1. Introduction
Iron and steel slags are produced as byproducts of steel making and refining. For instance, in 2018, Japan produced 13749 thousand tons of steel slag and 22737 thousand tons of blast furnace slag.1) In addition to the main uses of slag for terrestrial applications such as roadbed material and concrete aggregate1) (Fig. 1(a)), some of them such as calcium silicate and converter lime fertilizers have been used in agriculture (Fig. 1(b)). However, conventional applications of steel slag are currently facing competition from other raw materials. Furthermore, a small component of total steel slag, approximately 1.8% (w/w) corresponding to 255 thousand tons, is landfilled without being reused. This has caused a shortage of final disposal sites for industrial waste.
Therefore, it is crucial to develop additional applications of steel slag, including the use of slag in environments where it is exposed to water, and to induce the functional modification of the slag. Converter slag, which is a steelmaking slag containing various elements such as sulfur and iron, has been used on a trial basis as an environmental remediation agent for the anoxic water masses and anaerobic conditions of rocky coastal waters and marine sediments. For instance, JFE Steel Corporation’s demonstration tests2) improved the quality of marine sediment using the sediment conditioner “Marine Stone”, which is made from the steel slag. Further, Nippon Steel Corporation’s tests3) improved the growth of seaweed beds on the marine environment using materials made from steel slag and corrosive substances.
During the early stages of artificial material development, physical properties such as size and hardness, as well as chemical properties such as the composition of the material content, are emphasized. In addition, interactions with living organisms (biogenetic interactions with materials) are unavoidable when every material in the environment is used (Fig. 1(c)). Inadequate consideration of biogenetic interactions between materials and organisms can lead to environmental pollution caused by the leakage of components from the material. Therefore, to control the leakage of environmentally regulated metals contained in the slag, the use of slag in the environment is governed by various regulations, for instance soil environmental standards for terrestrial areas and sediment standards for marine areas.4,5,6) In addition to environmental pollution, a negative aspect of biogenetic interaction is the corrosion of materials (Fig. 1(c)), which is attributed to the formation of biofilms induced by microbial adsorption to materials.
Field trial tests of slag in environmental quality have succeeded in improving bottom sedimentary anaerobic conditions in tested areas, and in growing and maintaining seaweed such as kelp.7,8) The presence of microorganisms has been confirmed in the periphery of these slag-containing artifacts.9,10) Conversely, some microorganisms have useful applications, such as biological mining (bio-leaching),11,12,13) biological mineral formation (biomineralization),14,15) and biological environmental remediation (bioremediation).16,17) Therefore, biofilms that form on artificial materials in the environment could contribute to water purification and provide a basis for the easy growth of macroalgae and moss.18,19,20) Furthermore, after algae and moss propagate themselves on artificial materials, their photosynthetic organisms become food for other heterotrophic organisms, resulting in a positive effect on biodiversity maintenance.21) However, the function of the biofilm formed on the slag remains unclear because slags are chemically active.
To confirm whether the undesirable effects of slag can be improved using microbial functions,21,22) we investigated the chemical effects of slag biofilms by preparing slag-absorbing microbes, which are slags-coated with biofilm.22) Prior to determining the effect of these biofilm- coated slags, we developed quantitative evaluation techniques for microbial biofilms formed on the slags.21,22) This review outlines our findings to date, particularly emphasizing the evaluation and analysis methods for assessing microbial biofilm on slag.
