Monitoring Biohydrogen Production and Metabolic Heat in Biofilms by

Subscriber access provided by BUFFALO STATE

Article

Monitoring Biohydrogen Production and Metabolic Heat in Biofilms by Fiber-Bragg-Grating Sensors Ming Chen, Xin Xin, Huimin Liu, Yongwu Wu, Nianbing Zhong, and Haixing Chang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01559 • Publication Date (Web): 23 May 2019 Downloaded from http://pubs.acs.org on May 24, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Monitoring Biohydrogen Production and Metabolic Heat in Biofilms by Fiber-Bragg-Grating Sensors Ming Chen,† Xin Xin,† Huimin Liu,† Yongwu Wu,† Nianbing Zhong,*,† Haixing Chang,*,‡ †Intelligent

Fiber Sensing Technology of Chongqing Municipal Engineering Research Center of Institutions of Higher Education, Chongqing Key Laboratory of Modern Photoelectric Detection Technology and Instrument, Chongqing Key Laboratory of Fiber Optic Sensor and Photodetector, Chongqing University of Technology, Chongqing 400054, China ‡ School of Chemistry and Chemical Engineering, Chongqing University of Technology, Chongqing 400054, China

ABSTRACT: A fiber Bragg grating (FBG) was created to accurately and simultaneously monitor the biohydrogen and metabolic heat production in biofilms containing Rhodopseudomonas palustris CQK-01 photosynthetic bacteria (PSB). The proposed hydrogen sensor was made from an FBG unit separated into two regions by a wet etching process; a thin region with a diameter of 15 μm was employed to monitor the temperature. A smaller region of the etched FBG with a diameter of 8.0 μm was coated with a 50-nm-thick Pd film by sputtering to determine the responses to the temperature and hydrogen concentration. To monitor the biohydrogen production and metabolic heat within the biofilms, three FBGs were evenly distributed in a polydimethylsiloxane channel (biofilm carrier) with vertical distances of 80 μm. In addition, the thickness, surface morphology, active biomass, and porosity of the biofilms were investigated. The FBG sensor can rapidly and accurately determine the difference in Bragg wavelength shifts caused by changes in the hydrogen concentration and temperature. The measured biohydrogen concentration is highly correlated with the real biohydrogen production with a correlation of 0.9765. The biohydrogen production capacity of PSB in the surface layer is much higher than that internally because of sharp decreases in the active biomass and porosity from the surface to within the biofilm. The highest biohydrogen concentration is obtained at 12.18K ppm for a biofilm thickness of 165 μm, and the temperature difference from metabolic heat production is ~1.1 °C in the biofilm culture.

The production of biohydrogen by photosynthetic bacteria (PSB) biofilms has attracted a considerable amount of interest for its potential advantages, including the high conversion yield, the avoidance of biomass–liquid separation, and the dual functions of further wastewater biodegradation and continuous hydrogen production.1–3 Although promising, the biohydrogen production performance (rate and purity) with PSB biofilms is still low because the growth process has not been effectively and accurately controlled.4–6 Nevertheless, optimization and control of the biohydrogen process of biofilms depends on the information obtained while monitoring the inner biohydrogen and metabolic heat production.5,6 Thus, monitoring the concentration of biohydrogen produced and the temperature in the biofilm during the culturing process is very important to control the growth of the biofilm and enhance biohydrogen production. In the past 30 years, various techniques have been developed to monitor the hydrogen concentration online, and many technologies such as electrical impedance spectroscopy,7 dielectric spectroscopy,8 light intensity modulation fiber-optic sensors,9 and the wavelength demodulation of fiber-optic sensors10−13 have been designed. Of these methods, the wavelength demodulation of fiber-optic sensors, including fiber Bragg gratings (FBGs),10 Fabry–Perot interferometers,11 long-period fiber gratings,12 etc., have attractive properties due to their microstructure, corrosion resistance, immunity from electromagnetic interference and light intensity fluctuations, fast response speed, good biocompatibility, and easy installation.14,15 Particularly, FBG sensors are suitable for the

long-term operation of photobioreactors to obtain accurate information related to the hydrogen concentration and temperature because they can accurately and simultaneously monitor multiple parameters such as the refractive index (RI) and temperature, the pH and temperature, and the cell growth and temperature.15–17 However, there are no reports of the use of FBG sensors or the wavelength demodulation of fiber-optic sensors to monitor the biohydrogen production and metabolic heat in biofilms in the literature to the best of the authors’ knowledge. Furthermore, the biohydrogen production concentration and temperature in a biofilm have never been monitored online during biofilm cultivation. Hence, it is necessary to create an FBG sensor that can simultaneously monitor the concentration of biohydrogen produced and the temperature in a biofilm during the growth process. To accurately and simultaneously monitor the biohydrogen production and metabolic heat in biofilms containing Rhodopseudomonas palustris (R. palustris) CQK-01 PSB, we create a simple microstructured FBG (mFBG) sensor. The mFBG includes two regions: the temperature-sensing and temperature- and hydrogen-sensing regions. The offline performance of the proposed sensor was studied by measuring different hydrogen concentration and temperatures. Online measurement of the concentration of biohydrogen produced and the temperature using the sensor was investigated in a culture of the R. palustris CQK-01 biofilm. We also developed a theoretical model to demonstrate the measurement accuracy. In addition, the thicknesses of the biofilms at different culture times were monitored, and the surface morphology, porosity,

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 10

and active biomass of the biofilms at different layers in the biofilm were checked.

