Chemical compositions of EAF steel slags used for this study (mass %).
Abstract
Seaweed has long been an important kitchen ingredient and a functional food material. Microalgae have attracted the same attention as seaweed from food, pharmaceutical and cosmetic companies because several algae contain unique functional materials. Industry application of algae requires the selection of useful algal species, evaluation of their features and monitoring of their quality in culture. Taking Chlorella for example, this chapter presents a method using flow cytometry (FCM) to assess not only the number of algae but also algal quality. First, Chlorella was cultured in media containing eluate from steel slag as an experimental factor and trace metals. After the treatment of algae with eluate, the number and physiological features of algae were evaluated, respectively, using hemocytometry and FCM. Results show that eluate from slag induced neither lethality nor growth inhibition. Coupled with hemocytometry, FCM was used to estimate vigorous and aberrant algal status. Consequently, the eluate did not give rise to algae stresses. Interestingly, the addition of slag eluate increased the amounts of the carbonate species. The increase in the carbonate species actually triggered the potential increase in aqueous CO2 for photosynthesis, eventually inducing algal proliferation. These analyses can support evaluation of algal features and maintenance of their quality for industry application.
Keywords
- food science
- Chlorella
- Chlorophyll
- flow cytometry
- fluorescence spectroscopy
- trace elements
- steel slag
1. Introduction
Aqueous photosynthetic organisms such as algae are the foundation of aquatic ecosystems. The quantities of aqueous photosynthetic organisms as producers in the aqueous ecosystem support yields of both fish and shellfish. In addition to its role as a producer in aqueous ecosystems, seaweed has long been an important kitchen ingredient as a functional food material, providing functional nutrients such as carotenoids and anti‐oxidative fucoxanthin. A biorefinery that can take advantage of biofunctions presents an alternative concept to that of conventional refineries that manufacture materials using fossil fuels. Especially, autotrophic algal biorefineries present great advantages over conventional microbial biorefineries such as those using fermentation. Some microalgae species such as
The industrial application of algae demands the selection of useful algal
Taking green algae
2. Algal characteristics using FCM
Over the last few decades, FCM has become widely used as a powerful and valuable tool for studies of cell biology, microbiology, protein engineering and healthcare. Several functions of FCM include several procedures such as cell counting, biomarker detection and cell sorting through assessment of cell optical information. Figure 1 presents an outline of a flow cytometric instrument used for this study. This flow cytometer, which detects several optical properties, is equipped with a green laser operating at 532 nm. Forward scatter (FSC) signals were collected to ascertain the cell size. Red fluorescence is detected in the red fluorescence channel through a 680/30 nm band pass filter. Simultaneously, a yellow fluorescence channel through a 576/28 nm band pass filter is used [10–13]. Each fluorescence is converted into an electrical pulse. The electrical intensity is then quantified for each level of fluorescence intensity.
When heterotrophic cells, such as animal cells, are targeted for FCM measurement, fluorescence‐labelling antibodies against certain biomolecules are used to detect and quantify the biomolecules. When using phototrophic cells, such as phytoplankton and plant cells, a photosynthetic pigment, chlorophyll, can also function as a biomarker similar to a fluorescence labelling antibody. When exposed to appropriate excitation light, chlorophyll in each cell irradiates red fluorescence (Figure 2A and B) [12]. Figure 2C depicts emission spectra of
Chlorophyll is sensitive to physiological factors such as heat and acid. These physical factors eventually cause inactive chlorophyll because of degradation [11–13]. In fact, a previous study using
3. Features of steel slag used for this study
Iron and steel slags from blast furnace slag and steel making slag including converter slag and electric arc furnace (EAF) slag are produced as steel industrial by‐products. All blast furnace slag can be recycled completely for the use of steel making slag base and cement as soil aggregate [17, 18], although several volumes of steel making slag, particularly EAF slag, ultimately end up in landfill sites [19]. New applications of slag, such as depurative and sand capping materials in aquatic environments [20] have been regarded both as decreasing the amounts of discarded slag and as reducing high costs of discarding slag. Several environmental pollution laws, however, have restricted slag use in aquatic environments because steel slag contains environmentally hazardous substances. The toxicity of eluate from EAF slag for aquatic organisms remains poorly understood [11, 13, 21], but the physiochemical properties and effects of converter slag on organisms have been documented often [22–27].