2. Quantitative Evaluation Methods for the Biofilm Formed on Slags
2.1. Formation of Biofilm on Material Surfaces
Biofilms formed on material surfaces are generally defined as membranous aggregates of bacterial or other microorganismal aggregates, which are covered with extracellular polysubstances (EPS), as produced or secreted by either bacteria or microorganisms.18,19,20) EPS contains many organic carbon compounds such as polysaccharides, proteins, nucleic acids, lipids, and various ions. Many microbes remain inside biofilms. Compared with each free-living state of microbes suspended in the environment, microbes in biofilms have the advantage of efficient acquisition of dilute nutrients diffused in the environment. Their microbes can also escape from predators in the environment by staying in the biofilm.21) In addition, microbial physiological behavior and various physicochemical tolerances by biofilm formation are functionally altered compared to their free-living state.23,24)
Biofilms are formed on materials through various stages.21,25,26) First, organic matter present in the environment is adsorbed onto the material surface to form a conditioning film. Second, suspended microorganisms in the environment attach to the conditioning film, and grow on the material surface. Membranous aggregates are generated on the material surface when proliferating microorganisms produce and secrete EPS. Thus, the remaining floating microorganisms in the environment start to adsorb irreversibly. Here, microbes multiply further on the conditioning film, and the biofilm matures (Fig. 2(a)). For instance, when biofilms are formed on a flat base such as glass, microbial biofilms form partially multilayer structures such as tower-like structures18,22) (Fig. 2(b)). In addition, environmentally formed biofilms are exposed to physical stimuli, such as the force of water flux and physical contact with objects washed away by the water flux.21) When the overgrown biofilm is disrupted and partially collapsed by the physical impacts of water flow, microbes in the biofilm are released as suspended microbes. Their microbes adhere to the niche of the new conditioning film on the material surface. Thereafter, the microbes start to form a new biofilm.
Optical analysis, including microscopy, is a core technique in the analysis and study of various types of biological samples. Other optical measurements, such as spectrophotometers, turbidity and flow cytometers, weight measurement, and a plate count method for microbes using agar-cultured plates, have also been commonly used as evaluation methods for biological samples,19,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45) depending on their sample condition.
Microscope-based measurements have been used to confirm the presence of microbial biofilms formed on the material surface.18,46,47) In addition to the simplest optical microscopes such as a light microscope and electron microscopes capable of high-magnification observation, those with special functions, such as fluorescence and scanning probe microscopes (Fig. 2(b)), have been used to suit various research objectives.18,46,47) When only one side of the glass slide or flat material surface with biofilms can be analyzed and the entire material surface attached by microbes can be scanned, a microscope-based method can serve to quantify the microbial biofilm on the material surface. However, microorganisms do not always adhere to planar materials in the environment or to a certain aspect of the material surface. For instance, slags have a multifaceted granular morphology (Fig. 1(b)). Therefore, unlike in experiments using a glass plate, microbes adhere to not only one side surface of the slag, but also to every aspect of the slag. Thus, one-side surface observation by microscopy renders it difficult to obtain a comprehensive overview of microbial biofilm adsorption on the slag. In addition, slag grains have complex shapes with varied and uneven structures. However, microscopy generally excels in offering detailed analyses at partial magnifications. In terms of biofilms formed on flat materials, their tower-like three-dimensional structures are heterogeneous, showing locally high and low structures.18) Therefore, it is difficult to obtain and understand an entire biofilm overview by observing only a specific part microscopically. Thus, it is difficult to quantitatively evaluate the amount of microbes adsorbed on materials using the microscope-based method (Fig. 2(b)).
2.2.2. Characteristics of Biofilms Quantitative Methods Targeting Specific Biomolecules in Biofilm
In addition to various microscopic methods, biochemical analyses have been applied towards biofilm evaluation.18,19,20,21,22) Specific detection reagents, which reveal organic matter and cellular structures in biological objects, are generally used for quantitative evaluation in biochemical analyses. As mentioned above, biofilms contain various types of organic compounds constituting EPS (Fig. 2(c)), as well as microbial cells. Therefore, these compounds can be targeted for the quantitative analysis using molecular probes. Furthermore, some of the specific detection reagents can be observed at visible light or fluorescence. For instances in which quantitative biofilm evaluation is applied using observable molecule probes, qualitative evaluation of biofilms can also be simultaneously performed in combination with microscopy in principle.
