Biochemical Analysis of Microbial Adsorption Behavior on Iron and Steel Slag Using DNA-specific Fluorescent Reagent, and the Effect of Microbial Biofilm Attached to Slag on pH Buffering Action

Abstract

To evaluate microbial potentials for the material development of iron and steel slag, this study particularly investigated the chemical effect of slag, which was artificially coated with a microbial biofilm, on buffer action. Prior to evaluating the slag, this study also developed a method to determine the amount of microbes adhering to slag. To encourage the growth of Bacillus bacteria on slag, the slag was mixed with the bacteria in LB medium for 24 hours. After extracting microbial DNA using the hot-alkaline DNA extraction method, the microbial quantity attaching tightly to slag was determined from the concentration of the microbial DNA using Pico Green-based fluorometry. The adsorption isotherm between the microbial quantity attached to the slag and the corresponding reacting microbial amount was analyzed using the Langmuir and Freundlich adsorption models. To examine the buffering action of slag coated with and without microbes, each slag was immersed in distilled water for seven days. Next, both pH levels of each slag-containing solution and each amount of microbes attached to slag were determined. The pH increased in both solutions containing slag coated without biofilm and with partially desquamated one; in contrast, the slag coated with well-preserved biofilm showed a buffering action, resulting in an inhibited increase in pH. These results show that slag coated with biofilm is distinctively different from an original slag coated without biofilm in terms of buffer action. This processing technique using microbes could contribute to the development of a novel application of slag as a recycled material.

1. Introduction

Iron and steel slag are industrial byproducts of steel refining and thus are produced in large volumes. Although blast furnace slag and steelmaking slag are almost completely recycled for use as steelmaking slag base and cement for soil aggregate,¹⁾ in its conventional application, slag has faced serious competitive with other resources. To overcome the competitive situation, it is necessary to develop new uses for slag. To cover not only onshore uses but also uses in the presence of water, technical development based on functional changes of slag is essential. In fact, uses of slag in aqueous environment has already been discussed in some reports.^2,3,4)

The utilization of slag is restricted by several environmental lows to prevent contamination by heavy metals that may leak from the slag. For instance, environmental quality standards (EQS) for soil impose a limitation on onshore uses of slag, while EQS for dredge soil subject to the Act on the Prevention of Marine Pollution and Maritime Disaster limits its use in seawater environment. Slag components indeed leak out of slag in an aqueous environment, resulting in a pH increase in the surrounding waters. When using slag in the environment, engineers must consider not only physical (particle size and hardness) and chemical properties (the chemical composition of slag), but also the interaction of slag with environmental organisms. When using a complex mixture of slag and other materials in an aqueous environment, microbial presence was confirmed at the periphery of the slag mixture.^3,4)

From several instances of metallic corrosion, it is known that the interaction between materials and microorganisms occasionally changes the materials’ properties. Their interactions subsequently degrade the material function because of microbial adsorption on the material. However, some microbes have unique functions: bio-mining (bioleaching)^5,6,7) for harvesting metallic resources from the environment, biomineralization⁸⁾ for mineral formation, and bioremediation^10,11) for environmental cleanup. These microbes are sources of potential industrial and environmental benefits.

This study aimed at evaluating whether microbial function modifies slag as a material in some way. First, the slag was artificially coated with a microbial biofilm. Second, the chemical properties of the slag coated with biofilm were investigated. Prior to evaluating the slag, a method was developed to determine the amount of microbes adhering to the slag. In this study, we focused on pH change because it is not only easily assessable but also reflects the metal elution from slag.

2. Experimental

2.1. Slags, Their Chemical Compositions, and Microbes Used in This Study

2.1.1. Slags and Analysis of Their Chemical Composition

This study used two types of slag: slag from steel manufacturing and commercially available slag diverted for fertilizers (Figs. 1(a) and 1(b)). This study considered calcium silicate slag (Figs. 1(a) and 1(c)) as a model of the blast furnace slag and converter lime (Figs. 1(b) and 1(d)) as a model of the converter slag. To justify slag particle size between 1 mm and 5 mm, they were sieved. These sifted slags were subjected to microbial adsorption, and then the effect of microbial biofilm attached to the slag on pH buffering was determined. The chemical compositions of the slags were analyzed using fluorescent X-ray analysis by an external analytical institute. Table 1 shows the chemical compositions of the calcium silicate slag and converter lime. The basic constituent molecules of both slags were CaO, SiO₂, Al₂O₃ and MgO. The converter lime made from converter slag was black because converter lime had larger amounts of Fe₂O₃ than blast furnace slag (Figs. 1(c) and 1(d)).

Fig. 1.

Slag and bacteria used in this study. (a–d) Calcium silicate fertilizer (a & c) used as a model of blast furnace slag, and converter lime fertilizer (b & d) used as a model of converter slag, respectively. (e & f) Phase-contrast microscopic images of (e) B. subtilis cells and (f) B. circulans cells. The scale resolution is 1 mm in the panels (a & b). Each scale bar is 500 μm in the panels (c & d), and 50 μm in the panels (e & f), respectively. (Online version in color.)

Table 1. Chemical compositions of iron and steel slags used for this study [mass%]. (A) Chemical compositions of calcium silicate fertilizer used as a model of blast furnace slag. (B) Chemical compositions of converter lime fertilizer used as a model of converter slag.

(A)
	CaO	SiO2	Al2O3	MgO	SO3	TiO2	K2O	Fe2O3	MnO	Na2O
mass%	42.59	33.61	13.78	5.694	1.976	0.531	0.48	0.463	0.366	0.268

(B)
	CaO	SiO2	Al2O3	MgO	SO3	TiO2	Fe2O3	MnO	P2O5	V2O5	Cr2O3
mass%	50.6	13.52	2.188	3.385	0.17	0.355	23.36	4.148	1.591	0.241	0.22

2.1.2. Microorganisms

In this study, Bacillus subtilis (Strain JCM11003) and B. circulans (Strain JCM2504) were used as common bacteria in the environment for the subsequent adsorption treatment of the slag (Figs. 1(e) and 1(f)). These bacteria were purchased from the Japan Collection of Microorganisms, RIKEN BioResource Research Center (BRC).

