photobioreactor. Although the light conditions of the biomaterial for biofilm culturing can be improved by using the GSCM-coated HOE, it is still difficult to improve and regulate the SOF and GSCM-coated HOEs surface temperature because these fibers cannot perform function of spectral beam splitting.
Temperature is another important parameter here, as the enzyme activity for growth and metabolism of microorganisms are dependent on temperature [14]. Furthermore, the rates of cell adhesion and biochemical reaction are sensitive to temperature [15]. Hence, optimization of cell culture temperature is very important to enhance cell adhesion, biofilm growth and activity [16]. To date, the controlled biochemical reaction temperature is mainly based on air conditioning, circulating water bath, and heating rods [17,18]. These traditional methods have a high energy cost, low energy efficiency, and make direct control over the temperature of biofilm cells difficult, because heat from traditional heat sources is first transferred to the culture medium and then to the biofilm cells. To enhance the heat energy utilization efficiency and more accurately control the cell temperature, photothermal conversion nanomaterials which can directly heat the cell have been exploited in recent cancer treatments [19–21]. Unfortunately, few studies have reported these materials being applied to cell immobilization. More importantly, there is a lack of biofilm support materials that can directly control both temperature and light intensity using spectral beam splitting. Thus, to obtain high-quality biofilms, it is very important to create a biomaterial with temperature and light intensity control functionality.
In this work, to enhance the PSB adhesion capacity, biofilm growth and activity, a novel luminous-exothermic hollow optical element (LEHOE) with spectral beam splitting capabilities has been fabricated. A schematic of the preparation process and mechanism is shown in Fig. 1. The LEHOE is composed of a fiber core (air), cladding (SiO2), coating I (LaB6 film), coating II (SiO2-Agarose-Medium film, or SAM film), and a hemispherical tip. The fiber core and cladding are used to transmit light, while the coatings are used to control the temperature and luminous light intensity of the LEHOE surface. An LaB6 film was selected as the photothermal conversion material because LaB6 nanoparticles (NPs) have selective absorption spectrum characteristics [22–24] (absorptions around 380–510 nm and 660–780 nm, compared to the absorption spectrum of PSB around 590 nm for producing hydrogen [18]), and the generated heat can be directly used for biofilm growth. The SAM film is selected as coating II for the top-layer as it possesses a rough surface and chemoattractants, making PSB more likely to attach to the surface and enhancing the biofilm growth and activity. The hemispherical tip, with a Au film at 500 nm of the LEHOE, is employed to increase the internal light reflection. The temperature and luminous light intensity of the prepared biomaterial can be controlled by adjusting the LaB6 NP content. Overall, the LEHOE biomaterial is expected to improve the cell retention capacity, biofilm growth and activity. To clarify the performance of the LEHOE, we examined the surface morphology and composition of the LaB6 NPs, coating materials, and biofilm, and recorded the luminous intensity and heating rate at the surface of the LEHOE. We also analyzed the biocompatibility of the LEHOE for the PSB CQK-01, surveyed the PSB adhesion capacity and biofilm growth on the LEHOE, and tested the biofilm activity by using confocal laser scanning microscopy.
2. Materials and methods
2.1 Materials
Lanthanum hexaboride powders (LaB6, −325 mesh, 99.5% metals basis), ethylene glycol (analytically pure, 98%), tetraethoxysilane (TEOS, 99.9% + ), (3-Aminopropyl)trimethoxysilane (97%) and glacial acetic acid (analytically pure, 99.5%) were purchased from Sigma Chemical Co. (China). Agarose with a low melting point was purchased from Aladdin Industrial Inc. (China). The synthetic medium was the same as the reported by Zhang et al [25]. Absolute ethanol and deionized water were used in all experiments.
2.2 Preparation of LaB6 NPs
In order to take into account the spectral transmission and photothermal conversion properties of the prepared LaB6, spheroidal LaB6 NPs were synthesized as follows. The purchased LaB6 powder was ground using a planetary ball mill (QM-3SP4J; NanDa Instrument Plant, Nanjing, China). For the typical stirred bead milling process, 8 g LaB6 raw powder and 12 g agate grinding beads were mixed in 20 mL ethylene glycol. The mixture was then ground for 5 h at a speed of 300 rpm. The completed liquid samples were then placed in a vacuum drying oven for 12 h at 80 °C and then reheated to 150 °C for 3 h for complete drying. The dried LaB6 NPs were etched in a dilute nitric acid solution (1.5 wt%) for 5 min at 30 °C with ultrasonic agitation (DL-180D, Shanghai, China) at 180 W power and 20 kHz frequency. The nitric acid solution with the LaB6 NPs was neutralized by the addition of aqueous ammonia solution (10 wt%) to maintain a pH of 7.0. The supernatant of the neutralized solution was drained and the precipitate was washed three times with distilled water. The cleaned precipitate was then dried as previously discussed and the re-dried sample was milled for 30 min in an agate mortar for dispersion.
2.3 Synthesis of LaB6 film
The LaB6 film was prepared in a tube furnace (Central Furnace, Tianjin, China) using a high-temperature calcination method. To obtain a 0.10 wt% LaB6-ethylene glycol dispersion, 1 g LaB6 NPs having a hydrodynamic diameter of 296 nm was dispersed in 895 mL ethylene glycol at 30 °C. The mixture was processed by ultrasonic agitation (20 Hz, 180 W) for 180 min until the LaB6 was evenly dispersed in ethylene glycol. Next, 1 mL of the prepared LaB6-ethylene glycol dispersion was coated on a thoroughly washed quartz glass tube (hollow optical element, HOE), having length, outer diameter, and inner diameter of 160 mm, 6 mm, and 4 mm, respectively, using a dip coater (SYDC-100H, Shanghai SAN-YAN Technology Co. Ltd., China). The HOEs were sintered in a tube furnace under N2 for 20 min at 300 °C. Finally, the above steps were repeated to prepare samples with different concentrations of LaB6 NPs. In this work, the concentration of LaB6 NPs at the fiber surface is in the range of 0−9 g (m2)−1, and the weights of the LaB6 NPs and those on the fiber surface were calibrated using an ultra-microbalance (XP2U Automated-S, Mettler Toledo).