This review gives several examples of methods for biochemical analysis of biofilms. It also summarizes their characteristics: via a method using a crystal violet (CV) dye,48) which is often applied for microbial detection. This reagent reacts with negatively charged biomolecules such as polysaccharides on cell surfaces and stains the target cells purple (Figs. 3(a) and 3(b)).22) In addition, another method entails use of a fluorescent dye (PicoGreen),49) which reacts specifically with DNA in a microbial cell (Figs. 3(c) and 3(d)).22) Alternatively, total organic carbon (TOC) method measures all organic carbon compounds in microbial cells and EPS (Fig. 4(a)).21) Finally, A3 method measures ATPs, such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP), that are involved in energy metabolism in microbial cells (Fig. 4(b)).21,50) Unlike the CV dye and fluorescence methods, the TOC and A3 methods do not directly label specific biomolecules, but they convert the analytes of the biofilm into carbon dioxide (CO2) and luminescence, respectively. Thereafter, the products converted from biofilms are evaluated based on their amounts of CO2 or luminescence.
In the TOC method, all organic carbon compounds are collected and burned. Thereafter, amounts of CO2 from purgeable organic carbon and non-purgeable organic carbon (NPOC), as generated from the combustion, are measured and evaluated based on the amount of TOC contained in the biofilm21) (Fig. 4(a)). In the A3 method, ATP is converted into AMP by reacting ATP with the enzyme luciferase in the presence of luciferin and oxygen. Here, light energy, as bioluminescence, is released. Thereafter, the amount of luminescence is measured and considered to reflect the amount of ATP.21,50) The A3 method shows high sensitivity with high luminescence in that it detects not only ATP, but also both ADP and AMP. This is because ADP and AMP are converted into ATP by each enzymatic reaction21,50) (Fig. 4(b)). Therefore, the A3 method can detect all ATP-related molecules. The amount of luminescence depends on the number of viable microbes (Fig. 4(c)) because their luminescence reflects metabolic activity. Therefore, based on differences in the measurement principles, the TOC and A3 methods can be regarded as indirect evaluation methods of biofilms and their microbes, whereas the CV and fluorescence methods are direct labeling methods.
2.3. Experimental Examples of Quantitative Evaluation of Biofilms on Slag Surface
2.3.1. Issues in Evaluating Biofilms on Slag Using the CV Method
The CV method can be used to evaluate microorganisms using simple equipment, such as an optical microscope. Therefore, CV dye has been used for the qualitative evaluation of biofilms formed on glass and plastic substrates.18,19,20,22,46) In addition to qualitative analysis using CV dye via microscopy, after extraction of CV dye adsorbed on microorganisms using ethanol, the extracted CV dye can be analyzed spectroscopically. This allows for a quantitative determination of the CV concentration, which corresponds to the amount of microorganisms.19) A spectrophotometer is a basic research tool in bio and chemical laboratories. Therefore, spectroscopic analysis, such as the quantitation assay of extracts containing CV dye, is experimentally workable at low cost.29)
The CV method is a direct labeling method for biofilm evaluation. This is because the CV dye binds to polysaccharides in the microbial cell walls in a charge-dependent manner. However, CV dye also reacts with proteins and DNA as target molecules other than polysaccharides.51,52) In addition to organic molecules with negative charges, CV dye can react with inorganic matter. In other words, CV reacts with the slag that is not attached to microbes22) (Figs. 5(a), 5(b) and 5(c)). In particular, the reaction of blast furnace slag with CV dye was clear in appearance (Fig. 5(a)). Furthermore, the appearance of the CV dye extract adsorbed on slag using ethanol and spectral analysis revealed that the extracts of various slags treated with CV dye had absorption maxima at a wavelength similar to a pure CV solution (Figs. 5(c) and 5(d)). Thus, regardless of the type of slag, a reaction occurs between the CV dye and the slag. CV dyes have been used to quantitatively evaluate biofilms on materials such as glass substrates.21) This can be attributed to the fact that these glass substrates are not reactive to CV dye. The reaction of CV dye derived from microbes can be observed in the slag coated with microbes.22) However, it is difficult to accurately evaluate the amount of microbes adsorbed on the slag using the CV method. This is due to the clear reaction of the CV dye with the slag (Figs. 5(a)–5(d)) and the difference in the shape of each slag.