2.2. Adsorption of Microbes onto Slag and Estimation of Their Microbial Quantity Attached to the Slag

2.2.1. Adsorption Treatment of Microbes onto the Slag

Figure 2 is a flow diagram of the microbial adsorption treatment of the slags and the following experiment procedures. To increase the number of Bacillus bacteria for the following experiment in advance, bacteria were cultured in liquid Luria Broth (LB) medium for 24 h at 30°C. We used commercially available LB tablets to prepare the LB medium, which included tryptone, yeast extract and sodium chloride as basic components. The cell density of microbes precultured in the LB medium was determined as the optical density measured at 600 nm (OD₆₀₀) using spectrophotometry. To adsorb bacteria onto blast furnace slag or converter slag, each slag was mixed with bacteria in the LB medium for 24 h at 30°C. The ratio between the LB medium as a solvent and slag conformed to JIS K 0058-1: 2005 (Method for chemicals in slags Part 1: Leaching test). Thus, the amount of solvent used was 10 times the weight of the slag. After the adsorption of microbes onto the slag, culture supernatants containing suspended and non-adhering microbes were removed using a plastic pipette. After that, the slag was washed with a washing solution (10 times the weight of the slag). Then, fresh washing solution was poured into the slag using a plastic pipette and subsequently eliminated. The washing procedure was repeated three times to remove non-adhering microbes from the slag. The residue slag was considered the slag, which was artificially coated with a microbial biofilm. Then, the slag coated with microbes was used for all the experiments mentioned in sections 2.2.2 and further. Slag coated without microbes, which was prepared by mixing slag with the phosphate buffer (PBS; 136 mM NaCl, 2.6 mM KCl, 1.4 mM KH₂PO₄, 8 mM Na₂HPO₄ at pH 7.3), was used for comparison. The slag coated without microbes was also subjected to the same washing procedure as the slag coated with microbes.

Fig. 2.

Flow diagram of experimental procedures in this study. Here, the inset photograph is of the blast furnace slag. The treatment indicated with an asterisk was used for the adsorption isotherm analysis of the amount of microbes attached to slag. (Online version in color.)

Here, different washing solutions were used for different experiments. Slag changes the pH of an aqueous environment: thus, a rapid change in pH might lead to the detachment of microbes adhering to the slag. Therefore, PBS was used as the washing solution in an experiment to determine the quantity of microbes tightly attached to the slag. In the experiment for determining the pH buffering action of the slag coated with microbes, distilled water (DW), but not PBS, was used as the washing solution to exclude the residue of any buffer components.

In addition, a different reaction scale was used in different experiments. For an analysis of microbial adsorption, a small-scale experimental system (0.1 g of slag/1 mL of LB medium) was used for preparing several solutions with various densities of microbes, from low to high. In other analyses, such as the pH buffering effect of slag coated with microbes, the experimental scale was set to enable the pH measurement with an electrode (2 g of slag/20 mL of LB medium for the experimental procedure of microbial adsorption onto slag; 2 g of slag/20 mL of each solvent such as DW for pH measurement).

2.2.2. pH Measurement of Solvents in which Slag was Immersed

To confirm an impact of slag on solvent pH, after immersing the slag (blast furnace slag or converter slag) in DW, artificial pond water (APW; 0.1 mM NaCl, 0.1 mM KCl, 0.1 mM CaCl₂ at pH 6.17)^14,17) or van’t Hoff artificial seawater (ASW; 462 mM NaCl, 35.5 mM MgCl₂, 9.39 mM KCl, 99.8 mM MgSO₄, 8.50 mM CaCl₂, 59.5 mM NaHCO₃ at pH 8.50),¹⁴⁾ the pH of each solvent was measured using a pH meter. Furthermore, after 24 h of mixing the slag and LB medium with microbes, the culture supernatant was collected to measure pH. Herein, the pH of the LB medium without microbes was measured as a control reference.

To evaluate the effect of microbial adsorption onto the slag, the slag coated with microbes was immersed in DW for 7 days. After 7 days of immersion, the pH was measured. The pH of the solvent, in which the slag coated without microbes was immersed for 7 days, was used as a control.

2.2.3. Detection and Quantitative Estimation of Microbes

(1) Reaction of Crystal Violet Dye with Slag

The crystal violet (CV) dye¹⁸⁾ is often used to detect microorganisms as it reacts with negatively charged biomolecules, such as polysaccharides, on the cell surface (Figs. 3(a) and 3(b)). To determine the quantity of microbes adsorbed on the slag, it is important to know whether the slag itself reacts with CV. Herein, after the treatment of slag with CV, the reaction of slag with CV was examined. First, the PBS solution containing 0.2% (w/v) of CV (abbreviated as CV staining solution) was prepared. After adding 1 mL of CV staining solution to 0.1 g of slag using a plastic pipette, the slag was stained with CV dye at 30°C for 1 h. The reaction supernatant was removed using a plastic pipette to eliminate the unreacted CV dye from the slag. Then, 1 mL of DW was added to the slag using a plastic pipette, and the reaction supernatant was removed. This washing procedure was repeated four times to remove the unreacted CV dye from the slag. Further, the slag treated with CV was dried in an oven at 60°C overnight and examined. After examining the slag treated with CV, the slag was treated with 95% (v/v) ethanol and incubated for 10 min at room temperature. The CV dye absorbed on the slag was extracted from the slag with ethanol. To remove the slag from the ethanol extract, the reaction container containing the slag was centrifuged. Then, the extract supernatant was collected to evaluate CV-dependent absorbance using spectrophotometry at wavelengths from 400 nm to 700 nm. For the control experiment, only the PBS solution was added to the slag. Then, an ethanol extract from the slag treated with PBS was prepared. Furthermore, a CV solution (DW containing 10 mg/L of CV) was prepared as a reference for the CV dye in the ethanol extract.