2.4 Preparation of LEHOEs
A SAM sol was prepared using a previously-published procedure [7]. To prepare LEHOEs, the SAM sol was first coated on the surface of LaB6-coated HOE using the dip coater; the thickness of the coating II (SAM film) was controlled by the coating time. In this work, the thickness of the SAM film is around 57.5 μm. The SAM coated samples were then placed in a vacuum drying oven (Wuhuan Instrument Factory, Chongqing, China) at 85 °C for 8 h. Thereafter, LEHOEs were prepared and stored under vacuum.
2.5 Surface morphology, particle size and composition tests
Surface morphologies of the LaB6 NPs, LaB6 film and LEHOE were determined using a JSM-7800F electron microscope (JEOL Ltd.) at an acceleration voltage of 5.0 kV with a CCD camera, and the chemical composition analysis of the LaB6 film was subsequently determined using an energy dispersive X-ray spectrometer (EDS) attached to the FESEM. The optical profiles of the LaB6 film and LaB6-SiO2-agarose-medium film on the surface of the LEHOE were measured using a noncontact 3D optical profiler system (Wyko NT 1100, Veeco Instrument Inc., Plainview, NY, USA). The optical profiler data were collected using the Wyko Vision 32 ® analytical software package (Wyko Corporation, Tuscon, AZ, USA). The particle size distribution of the LaB6 NPs was determined using DLS on a Zetasizer Nano ZS90 (Malvern Instruments). Furthermore, the coating compositions of the fibers were analyzed using FT-IR (Nicolet iN10, Thermofisher, USA) and XPS (XSAM800, Kratos Co., UK).
2.6 Absorption spectra and luminous intensity tests
The absorption spectrum for the ethylene glycol dispersion of LaB6 NPs at 0.1 wt% (backgrounded to pure ethylene glycol) was measured using a UV7 Spectrophotometer (Mettler-Toledo Instrument Ltd., Shanghai, China). The absorption spectrum for the LaB6-SiO2-agarose-medium film (backgrounded to HOE) was determined in a similar manner. Furthermore, the luminous intensity along the surface of the HOEs and LEHOEs was detected using an optical power meter (UV 0.2, Newport Corporation, USA; obtained from NBeT Group Corp., China) with a wavelength range of 200−1100 nm, power range of 100 pW to 0.2 W, and uncertainty of 1−4%.
2.7 Photothermal tests
The photothermal conversion properties of the LaB6-ethylene glycol dispersion were determined using a light-emitting diode (LED) with a light irradiance medium value of 145.7 W (m2)−1 at 380–780 nm. 5 mL of LaB6-ethylene glycol dispersion was added to a quartz glass cuvette cell and then exposed to the LED. To determine the accuracy of the photothermal conversion data, we included a quartz glass cuvette cell full of ethylene glycol as the blank control group to quantify the interference of other environmental factors. The temperature of the LaB6-ethylene glycol and ethylene glycol solutions was measured with a fiber Bragg grate (FBG) sensor (center wavelength at 1595.14 nm, sensor length 3 mm) and an optical sensing interrogator (SM125; Micron Optics Inc.). Furthermore, the photothermal conversion performance of the LaB6 films and LaB6-SiO2-agarose-medium films were studied using the same LED light source. The light incident end (Fig. 1) of the prepared samples (SOF, HOE, and LEHOE) was directly coupled to the LED, the incident angle of light at the surface of the samples was 18 degrees, the distance between the LED and the incident end of the samples was 50 mm. The temperature along the surface of the HOEs and LEHOEs was detected using the FBG sensor and sensing interrogator. The distance between measurement points of the temperature along the surface of the HOEs and LEHOEs was 10 mm. All photothermal, absorption and luminous intensity experiments were carried out under vacuum at 25 °C and repeated three times, and each reported data point is the average of the three plus standard deviation.
2.8 Microorganism and cultivation
The Rhodopseudomonas palustris CQK 01 strain was employed as the PSB. The cells were cultivated anaerobically under Ar at 30 °C for 96 h under illumination from an LED emitting at 590 nm and 5 W (m2)−1. The initial pH value of the medium before incubation was adjusted to 7.0.
2.9 Biocompatibility, cell adhesion and biofilm culture
To investigate the biocompatibility of the SOF (fiber diameter of 6 mm, core material made of PMMA, core refractive index of 1.45, and an operating temperature range of −50 to 70 °C), HOE and LEHOE, the samples were immersed in bacterial suspensions (optical density OD600nm = 0.1). The cells had been deliberately starved by not refreshing the synthetic medium for 16 d at 25 °C, and the surface morphologies of the support materials with adhered cells were then checked using an environmental scanning electron microscope (ESEM, Quanta200, FEIr, USA). Thereafter, the biofilm photobioreactor system is the same as that in a previous report [13] and was operated in two stages. The first stage was the initial adhesion step, and the adsorption capacity of the materials to the cells was investigated during this stage (the medium is the same as that reported by Liao et al [26]. and the medium flow was kept at 70 mL∙h−1 and 25 °C). The other stage was undertaken to form a biofilm, where the medium flow was also kept at 70 mL∙h−1 and 25 °C, and was refreshed (50 mL) every day with the photobioreactors.