In general, the fluorescence method is superior to the common colorimetric methods in terms of specificity and sensitivity in the detection of target object.29) Furthermore, it is possible to distinguish whether the detected object is an object of interest using the detected fluorescence wavelength clearly.22,29) PicoGreen49) is a fluorescent dye that reacts selectively with biomolecule DNA. This fluorophore is commonly used to quantify DNA extracted from cells (Figs. 3(c) and 3(d)). The chemical structure of PicoGreen is similar to that of SYBR Green I, a well-known DNA-binding fluorophore.53) In addition, PicoGreen is more sensitive to DNA than Hoechst 33258, which is a DNA-binding fluorophore.53) PicoGreen is also cell-permeable because it reacts with nucleoids in bacterial cells.54) Therefore, PicoGreen could also be used as a direct-labeling evaluation method for biofilms similar to the CV method.
As mentioned above, the differences in the individual slag grain shape and the difficulties of scanning the entire surface of the slag grains should be considered in an experiment using slag. Thus, to evaluate the amount of microbes absorbed on the slag, extracts of DNA from microbes adsorbed on slags were prepared and quantified by the fluorescent method using the PicoGreen dye22) (Figs. 5(e) and 5(f)). In particular, microbial DNA can be easily extracted from microbial biofilms adsorbed on slags using the hot-alkaline DNA extraction method22,55) (Fig. 5(e)). Thus, the extracted DNA was quantified by reacting the DNA extract with the Qubit dsDNA BR Assay Kit56) reagent containing PicoGreen fluorescent dye. Both the amount of biofilm on the DNA concentration obtained from the DNA determination assay and the slag weight used in the study were assumed to quantitatively reflect the amount of microbes adsorbed on a certain amount of slag (MB: microbial binding amounts [DNA-μg]/slag-g).22)
The following study was conducted and presented in this review: Bacillus bacteria such as B. subtilis and B. circulans, which are ubiquitous in the environment, were adsorbed on commercially available and fine-grained slag derived from industrial substances such as calcium silicate fertilizer and converter lime fertilizer.22) In this study, calcium silicate fertilizer represented blast furnace slag. Meanwhile, converter lime fertilizer represented the converter slag. The formation of microbial biofilms using these slags was highlighted in a previous study.22) Considering the possibility of pH increase caused by the leakage of components from slag4,5,24) and subsequent microbial detachment into the experimental solvent from the slag surface, the adsorption behavior between slag and microbes was evaluated in a microbial culture medium, which has a strong buffering effect and could expect suppression of a rapid pH increase.22) Using various amounts of microbes (OD600 value) for the addition to blast furnace or converter slag, unadsorbed and suspended microbes were removed through appropriate slag washing after 24 h of adsorption of microbes to slag. Thereafter, the quantity of microbes adsorbed on the slag was evaluated using a fluorescence method.22) Figure 5(f) shows the results obtained using converter slag. Each circle denotes the experimental data for each microbe adsorbed per weight of slag, and their solid lines show the approximate curves using the experimental data derived from average values. Here, OD600 0 is the result of slag immersion in a microbe-free phosphate buffer (PBS) solution to confirm the accuracy of the quantification method. Changes in microbial adsorption on the slag include not only direct adsorption of microorganisms on the slag, but also complex phenomena such as multilayered growth of biofilm thereupon (Fig. 2). Although the interpretation of these results is complicated, the results for OD600 0 indicate that, unlike the CV method, the fluorescence detection method does not react with the slag (Fig. 5(f)). Thus, this method can evaluate only the microbes adsorbed on the slag without the influence of differences in the shape of individual slags.22)