Fig. 3.

Detection reagent of microbes. (a) Schematic diagrams of the microbial cell wall and its cell membrane used to detect molecules with negative potential. (b) Molecular structure of chromatically crystal violet reagent. (c) Schematic diagrams of microbial DNA. (d) Molecular structure of fluorescent PicoGreen dye that is used to detect DNA. (Online version in color.)

(2) Extraction of Microbial DNA from Slag Coated with Microbes and Determination of Microbial Quantity Adhering onto the Slag Using Fluorometry

PicoGreen¹⁹⁾ is a fluorescent dye that reacts selectively with DNA among biomolecules, and is used for the quantification of DNA extracted from cells (Figs. 3(c) and 3(d)). To determine the quantity of microbes adhering to the slag, PicoGreen dye was used in this study. Herein, the slag coated with microbes was prepared as described in section 2.2.1. The slag coated with microbes was immersed in an alkaline solution (50 mM Tris-HCl at pH 11) containing 10% (w/v) Chelex 100 resin²⁰⁾ and heated at 100°C for 10 min. After heating the slag, the slag was precipitated by centrifugation. Then, the reaction supernatant containing microbial DNAs was collected using a plastic pipette. To determine the DNA content of the extract using the hot-alkaline DNA extraction method described above, the DNA extracts were treated with Qubit dsDNA BR assay kit reagents²¹⁾ containing PicoGreen fluorescent dye. The quantities of microbes tightly attached to the slag (MB: Microbaial binding amounts [DNA·μg]/slag·g) were calculated from both the concentration of their microbial DNA and the slag weight used in the experiment.

(3) Adsorption Isotherm of the Amount of Microbial Biofilm Attached to the Slag

An adsorption isotherm was derived using each microbial quantity attached to the slag (MB), and the corresponding microbial quantity used for each reaction (OD₆₀₀). Herein, the microbial amount for each reaction indicates the corresponding density of microbes (OD₆₀₀ value) used to coat the slag. To determine the maximum adsorbed amount of microbial biofilm (MB_max) on the first layer surface of the slag, values obtained as MB were analyzed using three analysis plots: analysis plot I, $\frac{1}{\text{MB}}$ vs. $\frac{1}{{\text{OD}}_{600}}$; Analysis plot II, MB vs. $\frac{\text{MB}}{{\text{OD}}_{600}}$; Analysis plot III, log₁₀MB vs. log₁₀OD₆₀₀. To evaluate the validity of the MB_max values obtained from each analysis plot compared to the corresponding actual measured values, the values from the analyses plots were subjected to an outlier test using a t-test calculated from the statistic value (t_cat) (Eq. (1)) and the probability in the t-distribution table with n-1 degrees of freedom (Eqs. (2) and (3)).   

$$t_{cat}=\frac{\left|M{B}_{max.}-Av{g}_{exp.}\right|}{SD}\sqrt{\frac{n}{n+1}}$$(1)

  

$$P=P(\left|t\right|\ge t_{cat})$$(2)

  

$$t=t\left(n-1,\mathrm{\hspace{0.17em} }\alpha \right)$$(3)

The MB_max and Avg_exp values in Eq. (1) respectively represent the maximum adsorbed amount of microbial biofilm on the slag calculated from each analysis plot and the average value of the experimental measurements corresponding to the maximum adsorption of microbes on the slag; n denotes the number of experimental data points. Herein, the value, α=0.05, was used to infer significance in Eq. (3).

2.3. Analysis of the Correlation between the Amount of Microbial Biofilm Attached to the Slag and pH Variation Induced by the Slag

First, the slag coated with microbes was made by adding microbes corresponding to an amount of 0.3 of OD₆₀₀ to 2 g of the slag. After immersing the slag coated with microbes in 20 mL of DW for 7 days, the solvent pH was measured. After removing the supernatant using a plastic pipette, 20 mL of fresh DW was added using a plastic pipette. This washing procedure was repeated three times to completely remove the microorganisms not attached to the slag. The washed slag was dried in an oven at 60°C. Microbial DNA derived from microbes adhering to the slag was then extracted and quantified as described in section 2.2.3(2). Herein, the initial quantity of microbial DNA per slag weight was considered the amount of microbes adhering to the slag at day 0 before immersion in DW, and compared with the amount of microbes adhering to slag after immersion in DW for 7 days.

3. Results and Discussion

3.1. Manipulation of Microbial Adhesion to the Slag Surface and its Effects

To investigate the effect of microbial adhesion on slag, this study used commercially available calcium silicate slag and converter lime derived from actual steelmaking processes. These slags were selected as experimental materials because their particles are a few millimeters in size, which is convenient for microbial experiments. Slag particle clearly varied in size, as shown in Figs. 1(a) and 1(b). When slag is placed in an aqueous environment, its components leach out. Their elution concentration is related to the particle size.²²⁾ Therefore, both slags were sieved to adjust the particle size before the subsequent microbial adhesion experiments. It is commonly known that when slag is immersed in a solvent, the pH of the solvent increases (Fig. 4(a)). The pH increased rapidly in the case of freshwater, such as DW and APW, because freshwater generally has a weaker buffering action than ASW. When using a complex solvent containing several chemical substances, the individual effects of slag and bacteria adhering to the slag are difficult to quantify. Therefore, the effect of microbial adhesion to slag on pH was examined using DW, but not APW or ASW containing multiple salts, in the pH evaluation experiments.

Fig. 4.

Effect of microbial biofilm attached to the slag on buffer action. (a) The increase in pH of each solvent induced by slag. “DW” represents distilled water, “APW” represents artificial pond water, and “ASW” represents artificial sea water. (b) The changes in pH induced by each slag in LB medium. A pH value in LB medium was compared with each value in the media containing slag and microbes. (c & d) Bar graphs showing (c) the changes in the pH induced by the blast furnace slag coated without or with biofilm in DW, and (d) those by converter slag coated without or with biofilm in DW. A pH value induced by slag only was compared with the pH values by slag coated with biofilm. All error bars indicate standard deviation in panels b, c, and d. An asterisk denotes statistical significance using the t-test (P < 0.05). (Online version in color.)