2.10 Biofilm assay
The adhered cell number and biofilm dry weight were determined according to a previously reported method [13]. The biofilm morphology and thickness were analyzed by ESEM. The active biomass in the biofilm was examined using confocal laser scanning microscopy (CLSM) as follows: 1) the mature biofilm was sliced into three layers using an HM 505E Cryostat Microtome; 2) the sliced biofilms were stained using SYTO 63 (Molecular Probes, Carlsbad, CA); 3) CLSM (Leica TCS SP5 Confocal Spectral Microscope Imaging System, Mannheim, Germany) was employed to visualize the active biomass in the prepared biofilm samples at 5 μm sampling intervals, and the fluorescence of SYTO 63 was detected via excitation at 633 nm and emission at 650–700 nm. Furthermore, the protein of the biofilm was assayed using the CLSM as follows: 1) the sliced biofilm samples were stained using fluorescein isothiocyanate (FITC) (Sigma, China) was reconstituted at 1 g∙L−1 in dimethyl sulfoxide (Sigma, China); 2) CLSM was carried out to visualize the protein in the prepared biofilm samples at 5 μm sampling intervals, and the fluorescence of FITC was detected via excitation at 488 nm and emission at 500–550 nm.
3. Results and Discussion
3.1. Physical, optical, and photothermal properties of LaB6 NPs
To obtain a high-performance LaB6 photothermal conversion film, we first synthesized LaB6 NPs and recorded their physical, optical, and photothermal properties. The experimental results are shown in Fig. 2.
Fig. 2 Physical, optical, and photothermal properties of LaB6 NPs. (a) FESEM images, (b) EDX results and (c) DLS size distribution of LaB6 NPs. (d) Absorption spectra of the LaB6 NPs suspension in ethylene glycol (LaB6 NPs at 0.1 wt%, ethylene glycol background). (e) Variations in samples (ethylene glycol with and without LaB6 NPs) temperature with irradiation time under LED irradiation.
As shown in Fig. 2(a), under field-emission scanning electron microscopy (FESEM), LaB6 NPs appeared as spheroids, because the ground-up LaB6 NPs were etched in dilute nitric acid and the irregularly shaped patches were further corroded. To determine the elemental composition of the NPs, we carried out energy dispersive spectroscopy (EDS) analysis as shown in Fig. 2(b). La and B peaks originating from the core LaB6 are present here, and the Au peak observed in the EDS spectrum can be attributed to the conductive coating required for FESEM. These facts demonstrate that the NPs were composed of LaB6. Furthermore, the particle size of LaB6 NPs was statistically analyzed using dynamic light scattering (DLS) and the hydrodynamic diameter of 95% of the LaB6 NPs was distributed within the range of 240–360 nm, with a mean diameter of 296.0 nm as shown in Fig. 2(c). These facts reveal that the prepared LaB6 NPs have a uniform particle size distribution.
Figure 2(d) shows the optical properties of ethylene glycol dispersions of LaB6 NPs at 380–780 nm. The spheroidal LaB6 NPs show low spectral absorption at 550–610 nm, with the lowest absorption at 581 nm, suggesting that the LaB6 NPs have good light transmission properties at 581 nm. However, the LaB6 NPs had a higher absorption coefficient in the high-energy region of 380–510 nm and low-energy region of 660–780 nm. The strong light absorption within the high-energy region can be attributed to the interband transition from the B 2s, 2p bonding orbital to the unoccupied state in the conduction band of the LaB6 NPs [27,28]. The absorbance in the low-energy region can be explained by excitation of surface plasmon polaritons by free electrons [28]. These facts demonstrated that the LaB6 NP could be used as a material for transmission of light suitable for hydrogen production by PSB CQK 01 cells, as the most appropriate absorption spectrum for PSB CQK 01 cells is approximately 590 nm [18]. The strong light absorption of the LaB6 NPs in the range of 380–510 nm and 660–780 nm could be converted into heat energy for improving the environmental temperature and promoting cell growth.
Figure 2(e) shows the photothermal performance of sample solutions, indicating that the sample temperature increased with irradiation time. In particular, the temperature of the sample with 0.10 wt% LaB6 NPs increased by 8.7 °C after irradiation for 10 min; however, the ethylene glycol temperature was increased by 2.2 °C. The good photothermal properties of the spheroidal LaB6 NPs (296.0 nm diameter) were similar to those reported by Chen et al [22]. and could be attributed to free electrons on the surface, along with surface plasmon resonances caused by spectral absorption at 380–510 nm and 660–780 nm [28–30].
These results verified that the papered LaB6 NPs can perform spectral beam splitting, as the NPs show good light transmission at 550–610 nm as well as good photothermal conversion properties at 380–510 nm and 660–780 nm as shown in Fig. 2. Hence, the LaB6 NPs are suitable for providing light and heat for PSB CQK 01 growth.
3.2 Physical, optical, and photothermal properties of LaB6 and LaB6-SAM films
To investigate the physical, optical, and photothermal properties of LaB6 and LaB6-SAM films, we investigated the morphology, composition, and optical and photothermal properties of the films as shown in Fig. 3 (the physical, optical, and photothermal properties of LaB6 NPs are presented in Supplementary material B).
Fig. 3 (a) FESEM images of the LaB6 and LaB6-SAM films. (b–c) 2D and 3D images of the LaB6 and LaB6-SAM films, respectively. (d–e) FT-IR and XPS spectra of the GSCML film, respectively. (f–g) Visible transmittance spectra and photothermal conversion performance of the samples (the mean thickness of the LaB6 and LaB6-SAM films is respectively of 8.7 and 57.5 μm), respectively.
Figure 3(a) shows that the LaB6-SAM film exhibited high surface roughness, and the insert of Fig. 3(a) shows that the LaB6 NPs densely distributed on the surface of the HOE. To further detect the performance of the LaB6 and LaB6-SAM films, we performed 3D profile analysis as shown in Figs. 3(b) and 3(c). Figure 3(b) further demonstrates that the LaB6 NPs are densely distributed as a film on the surface of the HOE, with a mean coating thickness of 8.7 μm and mean roughness of 1.2 μm. Figure 3(c) shows that the mean coating thickness of the LaB6-SAM film is 57.5 μm with a mean roughness of 5.8 μm, which is similar to the size of the PSB cells; hence, the LaB6-SAM-caoted HOEs can enhance PSB adhesion capacity. The surface roughness of the LaB6-SAM film is attributed to the interaction between the SiO2 and agarose.