MATERIALS AND METHODS Fabrication of the mFBG Sensor. To monitor the biohydrogen production and metabolic heat within the biofilms, three FBGs (denoted as FBG_A, FBG_B, and FBG_C) were employed. The core diameter d, cladding diameter D, and grating length L of the three FBGs are 8.3 μm, 125 μm, and 50 mm, respectively. The grating pitches Λ of FBG_A, FBG_B, and FBG_C are 532.39, 535.84, and 539.25 nm, respectively. The central wavelengths of FBG_A, FBG_B, and FBG_C are of 1539.892, 1549.916, and 1559.992 nm, respectively, at a temperature of 25 °C. To enhance the mechanical strength and optical signal transmission and to obtain good FBG reflection spectra, mFBG sensors with two thinned regions and a smooth surface (see Figs. 1a and 1b), i.e., the temperature-sensing and temperature- and hydrogensensing regions, were prepared by wet etching.15,18 The length and diameter of the temperature-sensing region were 5 ± 0.2 mm and 15.0 ± 0.1 μm, respectively; the temperature- and hydrogen-sensing region had a length and diameter of 45 ± 0.2 mm and 8.0 ± 0.1 μm, respectively. When the FBG was etched into two regions, the Bragg reflection spectrum of the FBG changes to two Bragg reflection spectra, as shown in inset of Fig. 1a. One FBG reflection spectrum is used in response to the temperature, and the other is used to monitor the hydrogen concentration and temperature. Preparation of the temperature- and hydrogen-sensing region. To accurately and rapidly respond to changes in the hydrogen concentration, the temperature- and hydrogensensing region of the mFBGs (mFBG_A, mFBG_B, and mFBG_C) were sputtered using a sputter coater (SCD 500, Bal-Tec AG, Switzerland) using 99.999% pure Pd (Pd films have a high selectivity for and strong affinity towards H2 molecules.19). The preparation of the 50-nm-thick Pd coatings is described in section S1 of the Supporting Information. The thickness of 50 nm was selected because it can realize a high sensitivity and fast response time to hydrogen.20,21

Figure 1. (a) Schematics of the structure of the mFBG sensor and the Bragg wavelengths of the FBG and mFBG. SEM images of (b) the temperature- and hydrogen-sensing region of the mFBG, (c) the Pd-coated temperature- and hydrogen-sensing region, and (d) the Pd film. (e) EDX and (f) XRD results of the Pd coating (T denotes temperature; HT denotes hydrogen and temperature).

Figure 1c shows that the Pd layer has attached to the surface of the mFBG. The surface morphology of the Pd film (Fig. 1d), which was characterized by field-emission scanning electron microscopy (SEM; JSM-7800F, JEOL Ltd., Japan), appears uniform and dense, and there are no cracks on the surface of the Pd film. In Fig. 1e, Pd is observed on the surface of the Pd-coated mFBGs (the composition of the Pd film was analyzed by energy-dispersive X-ray (EDX) spectroscopy during SEM). In Fig. 1f, three primary X-ray diffraction (XRD) peaks appear, which were determined using a diffractometer (RINT 2500, Rigaku Corporation, Japan) with Cu Kα radiation. The three primary peaks at 2θ = 40.0°, 46.5°, and 68.1° were assigned to the (111), (200), and (220) reflection planes of the Pd0 crystal structure, respectively. These results demonstrate that the Pd film is pure, has not been oxidized, and can exhibit good mechanical performance during the hydrogen response. Microorganism and cultivation. The R. palustris CQK-01 strain was employed as the PSB for photoheterotrophic biohydrogen production. The cells were anaerobically cultivated with Ar gas at 30 °C for 96 h under illumination from a 590-nm light-emitting diode (LED) at 4000 lx because the appropriate illumination can trigger photosynthetic activity of bacteria to produce hydrogen.22,23 The synthetic medium was the same as that in our previous work,5,6 and the initial pH of the medium before incubation was adjusted to 7.0 using a NaOH solution with a pH of 14.0. Systems and operations. Figure 2 shows a schematic of the PSB biofilm culturing and measurement systems. A flat-panel biofilm photobioreactor was fabricated from polydimethylsiloxane (PDMS) with a working volume of 150 × 80 × 3 mm3. In particular, the bottom plate of the reactor