First, the slag was mixed with the quantity of bacteria equivalent to 0.3 of OD₆₀₀ in the LB medium using the procedure described in section 2.2.1. After 24 h of mixing the slag and LB medium containing bacteria (Fig. 2), the pH of the LB medium was determined using a pH meter (Fig. 4(b)). Here, the symbols (+) and (−) indicate the presence or absence of each item, as shown in Fig. 4(b). When the blast furnace slag was immersed in the LB medium containing bacteria, the LB medium showed little change in pH after 24 h of mixing. In contrast to the blast furnace slag, the immersion of the converter slag in the LB medium produced a statistically significant increase in pH compared to the pH of the LB medium without converter slag. Herein, the differences between the adsorptions of different microbial species on slag were small. The pH increase caused by both slags in the LB medium was smaller than that in DW (Fig. 4(a)) because of the high buffering action of the LB medium. The slag coated with microbes was obtained by removing unadsorbed microbes from each slag (Fig. 2).

The slag coated with microbes obtained using the above procedure was further immersed in DW. The pH was then evaluated after 7 days of immersion. The experiments with either slag showed a greater increase in pH in DW than in the LB medium (Figs. 4(c) and 4(d)) because of the luck of strong buffering capacity in DW (Fig. 4(b)). The blast furnace slag coated with microbes (except for the black bar in Fig. 4(c)), however, showed significant suppression of pH increase compared to the blast furnace slag without microbes (black bar in Fig. 4(c)). Although the pH values obtained in the experiments using converter slag coated with microbes (except for the black bar in Fig. 4(d)) were slightly lower than those of the converter slag alone (black bar in Fig. 4(d)), the effect of microbes adhering to the slag was not statistically significant. These results showed that the pH buffering capacity of the slag coated with microbes depended on the type of the slag rather than the microbial species used.

Herein, microorganisms adhering to a material surface are generally called a biofilm.^23,24,25) Biofilm is a biogenic film-like material, in which bacteria reside mainly on solid material surface.²³⁾ A large number of microbes are generally present in biofilms. Multiple kinds of microbes thrive at a neutral pH. The pH increase induced by the slag itself (Fig. 4(a)) might affect the adsorption of microbes on the material. In particular, the pH buffering effect of microbes adhering to the converter slag was weaker than that of microbes on the blast furnace slag (Figs. 4(c) and 4(d)). However, regardless of the microbial adsorption on the slag, the converter slag produced a greater pH increase than the blast furnace slag (Fig. 4(a)). Therefore, the amount of microbes adhering to the slag might vary between the blast furnace slag and converter one. To determine the correlation between microbial adsorption on the slag and its pH buffering capacity, it is essential to evaluate each change from the point immediately after the microbial adsorption on the slag to the other point after the subsequent immersion of the slag coated with microbes in solvents such as DW.

3.2. Determination of the Amount of Microbes, i.e. the Amount of Biofilm, Adhering to the Slag Surface

3.2.1. Reaction of CV Dye with the Slag

Various microscopic observations and biochemical analyses have been used to observe biofilms formed by the adsorption of microorganisms on material surfaces. In contrast to various microscopy methods that directly visualize microorganisms adhering to the surface of the material, biochemical methods of analysis use extracts containing specific biomolecules. These molecules are detected or quantified using specific reagents that quantitatively react with them. It is possible to quantify the microbial biofilm adhering to the material surface using microscopy only when the microbes adhering to just one side of a glass slide or a flat material surface are the targets of the analysis, and only when the entire surface can be scanned. In contrast, in the case of slag (Figs. 1(a)–1(d)), microorganisms can adhere to the entire surface of the slag particle. Thus, microscopic observation of only one surface of the slag cannot be used to evaluate microbial adsorption on the slag.

In this study, we developed a biochemical method for the evaluation of the amount of microbes adhering to the slag. The CV dye has been previously used for the visualization and quantification of microorganisms, especially for the evaluation of biofilms formed on glass and plastic plates.^23,24,25) Therefore, this study also considered the use of the CV dye for the evaluation of microbial adsorption on the slag. The reaction of CV with the slag, particularly the blast furnace slag, induced a clear color change even without microbial adsorption on the slag (Figs. 5(a) and 5(b)). In contrast to the blast furnace slag, the reaction of CV with the converter slag was not clear because the original color of the converter slag was black (Figs. 5(c) and 5(d)). Therefore, after extracting the CV absorbed on the slag using ethanol, the extract was assessed using spectral measurement. Even though the reaction of CV with the converter slag just could not be visually confirmed, it was clearly confirmed by ethanol extraction of CV from slag (Fig. 5(e)). Furthermore, each slag extract had an adsorption maximum at the same wavelength as the pure CV solution (Fig. 5(f)). These results indicate that CV reacted with the slag itself.

Fig. 5.

Reaction of slag with crystal violet (CV). (a & b) Photos of granulated blast furnace slag stained without (a) and with CV (b). (c & d) Granulated converter slag stained without (c) and with CV (d). (e) Ethanol extracts from each slag stained without and with CV, and that of CV solution (10 mg/L) as a reference. (f) Absorption spectra of an ethanol extract from each slag stained without and with CV, and the CV solution (10 mg/L). (Online version in color.)

CV generally reacts with polysaccharides on the microbial cell wall in a charge-dependent manner (Fig. 3(a)) and stains the target cells purple, enabling the evaluation of microbial quantity using simple equipment such as a microscope and a spectrophotometer. In addition to polysaccharides, CV also reacts with other biomolecules, such as proteins and DNA.²⁴⁾ Further, CV might react not only with organic materials but also with inorganic materials. In fact, CV reacted with the slag itself in this experiment (Fig. 5). Although the reaction of the slag coated with microbes with CV depended on microbial quantity in our preliminary experiments (data not shown), the absorbance of CV on slag alone varied because of differences in slag particle shapes. From the above results, it was difficult to accurately evaluate the amount of microbes adsorbed on slag using the CV dye method alone.

