**5. Analyses and discussion**

407 construction projects which constituted a category of the study population, was adopted for this study. Information on the project client were investigated. The distribution of projects sampled into the sub-variables of the project client characteristic was analyzed using percentage. The results are presented in **Table 1**.

On project client, **Table 1** shows that the projects sampled are distributed among Public (22.1%) and Private Individual (77.9%). These results show that the study covered short, average and long term projects.

#### **Table 1.**

*Descriptive results of the characteristics of project sampled.*

#### **5.1 Levels of adoption of communication process based on project client**

The third research hypothesis was tested for difference in the levels of adoption of communication process between public and private clients. The hypothesis was tested using Independent Sample t-test and Mann Whitney U test at p-value ≤0.05. The results reveal that the differences in the levels of adoption of nine (9) communication practices between projects procured by public and private clients are significant. The results are presented in **Table 2**.

**Table 2** shows that the p-values for the test of difference in the levels of adoption of written (0.032), audio-visual (0.023) and electronic communication media (0.001), print (0.001) and electronic (0.001) communication channels and upward (0.021) downward (0.001) lateral (0.007) and horizontal (0.021) communication flows between projects procured by public and private sector client are less than the critical p-value (0.05), therefore, the test rejects the hypothesis. The results indicate that the differences in the adoption of written, audio-visual and electronic communication media, print and electronic communication channels and upward, downward, lateral and horizontal communication flows between public and private sector project are significant. The implication is that project clients have significant effect on the levels of adoption of the two communication media, two communication channels and four communication flows.


*Article* **Optimization-Driven Powertrain-Oriented Adaptive Cruise Control to Improve Energy Saving and Passenger Comfort**

**Pier Giuseppe Anselma 1,2**

**Abstract:** Assessing the potential of advanced driver assistance systems requires developing dedicated control algorithms for controlling the longitudinal speed of automated vehicles over time. In this paper, a multiobjective off-line optimal control approach for planning the speed of the following vehicle in adaptive cruise control (ACC) driving is proposed. The implemented method relies on the principle of global optimality fostered by dynamic programming (DP) and aims to minimize propelling energy consumption and enhance passenger comfort. The powertrain model and onboard control system are integrated within the proposed car-following optimization framework. The retained ACC approach ensures that the distance between the following vehicle and the preceding vehicle is always maintained within allowed limits. The flexibility of the proposed method is demonstrated here through ease of implementation on a wide range of powertrain categories, including a conventional vehicle propelled by an internal combustion engine solely, a pure electric vehicle, a parallel P2 hybrid electric vehicle (HEV) and a power-split HEV. Moreover, different driving conditions are considered to prove the effectiveness of the proposed optimization-driven ACC approach. Obtained simulation results suggest that up to 22% energy-saving and 48% passenger comfort improvement might be achieved for the ACC-enabled vehicle compared with the preceding vehicle by implementing the proposed optimization-driven ACC approach. Engineers may adopt the proposed workflow to evaluate corresponding real-time ACC approaches and assess optimal powertrain design solutions for ACC driving.

**Keywords:** adaptive cruise control; automated driving; energy-saving; fuel-saving; optimal control; passenger comfort


Contents

**Preface XI**

#### **Section 4 Key Competences for Employment 85**

#### Chapter 6 **Traceability of Intra- and Interpersonal Skills: From Education to Labor Market 87**

Alberto Cerezo-Narváez, María José Bastante Ceca and José Luis Yagüe Blanco


*2.3. Role of Parathyroid Hormone*

Secondary hyperparathyroidism is a frequent complication of CKD characterized by an increase in PTH synthesis and secretion and by parathyroid gland hyperplasia. High levels of PTH have an impact on the cardiovascular system apart from the regulation of calcium and phosphate homeostasis [54]. Elevated PTH levels are a common finding in uremic patients which appears much earlier than hyperphosphatemia. PTH and FGF23 have both phosphaturic effects. The difference remains that only PTH has an impact on increased serum calcium. While PTH receptor (PTH1R), is present in bone and kidney, the klotho coreceptor is only expressed in the kidney [7,31]. In addition, PTH stimulates calcitriol synthesis that further contributes to increased serum calcium, whereas FGF23 has an opposite effect on vitamin D and calcium. In the physiologic state, FGF23 acts on the parathyroid gland by reducing gene expression and secretion while in the absence of Klotho, the parathyroid gland shows resistance to FGF23, so enhances PTH secretion.