Page 3 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with a channel (the length, width, and height are 100 mm, 60 mm, and 240 μm, respectively) was fabricated and used as the biofilm carrier. The LED light source (4000 lx; 590 nm) for biofilm growth was mounted on one side of the biofilm photobioreactor. Glucose-based synthetic wastewater with a temperature of 30 ± 0.01 °C, which was controlled using a water bath (GDH-0506, SHUNMATECH, China), continuously passed through the reactor using a peristaltic pump at a volumetric flow rate of 100 mL/h. Before the PSB suspension [optical density (OD600nm) and pH of 0.12, and 7.0, respectively] was prepared using cultivated bacteria and sterilized distilled water and inoculated in the bioreactor, the photobioreactor system was sterilized with 3.0% formalin and then thoroughly washed with deionized water. Thereafter, the reactor was operated in two stages, as shown in Fig. 2. During the first stage (lasting 6 h), the medium was recycled to avoid the loss of activated PSB cells in the circulated liquid solution. In the second stage, the photobioreactor became an open-loop system; the evolved biogas and effluent liquid flowed into a gas–liquid separator, which was coupled with a filter with a pore size of 0.22 μm. The online measurement system consists of three subsystems for measuring the biohydrogen production and metabolic heat with mFBGs, the biofilm thickness with an optical microscope, and the hydrogen concentration with a microelectrode. The subsystem for measuring the biohydrogen production and metabolic heat consists of three fibers, three mFBGs, three 3-dB couplers, an optical splitter, an optical coupler, full spectral scanning (1510–1590 nm) equipment, and an FBG interrogator (SM125-500, Micron Optics Inc.) with a high accuracy (1 pm). Fiber_1, Fiber_2 and Fiber_3 are connected to mFBG_A, mFBG_B, and mFBG_C, respectively. In particular, the vertical and horizontal spacings between two mFBGs are 80 μm and 2 mm, respectively. mFBG_A has a vertical distance of 80 μm from the bottom surface of the PDMS biofilm carrier. mFBG_B is located at the center of the PDMS channel. To monitor the biofilm thickness, an optical microscope (IX81, Olympus, Japan) with a resolution of ±1 μm was employed. The procedure for measuring the biofilm thickness can be found in the literature.24 To calibrate the hydrogen concentration, the subsystem utilizing the microelectrode was applied, which comprises a H2 sensor (microelectrode) with a tip diameter of 10 µm (OX10, Unisense A/S), a H2 reference microelectrode, a high-resolution analog-to-digital (AD) converter (ADC16, PicoTech), and a picoammeter (PA2000, Unisense, Denmark). The subsystem with the microelectrode has a H2-concentration resolution of 100 ppb.

Figure 2. Schematic of the experimental system (PDMS: polydimethylsiloxane, PC: personal computer, GL: gas–liquid).

PSB and biofilm characteristic analysis. A PSB cell was examined by environmental scanning electron microscopy (ESEM; FEI Quanta 600 FEG). The biofilm structure at different thicknesses (80 μm, 160 μm, and the surface-layer biofilm at 298 μm) was also characterized by ESEM. To assess the changes in the porosity within biofilms, a mature biofilm was first sliced into three layers using an HM 505E Cryostat Microtome. Second, to visualize bacteria, the sliced biofilms were stained using a BacLight™ Bacterial Viability Kit (Invitrogen, Paisley, UK). Third, optical micrographs of the bacteria were acquired using confocal laser scanning microscopy (CLSM; TCS SP5 Confocal Spectral Microscope Imaging System, Leica, Mannheim, Germany). Fourth, to obtain quantitative data about the porosity of the biofilms, the acquired optical images were processed as binary images. Fifth, in the binary images, white pixels were considered to be the distribution area of the PSB bacteria within the biofilms, and black pixels were considered to be the distribution area of the pores in the biofilms. Sixth, the porosity of the biofilms was determined from 30 images consisting of 640 × 480 pixels, which were taken at random locations within the biofilm, as the ratio of the surface area of black pixels to the total surface area (total number of white and black pixels) with Image J 1.37v (Wayne Rasband, National Institutes of Health, USA). To obtain the active biomass, the sliced biofilm samples were stained using SYTO 63 (Molecular Probes, Carlsbad, CA); then, CLSM was employed to visualize the active biomass in the sliced biofilms. Thereafter, the fluorescence of SYTO 63 was analyzed via excitation at 633 nm and emission at 650– 700 nm. Measurement Principles. Of an mFBG unit with two sensing regions, the mFBG region with a diameter of 15.0 ± 0.1 μm is used to sense the temperature T. The shift in the Bragg wavelength with the change in T can be expressed as25

ΔλB_1 = λB_1(αΛ + αn)ΔT = 𝐾T_1ΔT

(1)

where λB_1, αn, and αΛ are the Bragg wavelength, thermo-optic coefficient, and thermal expansion coefficient of the mFBG sensing region, respectively.



The Pd-coated mFBG region is used to sense T and the hydrogen concentration CH. When T was maintained at 30 °C, the shift in the Bragg wavelength with CH can be expressed as26 𝑋 𝑃 𝑋𝐹

𝛥𝜆𝐵_2 = 𝐾(𝑋𝑃𝑋𝐹 + 𝑌𝑃𝑌𝐹 + 𝐴𝑃𝐴𝐹)𝜆𝐵_2𝛥𝐶𝐻

(2)

where K is a constant of the Er-doped fiber; XP and XF are the axial stresses of the Pd-coated fiber layer and unperturbed fiber, respectively; YP and YF are the elastic moduli of the Pdcoated fiber layer and unperturbed fiber, respectively; and AP and AF are the cross-sectional areas of the Pd-coated fiber layer and unperturbed fiber, respectively. However, the temperature changes with the culture time because the metabolic heat produced by the PSB in the biofilm changes. Thus, ΔλB_2 for the Pd-coated mFBG region is a function of the changes in T and CH during biofilm growth and can be expressed as

∂λB_2 ∂2λB_2 ΔλB_2 = ΔCH + ΔT + ΔCHΔT ∂CH ∂T ∂CH∂T ∂λB_2

∂2λB_2

1

+ 2(ΔC2H

∂C2H

∂2λB_2

+ΔT2

∂T2

) + ⋅⋅⋅

(3)

For a Pd-coated mFBG coated with a biofilm, eq. 3 shows that ΔλB_2 changes with ΔCH, ΔT, the cross-term ΔCHΔT, and the higher-order terms of ΔCH, ΔT, and ΔCHΔT. However, according to our previous work, the biohydrogen production and metabolic heat during the biofilm growth process showed a very slight variation within 10 min.5,6 Thus, in this work, the effect of the cross-term and higher-order terms on ΔλB_2 can be ignored, and ΔλB_2 can be expressed as