Figure 3(d) shows the Fourier transform infrared (FT-IR) spectrum of the LaB6-SAM film. The band at 1450 cm–1 is characteristic of LaB6 [31], it indicates that the LaB6-SAM film can carry out the function of spectral beam splitting use because of the presence of LaB6. The bands observed at 1070, 1654, 1156, and 930 cm–1 are characteristic of agarose, indicating that the LaB6-SAM film contained agarose. A strong and broad absorption band appeared around 1085 cm–1 due to overlapping of Si-O-Si asymmetric stretching at 1130–1000 cm–1 and Si-O-C stretching at 1100–1050 cm–1, and the peak at 876 cm–1 was assigned to the Si-C stretching vibration. These facts reveal that the LaB6-SAM film contained SiO2; doping with SiO2 can increase compatibility between the SAM coating film and LaB6 film, increasing the surface roughness, and improving the mechanical strength of the SAM coating film. Furthermore, the peak around 1715 cm–1 corresponds to the stretching vibration of C = O from CO(H2N)2, the band near 1370 cm–1 is the most pronounced saccharide band indicating glucose in the film [32], and the band observed at 654 is the PO3- 4inorganic vibration bands (from KH2PO4 and K2HPO4·3H2O)· These results illustrate that the film contains nutrient medium and can be used to improve chemotaxis for PSB and promote PSB growth and metabolism.
To further analyze the chemical components of the LaB6-SAM film, we carried out X-ray photoelectron spectroscopy (XPS) analysis as shown in Fig. 3(e). The film exhibits three strong peaks at 286, 534, and 979 eV corresponding to C 1s, O 1s, and O KLL Auger emissions, respectively. Two weak peaks at 103 and 155 eV are ascribed to Si 2p and Si 2s, further suggesting that SiO2 exists in the film. Two weak peaks at 350 and 441 eV are ascribed to Ca 2p and Ca 2s, and the four other weak bands appear at 233, 380, 402, and 524 eV, which correspond to S 2s, K 2s, N 1s, and Na A, respectively. These peaks further indicate the synthetic medium in the film. Furthermore, the weak peak at 194 eV is assigned to B 1s, and the two weak peaks at 838 and 853 eV are ascribed to La 3d5 and La 3d3 [33]; these three peaks further imply that LaB6 NPs exist in the film.
Figure 3(f) shows the transmittance spectra of these samples. Although the absorbance gradually decreased over the sequence of LaB6 NPs, LaB6 film and LaB6-SAM film, the LaB6 and LaB6-SAM films exhibit the same spectral transmission trend as the LaB6 NPs in the range of 380–780 nm. This confirmed that the LaB6-SAM film can be used as a material for transmission of light suitable for the PSB CQK 01 cell cultures.
As shown in Fig. 3(g), irradiation of the HOE, LaB6-coated HOE and LaB6-SAM-coated HOE for 10 min caused the temperature to increase by 2.7, 6.8 and 7.4 °C, respectively. The temperature of the coated HOEs is higher than that of the HOE, because of the collective heating effects of the coating films with LaB6 NPs leading to increased conversion of light into heat energy. In addition, the temperature at the surface of the LaB6-coated HOE is higher than that of the LaB6-SAM-coated HOE, which can be attributed the thermal resistance of the SAM film. Although the thermal resistance exists in the LaB6-SAM film, the LaB6-SAM film still has good visible spectral photothermal conversion performance as indicated by the small (0.6 °C) difference between the LaB6-coated HOE and LaB6-SAM-coated HOE after 10 min. The data in Fig. 3, indicate that the LaB6-SAM film could perform spectral beam splitting, increase the adhesion of PSB cells and enhance the PSB biofilm growth because of good light transmission at 581 nm, photothermal conversion, appropriate surface roughness and carbon sources and inorganic elements.
3.3. Temperature and luminous intensity at fiber surface
Although the optical and photothermal properties of the LaB6-SAM films have been clarified, the optical and photothermal properties of LEHOEs have not been verified. Hence, to obtain high-performance LEHOEs, we first checked the effect of LaB6 NP mass on the temperature and luminous intensity distribution along the surface of the sample fibers. Thereafter, an improved LEHOE with a gradient distribution LaB6 film was further investigated. The experimental results are shown in Fig. 4.
Fig. 4 Temperature (10 min) and luminous intensity curves (the thickness of the SAM film is 57.5 μm). (a)–(b) Temperature and luminous intensity along the axial direction of the sample fibers (LaB6 film with uniform distribution along the surface of the LEHOEs). (c)–(d) Temperature and luminous intensity at the surface of the samples (LaB6 NPs shows graded distribution along the surface of the LEHOEs, and the quality of LaB6 NPs is in the range of 1.5−9 g (m2) −1). MP, measurement points.
Figure 4(a) shows the temperature of a commercial light-emitting SOF, uncoated HOE and LaB6-SAM-coated HOEs (LEHOEs) at the fiber surface. The inset of Fig. 4(a) shows the measurement points along the fibers. The temperature decreases along the axial direction of the sample fibers in the range of 0–130 mm. This can be attributed to the fact that the luminous intensity along the axial direction of the fibers decreases as shown in Fig. 4(b), and decreasing the light intensity decreases the photothermal conversion rate. However, close to the end of the fiber, the fiber temperature increases because the internal light reflection at the hemispherical tip of the fiber ending increases the surface luminous intensity [Fig. 4(b)] and photothermal conversion rate. Furthermore, one can see that the surface temperature of SOF and uncoated HOE remains at a low level, because these surfaces lack the photothermal conversion LaB6-SAM film. In particular, the temperature of the LEHOEs increase with increasing LaB6 NP contents, owing to the increased irradiation absorbed by the LaB6 NPs in the film and conversion of this irradiation into heat energy. Thus, the variations in temperature increases of the LEHOEs can be regulated by controlling the coating qualities of the LaB6 NPs in the LaB6 film.