To briefly explain the adsorption behavior of microorganisms on the slag, the amount of microbes adsorbed on the slag increased when low amounts of microbes (OD600 < 0.3) were added to the slag in the range of microbial amounts used in the experiment. When high amounts of microbes (OD600 > 0.3) were added, the amount of microbes adsorbed on the slag decreased (Fig. 5(f)). Looking at varying degrees, this trend observed in the results using converter slag is generally similar to that observed in the results using the blast furnace slag.22)
2.3.3. Characteristics of Quantitative Evaluation of Biofilms on Slag Using the TOC Method
Biomolecules of microbial cells constituting biofilms and extracellular organic carbon compounds, such as EPS secreted by microorganisms, were analyzed using the TOC method. It is necessary to extract all analytes from the biofilm adsorbed on the material surface because the TOC method is not a direct labeling method for specific biomolecules. In the measurement presented in this study (Fig. 6(a)), the material with microbial attachment was placed in a centrifuge tube filled with pure water. Thereafter, the centrifuge tube was vibrated with a vortex mixer for 2 min to detach and suspend the biofilm on the material surface in the solvent. To disrupt the biofilm and microbial cells, the biofilm suspension was subjected to ultrasonic disruption for 5 min using an ultrasonic homogenizer and then centrifuged. Thereafter, the supernatant was measured using the TOC method based on the high-temperature oxidation combustion method21) (Figs. 4(a) and 6(a)).
Figure 6(b) shows the results using the TOC method to evaluate biofilms on slags.21) Converter (the label “Slag F” in Fig. 6(b)) and hot metal pretreatment (the label “Slag 5-2” in Fig. 6(b)) slags were used in this study. For reference, the origin of the hot-metal pretreatment slag is briefly explained. When steel is smelted from scrap, a hot metal is produced. Thereafter, the hot-metal pretreatment slag is made from hot metal treated through desulfurization, desiliconization, and dephosphorization, and is the raw material for the converter process. Biofilms were produced on these slag surfaces using a laboratory biofilm reactor (LBR)21,47,57) (Fig. 6(a)). Herein, artificial seawater with a salinity of approximately 3% (w/v) was circulated at 30°C for 2 weeks. The LBR system induces biofilm formation by allowing atmospheric indigenous bacteria to enter the LBR and produces an indigenous biofilm on the slag surface. The results of TOC analysis indicate that, compared to the condition without biofilm (pure water, slag 5-2 without biofilm, and slag F without biofilm in Fig. 6(b)), the organic carbon content (TOC value) increased substantially depending on the formation of biofilm on the slag (slag 5-2 with biofilm and slag F with biofilm in Fig. 6(b)). Thus, TOC analysis can quantitatively evaluate the biofilm on slag with low interference to the slag. Here, the biofilm formed on slag 5-2 of the hot-metal pretreatment slag was greater than that formed on slag F of the converter slag. This is attributed to the slag composition in this study.21)
2.3.4. Characteristics of Quantitative Evaluation of Biofilms on Slag Using the A3 Method
Generally, ATP, ADP, and AMP are crucial for energy metabolism in organisms (cellular metabolism in Fig. 4(b)). There is a similar need amongst microbes with biofilms. In addition to microbial extracellular substances such as EPS in the biofilm, the greater the biofilm volume, the higher the number of viable microbes expected to be in the biofilm. Thus, the amount of ATPs could be highly dependent on the number of viable bacteria in the biofilm. In this sense, the biofilms on the material surface could be quantified by measuring the ATPs using the A3 assay. Similar to the TOC method, the A3 method, which evaluates bioluminescence, is not a direct labeling method for specific biomolecular structures. Thus, it is necessary to extract all analytes from the biofilm that is adsorbed on the material surface. During the experimentations presented in this paper (Fig. 6(a)), materials with microbial attachment were placed in a centrifuge tube filled with pure water, similar to the TOC method. Thereafter, the centrifuge tube was vibrated with a vortex mixer for 2 min to detach and suspend the biofilm in the solvent. The biofilm suspension was then mixed with specific reagents containing luciferase and luciferin. Thereafter, the amount of luminescence was measured as a relative light unit (RLU) using a portable luminometer (Lumitester Smart: Kikkoman Biochemifa Company, Japan)21) (Fig. 6(a)).