This study specifically examined stainless steel slag (designated as slag A) and common steel slag (slag B), exhausted respectively from oxidation processes of stainless and common steelmaking in EAF processes [11, 13]. Table 1 presents compositions of EAF slags used for this study [11, 13, 21, 28, 29]. In brief, slag A contains more SiO2, CaO, and Cr2O3 than slag B does, whereas slag A contains less FeO than slag B. All Fe and Cr compounds are described respectively as FeO or Cr2O3 because it is generally difficult to distinguish FeO and Cr2O3 formed form Fe and Cr in a suspended metal solution after alkali fusion of stainless steel slag [11, 13].
FeO | SiO2 | CaO | Al2O3 | MgO | MnO | Cr2O3 | ZnO | NiO | CuO | |
---|---|---|---|---|---|---|---|---|---|---|
Slag A | 0.74 | 44.1 | 33 | 5.39 | 7.68 | 4.09 | 3.29 | 0.01 | 0.06 | 0.024 |
Slag B | 35.1 | 19.2 | 20.8 | 15.2 | 4.1 | 5.1 | 0.43 | 0.071 | 0.028 | 0.025 |
4. Research methods
The author used
Steel slag was subjected to a leaching test based on JIS K0058‐1: 2005 (Method for chemicals in slags Part 1: Leaching test) to elute metal components of slag with HCl [11, 13, 21, 28, 29, 32]. After elution, the solution was filtrated with a 0.45 μm pore filter to eliminate slag particles as described in previous reports. The filtrated eluate from the slag (designated respectively as eluate A and eluate B) was used for bioassay with
To assess the eluate effects on algal growth,
Algae (initial density of 1.0 × 104 cells/ml adjusted using hemocytometry) were cultured with CA medium containing eluate from each slag for 1 week in a plastic tube under an LD cycle (12 h light/12 h dark) at approximately 1100 lux of natural white fluorescent light and 23 ± 2°C as described in previous reports [11, 13]. After treatment of algae with eluate, the number and physiological features of algae were quantified respectively using hemocytometry and FCM. The algal proliferation ratio (average ± standard error) was expressed as a proportion of the number of algae treated with eluate to that of control without eluate [11, 13].
To investigate algal status using FCM, the algal status was analysed and estimated based on the corresponding fluorescence. In brief, the stress of each alga is portrayed as a two‐dimensional graph (2D map) of red fluorescence intensity (665–695 nm) as the index of vigorous algae and yellow fluorescence intensity (562–590 nm) as the index of variant algae [11, 13]. To facilitate comparison of vigorous algae with stressed and dying algae, a reference standard of algae subjected to stress was prepared by treatment of algae with heat for 5 min at 100°C (designated as heated algae) [11, 13]. For FCM analyses, FSC signals detected only in the culture medium were removed as technical noise from FCM measurements as described in previous reports [11, 13]. The remaining signals were re‐analysed as algal signals.
Aquatic CO2 (CO2(aq)) concentrations are related directly with photosynthesis and algal proliferation. CO2(aq), HCO3− and CO32− are present as the carbonate species in solution, as presented in Eq. (1)–(3) [11, 13].
For aqueous photosynthetic organisms, CO2(aq) of these carbonate species is particularly necessary to support photosynthesis. We ascertained the concentration of CO2(aq) in slag eluate using potentiometry with a diaphragm‐type electrode to measure the CO2(aq) concentrations [11, 13]. Both HCO3− and CO32- can be estimated as CO2(aq) in acidic conditions (≤ pH 4.0) as portrayed in Figure 4 resulting from the following Henderson–Hasselbalch Eqs. (4) and (5) [11, 13].
The respective pK values of pK1 = 6.35 and pK2 = 10.33 [33] were used for this study. The carbonate species aside from CO2(aq) were converted into CO2(aq) by adding a pH‐adjustable solution. Then they were estimated as the amounts of total carbonate species. Each concentration of CO2(aq), HCO3−, and CO32- was calculated from the amounts of total carbonate species and pH values using Eqs. (4) and (5) above. Here, the [H2CO3(aq)] given by Eq. (2) was expressed as [CO2(aq)] in Eq. (4) because it was difficult to distinguish CO2(aq) from H2CO3(aq) in solution, as described in previous reports [11, 13]. The result was expressed as the concentration of CO2(aq) (average ± standard error) under each condition [11, 13].