As shown in Fig. 4(b), in the range of 0–50 mm, the luminous intensity of the HOE and LEHOEs is significantly higher than that of the SOF because the HOE and LEHOEs can capture a bundle of rays in any direction, whereas the light rays entering the SOF are restricted by the numerical aperture of the fiber. The luminous intensity of the HOE and LEHOEs rapidly decreases in the range of 0–60 mm, as the captured partial light beam is not suitable for total reflection and propagates into the external environment (HOE) or LaB6-SAM film (LEHOEs) via scattering and refraction. Comparing the HOE and LEHOEs, when close to the light source (10 mm), the luminous intensity of the LEHOEs is lower than that of the HOE, because the partial escaped light is absorbed by the LaB6 film and converted into heat energy [Fig. 4(a)]. Furthermore, one can see that the luminous intensity of the LEHOEs decreases with increasing LaB6 NP mass, because of the increased irradiation absorbed by increased numbers of LaB6 NPs and conversion of this irradiation into heat energy as shown in Fig. 4(a). Importantly, when the fiber is longer than 20 mm, the luminous intensity of the LEHOE with the LaB6 NPs quality at 3 g (m2) −1 shows the highest level in this regard. These good luminescence properties can be explained using a few different observations. First, the refractive index of the LaB6 NPs (n = 14.803) is high enough that the cladding of the LEHOEs leading to the light rays at the cladding-coating interface is no longer suitable for total reflection, so they propagate into the coating II via scattering and refraction. Second, although the LaB6 film of the LEHOEs partially absorbs luminescence to convert to heat energy, the spheroidal LaB6 NPs shows high Mie scattering at 380–780 nm, which can increase the luminous intensity. Third, a LaB6 film with an appropriate quality of LaB6 NPs also can enhance the luminous intensity on the surface of the LEHOEs.
Furthermore, as shown in Figs. 4(a) and 4(b), although the temperature and luminous intensity of the LEHOEs are enhanced, uneven distributions are also demonstrated at the fiber surface. In particular, comparing Figs. 4(a) and 4(b) shows that the temperature increase and luminous intensity of the LEHOEs exhibited the opposite trend as the increase in the amount of LaB6 NPs in the LaB6 film. Thus, an appropriate amount of LaB6 NPs is critical when considering the distributions of the temperature and luminous intensity along the axial direction of the LEHOEs for fixing PSB cells and enhancing growth and activity of the PSB biofilm. Hence, in this work, to obtain a high-performance of the LEHOE, we also fabricated a LEHOE with a graded distribution of LaB6 NPs (the quality of LaB6 NPs is in the range of 1.5−9 g (m2) −1) as shown in the insert of Fig. 4(c).
Figures 4(c) and 4(d) show that the unevenness of temperature and light intensity are reduced in LEHOEs with graded distributions of LaB6 NPs, compared to those of the LEHOE (4.5 g (m2) −1), HOE and SOF. The facts can be attributed to the facts that the LaB6 NPs content increases from the fiber incident end to the fiber end, leading to the light attenuation and Mie scattering increase with increase in LaB6 NPs content. This implies that the captured light energy in the fiber insight end of the LEHOE was effectively utilized by graded LaB6 NPs for conversion to heat energy and propagation into the SAM film via scattering and refraction; the heat and light energy are finally absorbed by the biofilm cells as a result. Furthermore, the temperature and luminous intensity of the improved LEHOE reaches to 28.0 °C and 77.0 μW (cm2) −1 respectively at a fiber length of 120 mm. The temperature of the improved LEHOE is 0.11 × , 0.11 × , and 0.03 × higher than that of the SOF, HOE, and LEHOE (4.5 g (m2) −1), while the luminous intensity of the improved LEHOE is 1.13 × , 1.29 × , and 1.52 × higher than that of the SOF, HOE, and LEHOE (4.5 g (m2) −1), the detailed results are shown in Tab. 2. These findings show that the LEHOE with a graded distribution of LaB6 NPs has good visible spectral photothermal conversion and light-emission performances and that could improve the cells culture conditions, enhance the cell adhesion capacity, biofilm growth and activity. Hence, in this work, we chose the improved LEHOE with a graded distribution of LaB6 NPs for subsequent bio-experiments of PSB adhesion and PSB biofilm growth.
Tab. 2. Experimental data of the prepared samples at an optical element length of 120 mm
3.4. Biocompatibility, cell adhesion, and biofilm growth
Although the improved LEHOE possesses good photothermal conversion and light-emission properties, the biotoxicity, cell adhesion capacity and biofilm growth have not been examined in detail. Thus, we carried out experiments to examine the biocompatibility of the LEHOE, SOF and HOE biomaterials. In addition, we investigated the cell adhesion rate and biofilm growth on different support materials.
Figures 5(a) and 5(b) show that PSB cells on the surface of the SOF and HOE were significantly decreased in size, owing to the smooth surface and lack of nutrients. However, as shown in Fig. 5(c), the adhered PSB cells on the LEHOE surface are of normal size, with an average length of 4.45 μm and diameter of 2.16 μm, and form many bacterial colonies. These facts reveal that the support material LEHOE is biocompatible and nontoxic. The LEHOE surface contained nutrients and had a more suitable temperature, enhancing cell growth and reproduction. These findings suggest that this biomaterial was effective for immobilizing PSB cells and supporting PSB biofilm growth.
Fig. 5 FESEM images of surface morphology of (a) SOF, (b) HOE, and (c) LEHOE with graded distribution of LaB6 NPs attached PSB cells. (d–e) Initial adhered cell numbers and biofilm dry weights on SOF, HOE, and LEHOE, respectively.