Figure 6(c) shows the results obtained from the A3 method. Here, the biofilm in Fig. 6(c) was formed on the slag using the LBR apparatus, as described in section 2.3.3. After the formation of biofilm on the slag, the biofilm on the slag surface was detached and suspended in pure water as described above. Then, the luminescence derived from the microbial ATPs was evaluated using a luminometer. The results from the A3 method indicate that, compared to conditions without biofilm (pure water, slag 5-2 without biofilm, and slag F without biofilm in Fig. 6(c)), the bioluminescence (RLU value) increased considerably depending on the formation of biofilm on the slag (slag 5-2 with biofilm and slag F with biofilm in Fig. 6(c)). Thus, the A3 method can quantitatively evaluate the biofilm on slag with low interference to the slag. Using the same slag as that described in section 2.3.3, the amount of biofilm between slag 5-2 and F showed a similar trend to the results in Fig. 6(b).
2.3.5. Differences in Interpreting the Amount of Biofilm Obtained Using the CV Method, the Fluorescence Method Targeting DNA, TOC Method, and A3 Method
As mentioned earlier, the CV and fluorescence methods, as direct labeling methods for specific biomolecular structures of microorganisms, are directly correlated with the number of microbes present in the biofilm. However, the CV method cannot be used when the base for biofilm formation is slag due to the reaction of the chemically active slag with the CV dye (Figs. 5(a)–5(d)). The fluorescence method targets DNA in microbes (Fig. 3). As observed in the experiment using the model microorganism shown in Fig. 5, the genome size of the microbe is also likely to be revealed when the microbe added to the slag is an already-known organism. Thereafter, the number of microbes can be calculated from the amount of DNA detected in the measurements. If the microbes that react with the slag are unknown, such as environmental microbes, the microbial species should be able to identify by sequencing DNA extracted from microbes in the biofilm. Here, when the genome size of the microorganisms identified by DNA sequencing is determined from the available database, the number of microbes constituting the biofilm can be evaluated.
In addition to microbial cells, biofilms contain extracellular secretion products such as EPS, which are important for maintaining the three-dimensional structure of biofilms on the material surface. Furthermore, biofilms contain a significant number of dead cells and their cell debris because each life cycle of an individual microorganism is significantly faster than that of multicellular organisms. The TOC method is considered the best method for evaluating biofilms on material by way of its capacity to cover all of these components of the biofilm.
Compared to the free-living state of microbes suspended in a solvent, microbial physiological behavior and various physicochemical tolerances change after the formation of biofilms on the material surface.18,22,24) Using this feature of biofilm, slag coated with microbes might change the leaching behavior of components from the slag in the environment.22) However, when the microbes absorbed on the slag are not alive or have low viability, their changes might be induced by the physicochemical properties of the material coated with microbes. Therefore, to verify the effect of biofilms on the function of materials coated with microbes, it is not sufficient to evaluate the absolute amount of microbes containing both viable and dead cells using the CV and fluorescence methods, as well as to quantify the amount of microbial-derived organic carbon compounds using the TOC method. Therefore, it is necessary to evaluate the number of viable microbes in biofilms. That is, apart from biofilm evaluations such as the CV method, the fluorescence method targeting DNA and the TOC method regardless of whether microbes in the biofilm are viable or dead, there might be analytical situations in which a technique becomes necessary for evaluating only the number of viable microbe. The A3 method, which evaluates only substances produced by viable cell metabolism, is suitable for this type of biofilm evaluation.
Nonetheless, the above features do not mean that one method is better than the other. Each method should be used according to each purpose with respect to biofilm evaluation. When a combination of several methods is used, biofilms can be evaluated from multiple perspectives.