Experimental data have shown that PTH may directly affect the myocardium although the effect of PTH on the CV system is still under study.

PTH was shown to affect directly rat myocardial cells causing early death of cells by increasing calcium entry into the heart cells [55]. Calcium ions are crucial to myocardial excitation–contraction coupling and cardiac contraction and relaxation [56].

There are early reports by Amman et al. regarding the non-hemodynamic e ffect of PTH on cardiac fibrosis which was related to diastolic LV function [9]. An experimental rat model of CKD (5/6 nephrectomy) reported that continuous infusion of supraphysiological rates of synthetic PTH in animals with parathyroidectomy was associated with an extensive progression of VC—independently of serum Pi levels or the presence of uremia [57]. Moreover, the higher PTH levels have direct trophic effects on cardiomyocytes, interstitial fibroblasts, and smooth muscle cells of intramyocardial arterioles, promoting cardiac hypertrophy and fibrosis. PTH activates fibroblasts and regulates pro-fibrotic factors, such as aldosterone and angiotensin II (PTH stimulates aldosterone secretion by increasing the calcium concentration in the cells of the adrenal zona glomerulosa directly by binding to the PTH/PTH-rP receptor and indirectly by potentiating angiotensin 2 induced e ffects) [54,58]. Additionally, PTH potentially would activate protein kinase C, which further on activates other proteins, such as TGF-b, that in turn, promote the proliferation of fibroblasts, collagen synthesis, and fibrosis [59,60]. In vitro studies have found that PTH shows to have chronotropic, inotropic, as well as hypertrophic e ffects on cardiomyocytes [55] and based on research it was represented that there is a source for a direct role of PTH on cardiac electrophysiology outside of its e ffect on serum calcium [61].

Furthermore, ex-vivo experiments have shown the interaction between PTH and norepinephrine release in isolated human atria and renal cortex tissue through activation of the PTH1-receptor subtype. This e ffect would be an explanation for another potential underlying mechanism of the sympathetic overactivity and the associated cardiovascular mortality seen in patients with ESRD [62].

In hemodialysis patients, like in the rat model, the e ffect of PTH on the myocardium and cardiac fibrosis was well perceived. The hormone was shown to raise the beating rate of myocardial cells and induced their death after prolonged hormonal exposure; PTH stimulates the cyclic AMP production and impairs energy production, transfer, and utilization by myocardial cells [63] and myofibrillar activity of creatine kinase [64]. The presence of sHPTH has also been shown to correlate with enhanced myocardial calcium content and impaired ventricular systolic and diastolic function [65].