Figure 5(d) shows that the adhesion capacity of the HOE is slightly higher than that of the SOF. This can be attributed to the difference in the hydrophobicity and the fact that the PSB strain is easily adsorbed on hydrophilic surfaces. Furthermore, the number of adhered PSB cells on the LEHOE is significantly higher than that on the uncoated HOE. The improved bacterial retention capacity can be explained by the fact that the LaB6-SAM-coated HOE has an appropriate surface roughness to increase the contact area and binding potential for the PSB cells. The high-level luminous intensity, suitable temperature and chemoattractants in the LaB6-SAM coating induce bacterial adsorption. Consequently, after 30 min of adsorption, the adhesion density on the LEHOE was about 5.53 × and 3.87 × higher than on the SOF and normal HOE, respectively.
Figure 5(e) shows that the dry weights increased over time, then trended toward stable values. The initial increase shows that the adhered PSB form a biofilm; the steady state of the biofilms indicates a plateau in the biofilm growth process, in which the cells appear to be in dynamic adsorption-desorption. Furthermore, the biofilm with low dry weights on the SOF and uncoated HOE materials can be explained as follows. The temperature and luminous intensity at the surface of the SOF and HOE both maintain low levels. Because the SOF and HOE surfaces are rather smooth and lack the medium, it is difficult for cells to attach and develop a biofilm. Furthermore, the biofilm dry weight was lowest on the SOF because it is difficult for the hydrophobic surface to retain the liquid culture medium for PSB adhesion and growth. However, the dry weight of the biofilm on the LEHOE was very high; after 16 days, it was 3.01 × and 2.35 × those of the SOF and uncoated HOE, respectively. There are a few observations that can explain this behavior. First, the appropriate surface roughness enhances the initial PSB adhesion capacity and supports biofilm formation and stabilization. Second, the improved temperature and luminous intensity of the solid medium in the fiber coating II accelerate biofilm growth. In addition, as shown in Fig. 5(e), the biofilm on the SOF and HOE surfaces approached a stable state after approximately 12 days, whereas the stabilization time of the biofilm on the LEHOE surface was only 8 days. The high biofilm growth on the LEHOE surface can also be attributed to the appropriate surface grooves, presence of nutrients, suitable temperature and high luminous intensity.
3.5. Biofilm morphology, activity and protein
Although this novel LEHOE biomaterial enhances the PSB adhesion and biofilm development, the internal information in the biofilm is still unclear. In this section, we further examined the surface and internal morphology, active biomass and protein of the biofilms, using mature biofilms from the HOE and LEHOE sliced into three layers. In particular, for the biofilms from the HOE surface, the slice dimensions are as follows: from the bottom to the top of the biofilm, with layer thicknesses of 0–20 μm (Sample A), 20–40 μm (Sample B), and 40–72 μm (Sample C). For the biofilms from the improved LEHOE surface, the slice dimensions are as follows: from the bottom to the top of the biofilm, with layer thicknesses of 0–20 μm (Sample D), 20–92 μm (Sample E), and 92–185 μm (F). The experimental results are shown in Fig. 6.
Fig. 6 FESEM images of surface morphology of (a) original biofilm from HOE, (b) sliced biofilm from HOE, (c) original biofilm from LEHOE, and (d) sliced biofilm from LEHOE. (e_1)–(e_3) and (g_1)–(g_3) Distribution images of active biomass in biofilm samples which are from the surface of the HOE and LEHOE, respectively. (f_1)–(f_3) and (h_1)–(h_3) Distribution image of protein in biofilm samples which are from the surface of the HOE and LEHOE, respectively. Figures 6(a), 6(e_1) and 6(f_1) from Sample A. Figures 6(e_2) and 6(f_2) from Sample B. Figures 6(b), 6(e_3) and 6(f_3) from Sample C. Figures 6(c), 6(g_1) and 6(h_1) from Sample D. Figures 6(g_2) and 6(h_2) from Sample E. Figures 6(d), 6(g_3) and 6(h_3) from Sample F.
Figures 6(a) and 6(b) show that the biofilms on the HOE developed into a denser structure, but the samples from the top and bottom of the biofilms on the LEHOE exhibit a porous structure as shown in Figs. 6(c) and 6(d). The denser structure is due to the high content of EPS in the biofilms, as the surface of the HOE is very smooth and the biofilm can adhere to the fiber surface by producing large amounts of EPS.
Figures 6(e_1)–6(e_3) show that the biofilm activity from the top to bottom significantly decreases (in Fig. 6(e_3), slightly red regions indicate substantial bacteria inactivation); however, Figs. 6(g_1)–6(g_3) show the biofilm activity has a slight decrease. These facts can be attributed to a few different observations. First, the dense biofilm on the HOE increases the mass transfer resistance and light decay, which causes the bottom of the biofilm to suffer from substrate and light limitations as well as product inhibition [34]. Second, improved biofilm activity arises from the desirable properties of the LaB6-SAM-coated HOE, such as surface roughness, suitable temperature, high luminous intensity, uniform light distribution, and the presence of the culture medium, so the biofilm cells exhibit a high metabolism and activity.
As shown in Figs. 6(f_1)–6(e_3), the protein content of the biofilms (from HOE) decreases slightly from the top slice to the bottom. However, the protein content exposes a rapid decrease in the biofilms on the LEHOE as shown in Figs. 6(h_1)–6(h_3). As shown previously, this protein is mainly produced by active PSB cells, and the decrease of protein metabolite in the biofilms indicates that the active biomass decreases; these facts further verified our results (i.e., decrease in biofilm activity) as shown in Figs. 6(e) and 6(g). Of note when comparing Sample C and Sample F, although Sample F contains higher active biomass than Sample C [Figs. 6(e_3) and 6(g_3)], Sample F shows a lower protein content as shown in Figs. 6(f_3) and 6(h_3). The LaB6-SAM-coated HOE exhibited an appropriate temperature, high luminous intensity and a uniform light distribution, improving the biofilm culture conditions, and the LEHOE surface exhibited pits and the culture medium. Thus, even though the protein content was low, the biofilm had long-term adhesion and stability on the LEHOE. However, for the biofilm on the HOE, the protein content of the HOE was higher than that of the LEHOE, because the high protein content and ESP can form denser structures [Figs. 5(a) and 5(b)] and a protective layer for cells against harsh external conditions, prevent cell detachment from the support material surface, and also serve as carbon and energy reserves during starvation [35]. For biofilm culture, if the LaB6-SAM-coated HOE is employed as the support material, the biofilm produces a low protein content and porous biofilm structure, which can reduce the mass transfer resistance and increase the substrate, product, heat, and light transfer, enhancing the biofilm growth and activity. Accordingly, we concluded that the LEHOE biomaterial (LaB6-SAM-coated HOE) was a good material for improving the microorganism adhesion, biofilm growth and activity because of good luminous-exothermic properties, suitable surface roughness and chemoattractants.