3. Significance of the Biofilm Formed on Slag
3.1. Buffering Effect of the Biofilm Formed on Slag on pH Increase Induced by Slag
This review section presents the study22) performed under conditions described in section 2.3.2. This study examined the chemical effects of biofilms formed on slag, using Bacillus bacteria such as B. subtilis and B. circulans. Furthermore, the researcher used calcium silicate fertilizer to represent the blast furnace slag, and converter lime fertilizer to represent the converter slag. Figure 7(a) shows the experimental flow of this study. The experimental operations were as follows: First, the slag coated with microbial biofilm was prepared using experimental procedures described in section 2.3.2. Here, the amount of microbes reacting with 2 g of slag was equivalent to an OD600 of 0.3. Second, the slag coated with biofilm was immersed in 20 ml of distilled water (DW) for 7 days. Then, the pH of the solvent was measured using a pH meter (Figs. 7(b) and 7(c)).
Slag increases pH in aquatic environments because of the elution of chemical constituents from the slag. Therefore, the use of slag in the environment is regulated.4,5,6) Figures 7(b) and 7(c) show results obtained using the blast furnace slag and converter slag, respectively. The + or - symbols in the figure denote the presence or absence of each element, and DW in Fig. 7 denotes distilled water.
As in several previous reports, the pH of the solvent increased in those experiments using only the slag coated without biofilm; whether the blast furnace (BF) slag or the converter slag (black bars in Figs. 7(b) and 7(c)). The blast furnace slag absorbing microbes (other than the black bar in Fig. 7(b)) significantly suppressed pH increase compared to the slag without biofilm (black bar in Fig. 7(b)). The pH value in the experiment using the converter slag-absorbing microbes (other than the black bar in Fig. 7(c)) was slightly lower than that in the experiment using the corresponding slag alone (black bar in Fig. 7(c)). However, the buffering effect of the converter slag coated with biofilm at the pH was not significant.22) In this experiment, blast furnace and converter slags, as well as two types of microbes of Bacillus bacteria, were used. The results indicated that the effective intensity of buffering by the slag-absorbing microbes depends on the type of slag used.22) The presumed causes of these results are discussed in section 3.2.
3.2. Relationship between the Amount of Biofilm on Slag and its Intensity in pH Buffering Effect
As mentioned above, many microbes remained in the biofilm. However, although there are exceptions that some microbes can grow under severe conditions, most microbes generally grow in a neutral pH environment. The pH increase induced by the slags (Fig. 7(b)) could affect microbial adsorption on each slag, assuming impacts on biofilm formation and maintenance. As shown in Figs. 7(b) and 7(c), the effect of the converter slag-absorbing microbes was weaker than that of the blast furnace slag absorbing microbes. Here, the converter slag had a significant effect on the pH increase compared to the blast furnace slag (black bars in Figs. 7(b) and 7(c)). Therefore, the amount of biofilm formed on each slag is different. Thus, to clarify causal relationships between the formation of biofilm on slag and its pH buffering effect, it is necessary to evaluate whether the amount of biofilm formed on slag is changed by immersion in DW quantitatively.
To elucidate the relationship between amount of biofilm and pH buffering effect, Takahashi22) prepared blast furnace slag or converter slag granules coated with Bacillus bacteria (B. subtilis or B. circulans) under conditions described in section 2.3.2; as described previously, the amount of microbes equivalent to OD600 0.3 was reacted with 2 g of the slag. Thereafter, the slag was immersed in 20 ml of DW for 7 days, as described in section 3.1 (Fig. 7(a)). The supernatant was then removed using a plastic pipette; thereafter, DW (20 ml) was added several times using a plastic pipette to remove microbes detached from the slag. Microbial DNA was extracted from the residue biofilm attached to the slag through hot-alkaline extraction, and the extracted DNA was quantified using the fluorescence method with PicoGreen. The amount of microbes adsorbed per weight of slag before immersion in DW was indicative of the amount of microbes adsorbed on day 0, corresponding to the results in Fig. 5(f). Thereafter, the amount of microbes adsorbed on day 0 was compared with that adsorbed after 7 days of immersion in DW (Fig. 7(d)). Here, microbial adhesion volumes ([DNA-μg]/slag-g) were calculated from the concentration of DNA obtained from the quantitative assay and slag weight. This value was evaluated as the relative value of the amount of microbes adsorbed on a certain amounts of slag.