Despite a theoretical inverse association between plasma PTH concentration and left ventricular function, parathyroidectomy is not consistently associated with improvement in cardiac contractile function [66]. This suggests that changes induced by PTH could be irreversible in the case of long-standing severe hyperparathyroidism, or other factors contributing to myocardial dysfunction were more important than PTH excess or PTH interferes with the other risk factors of CVD. Despite a theoretical inverse relation between plasma PTH concentration and left ventricular function, parathyroidectomy is not consistently associated with improvement in cardiac contractile function [66]. This suggests in the case of long-standing severe hyperparathyroidism changes induced by PTH could be irreversible, or other factors with an impact on myocardial dysfunction are more important than PTH excess or PTH interferes with the other risk factors of CVD. Furthermore, the inconclusive results of the EVOLVE trial have been linked with this uncertainty since in intent-to-treat analysis a significant advantage of cinacalcet treatment over best presently available standard treatment in the combined primary endpoint (cardiovascular events plus death) was not shown, despite a marked decrease in serum PTH [67]. However, in the subanalysis, when it was adjusted for major confounders such as age and study drug discontinuation, the better control of hyperparathyroidism correlated with a significant advantage in hard outcomes. It was reported that PTH increases with age, weight, BMI, SBP, and LDL, all risk factors for CVD. Increased SBP would be a hemodynamic e ffect of PTH on cardiac remodeling [68,69]. Evidence suggests that PTH has vascular e ffects [68]. Here its potential e ffects on endothelial dysfunction, and increased serum levels of endothelin-1 and IL-6 could be mentioned. In addition, PTH may stimulate the vascular smooth muscle cells to produce factors including collagen and beta-1 integrin which could, in turn, remodel the peripheral vasculature. Another potential e ffect of PTH would be the increase of renin release and activation of the renin–angiotensin system, a process mediated by serum calcium and renal 1-alpha hydroxylase [69]. Aman et al. have underlined the e ffect of PTH as the major determining factor of coronary artery lesions, ranging from the discontinuity of the elastic lamina to the calcification of the medial layer, confirming the agreeable action of PTH [70].

In conclusion, there are clinical and experimental reports which support the hypothesis that PTH behaves as a systemic uremic toxin, with direct and indirect e ffects on uremic cardiomyopathy. PTH acts through four major cardiovascular e ffects; contractile disturbance, cardiomyocyte hypertrophy, cardiac interstitial fibrotic, and vasodilator e ffect. Severe sHPTH is an important threat to CKD patient outcomes a ffecting CV morbidity and mortality and remains an important therapeutic target to prevent bone and CV complications in such patients.


 The prevalence of dementia rises with age, doubling every 5 years between the ages of 60 and 90 (Corrada et al., 2008). Based on the epidemiological data, dementia is widely recognized as a major medical, social and economic problem in developed countries where the age over 65 accounts for an increasingly high percentage of the dementic population (Breitner et al., 2009). Unfortunately, dementia is now becoming a major problem in developing countries where it did not exist 50 years ago (Zilkens et al., 2009). More than 50 million people worldwide have dementia and the most common and irreversible cause of this dementia is Alzheimer's disease (AD) (Adlard et al., 2009). AD is a neurodegenerative disorder divided into two forms namely familial (FAD) and sporadic (SAD) cases characterized by cognitive deficits and extensive neuronal loss in the central nervous system (CNS) (Michon et al., 2009; Reed et al., 2009) and at the molecular level by the presence of specific cytoskeletal abnormalities, including intracellular neurofibrillary tangles (NFT) formed by hyperphosphorylated tau protein and the presence of high levels of the 40- and 42-amino acid long amyloid beta (A) (Woodhouse et al., 2009). The early onset form (i.e. FAD) has a strong genetic correlation that exists between characteristic features of AD pathogenesis and mutations in amyloid precursor protein (APP), (Bernardi et al., 2009), presenilin (PS-1) and PS-2 (Huang et al., 2009). Of particular interest, the other form of AD, SAD is a multifactorial disease to which both genetic and epigenetic factors contribute (Zawia et al., 2009). The well confirmed genetic factors for SAD are apolipoprotein E (APOE) epsilon 4 allele (Wharton et al., 2009) and PS-2 promoter polymorphism (Liu et al., 2008). Accumulating data indicates that disturbances of several aspects of cellular metabolism appear pathologically important in SAD. Among these, increased brain insulin resistance (Salkovic-Petrisic, 2008), decreased glucose utilization and energy metabolism are observed in the early stages of the disease (De la Torre, 2008),