In general, the amounts of Ca2+ and Mg2+ are related to the water hardness and are highly reactive with carbonate species. To examine whether these elements contribute to the concentration of CO2(aq) in solution, the amounts of Ca2+ and Mg2+ were measured before and after treatment of algae with CA medium containing slag eluate [11, 13]. After treatment of algae with CA medium containing eluate, the culture tube including the algae was centrifuged. The supernatant, which no longer included algae, was collected and subjected to elemental analysis. Several organic compounds, such as biomolecules reportedly interfere with these measurements [34]. Therefore, this study applied colorimetric determination using specific chelate reagents to elucidate the amounts of Ca2+ and Mg2+ in the culture supernatant. In practice, the chlorophosphonazo‐III method [35] and the xylidyl blue‐I method [36] were used for the evaluation of the concentration of Ca2+ and that of Mg2+. Moreover, elemental concentrations before treatment of algae with eluate were compared statistically with those after treatment using
5. Results and discussion
Before evaluating the effects of slag eluates on algae, the concentrations of elements in each slag eluate were analysed and discussed (Table 2). In addition to the results of leaching tests for slag, the environmental quality standards (EQSs) for soil pollution and for marine and water pollution are shown as reference values in Table 2 [13]. In brief, concentrations of Ca, Mg and Si eluted from the slag samples were high because these slags contained large amounts of those materials (Table 1). In contrast to the elements above, the eluted concentrations of Al and Fe were quite low in spite of their high concentrations in the slag particles. An earlier report [32] explained this contradictory phenomenon as a difference of these elements in terms of solubility. Leaching tests revealed that concentrations of components eluted from two slags used for this study were almost all lower than the respective EQSs, except for selenium (Se) in eluate from the slag A (eluate A), as described in earlier reports [11–13].
This study examined the effects of slag eluates as an experimentally stress factor on algal growth, particularly that of
Origin of slag | Eluate of EAF stainless steel oxidation slag (Slag A) | Eluate of EAF normal steel oxidation slag (Slag B) | Environmental quality standards | |||||
---|---|---|---|---|---|---|---|---|
Soil pollution | Marine pollution | Water pollutant | Effluent standard | Drinking water standard | ||||
Total As | ND1 (RDL2: 0.001) | ND3 | 0.01 | 0.1 | 0.01 | 0.1 | 0.01 | |
Total B | 0.16 | 0.283 | 1 | 14 | 104, 2305 | 1 | ||
Total Be | ND (RDL: 0.0005) |
ND3 | 2.5 | |||||
Total Cd | ND (RDL: 0.0001) | ND3 | 0.01 | 0.1 | 0.01 | 0.036 | 0.003 | |
Chromium (VI) |
ND (RDL: 0.005) |
ND3 | 0.05 | 0.5 | 0.05 | 0.5 | 0.05 | |
Total Cu | 0.003 | ND | 0.001 | 3 | 3 | 1 | ||
Total Pb | ND (RDL: 0.0005) | ND3 | 0.01 | 0.1 | 0.01 | 0.1 | 0.01 | |
Hg | ND (RDL: 0.0001) | ND3 | 0.0005 | 0.005 | 0.0005 | 0.005 | 0.0005 | |
Total Ni | 0.001 | ND | 0.001 | 1.2 | 0.02 | |||
Total Se | 0.012 | 0.0033 | 0.01 | 0.1 | 0.01 | 0.1 | 0.01 | |
Total V | ND (RDL: 0.001) |
0.013 | 1.5 | |||||
Total Zn | 0.099 | 0.0143 | 2 | 0.037, 0.029, 0.019 | 2 | 1 | ||
F− | ND (RDL: 0.1) | 0.53 | 0.8 | 15 | 0.84 | 84, 155 | 0.8 | |
Total Al | ND | 1.8 | ||||||
Total Ca | 9.3 | 10.1 | 30010 | |||||
Total Fe | ND | 0.23 | 10 | 0.3 | ||||
Total Mg | 0.9 | 1.1 | 30010 | |||||
Total Mn | 0.028 | ND | 10 | 0.05 | ||||
Total Si | 1.8 | 1.9 | ||||||
Total N | 0.4 | 0.3623 | 0.1–111 0.2–18 |
100 | 0.0412, 1013 | |||
Total P | ND (RDL: 0.1) | ND3 | 0.005–111 0.02–0.098 |
16 |