The buffering effect of the remaining amounts of biofilm on the pH increase induced by the slag was examined. When either of the two types of microbes was used, the blast furnace slag, after 7 days of immersion in DW, maintained almost the same amount of biofilm as the amount of microbes on day 0. Compared to that of the blast furnace slag, the biofilm of B. subtilis or B. circulans on the converter slag was removed from the slag after immersion in DW: removal by approximately 40% of the amount of B. subtilis biofilm compared to the corresponding biofilm on day 0, and more than 70% of the amount of B. circulans biofilm compared to the corresponding biofilm on day 0 (Fig. 7(d)). The pH increase induced by the slag results from the component elution from the slag. However, when using the converter slag with a large detachment of microbes, it retained almost the original slag grain shape after 7 days of immersion in DW.22) Therefore, the collapse of slag grains was not the main cause of the detachment of the biofilm from the slag. Although both slags increased the pH in DW, the effect of the converter slag on the pH increase was greater than that of the blast furnace slag (Figs. 7(b) and 7(c)). Thus, the biofilm formed on the slag was significantly removed from the converter slag because of the rapid increase in pH. Thereafter, the slag surface was exposed to the solvent in some parts of the slag (Fig. 7(d)). Several studies, besides research using slags, have also shown that a highly alkaline environment promotes high detachment of biofilms compared to acidic conditions.58,59) When the biofilm has a buffering effect on the pH induced by slag, the pH buffering effect of the biofilm did not function effectively under the rapid pH increase (Fig. 7(c)).
As shown in Fig. 7, the effect of biofilm on slags was evaluated by the change in pH, which reflects the results of leaching various components from slag.4,5,37) However, it is unclear how the biofilm and the individual microorganisms constituting the biofilm alter the elution of the various components responsible for the pH change. In addition to Fig. 7, the difficulty of biofilm maintenance on the converter slag is also observed in Figs. 6(b) and 6(c). Therefore, to use microbial biofilm as a technology for slag modification effectively, it is important to develop a mechanism to maintain the microbial biofilm on slag under the rapid pH increase caused by the converter slag.
3.3. Potential of Biofilm as a Surface Processing Technology
As mentioned above, the use of steel slag in aquatic environments has been tested as an environmental remediation agent for rocky coastal marine sediments.2,3,7,8,9,10,60) In addition, the formation of biofilms on the slag surface during slag usage in these environments is inevitable. Moreover, microorganisms have been confirmed at the periphery of artifacts containing slag in the water environment.9,10) The change of free-living microorganisms into biofilms not only alters microbial behavior and various physicochemical tolerances18,24) but also modifies the properties of the material. In actuality, the functional change of the material induced by microbes can be inferred from the pH buffering capacity of the slag coated with biofilm as presented in this study22) (Fig. 7). The effects and functional properties of biofilms are poorly understood because of experimental difficulties in conducting detailed analysis of biofilms on the material surface, particularly for chemically active slag. However, environmental trials in the environment have also been conducted without considering effects of biofilms.
Biofilms have been studied due to their negative industrial aspects, such as metal corrosion induced by microbes. If the characteristics of the biofilm, such as pH buffering ability (Fig. 7), can be well utilized, biofilm techniques could contribute to the development of novel slag products by tailoring the elution of slag componets to each purpose. Thus, technology using microbial biofilms could be applied not only to slag but also to other materials as a material processing and surface treatment approach to developing novel materials.