3.2.2. Microbial Detection Using a Fluorescence Method

Fluorescence methods are generally superior to most colorimetric methods in terms of specificity and sensitivity.^28,29) Here, we used a fluorescence method to detect microbes because CV was not suitable in the presence of slag. After extracting DNA from microbes adhering to the slag using the hot-alkaline DNA extraction method, the concentration of the extracted DNA was determined and evaluated as the amount of microbes adhering to the slag (Fig. 6(a)).

Fig. 6.

Adsorption isotherm analysis of the amount of microbial biofilm attached to slag. (a) Flow diagram of experimental procedures for microbial DNA measurement. (b & c) Absorption isotherm of microbes onto (b) the blast furnace slag and (c) the converter slag. (b’) A partially magnified view of panel (b). (Online version in color.)

The amount of microbes adhering to the slag was evaluated after 24 h of treatment using different amounts of each bacterium (OD₆₀₀ value). Figures 6(b) and 6(c) show the results for the blast furnace slag and the converter slag, respectively; Fig. 6(b’) is a partially enlarged view of Fig. 6(b). The points on the graph represent the measured values expressed as the amount of each bacterium adsorbed per weight of slag. The solid lines are the results of calculation using the measured values. To confirm the accuracy of this method, the 0 OD₆₀₀ point, obtained using slag immersed in microbe-free PBS, was prepared. Even if Bacillus sp. bacteria were not inoculated in LB medium at the beginning of the experiment, unforeseen microorganisms developed using the nutrients in LB medium. These unforeseen microbes might have been introduced into the reaction medium during the experimental procedures or they might have adhered to the slag during the storage. In any case, an experiment using LB medium failed to establish an experimental condition of microbial concentration at 0 OD₆₀₀. Therefore, to determine the microbial concentration at 0, PBS was used instead of the LB medium in this study. Unlike the results obtained using the CV method, the results at 0 OD₆₀₀ revealed that the PicoGreen dye did not react with slag. Thus, this method could be used to evaluate only the microbes adhering to the slag without being affected by the difference in the shape of slag particles.

Furthermore, the results showed that the lower the amount of microbes added to the reaction medium, the more the quantity of microbes adhering to the slag increased, at least in the range of microbial amounts less than 1.0 OD₆₀₀. In contrast, at high values of OD₆₀₀, the number of microbes adhering to the slag tended to decrease. Although there was a difference in the degree, this trend was roughly similar between the experiments using blast furnace slag and converter slag.

3.3. Analysis of the Adsorption Isotherm of Microbes Attached to Slag

Microbial biofilms form partially multilayer structures, such as tower-like structures, even when biofilms are formed on a flat substrate, such as glass. The tower-like structure of biofilm is not homogeneous but heterogeneous, with locally high and low structures.²³⁾ Considering the geometrical characteristics of the biofilm, the microbial biofilm formed on the slag by the adsorption of Bacillus sp. bacteria might have a multilayered structure. Thus, it was assumed that the biofilm might contain not only microbes in direct contact with the slag but also microbes layered on microbes and thus in indirect contact with slag. However, it was considered that an interface between the slag surface and microbes could be analyzed using the concept of adsorption isotherms. Herein, the results obtained in Figs. 6(b) and 6(c) were considered as adsorption isotherms and were further analyzed. Several analytical models, such as the Langmuir and the Freundlich equations, were used to analyze the adsorption behavior between an adsorbent and its corresponding acceptor. The Langmuir equation is mainly used to analyze monolayer adsorption events between an adsorbent and its acceptor, while the Freundlich equation is an experimental equation for reversible adsorption and is not generally limited to monolayer adsorption.³⁰⁾ In addition, the Dubinin-Radushkevich equation and the Temkin equation are derived based on the empirical rules for specific phenomena.³⁰⁾ To calculate the amount of microbes adhering to the slag surface, the experimental data were analyzed using the plots for the Langmuir and the Freundlich models. More specifically, the analytical plot I ($\frac{1}{\text{MB}}$ vs. $\frac{1}{{\text{OD}}_{600}}$) (Fig. 7(a)) and analytical plot II (MB vs. $\frac{\text{MB}}{{\text{OD}}_{600}}$) (Fig. 7(b)) were used as the Langmuir model, while analytical plot III (log₁₀MB vs. log₁₀OD₆₀₀) (Fig. 7(c)) was used as the Freundlich model. Particularly, the above analysis was applied to the results obtained using the converter slag (Fig. 6(c)), as shown in Fig. 7. It is notable that the graphs shown in Figs. 6(b) and 6(c) are different in shape from the general adsorption isotherms between an adsorbent and its acceptor. The graphs (Figs. 6(b) and 6(c)) showed a high adsorption capacity for microbes adhering to the slag at low OD₆₀₀ values and relatively low capacity at high OD₆₀₀ values. Therefore, the slopes of the lines for each approximation formula calculated using any of the above analytical plots in the graphs (Figs. 7(a)–7(c)) were opposite to those obtained by analyzing general adsorption isotherms. The intersection of each approximation formula with the corresponding vertical axis (i.e., the intercept with the vertical axis) is expressed as $\frac{1}{{\text{MB}}_{\text{max}}}$ for analytical plot I, MB_max for plot II and log₁₀(MB_max) for plot III. It was computationally possible to calculate the maximum amount of microbes adhering to the outermost layer of the slag surface (MB_max) using the intercepts. As previously described, when the amount of microbes added to the slag was higher than a certain OD₆₀₀ value, the number of microbes adhering to the slag tended to be lower (Figs. 6(b) and 6(c)). Therefore, it was assumed that microbial biofilm did not grow excessively, after the microbial adsorption to the outermost layer of the slag surface at the maximum added amount of microbes (OD₆₀₀ = 1.0). Based on this hypothesis, the amount of microbial adsorption on the slag under this condition (OD₆₀₀ = 1.0) was assumed to be equivalent to MB_max. To statistically evaluate whether the computational value calculated from each analytical plot was numerically valid as MB_max under this assumption, the MB_max value using the analytical plots was compared with the actual microbial adsorption on the slag at 1.0 OD₆₀₀ (Fig. 7(d)). As a result, in addition to not being rejected statistically against the measured values, all computational MB_max values calculated from all analytical plots fell within the standard deviation of the measured values. In summary, all computational MB_max values were equivalent to the corresponding measured values.