𝛥𝜆𝐵_2 = 𝛥𝐶𝐻 ⋅ 𝐾𝐶 +𝛥𝑇 ⋅ 𝐾𝑇_2 ∂𝜆𝐵

∂𝑛𝑒𝑓𝑓

∂𝛬

𝐾𝐶 = ∂𝐶𝐻 = 2 ⋅ ( ∂𝐶𝐻 ⋅ 𝛬 + ∂𝐶𝐻 ⋅ 𝑛𝑒𝑓𝑓) 𝐾𝑇_2 =

∂𝜆𝐵 ∂𝑇

∂𝑛𝑒𝑓𝑓

=2⋅(

∂𝑇

∂𝛬

⋅ 𝛬 + ∂𝑇 ⋅ 𝑛𝑒𝑓𝑓)

To accurately and simultaneously monitor the biohydrogen concentration and temperature in the biofilm culturing process, two types of experiments should be conducted to separate their effects on the rate of change in the Bragg wavelength by holding one parameter constant and varying the other for each parameter. Using the experimental data, the following matrix can be written:

0 [Δ𝜆 Δ𝜆 ] = [ 𝐾 𝐵_1 𝐵_2

(6)

where KC is the hydrogen-concentration sensitivity coefficient and is a function of the axial stress, elastic modulus, and crosssectional area of the Pd film. KT_2 is the temperature sensitivity coefficient and is a function of the thermal expansion coefficient and thermo-optic coefficient of the fiber.

C

][ ]

𝑘T_1 Δ𝐶 𝑘T_2 Δ𝑇

(7)

RESULTS AND DISCUSSION Sensor performance during offline measurement. To evaluate the sensitivity and feasibility of the prepared mFBG sensors for accurate and simultaneous detection of the temperature and hydrogen concentration, the sensor performance in offline measurements are described in sections S2 and S3 of the Supporting Information. The offline measurements include the response of the sensors to temperature, the response of the sensors to hydrogen concentration, response time of the sensors with the change in the hydrogen concentration, and the response of the sensors to CO2, and the experimental results are shown in Figs. S1–2. According to the offline measurements, the matrices of the mFBG sensors can be directly obtained; for example, the matrix of the mFBG_B sensor is

[Δ𝜆Δ𝜆 ] = [0𝐾 𝐵1 𝐵2

0 [0.0042

(4) (5)

Page 4 of 10

C

][ ]

𝑘T1 Δ𝐶 𝑘T2 Δ𝑇

][ ]

9.6701 Δ𝐶 15.3430 Δ𝑇

= (8)

Distributed online monitoring of biohydrogen production and metabolic heat in biofilms. The PSB biofilm culturing and measurement systems are shown in Fig. 2. Prior to online experiments, a PSB cell was examined by ESEM, as shown in Fig. 3a; then, the changes in the Bragg wavelengths of FBG_A, FBG_B, and FBG_C with the biofilm culture time were checked, and the actual biofilm thickness was monitored by using a microscope, as shown in Fig. 3b.


Page 5 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. (a) ESEM image of CQK 01 PSB. (b) Output signals of FBG_A2, FBG_B2, and FBG_C2 and the biofilm thicknesses as a function of the culture time. (c) Hydrogen concentrations measured by mFBGs and a H2 microelectrode as a function of the biofilm thickness (HMS: H2 microelectrode sensor). (d) Temperature measured by mFBGs as a function of the biofilm thickness.

Fig. 3a shows the CQK-01 PSB with an average length of 4.25 μm and a diameter of 1.28 μm because they were cultured under optimal conditions of 4000 lx and 590 nm. The good morphology of the bacteria is due to the high chlorophyll α concentration and cell activity, which allowa large amount of biohydrogen to be produced.27,28 In Fig. 3b, biofilm growth occurred in three phases: adsorption, exponential, and stationary. The highest biofilm thickness was about 298 nm at a culture time of 198 h. Although the biofilm shows a complete biofilm growth curve in culture-time range of 0–240 h, the values of ΔλB of FBG_A2, FBG_B2 and FBG_C2, which were used to monitor the biohydrogen production, showed different trends. The different trends can be explained as follows. (1) FBG_A2, FBG_B2 and FBG_C2 were distributed at different heights in the biofilm carrier (see Fig. 2) and can simultaneously monitor the hydrogen concentration and temperature. (2) The hydrogen and metabolic heat production changes as the biofilm thickness changes (culture time). (3) When the biofilm thickness on the carrier is higher that of the mFBG, the mFBG was coated by the biofilm, and the Bragg wavelengths of FBG_A2, FBG_B2, and FBG_C2 will be affected by changes in the temperature of and hydrogen concentration in the biofilm. However, when the thickness of the mFBG was higher than that of the biofilm and not coated by the biofilm, ΔλB only responded to the biohydrogen

concentration. In Fig. 3b, the values of ΔλB of FBG_A1, FBG_B1 and FBG_C1 also show different trends and can be explained as follows. (1) FBG_A1, FBG_B1, and FBG_C1 maintain the same horizontal and vertical heights as FBG_A2, FBG_B2 and FBG_C2, respectively. (2) Biofilms with different thicknesses will produce different amounts of heat. (3) FBG_A1, FBG_B1, and FBG_C1 can only respond to the temperature when they were coated by a biofilm. Although ∆λB of the mFBGs varies with the changes in the hydrogen concentration and temperature for different layers of the biofilm, it is complex and incomprehensible. Thus, it is necessary to process the original data using the matrix in eq. 8 and to distinguish and reveal the changes in the biohydrogen concentration and temperature in different layers of the biofilm. The processed results are shown in Figs. 3c and 3d. In Fig. 3c, the biohydrogen concentrations of different layers of the biofilm were measured by FBG_A2, FBG_B2 and FBG_C2 and experienced four phases as the biofilm thickness changes: stable, quickly increasing, decreasing, and stationary phases. In the stable phase, the CQK-01 PSB were maintained in the adsorption phase to form a biofilm (see Fig. 3b). Subsequently, the measured hydrogen concentration increased as the biofilm thickness increased in the ranges of 7–75 μm (FBG_A2), 7–153 μm (FBG_B2), and 7–153 μm (FBG_C2) because the biofilm is in the exponential phase and the active