**1. Introduction**

 \*

Corresponding Author


**3. Results**

#### *3.1. Determination of the Weighting of the Indicators*

Emergency preparedness planning comprises the five process steps preliminary planning (1), risk analysis (2), preventive measures (3), crisis management (4) and evaluation (5). In case of equal distribution, the weighting of the five process steps corresponds to 20% or *xj* = 0, 2 each. The expert-based weights are between 17 and 22% (Figure 4). The mean value of the expert-based weight of the process step Preliminary Planning (1) is 0.22, the highest value, and the mean value of the expert-based weight of the process step Evaluation (5) is 0.17, the lowest value of the five weight. The expert opinion regarding the weight of the process step Evaluation (5) varies the strongest. The difference in the mean values of the expert-based weightings is statistically significant (*p* < 0.05).

**Figure 4.** Expert-based weighting of the five process steps of emergency preparedness planning (*n* = 14).

#### *3.2. Assessment of the Applicability and Significance of the Emergency Preparedness Planning Indicator Based on the Case Study*

In order to determine the status quo of emergency preparedness planning, a data collection form was compiled from the indicators listed. This was answered by the water supply company and the responsible disaster control authority from the case study and the data was evaluated.

For the case study, this results in an EPPS of 0.66 and an EPPE of 0.67 (Figure 5). The main indicator PP with a value of 0.96 corresponds to the highest result of the five main indicators. The lowest value is obtained for the main indicator CM with 0.43 and 0.44. This means that in the area of preliminary planning almost all aspects have been implemented in the company and district, but in the area of preventive crisis management aspects are not yet sufficiently practiced.

**Figure 5.** Results of the EPP and the main indicators for the case study based on the different weightings (**a**) Expert-based Weights (**b**) Equal Weights.

The methodology developed in this study to assess the status of emergency preparedness planning is applicable in practice. The applied questionnaire captures the relevant aspects for assessing the status quo and deriving the need for action. The quantitative results also enable a comparison between municipalities and the prioritisation of planned measures to address the need for action.

#### *3.3. Status of Emergency Preparedness Planning in Germany*

#### 3.3.1. Data Basis for the Analysis of the Status Quo of Emergency Preparedness Planning

In order to determine the status of emergency preparedness planning in the water supply sector, a total of 360 data sets were analysed and evaluated. These comprise 194 data sets from districts and 166 data sets at the municipal level. Due to the different responsibilities in Germany, the data sets of the districts and municipalities are evaluated separately from the data sets at the municipal level (Table 2). The data sets are each divided into four groups of approximately equal size according to the number of inhabitants covered. In addition, a distinction was made between the areas of responsibility of the senders of the data collection forms. It is therefore necessary to examine, whether differences can be identified with regard to the level of preparation of the different senders and the size of the municipality.

**Table 2.** Size of the municipalities and districts as well as the field of activity of the senders of the survey forms.

**Table 2.** *Cont.*

#### *3.4. Assessment of the Status of Emergency Preparedness Planning in Germany*

In order to assess the status of the emergency preparedness planning of the districts as well as the municipalities, the composite indicator EPP and the main indicators PP, RA, PM, CM and E were determined on the basis of the data sets presented. For this purpose, the results of the emergency preparedness planning indicators with weighting according to expert opinion (EPPE) and with equally distributed weighting (EPPS) are presented below.

The results of the EPPE and EPPS vary for the districts in a few cases (Figure 6) The mean value of the EPPE as well as the EPPS is equal to 0.42 (Table 3). The small differences in the weighting show only a little effect in the result. Moreover, these differences are not statistically significant (*p* > 0.05).

**Figure 6.** Emergency preparedness planning indicator according to the size of the districts.

**Table 3.** Results of the emergency preparedness planning indicator with expert based and equally distributed weighting and the main indicators.

The participating districts show a different level of preparation. Districts with more inhabitants achieve a higher EPP (Figure 7). The range of EPP increases with the number of inhabitants, so that the largest districts show the greatest difference in the level of emergency preparedness within a size group. The differences in the mean values by size of the districts are significant in both cases (*p* < 0.05).