Fig. 7.

Analysis of the maximum adsorbed amount of microbial biofilm onto the converter slag. (a–c) Analytical plots of (a) $\frac{1}{\text{MB}}$ vs. $\frac{1}{{\text{OD}}_{600}}$, (b) MB vs. $\frac{\text{MB}}{{\text{OD}}_{600}}$, and (c) log₁₀MB vs log₁₀OD₆₀₀. Here, MB indicates the amount of microbial biofilm attached to slag. (d) A comparison of each maximum adsorbed amount of microbial biofilm onto the slag, calculated using each analytical plot with an average value from the actual measured values. The circle, rhombus, triangle, and square symbols represent data for the actual measured values for panel (a), panel (b), and panel (c), respectively. (Online version in color.)

Using MB_max values, adsorption isotherms generated from the measured data (Figs. 6(b) and 6(c)) and the following Eqs. (4) and (5), a new adsorption isotherm confined to the outermost layer of slag surface was derived by subtracting values corresponding to a growth of microbial biofilm on slag (Figs. 8(a) & 8(b)). The MB_max value obtained from the analysis using the Freundlich model was used in Fig. 8.   

$$\begin{array}{l}\mathrm{E}\mathrm{a}\mathrm{c}\mathrm{h}\mathrm{m}\mathrm{i}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{b}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{d}\mathrm{h}\mathrm{e}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{l}\mathrm{a}\mathrm{g}=\\ \mathrm{M}{\mathrm{B}}_{\mathrm{m}\mathrm{a}\mathrm{x}}(\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{m}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{u}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}\ge \\ \mathrm{M}{\mathrm{B}}_{\mathrm{m}\mathrm{a}\mathrm{x}}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{f}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{a}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s})\end{array}$$(4)

  

$$\begin{array}{l}\mathrm{E}\mathrm{a}\mathrm{c}\mathrm{h}\mathrm{m}\mathrm{i}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{b}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{a}\mathrm{d}\mathrm{h}\mathrm{e}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{l}\mathrm{a}\mathrm{g}=\\ \mathrm{t}\mathrm{h}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{m}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{u}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}\\ (\mathrm{i}\mathrm{n}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{c}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{m}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{u}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}<\\ \mathrm{M}{\mathrm{B}}_{\mathrm{m}\mathrm{a}\mathrm{x}}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}\mathrm{\hspace{0.17em} }\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{c}\mathrm{u}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{f}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{a}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s})\end{array}$$(5)

Simulated curves for the new adsorption isotherms were derived using the above calculations, and the curves were similar to the general adsorption isotherms for monolayer adsorption. In these simulated curves, the microbial adhesion increased as the microbial concentration (OD₆₀₀ value) increased, and the adhesion gradually saturated (dotted lines for B. subtilis and B. circulans in Fig. 8(a), and dotted line for B. circulans in Fig. 8(b)). Furthermore, even when using MB_max values obtained from analytical plots other than the Freundlich model, the graphs had approximately the same shape as those obtained using the Freundlich model. The differences between the adsorption isotherms for monolayer adsorption (dotted lines in Figs. 8(a) and 8(b)) calculated from the MB_max values and the measured data (solid graphs in Figs. 8(a) and 8(b)) might represent the growth of microbial biofilm on the slag. However, using this analysis method, no range, where the amount of microbial adsorption on slag would be completely stable, was found for B. subtilis on the blast furnace slag (Fig. 8(b)). Therefore, the graph shape was slightly different from the standard adsorption isotherm, reflecting that the amount of B. subtilis adsorbed on the blast furnace slag increased even in the range of high microbial concentration (up to 1.0 OD₆₀₀). The appropriate MB_max value could not be calculated in this analysis because this study did not acquire data containing the corresponding MB_max value without overgrowth of microbial biofilm on the blast furnace slag. To solve this experimental problem and derive an accurate adsorption isotherm for monolayer adsorption of microbes on the blast furnace slag, it is necessary to conduct experiments at concentrations of microbes higher than 1.0 OD₆₀₀ or with shorter incubation times before the overgrowth of the microbial biofilm to obtain a value corresponding to MB_max on the outermost layer of the slag surface.

Fig. 8.

Adsorption isotherm using actual measured values only (shown with solid lines) and a simulated curve using values calculated from an analytical plot using the Freundlich model (shown with dot-lines). (a & b) Adsorption isotherm of the amount of microbial biofilm attached to (a) the converter slag and (b) the blast furnace slag. (b’) A partially magnified view of panel (b). (Online version in color.)

3.4. Relationship between the Amount of Microbes, i.e. Biofilm Volume, Attached to the Slag and the Buffering Action Against the pH Increase Induced by Slag

The microbial adsorption on the blast furnace slag demonstrated a high buffering action meaning offsetting of the pH increase induced by slag (Fig. 4), while the effect of microbial adsorption was not clear in the case of the converter slag. Considering the amount of microbial adsorption on the slag, this study re-examined the differences between both conditions using each slag in the buffering experiment. The slag coated with microbes was treated using the quantity of microbes equivalent of 0.3 OD₆₀₀. Immediately after the washing of the slag, the microbial amount quantified from the slag was considered the amount of microbial adsorption on the slag at day 0 before the immersion in the solvent. The other slag coated with microbes, which was not subjected to the DNA extraction after washing, was then immersed in DW for 7 days. The amount of microbes adhering to the slag after 7 days of immersion was compared with that before the immersion (the amount of microbial adsorption on slag at day 0). As a result, regardless of the species of Bacillus bacteria used, the blast furnace slag continued to hold almost the same amount of microbes after 7 days of immersion in DW compared with the amount at day 0. In contrast, approximately 40% of B. subtilis biofilm and 70% of B. circulans biofilm were removed after 7 days of immersion of the converter slag in DW compared to the respective microbial amount on day 0 (Fig. 9(a)). Although both slags increased the pH in DW, the effect for the converter slag was greater than the blast furnace slag (Fig. 4(a)), suggesting that microbes adsorbed on converter slag were dissociated from the slag because of the rapid pH increase. Because of the leakage of slag components, the slag was considered to be partially dissolved in the aqueous environment. However, even after the large detachment of microbes from the converter slag, after being immersed in DW for 7 days, its particles had almost the same shape as the initial slag grains before the immersion (Fig. 9(b)).