biofilm biomass increased with increasing biofilm thickness. Thus, the biohydrogen concentration increased. The hydrogen concentration measured by FBG_A2 quickly increased in the biofilm thickness range of 7–75 μm for the following reasons. (1) FBG_A2 is mounted at a height of 80 μm; thus, it can only respond to the biohydrogen concentration. (2) The 75-μmthick CQK-01 PSB biofilm did not reach its active thickness of 165 μm (this active thickness was obtained from the results measured by the H2 microelectrode; the biofilm achieves the highest biohydrogen concentration of 12.18K ppm at a thickness of 165 μm, as shown by the “HMS” curve in Fig. 3c), and the H2 production rate increases as the biofilm thickness increases within its active thickness range.6,29,30 The hydrogen concentration measured by FBG_B2 increased in the biofilm thickness range of 7–153 μm, which can be explained as follows. First, FBG_B2 is mounted at a height of 160 μm; thus, the biohydrogen concentration measured by FBG_B2 was from the entire biofilm in the thickness range of 0–153 μm. Second, the biohydrogen production increases as the active biofilm thickness measured in the range of 0–165 μm increases. However, for FBG_C2 mounted at a height of 240 μm, the measured biohydrogen concentration does not increase when the biofilm thickness is above an active thickness of 165 μm because thicker biofilms are more likely diffusion- and light-intensity-limited,6,31,32 leading to a decrease in the rate of H2 production. In Fig. 3c, the biohydrogen concentration measured by FBG_A2 sharply decreased in the biofilm thickness range of 80–122 μm, which can be explained as follows. The sensing region of FBG_A2 is coated by the biofilm, and the measured biohydrogen concentration is that produced by a biofilm with a thickness less than 80 μm because the hydrogen concentrations measured by FBG_B2, FBG_C2, and the H2 microelectrode did not sharply decrease at the layer with a height of 80 μm (Position of the mFBG_A). The biohydrogen production activity of the PSB in the biofilm decreased as the biofilm thickness (culture time) increased because the PSB have to produce large amounts of extracellular polymeric substance (EPS).33 As the amount of EPS and the biofilm thickness increase, the biofilm at the layer with a height of 80 μm changed into a very dense structure (see Fig. 4a) with a very low porosity (average porosity ~ 26.17±8.67%; see Fig. 4d), and the CQK-01 PSB of the inner biofilm were inactive (see Fig. 4g; in Figs. 4g–i, slightly red regions indicate substantial bacteria inactivation or death34). Thereafter, the biohydrogen concentration measured by FBG_A2 slowly decreased and was maintained at very low level when the biofilm thickness was greater than 236 μm. The low biohydrogen concentration can be explained as follows. When the biofilm developed into a mature biofilm with a thickness of 298 μm (see Fig. 3b), the PSB in the biofilm at the layer with a height of 80 μm are inactive or dead, as shown in Fig. 4g. In Fig. 3c, the biohydrogen concentration measured by FBG_B2 quickly decreased in the biofilm thickness range of 153–187 μm. This can be explained as follows. First, FBG_B2 was mounted at a height of 160 μm (Position of the mFBG_B). The biohydrogen concentration was produced by a biofilm with a thickness less than 160 μm because the hydrogen concentrations measured by FBG_C2 and the H2 microelectrode did not sharply decrease at the layer with a height of 160 μm. Second, the rate of H2 production and the