**Figure 7.** EPP according to the size of the districts. (**a**) Expert-based weighting of the main and sub-indicators; (**b**) Equally distributed weighting of the main and sub-indicators.

The mean values of the EPPE and EPPS for municipalities are 0.32. Some municipalities have thus already implemented certain aspects of emergency preparedness planning. However, these implementations are still in their beginnings. Differences between the EPPE and EPPS are only evident in a few cases (Figure 8), but do not show statistical significance (*p* > 0.05).

Municipalities with an increasing number of inhabitants achieve higher results in the EPPE as well as in the EPPS. The differences in the mean values between the size of the municipalities are significant (*p* < 0.05). However, the range of the EPPE does not increase with a growing number of inhabitants in the municipality, which can be seen at the level of districts.

**Figure 8.** Emergency preparedness planning indicator by size of municipalities.

The Figure 9 shows the differences of the EPPE and EPPS between expert-based and equally distributed weighting. The colored variance of the rectangles shows that the different weightings affect the results of the EPPE and EPPS. In addition, the figure shows that the difference between EPPE and EPPS is in the range of ± 0.018 and is therefore only noticeable in a few cases when the result is rounded to the second decimal place.

**Figure 9.** Difference between the emergency preparedness planning indicators EPPE and EPPS according to the size of the data sets taken into account.


Mode

Copper ore from Legnica consists of 17% copper minerals in a quartz (64%) and calcite matrix (19%) (see Figure 4).

**Figure 4.** Copper ore (green sandstone ore): (**a**) hand specimen, (**b**) microphotograph under reflected light.

### Grain Size

The quartz shows a mean grain size of 265 μm and the copper minerals with 277 μm are of similar size. The standard deviation σln of the diameter distribution of the copper minerals shows a scatter parameter of 0.504. Figure 5 shows the grain size distribution of both minerals as identified by QMA.

**Figure 5.** Mineral grain size distribution of the copper ore according to QMA.

### Grain Shape

The shape of the copper minerals of the copper ore is predominantly platy-like. This is confirmed by the values for their elongation E and flatness F (E = 1.224; F = 1.156). In comparison, the shape of the quartz is predominantly needle-like. This is confirmed by respective elongation and flatness values (E = 1.458; F = 1.162).

#### Roughness

The roughness KR of the copper minerals is around 62% and is therefore in a very high range compared to minerals of other rock fabrics.

#### Specific Surface Area

The specific surface area SV of the copper minerals is in the medium range and is around 53 mm2/mm3 per mineral grain. The specific surface area and the roughness characterize the "intergrowth" of the minerals with each other. This intergrowth is an important information since it allows to draw conclusions on the breakage behaviour of a material, the specific energy consumption, wear of comminution machine parts etc.

#### Degree of Clustering

The degree of clustering C shows whether a mineral grain forms an agglomeration in the rock with another mineral grain of the same type. The quartz shows a 6% cluster formation, that is, 6% of all quartz grain surfaces lie next to another quartz grain surface.

#### Space Filling

The very good space filling means that moisture can only adhere to the surface of particles, as there are no pores in the ore sample.