Fig. 9.

Relationship between the amount of microbes attached to slag and buffering action. (a) A comparison of the amount of microbes attached to slag just after the adsorption treatment and the amount after immersion of the corresponding slag in DW for seven days. An asterisk denotes statistical significance using the t-test (P < 0.05). (b) Converter slag after immersion treatment of slag in DW for 7 days and the following microbial DNA extraction. A scale bar is 5 mm in the panel (b). (c) The relationship between the amount of microbial biofilm attached to slag and buffering action against the increase in pH induced by slag. The error bars, a color bar, and a dotted line indicate standard deviation, biofilm density onto slag, and the microbial amounts on the slag just after the attachment treatment, respectively. The black and white vertical lines indicate the pH values using blast furnace slag coated without biofilm and the values using converter slag coated without biofilm, respectively. (Online version in color.)

Figure 9(c) shows the relationship between the residual ratio of microbes adhering to the slag and the pH increase induced by the slag. The black and white vertical lines in Fig. 9(c) indicate the pH values after immersion of the slag without microbes in DW. The circles and triangles indicate the results for the experiments using the blast furnace slag coated with microbes and converter slag done with microbes, respectively. The dotted line indicates the amount of microbes adsorbed on the slag at day 0 before the immersion of the slag in DW. As a result, the higher the residual amount of microbial adsorption on the slag became, the more inhibited was the pH increase. However, in the case of the converter slag, the biofilm was partly dissociated into the solvent during immersion of the slag coated with microbes in DW for 7 days (Fig. 9(a)). Thus, it was inferred that some areas on the slag surface, which were no longer coated with enough microbes, got exposed to the solvent after the dissociation of microbes from the slag.

This study evaluated the effect of slags coated with microbes on the pH of the solution. The changes in pH were induced by the slag because of the leaching of various components from the slag. In future studies, in addition to monitoring of the pH change, it is necessary to analyze how the leakage of components from the slag is affected by microbes attached to the slag. In the case of the converter slag, microbes absorbed on the slag were dissociated from slag because of the rapid pH increase. Therefore, it is necessary to develop a method for maintaining the microbial biofilms on the slag even under high pH conditions.

3.5. Significance of Microbial Adsorption on Slag

This study revealed that microbial adsorption on the slag could be analyzed using the concept of reaction kinetics (Figs. 6, 7, 8). In other words, the adsorption of microbes on the slag can be well controlled. In addition to evaluating microbial adsorption on an interface between the slag and microbial biofilm, this study devised a quantitative method of analysis for estimating the growth of multi-layered biofilms on the slag using adsorption isotherms. From these results, regardless of the type of slag, the lower was the amount of microbes added to the reaction, the greater was the amount of microbes adsorbed on the slag. However, at high amounts of microbes added to the reaction, the amount of microbes adsorbed on the slag tended to decrease (upper part of Fig. 10).

Fig. 10.

Explanatory diagram of both adsorption behavior of microbes to slag and the effect of microbial biofilm attached to slag on pH. (Online version in color.)

When microbes form biofilms, their physiological behavior and various physicochemical tolerances change compared to their original free-living state, which they are suspended in liquid medium.^22,23) Therefore, pre-coating of slag with microbes may change the elution behavior of the slag components. This can be inferred from the pH buffering effect of the slag coated with microbes in both the observation and measurement results in this study (Figs. 4, 9, and 10). Making good use of these features of the slag coated with microbes, this technique might contribute to the development of new slag products with varying component elution according to the purpose of use. Furthermore, this technique might have the potential to expand into the material development field as a novel material processing and surface treatment technology using microorganisms.

4. Conclusion

This study investigated the effect of microbial adsorption on the slag on the pH change induced by the slag. In addition, this study developed a method for evaluation of the amount of microbes adsorbed on the slag surface. The results obtained from this study are summarized as follows:

(1) Fluorescent DNA detection using PicoGreen could selectively evaluate only the microbes adsorbed on the slag without reacting with the slag.

(2) Depending on the experimental conditions, the adsorption of microbes on the slag surface included not only monolayer adsorption but also multiple-layered growth of the biofilm. The graph for microbial adsorption on the slag was different in shape from the general adsorption isotherm for an adsorbent and its acceptor.

(3) After the absorption of microbes on the slag, the microbial biofilm formed on the slag surface dissociated depending on the type of slag. Furthermore, the maintenance of microbial biofilms on the slag was important for buffering the pH increase induced by slag.

Acknowledgements

This research was supported by the Iron and Steel Institute of Japan (ISIJ) Research Promotion Grant from ISIJ, a Grant for the Reserch Group I entitled “New functionalities of iron and steelmaking slags by biofilm coating” from the ISIJ, and a Grand for the Reserch Forum entitled “Analysis and evaluation of slag with functional changes induced by a chemical or biological treatment” from the ISIJ.