In addition to algal population estimation using hemocytometry, estimation of the cellular status of algae using FCM was conducted. The results are presented two‐dimensionally in Figure 6 [11–13]. Here, each single dot on the 2D map represents optical information of a single alga. To compare the status of vigorous algae with that of stressed and dying algae, algae treated with heat (heated sample) were prepared as a reference standard of algae subjected to stress [11–13]. Results show that the 2D map of the red versus the yellow fluorescence intensity for control algae differs clearly from that for the heated algae (Figure 6). The 2D map of the red versus yellow fluorescence intensity for control algae showed respectively 102–103 on the red channel and 101–102 on the yellow, whereas that for the heated algae did 101–102 on the red channel and 101–103 on the yellow. It is particularly interesting that the dot distribution of algae treated with each slag eluate closely resembled that of control, although that with each eluate shifted slightly upward relative to that of control algae [11–13].
Quantitative analysis of algal distribution patterns (Table 3) was conducted along with qualitative analysis of those patterns (Figure 6). Each graph in Figure 6 is divisible into four subareas (regions I–IV) based on algal viability [11–13]: region I represents an area for vigorous algae; region II includes dead and variant algae such as heated algae; region III includes algae with low red fluorescence intensity; and region IV includes data from which algae are virtually absent. Algal distribution patterning revealed clear differences between the algal distribution in control samples and those in heated algae. The signals of algae treated with eluate were also distributed almost entirely to region I. The ratio was 96.81 ± 2.60% in control, 98.15 ± 0.31% in eluate A, and 98.13 ± 0.24% in eluate B. Ratio analysis shows that the percentages of algae treated with slag eluates were slightly higher than those of control algae. Components dissolved from slags did not apparently give rise to algae stress because the quantities of algae in media containing the respective eluates were equal to or greater than those in media with no eluate (Figure 5). The tested slags contain metals such as copper, zinc and aluminium (Table 2). Aluminium, which is also not contained in the CA medium, has been particularly reported as inhibiting plant growth [37, 38]. Although the culture medium containing aluminium and other metal elements was predicted to affect algal growth and status, they caused no effect on algal growth directly. The data demonstrated that components eluted from slag showed no marked toxicity to algae. This assessment system using FCM, which estimates chlorophyll fluorescence of photosynthetic pigments, might be applicable to other algae, other phytoplankton and aquatic plants with chlorophyll, although this report presents data only for
I | II | III | IV | |
---|---|---|---|---|
Control | 96.81 ± 2.60 | 2.40 ± 2.91 | 0.73 ± 0.31 | 0.07 ± 0.08 |
Heated sample | 0.28 ± 0.36 | 97.27 ± 0.81 | 2.59 ± 0.42 | 0.02 ± 0.03 |
Eluate A | 98.15 ± 0.31 | 0.17 ± 0.08 | 1.67 ± 0.21 | 0.02 ± 0.03 |
Eluate B | 98.13 ± 0.24 | 0.52 ± 0.51 | 1.29 ± 0.33 | 0.05 ± 0.05 |
It remains unclear why algae in media containing eluate proliferated more than algae in media without eluate (Figure 5). In general, the growth and proliferation of photosynthetic organisms, such as land plants and algae, depend strongly on photosynthetic efficiency. Photosynthesis is divisible mainly into two metabolizing systems: light‐dependent reactions, which harvest light energy from sunlight and which perform electron transport; and the Calvin cycle, which performs CO2 fixation to synthesize glucose. This study particularly examined the concentrations of CO2, which are related to the Calvin cycle, because all experiments in this study were conducted under constant light conditions [11, 13]. This CO2(aq) can be detected as infrared absorption near 2350 cm-1 using FT‐IR [11, 13], which is identical to the infrared absorption attributable to anti‐symmetric stretching of CO2 [39]. To examine the relation between algal proliferation and the concentration of CO2(aq) under treatment of algae with slag eluate, this study directly evaluated CO2(aq) in medium containing eluate using FT‐IR [11, 13]. The result shows that both eluates had higher infrared absorption identical to CO2 than that of controls without slag eluate [11, 13]. Next, concentrations of CO2(aq) under each test condition were quantified from the amounts of total carbonate species using a