Page 6 of 10

hydrogen yield decreased because the biofilm thickness is above the active thickness of 165 μm. Thereafter, the biohydrogen concentration measured by FBG_B2 was maintained at a constant level when the biofilm thickness was greater than 255 μm. This can be explained as follows. The biofilm at the layer with a height of 160 μm has a dense structure (see Fig. 4b), low porosity (average porosity ~ 61.52±6.94%; see Fig. 4e), and low biomass inside the biofilm (see Fig. 4h). Fig. 3c shows that the biohydrogen concentration measured by FBG_C2 in the layer with a height of 240 μm first slowly decreased, then quickly decreased, and subsequently slowly decreased. The first slow decrease can be attributed to the biofilm thickness, which is greater than the active thickness of 165 μm, leading to a decrease in the biohydrogen yield as the biofilm thickness increases. The results measured by FBG_C2 are the same as those measured by the H2 microelectrode. The fast decrease in the biohydrogen concentration at a biofilm thickness of 254 μm is due to the coating of FBG_C2 by the biofilm. This means that the measured biohydrogen concentration originates from the internal-layer biofilm having a thickness less than 240 μm because the hydrogen concentration of the photobioreactor measured by the H2 microelectrode did not sharply decrease. When the biofilm thickness is greater than 254 μm, the biohydrogen concentration slowly decreased again; this is attributed to the further decrease in the activity of the hydrogen produced by the biofilm, which has a thickness less than 240 μm. In Fig. 3c, the different sensors show different measured hydrogen concentrations when the biofilm was developed to a mature biofilm with a thickness of 298 μm. This indicates that the biohydrogen yield is different at different thicknesses for the same biofilm because the CQK-01 PSB in the same biofilm will produce a large amount of EPS with increasing biofilm thickness. As the amount of EPS increased, the porosity and active biomass in different layers of the biofilm decreased, as shown in Figs. 4d–f and Figs. 4g–i, respectively. Thus, the mass transfer resistance and light decay increased, meaning that the PSB bacteria in the biofilm were substrateand light-limited and production was inhibited. Further, the hydrogen yield decreased; therefore, the biohydrogen concentration in different layers of the biofilm decreased. Although the bottom-layer biofilm is inactive, the surfacelayer biofilm shows a high hydrogen production activity according the results measured by the H2 microelectrode because the surface-layer biofilm has a loose structure, good porosity (average porosity ~ 88.03±4.96%), and high active biomass, as shown in Figs. 4c, 4f, and 4i. Thus, the hydrogen concentration measured by the H2 microelectrode is the highest of all sensors. In Fig. 3d, FBG_A1, FBG_B1, and FBG_C1 show different measured temperatures. For FBG_A1, the measured temperature first increased and then decreased in the biofilm thickness range of 75–255 μm. The increase in the temperature, which was measured by FBG_A1, is due to the gradual coating of the sensing region of FBG_A1 by the biofilm. The metabolic heat of the biofilm increased as the biofilm thickness increased in the range of 75–108 μm. However, when the biofilm thickness is greater than 108 μm, the active biomass in the biofilm


Page 7 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. (a)–(c) Surface morphologies, (d)–(f) porosities, and (g)–(i) active biomasses of different biofilm layers, which are from a mature biofilm with a thickness of 298 μm. Subfigures a, d, and g are from the bottom-layer biofilm (the vertical distance between the sliced biofilm sample and the biofilm carrier is 80 μm), subfigures b, e, and h are from the inner-layer biofilm (the vertical distance between the sliced biofilm sample and the biofilm carrier is 160 μm), and subfigures c, f, and i are from the surface-layer biofilm.

decreased as the biofilm thickness increased; thus, the metabolic heat of the biofilm at the layer with a height of 80μm decreased. When the biofilm was further developed and was thicker than 255 μm, the measured temperature is very low because the CQK-01 PSB in biofilm were inactive or dead, as shown in Fig. 4g. In Fig. 3d, FBG_B1 also first increased and then decreased in the biofilm thickness range of 153–298 μm. The increase in the temperature can be attributed to the gradual coating of the sensing region of FBG_B1 by the biofilm in its thickness range of 153–175 μm. The active biomass increased as the biofilm thickness increased; thus, the metabolic heat of the biofilm increased. However, when the biofilm thickness is greater than 175 μm, the active biomass in the biofilm at the layer with a height of 160 μm decreased. Hence, the metabolic heat decreased, leading to a decrease in the measured temperature. Furthermore, in Fig. 3d, FBG_C1 first increased and then decreased in the biofilm thickness

range of 236–298 μm. The increase in temperature can be attributed to the gradual coating of the sensing region of FBG_C1 by the biofilm in its thickness range of 153–274 μm. The active biomass increased as the biofilm thickness increased; thus, the metabolic heat of the biofilm increased. However, when the biofilm thickness is greater than 274 μm, the active biomass in the biofilm at the layer with a height of 240 μm decreased. Therefore, the metabolic heat decreased, leading to a decrease in the measured temperature. For FBG_A1, FBG_B1, and FBG_C1, the measured temperature was in the following order: FBG_A1 < FBG_B1 < FBG_C1; the highest measured temperature was produced by the metabolic heat of the biofilm and is about 1.1 °C at a biofilm thickness of 274 μm. This can be attributed to the different active biomasses at different heights in the same biofilm, as shown in Figs. 4g–i.



Furthermore, to analyze the accuracy of the biohydrogen concentration measurement, we use the degree of correlation between the biohydrogen concentrations measured by FBG_C2 and the H2 microelectrode in the biofilm thickness range of 0– 236 μm (Fig. 3c) based on the Grey absolute degree of correlation [absolute degree of Grey incidence (ADGI)].15 The ADGI and data evaluation algorithms are shown in section S4 of the Supporting Information. According to the calculations, we obtained an absolute degree of correlation of 0.9765 between the curves of FBG_C2 and the H2 microelectrode in Fig. 3c. The high degree of correlation demonstrates that FBG_A2, FBG_B2, and FBG_C2 can be used to accurately monitor the biohydrogen concentrations at different heights in the biofilm. Furthermore, these sensors can accurately monitor the temperatures of different layers of the biofilm because the measured biohydrogen concentration needs to use the results measured by these sensors, as shown in the matrix in eq. 8.

CONCLUSION A simple mFBG sensor based on a normal FBG unit demonstrated accurate simultaneous measurement of the hydrogen concentration and temperature. The distributions of the hydrogen concentration and temperature in the biofilm were monitored by using three mFBGs. We discovered that the hydrogen production and metabolic heat from the surfacelayer biofilm to bottom-layer biofilm gradually decreased because of the increase in the amount of EPS and the decreases in the porosity and active biomass. We also found that the active biofilm thickness was 165 μm and that the highest hydrogen concentration was at 12.18K ppm because of the highest active biomass and good porosity. The measured temperature difference from metabolic heat production reached 1.1 °C at a biofilm thickness of 274 μm. In conclusion, the proposed sensor can be used to accurately and simultaneously monitor the biohydrogen concentration and temperature, and it can also be applied in other fields including chemistry, biochemistry, the life sciences, and the environmental sciences.