4. Conclusions
In conclusion, we fabricated a novel LEHOE biomaterial, a LaB6-SAM-coated HOE that is capable of spectral beam splitting. The LEHOE graded distribution of the LaB6 NPs showed good light transmission at 550–610 nm, superb photothermal conversion properties at 380–510 nm and 660–780 nm, and the temperature and luminous intensity along the axial direction of the LEHOE are respectively in the range of 28–30 °C and 80–170 μW (cm2)−1. Furthermore, the LEHOE has suitable surface roughness and chemoattractants. These properties enhance the PSB cell adhesion, biofilm growth and biofilm activity. The biofilm on the surface of the LEHOE presents a porous structure, and the dry weight of the biofilm on the LEHOE was 3.01 × and 2.35 × those of the commercial light-emitting SOF and uncoated HOE, respectively. These excellent features conferred the LEHOE biomaterial with the potential for future large-scale immobilized photosynthetic microorganism cultures, wastewater biodegradation and bioenergy production.
Funding
This work was supported by the National Natural Science Foundation of China (grant No. 51406020), Scientific and Technological Research Program of Chongqing Municipal Education Commission of China (Grant No. KJ1600901), Foundation and Frontier Research Project of Chongqing of China (grant No. cstc2016jcyjA0311) and University Innovation Team Building Program of Chongqing (grant No. CXTDX201601030).
References and links
1. M. A. Shannon, P. W. Bohn, M. Elimelech, J. G. Georgiadis, B. J. Mariñas, and A. M. Mayes, “Science and technology for water purification in the coming decades,” Nature 452(7185), 301–310 (2008). [CrossRef] [PubMed]
2. C. Zhou, A. Ontiveros-Valencia, Z. Wang, J. Maldonado, H. P. Zhao, R. Krajmalnik-Brown, and B. E. Rittmann, “Palladium recovery in a H2-based membrane biofilm reactor: formation of Pd (0) nanoparticles through enzymatic and autocatalytic reductions,” Environ. Sci. Technol. 50(5), 2546–2555 (2016). [CrossRef] [PubMed]
3. N. Zhong, Q. Liao, X. Zhu, and R. Chen, “A fiber-optic sensor for accurately monitoring biofilm growth in a hydrogen production photobioreactor,” Anal. Chem. 86(8), 3994–4001 (2014). [CrossRef] [PubMed]
4. Y. Su, A. Mennerich, and B. Urban, “The long-term effects of wall attached microalgal biofilm on algae-based wastewater treatment,” Bioresour. Technol. 218, 1249–1252 (2016). [CrossRef] [PubMed]
5. Q. Liao, N. B. Zhong, X. Zhu, Y. Huang, and R. Chen, “Enhancement of hydrogen production by optimization of biofilm growth in a photobioreactor,” Int. J. Hydrogen Energy 40(14), 4741–4751 (2015). [CrossRef]
6. P. Praveen, D. T. T. Nguyen, and K. C. Loh, “Biodegradation of phenol from saline wastewater using forward osmotic hollow fiber membrane bioreactor coupled chemostat,” Biochem. Eng. J. 94, 125–133 (2015). [CrossRef]
7. N. B. Zhong, X. Zhu, Q. Liao, Y. Z. Wang, and R. Chen, “GeO2-SiO2-chitosan-medium-coated hollow optical fiber for cell immobilization,” Opt. Lett. 38(16), 3115–3118 (2013). [CrossRef] [PubMed]
8. K. Wagner, K. Besemer, N. R. Burns, T. J. Battin, and M. M. Bengtsson, “Light availability affects stream biofilm bacterial community composition and function, but not diversity,” Environ. Microbiol. 17(12), 5036–5047 (2015). [CrossRef] [PubMed]
9. W. R. Hill and I. L. Larsen, “Growth dilution of metals in microalgal biofilms,” Environ. Sci. Technol. 39(6), 1513–1518 (2005). [CrossRef] [PubMed]
10. H. R. Bonomi, D. M. Posadas, G. Paris, M. C. Carrica, M. Frederickson, L. I. Pietrasanta, R. A. Bogomolni, A. Zorreguieta, and F. A. Goldbaum, “Light regulates attachment, exopolysaccharide production, and nodulation in Rhizobium leguminosarum through a LOV-histidine kinase photoreceptor,” Proc. Natl. Acad. Sci. U.S.A. 109(30), 12135–12140 (2012). [CrossRef] [PubMed]
11. D. Erickson, D. Sinton, and D. Psaltis, “Optofluidics for energy applications,” Nat. Photonics 5(10), 583–590 (2011). [CrossRef]
12. S. C. Chu, H. L. Yang, Y. H. Liao, H. Y. Wu, and C. Wang, “One-time ray-tracing optimization method and its application to the design of an illuminator for a tube photo-bioreactor,” Opt. Express 22(5), 5357–5374 (2014). [CrossRef] [PubMed]
13. Q. Liao, N. B. Zhong, X. Zhu, and R. Chen, “High-performance biofilm photobioreactor based on a GeO2–SiO2–chitosan-medium-coated hollow optical fiber,” Int. J. Hydrogen Energy 39(19), 10016–10027 (2014). [CrossRef]