**2.2 Determination of kunitz trypsin inhibitor genotype**

98 F2 seed from F1 plants for population 1 and 243 F2 seed from F1 plants for population 2 was analysed electrophoretically to determine the presence (SKTI-`+') or absence (SKTI- 'null') of kunitz trypsin inhibitor. A piece of cotyledon from each F2 seed was removed and the remaining embryo germinated to given a F2 mapping population. The separated cotyledon tissue was incubated for 30 min (room temperature) in 1 ml Tris-HCl, pH 8.0, containing 1.56 % v/v β-mercaptoethanol. After centrifugation, 50 l of the supernatant were added to an equivalent amount of 5X sample buffer [10% w/v sodium dodecyl sulphate (SDS), 50% v/v glycerol, 1.96% v/v β-mercaptoethanol, 1 M Tris-HCl, pH 6.8]. The samples were boiling at 97°C for 5 min and then centrifuged. Two microlitre of the supernatant were used for electrophoresis on 12% acrylamide SDS polyacrylamide gel electrophoresis (SDS-PAGE) medium gels in Owl Separation Systems. Inc(Model: P9DS, Portsmouth, NH USA). Electrophoresis was practiced at 120 V for 7 hr. Gels were stained overnight in an aqueous solution of 0.25 g coomassie brilliant blue R250, 10% acetic acid, 45% methanol and destaining solution (5% acetic acid, 14 methanol) for several hours. A Wide-Range SDS-PAGE molecular mass standard (Sigma MarkerTM, Product Code : M4038) containing the 21.5kDa soybean trypsin inhibitor protein, was used to aid recognition of samples lacking the kunitz trypsin inhibitor.


**5. Conclusions**

Based on the findings in the present research, the following conclusions were drawn:

6. The future work should be focused to understand the pelleting characteristics of high mositure pine residue, switchgrass, and blends of pine and switchgrass in a pilot scale ring die pellet mill. Evaluate how the pelleting process variables impact the physical properties, proximate and ultimate composition, and energy properties.

**Acknowledgments:** This work was supported by the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy under DOE Idaho Operations Office Contract DE-AC07-05ID14517. Accordingly, the U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript or allow others to do so, for U.S. Government purposes. The author would like to thank the project partners Genera Bioenergy, Vonore, TN, University of Tennessee, Knoxville, TN Auburn University, Auburn, AL and Herty Advanced Biomaterials, Savannah, GA, USA for providing 3/16-inch grind switchgrass and 2-inch top pine residue samples for pelleting tests. The author also wants to thank Matt Dee, Research Engineer and Craig. C. Conner, retired INL employee, for supporting the experimental work.

**Conflicts of Interest:** The authors declare no conflict of interest.

**U.S. Department of Energy Disclaimer:** This information was prepared as an account of work sponsored by an agency of the U.S. Government. Neither the U.S. Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. References herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the U.S. Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the U.S. Government or any agency thereof.


**1. Introduction**

Architectural acoustics is an important consideration in the overall comfort of openplan offices. Researchers [1–7] have made considerable effort to investigate the acoustics of open-office spaces and present potential solutions for improving their indoor environmental quality. Examples of these solutions are (1) configuring the floor plan to effectively balance and acoustically separate areas; (2) designing collaborative and transition spaces with sound-absorbing surfaces; (3) employing high-performance, noise-reducing interior partitions, and exterior facades; and (4) adding background noise control and enhancement measures for selective workstation. While these measures have proven to help achieve a comfortable indoor work environment, the lack of approaches to visualize their effectiveness have resulted in limitations of their applications in practice. This paper reports on the applicability of a novel visualization approach to aid in the interpretation and comparison of various open-office acoustic solutions from a human-centric acoustic environment perspective.

Poor acoustic indoor environments have been shown [8–11] to negatively impact the cognitive performance and well-being of occupants, thus negatively affecting their productivity. The presence of irrelevant speech from single and multi-talkers in an openplan office, along with noise from printers, phones, and HVAC systems can lead to a reduction in the quality of the acoustic environment. However, in some cases, HVAC systems can improve the acoustic comfort in open-plan offices by providing sound masking. Yadav et al. [12,13] have studied the impact of both single and multi-talkers on sound pressure level and their effect on the cognitive and task performance of occupants in an open-office environment. Their studies [13] investigated the potential benefit of the "babble effect", acting as a sound-masking source, thus improving the acoustic comfort of

**Citation:** Glean, A.A.; Gatland, S.D., II; Elzeyadi, I. Visualization of Acoustic Comfort in an Open-Plan, High-Performance Glass Building. *Buildings* **2022**, *12*, 338. https:// doi.org/10.3390/buildings12030338

Academic Editors: Yue Wu, Zheming Liu and Zhe Kong

Received: 29 January 2022 Accepted: 27 February 2022 Published: 11 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the space, but its impact was found to be limited. Sound masking, which is the addition of background noise to reduce the impact of distracting sound sources, is one of the common solutions [14–16] for improving the acoustic comfort within open-plan offices. In some instances, multiple voices in open-plan offices can be a potential source of sound masking [17], thus improving the acoustic environment.