diaphragm‐type electrode to measure CO2(aq) and from calculation using the Henderson–Hasselbalch equations (Figure 7) [11, 13]. The result also showed that concentrations of CO2(aq) in media containing eluate were higher than those of control samples. Moreover, the concentration in the medium containing eluate B had higher concentrations than that in eluate A. Speculating based on these obtained data, the addition of slag eluate appears to improve aqueous environments for photosynthetic organisms. It might facilitate algal photosynthesis more than CA medium alone. Consequently, increasing concentrations of CO2(aq) by adding slag eluates induced greater algal proliferation than that in the control sample (Figure 5). Figure 5 shows that this study also stumbled on the fact that the addition of eluate B to the culture medium induced greater proliferation of algae than that of eluate A. Accounting for the different concentrations of CO2(aq) between eluate A and eluate B, the greater effects of eluate B than those of eluate A on algal proliferation might also be explained.
Formation of the greater concentrations of CO2(aq) by addition of slag eluate than the control condition has been discussed in the literature [11, 13]. Ca2+ and Mg2+ are highly reactive substrates with the carbonate species. These elements conveniently existed in higher contents of eluates than other elements (see Table 2). As portrayed in Figure 4, the fraction of HCO3− is the highest content of the carbonate species at pH 7.2, which were experimental conditions used for this study. Therefore, Ca2+ in eluate, for instance, might be presumably reacted with HCO3− as described in the following Eq. (6).
Ca(HCO3)2 can interfere in the chemical equilibrium of the carbonate species as HCO3− because Ca(HCO3)2 is ionized completely. Here, the ratios of concentrations of the respective carbonate species must be constant in solution. Consequently, the increase in HCO3− concentrations in solution prompts Eq. (2) to proceed leftward, resulting in increasing concentrations of CO2(aq). Increased CO2(aq) might be consumed by algae as a raw material of photosynthesis. Assessment of the transitional change of concentrations of Ca2+ and Mg2+ revealed no significant difference between the concentrations of these elements before and after incubation of algae with eluates, even at 7 days after incubation (Table 4). These obtained data support the hypothesis that the addition of slag eluate, particularly Ca2+ in eluate, increases the amounts of the total carbonate species and that the increase in the total amounts of the carbonate species by adding slag eluates triggers the potential increase of CO2(aq), eventually inducing algal proliferation.
Before incubation | After incubation | ||
---|---|---|---|
Concentration of Ca2+ (mg/L) | |||
Eluate A | 9.904 | 8.945 ± 0.917 | |
Eluate B | 10.464 | 9.763 ± 1.056 | |
Eluate A | 2.602 | 3.362 ± 0.381 | |
Eluate B | 2.742 | 2.931 ± 0.075 |
The biochemical importance of CO2(aq) increased by slag eluates was discussed as described in earlier reports [11, 13]. In general, the increase of CO2(aq) can promote carbon dioxide assimilation by photosynthesis on an algal cellular level. However, the present concentrations of CO2(gas) in air determine the concentrations of CO2(aq) that can be dissolved in water. Consequently, the concentrations of CO2(gas) are regarded as a rate‐determining factor of photosynthesis [40]. Its action as the rate‐determining factor of CO2(gas) is common not only to land plants using CO2(gas) directly but also to aquatic organisms such as phytoplankton using CO2(aq) dissolved into water. This study indicates that slag components in solution did not cause toxicity to
6. Conclusion
For autotrophic algal biorefineries, biofuel materials, and bioremediation materials, it is important to evaluate features of interesting algae and their qualities in culture. In this study,
Acknowledgments
This research was mainly supported by a Grant for Young Scientists from the Iron and Steel Institute of Japan and partly by a Grant‐in‐Aid for Young Scientists (B) (Grant No. 26870820).
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