■ ASSOCIATED CONTENT Supporting Information Additional materials as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org/. Corresponding Author * Tel.:+86-023-62563277, Fax: +86-023-62563277, E-mail address: [email protected] (N.B.Z.), [email protected] (H.X.C.)

Author Contributions N.B.Z. and H.X.C. designed the experiments. M.C. and X.X. performed the experiments. N.B.Z. performed the analysis with the support of H.X.C., H.M.L. and Y.W.W. of the sensor performance and biofim characterization. N.B.Z. and H.X.C. interpreted the results. N.B.Z. wrote the paper. H.X.C., M.C., X.X., H.M.L.and Y.W.W. revised the paper.

ACKNOWLEDGMENT The authors gratefully acknowledge support from the Foundation and Frontier Research Project of Chongqing, China

Page 8 of 10

(cstc2016jcyjA0311); the National Natural Science Foundation of China (NSFC) (51876018, 51806026); the Foundation and Frontier Research Project of Chongqing, China (cstc2015jcyjA40051, cstc2017jcyjAX0268, cstc2018jcyjA2331); the Scientific and Technological Research Program, Chongqing Municipal Education Commission Foundation (KJQN201801117); and the Postgraduate Research Innovation Project of Chongqing (CYS18309).

REFERENCES (1) Tian, X.; Liao, Q.; Liu, W.; Wang, Y. Z.; Zhu, X.; Li, J.; Wang, H., Photo-hydrogen Production Rate of a PVA-Boric Acid Gel Granule Containing Immobilized Photosynthetic Bacteria Cells. Int. J. Hydrogen Energy 2009, 34, 4708–4717. (2) Tian, X.; Liao, Q.; Zhu, X.; Wang, Y., Zhang, P.; Li, J.; Wang, H., Characteristics of a Biofilm Photobioreactor as Applied to Photo-hydrogen Production. Bioresour. Technol. 2010, 101, 977–983. (3) Tomczak, W.; Ferrasse, J.-H.; Giudici-Orticoni, M.-T.; Soric, A., Effect of Hydraulic Retention Time on a Continuous Biohydrogen Production in a Packed Bed Biofilm Reactor with Recirculation Flow of the Liquid Phase. Int. J. Hydrogen Energy 2018, 43, 18883–18895. (4) Märk, J.; Karner, M.; Andre, M.; Rueland, J.; Huck, W. C., Online Process Control of a Pharmaceutical Intermediate in a Fluidized-Bed Drier Environment Using Near-Infrared Spectroscopy. Anal. Chem. 2010, 82, 4209–4215. (5) Zhong, N.; Liao, Q.; Zhu, X.; Chen, R., A Fiber-Optic Sensor for Accurately Monitoring Biofilm Growth in a Hydrogen Production Photobioreactor. Anal. Chem. 2014, 86, 3994– 4001. (6) Liao, Q.; Zhong, N.; Zhu, X.; Huang, Y.; Chen, R., Enhancement of Hydrogen Production by Optimization of Biofilm Growth in a Photobioreactor. Int. J. Hydrogen Energy 2015, 40, 4741–4751. (7) Walter, E. C.; Favier, F.; Penner, R. M., Palladium Mesowire Arrays for Fast Hydrogen Sensors and Hydrogen-Actuated Switches. Anal. Chem. 2002, 74, 1546–1553. (8) Hübner M.; Pavelko R.G.; Barsan, N.; Weimar, U., Influence of Oxygen Backgrounds on Hydrogen Sensing with SnO2 Nanomaterials. Sensors Actuators B: Chem. 2011, 154, 264– 269. (9) Schultz, A. M.; Brown, T. D.; Buric, M. P.; Lee, S.; Gerdes, K.; Ohodnicki, P. R., High Temperature Fiber-Optic Evanescent Wave Hydrogen Sensors Using La-Doped SrTiO3 for SOFC Applications. Sensors Actuators B: Chem. 2015, 221, 1307–1313. (10) Yang, S.; Dai, J.; Qin, Y.; Xiang, F.; Wang, G.; Yang, M., Improved Performance of Fiber Optic Hydrogen Sensor Based on MoO3 by Ion Intercalation. Sensorors Actuators B: Chem. 2018, 270, 333–340. (11) Wang, Y.; Yang, M.; Zhang, G.; Dai, J.; Zhang, Y.; Zhuang, Z.; Hu, W., Fiber Optic Hydrogen Sensor Based on Fabry– Perot Interferometer Coated With Sol-Gel Pt/WO3 Coating. J. Lightwave Technol. 2015, 33, 2530–2534. (12) Wei, X.; Wei, T.; Xiao, H.; Lin, Y. S., Nano-structured PdLong Period Fiber Gratings Integrated Optical Sensor for Hydrogen Detection. Sensorors Actuators B: Chem. 2008, 134, 687–693. (13) Zhong, N.; Chen, M.; Wang, Z.; Xin, X.; Li, B., Photochemical Device for Selective Detection of Phenol in Aqueous Solutions. Lab Chip 2018, 18, 1621–1632. (14) Malachovská, V.; Ribaut, C.; Voisin, V.; Surin, M.; Leclère, P.; Wattiez, R.; Caucheteur, C., Fiber-Optic SPR Immunosensors Tailored To Target Epithelial Cells through Membrane Receptors. Anal. Chem. 2015, 87, 5957–5965.