14. C. Salthouse, S. Hilderbrand, R. Weissleder, and U. Mahmood, “Design and demonstration of a small-animal up-conversion imager,” Opt. Express 16(26), 21731–21737 (2008). [CrossRef] [PubMed]
15. N. Zhong, Q. Liao, X. Zhu, and M. Zhao, “Fiber Bragg grating with polyimide-silica hybrid membrane for accurately monitoring cell growth and temperature in a photobioreactor,” Anal. Chem. 86(18), 9278–9285 (2014). [CrossRef] [PubMed]
16. P. Carlozzi and M. Lambardi, “Fed-batch operation for Bio-H2 production by Rhodopseudomonas Palustris (Strain 42OL),” Renew. Energy 34(12), 2577–2584 (2009). [CrossRef]
17. F. Lahoz, I. R. Martín, J. Gil-Rostra, M. Oliva-Ramirez, F. Yubero, and A. R. Gonzalez-Elipe, “Portable IR dye laser optofluidic microresonator as a temperature and chemical sensor,” Opt. Express 24(13), 14383–14392 (2016). [CrossRef] [PubMed]
18. X. Tian, Q. Liao, X. Zhu, Y. Wang, P. Zhang, J. Li, and H. Wang, “Characteristics of a biofilm photobioreactor as applied to photo-hydrogen production,” Bioresour. Technol. 101(3), 977–983 (2010). [CrossRef] [PubMed]
19. P. Poizot, S. Laruelle, S. Grugeon, L. Dupont, and J. M. Tarascon, “Nano-sized transition-metal oxides as negative-electrode materials for lithium-ion batteries,” Nature 407(6803), 496–499 (2000). [CrossRef] [PubMed]
20. A. Nel, T. Xia, L. Mädler, and N. Li, “Toxic potential of materials at the nanolevel,” Science 311(5761), 622–627 (2006). [CrossRef] [PubMed]
21. M. Sakemoto, Y. Kishi, K. Watanabe, H. Abe, S. Ota, Y. Takemura, and T. Baba, “Cell imaging using GaInAsP semiconductor photoluminescence,” Opt. Express 24(10), 11232–11238 (2016). [CrossRef] [PubMed]
22. C. J. Chen and D. H. Chen, “Preparation of LaB6 nanoparticles as a novel and effective near-infrared photothermal conversion material,” Chem. Eng. J. 180, 337–342 (2012). [CrossRef]
23. Q. Shao, L. Ouyang, L. Jin, and J. Jiang, “Multifunctional nanoheater based on NaGdF4: Yb3+, Er3+ upconversion nanoparticles,” Opt. Express 23(23), 30057–30066 (2015). [CrossRef] [PubMed]
24. T. M. Mattox, A. Agrawal, and D. J. Milliron, “Low temperature synthesis and surface plasmon resonance of colloidal lanthanum hexaboride (LaB6) nanocrystals,” Chem. Mater. 27(19), 6620–6624 (2015). [CrossRef]
25. C. Zhang, X. Zhu, Q. Liao, Y. Z. Wang, J. Li, Y. D. Ding, and H. Wang, “Performance of a groove-type photobioreactor for hydrogen production by immobilized photosynthetic bacteria,” Int. J. Hydrogen Energy 35(11), 5284–5292 (2010). [CrossRef]
26. Q. Liao, Y. J. Wang, Y. Z. Wang, X. Zhu, X. Tian, and J. Li, “Formation and hydrogen production of photosynthetic bacterial biofilm under various illumination conditions,” Bioresour. Technol. 101(14), 5315–5324 (2010). [CrossRef] [PubMed]
27. S. Kimura, T. Nanba, S. Kunii, and T. Kasuya, “Low-energy optical excitation in rare-earth hexaborides,” Phys. Rev. B Condens. Matter 50(3), 1406–1414 (1994). [CrossRef] [PubMed]
28. H. Takeda, H. Kuno, and K. Adachi, “Solar control dispersions and coatings with rare-earth hexaboride nanoparticles,” J. Am. Ceram. Soc. 91(9), 2897–2902 (2008). [CrossRef]
29. S. Schelm and G. B. Smith, “Dilute LaB6 nanoparticles in polymer as optimized clear solar control glazing,” Appl. Phys. Lett. 82(24), 4346–4348 (2003). [CrossRef]
30. Y. Li, N. B. Zhong, Q. Liao, Q. Fu, Y. Huang, X. Zhu, and Q. L. Li, “A biomaterial doped with LaB6 nanoparticles as photothermal media for enhancing biofilm growth and hydrogen production in photosynthetic bacteria,” Int. J. Hydrogen Energy, in press (2017).
31. G. K. Mehta, S. Kondaveeti, and A. K. Siddhanta, “Facile synthesis of agarose-l-phenylalanine ester hydrogels,” Polym. Chem.-UK 2(10), 2334–2340 (2011). [CrossRef]
32. C. Krafft, L. Neudert, T. Simat, and R. Salzer, “Near infrared Raman spectra of human brain lipids,” Spectrochim. Acta A Mol. Biomol. Spectrosc. 61(7), 1529–1535 (2005). [CrossRef] [PubMed]
33. M. F. Zhang, L. Yuan, X. Q. Wang, H. Fan, X. Y. Wang, X. Y. Wu, H. Z. Wang, and Y. T. Qian, “A low-temperature route for the synthesis of nanocrystalline LaB6,” J. Solid State Chem. 181(2), 294–297 (2008). [CrossRef]
34. T. R. Neu, U. Kuhlicke, and J. R. Lawrence, “Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms,” Appl. Environ. Microbiol. 68(2), 901–909 (2002). [CrossRef] [PubMed]
35. H. Liu and H. H. P. Fang, “Extraction of extracellular polymeric substances (EPS) of sludges,” J. Biotechnol. 95(3), 249–256 (2002). [CrossRef] [PubMed]