References

• 1)  Nippon Slag Association: Tekko Sulagu Toukeinenpou (Iron and Steel Slag Statistics), (FS-169), 2, (accessed 2019-12-01) (in Japanese).
• 2)   N.  Kinjo,  N.  Touch and  T.  Hibino: J. Jpn. Soc. Civ. Eng., Ser. B2 (Coast. Eng.), 72 (2016), I_1333. https://doi.org/10.2208/kaigan.72.I_1333
• 3)   O.  Miki,  C.  Ueki and  T.  Kato: J. Water Environ. Technol., 11 (2013), 101. https://doi.org/10.2965/jwet.2013.101
• 4)   A.  Aneksampant,  A.  Tanaka,  X.  Tu,  H.  Iwai,  M.  Yamamoto,  K.  Nakashima and  M.  Fukushima: Anal. Sci., 34 (2018), 1303. https://doi.org/10.2116/analsci.18P147
• 5)   H.  Brandl: Biotechnology, Special Processes, Vol. 10, ed. by H.-J. Rehm and G. Reed, Wiley-VCH, Weinheim, (2001), 191. https://doi.org/10.1002/9783527620937.ch8
• 6)   N.  Okibe: J. Environ. Biotechnol., 11 (2011), 39 (in Japanese).
• 7)   H.  Jin,  D. W.  Reed,  V. S.  Thompson,  Y.  Fujita,  Y.  Jiao,  M.  Crain-Zamora,  J.  Fisher,  K.  Scalzone,  M.  Griffel,  D.  Hartley and  J. W.  Sutherland: ACS Sustain. Chem. Eng., 7 (2019), 15311. https://doi.org/10.1021/acssuschemeng.9b02584
• 8)   W.  Lin,  K.  Benzerara,  D.  Faivre and  Y.  Pan: Front. Microbiol., 5 (2014), 293. https://doi.org/10.3389/fmicb.2014.00293
• 9)   D.  Arias,  L. A.  Cisternas and  M.  Rivas: Crystals, 7 (2017), 345. https://doi.org/10.3390/cryst7110345
• 10)   O. B.  Ojuederie and  O. O.  Babalola: Int. J. Environ. Res. Public Health, 14 (2017), 1504. https://doi.org/10.3390/ijerph17051504
• 11)   P.  Bhatt,  Y.  Huang,  H.  Zhan and  S.  Chen: Front. Microbiol., 10 (2019), 1778. https://doi.org/10.3389/fmicb.2019.01778
• 12)   S.  Yokoyama,  T.  Shimomura,  M. N. N.  Hisyamudin,  T.  Takahashi and  M.  Izaki: J. Phys. Conf. Ser., 352 (2012), 012051. https://doi.org/10.1088/1742-6596/352/1/012051
• 13)   S.  Yokoyama,  A.  Suzuki,  Nik  H. B. M. N.,  H.  Kanematsu,  A.  Ogawa,  T.  Takahashi,  M.  Izaki and  M.  Umemoto: ISIJ Int., 50 (2010), 630. https://doi.org/10.2355/isijinternational.50.630
• 14)   T.  Takahashi,  Y.  Ogura,  A.  Ogawa,  H.  Kanematsu and  S.  Yokoyama: J. Water Environ. Technol., 10 (2012), 347. https://doi.org/10.2965/jwet.2012.347
• 15)   T.  Takahashi and  S.  Yokoyama: ISIJ Int., 56 (2016), 1497. https://doi.org/10.2355/isijinternational.ISIJINT-2015-539
• 16)   T.  Takahashi: Monitoring Artificial Materials and Microbes in Marine Ecosystems, Interactions and Assessment Methods, Marine Ecology, Current and Future Developments, Vol. 2, ed. by T. Takahashi, Bentham Science Publishers, Sharjah, (2020), 255.
• 17)   Y.  Tominaga,  K.  Kuchitsu,  M.  Katsuhara,  M.  Tazawa and  S.  Miyachi: Plant Cell Physiol., 32 (1991), 261. https://doi.org/10.1093/oxfordjournals.pcp.a078072
• 18)   T. J.  Beveridge: Biotech. Histochem., 76 (2001), 111. https://doi.org/10.1080/bih.76.3.111.118
• 19)   A. I.  Dragan,  J. R.  Casas-Finet,  E. S.  Bishop,  R. J.  Strouse,  M. A.  Schenerman and  C. D.  Geddes: Biophys. J., 99 (2010), 3010. https://doi.org/10.1016/j.bpj.2010.09.012
• 20)  Bio-Rad Laboratories, Inc.: Chelex 100 Resin, https://www.bio-rad.com/en-jp/product/chelex-100-resin?ID=6448ab3e-b96a-4162-9124-7b7d2330288e, (accessed 2020-02-29).
• 21)  Thermo Fisher Scientific Inc.: Nucleic Acid Quantitation in Solution—Section 8.3, https://www.thermofisher.com/jp/ja/home/references/molecular-probes-the-handbook/nucleic-acid-detection-and-genomics-technology/nucleic-acid-detection-and-quantitation-in-solution.html, (accessed 2020-02-29).
• 22)   K.  Nakamura and  M.  Yoneda: J. Jpn. Soc. Mater. Cycles Waste Manag., 25 (2014), 25 (in Japanese). https://doi.org/10.3985/jjsmcwm.25.25
• 23)   H.  Kanematsu and  M.  Tanaka: Bunseki Kagaku, 63 (2014), 569 (in Japanese). https://doi.org/10.2116/bunsekikagaku.63.569
• 24)   G. A.  O’Toole: J. Vis. Exp., 47 (2011), 2437. https://doi.org/10.3791/2437
• 25)   J. H.  Merritt,  D. E.  Kadouri and  G. A.  O’Toole: Curr. Protoc. Microbiol., (2005), Unit-1B.1. https://doi.org/10.1002/9780471729259.mc01b01s00
• 28)   T.  Takahashi: Molecules, 24 (2019), 4441. https://doi.org/10.3390/molecules24244441
• 29)   T.  Takahashi: Sci. Rep., 6 (2016), 31638. https://doi.org/10.1038/srep31638
• 30)   K. Y.  Foo and  B. H.  Hameed: Chem. Eng. J., 156 (2010), 2. https://doi.org/10.1016/j.cej.2009.09.013