8


Page 9 of 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry (15) Zhong, N.; Liao, Q.; Zhu, X.; Zhao, M., Fiber Bragg Grating with Polyimide–Silica Hybrid Membrane for Accurately Monitoring Cell Growth and Temperature in a Photobioreactor. Anal. Chem. 2014, 86, 9278–9285. (16) Wang, X.-D.; Wolfbeis, O. S., Fiber-Optic Chemical Sensors and Biosensors (2013–2015). Anal. Chem. 2015, 88, 203–227. (17) Fernandes, D.; Barreto, R. C.; Macedo, A. G.; Da Silva, J. C. C.; Kamikawachi, R. C., A Simple Equation to Describe Cross-Sensitivity Between Temperature and Refractive Index in Fiber Bragg Gratings Refractometers. IEEE Sensors J. 2018, 18, 1104–1110. (18) Zhong, N.; Liao, Q.; Zhu, X.; Wang, Y. Z.; Chen, R., HighQuality Fiber Fabrication in Buffered Hydrofluoric Acid Solution with Ultrasonic Agitation. Appl. Opt. 2013, 52, 1432–1440. (19) Yoo, H.-W.; Cho, S.-Y.; Jeon, H.-J.; Jung, H.-T., WellDefined and High Resolution Pt Nanowire Arrays for a High Performance Hydrogen Sensor by a Surface Scattering Phenomenon. Anal. Chem. 2015, 87, 1480–1484. (20) Ttouillet, A.; Marin, E.; Veillas, C., Fibre Gratings for Hydrogen Sensing. Meas. Sci. Technol. 2006, 17, 1124. (21) Schroeder, K.; Ecke, W.; Willsch, R., Optical Fiber Bragg Grating Hydrogen Sensor Based on Evanescent-Field Interaction with Palladium Thin-Film Transducer. Opt. Lasers Eng. 2009, 47, 1018–1022. (22) Haryanto, A.; Fernando, S.; Murali, N.; Adhikari, S., Current status of hydrogen production techniques by steam reforming of ethanol: a review. Energ. Fuel. 2005, 19, 2098. (23) Chang, H.; Quan, X.; Zhong, N.; Zhang, Z.; Lu, C.; Li, G.; Cheng, Z.; Yang, L., High-efficiency nutrients reclamation from landfill leachate by microalgae Chlorella vulgaris in membrane photobioreactor for bio-lipid production. Bioresource Technol. 2018, 266, 374. (24) Bakke, R.; Olsson, P.Q., Biofilm Thickness Measurements by Light Microscopy. J. Microbiol. Methods 1986, 5, 93–98. (25) Xu, M. G.; Archambault, J.-L.; Reekie, L.; Dakin, J. P., Discrimination Between Strain and Temperature Effects Using Dual-Wavelength Fibre Grating Sensors. Electron. Lett. 1994, 30, 1085–1087. (26) Zhou, X.; Dai, Y.; Zou, M.; Karanja, J. M.; Yang, M., FBG Hydrogen Sensor Based on Spiral Microstructure Ablated by Femtosecond Laser. Sensors Actuators B: Chem. 2016, 236, 392–398. (27) Budiman, P. M.; Wu, T. Y.; Ramanan, R. N.; Jahim, J. Md., Improvement of Biohydrogen Production through Combined Reuses of Palm Oil Mill Effluent Together with Pulp and Paper Mill Effluent in Photofermentation. Energy Fuels 2015, 29, 5816–5824. (28) Li, Y.; Zhong, N.; Liao, Q.; Fu, Q.; Huang, Y.; Zhu, X.; Li, Q., A Biomaterial Doped with LaB6 Nanoparticles as Photothermal Media for Enhancing Biofilm Growth and Hydrogen Production in Photosynthetic Bacteria. Int. J. Hydrogen Energy 2017, 42, 5793–5803. (29) LaMotta, E. J., Internal Diffusion and Reaction in Biological Films. Environ. Sci. Technol. 1976, 10, 765–769. (30) Cheng, K. C.; Demirci, A.; Catchmark, J. M., Advances in Biofilm Reactors for Production of Value-Added Products. Appl. Microbiol. Biotechnol. 2010, 87, 445–456. (31) Hubaux, N.; Wells, G.; Morgenroth, E., Impact of Coexistence of Flocs and Biofilm on Performance of Combined Mitritation-Anammox Granular Sludge Reactors. Water Res. 2015, 68, 127–139. (32) Torresi, E.; Fowler, S. J.; Polesel, F.; Bester, K.; Andersen, H. R.; Smets, B. F.; Plósz, B. G.; Christensson, M., Biofilm Thickness Influences Biodiversity in Nitrifying MBBRs— Implications on Micropollutant Removal. Environ. Sci. Technol. 2016, 50, 9279–9288.

(33) Ding, Y.; Zhou, Y.; Yao, J.; Szymanski, C.; Fredrickson, J.; Shi, L.; Cao, B.; Zhu, Z.; Yu, X.-Y., In Situ Molecular Imaging of the Biofilm and Its Matrix. Anal. Chem. 2016, 88, 11244–11252. (34) Zhong, N.; Zhao, M.; Li, Y., U-Shaped, Double-Tapered, Fiber-Optic Sensor for Effective Biofilm Growth Monitoring. Biomed. Opt. Express 2016, 7, 335–351.

9



Page 10 of 10

For Table of Contents Only

10


Monitoring Biohydrogen Production and Metabolic Heat in Biofilms by

Recommend Documents