Acoustic comfort within an open-plan office can be characterized by the calculated distraction distance, which is based on sound transmission index (STI) measurements. This relates to speech intelligibility as a function of distance from the source. Haapakangas et al. [18] conducted an analysis of 21 open-plan offices using surveys and room acoustic measurements of distraction distance, spatial decay of speech, speech level at 4 m from the speaker, and background noise level. Their findings showed that the perception of noise was mainly related to background speech and that an increase in distraction distance predicted an increase in disturbance by noise. The other measured quantities were not significant standalone indicators of noise disturbances. Distraction distance is an important metric in characterizing acoustic comfort. Despite the apparent benefits of visualizing distraction distance as a tool to understand and compare the acoustic environment, there is a current gap in the literature of examples suggesting visualization procedures and techniques for practical application. This limitation impacts the comparative analysis of acoustical treatment evaluations and presents barriers to employing innovative acoustic treatments to open-plan office environments.

This study investigates the utility of novel visualization and a building performance evaluation technique to document the interactional impact of acoustical comfort on the health and well-being of occupants in an open-office environment. In this study room acoustic measurements were conducted from which the distraction distance was calculated. The employee experience was captured using a comprehensive Pre-Post Occupancy Evaluation (PPOE) protocol. This paper illustrates a visualization strategy to aid in the interpretation and comparison of various open-office acoustic solutions, which is also correlated to survey responses by occupants from a human-centric acoustic environment perspective.

#### **2. Materials and Methods**

*2.1. Building Design-Acoustic Comfort*

#### 2.1.1. Traditional Building

A corporate headquarters was relocated from a traditional cellular private office complex (pre-move) to a LEED platinum-certified open-concept plan office (post-move) in 2015. The traditional late 1960's corporate campus consisted of four low-rise, multistory buildings having perimeter offices and centrally located cubicle workstations. Transparent glass and gypsum wall partitions separated the perimeter and central spaces. The building envelope consisted of a brick facade and large single-glazed fixed casement windows. Workstations were divided using 1524 mm tall fabric covered partitions. Space lighting was created using ballasted tube florescent lights integrated into the suspended acoustical ceiling. Numerous hard sound reflective surfaces were created in the open plan space by lighting fixtures, furniture, metal ceiling panels, gypsum and glass partitions. The traditional building exterior is presented in Figure 1a along with a typical workstation in Figure 1b.

**Figure 1.** (**a**) Traditional building complex. (**b**) Typical workstation of traditional building. (**c**) LEEDcertified platinum building. (**d**) Workstations in new LEED certified building.

#### 2.1.2. LEED-Certified Platinum Building

The renovated four-story, all-glass-facade LEED platinum-certified building consisted of large, centrally located workstations with perimeter hallways and collaborative areas. Transparent glass and gypsum wall partitions separated the perimeter collaborative and central spaces. Individual workstations were separated by fabric-covered partitions with colored glazing extensions having varying heights of 1047 mm, 1448 mm, and 1625 mm. Slim profile LED luminaries with sharp cut-off angles were integrated into the suspended acoustical ceiling to provide additional space lighting. Hard reflective surfaces were limited to the perimeter of the large open-plan office space. An active sound masking system was implemented in all open-plan spaces to increase acoustic comfort at the 800 plus workstations. The LEED certified platinum building exterior is presented in Figure 1c along with a typical workstation in Figure 1d.

Table 1 provides a comparison of the traditional building complex and the new LEEDcertified building, mainly from an acoustic surface characteristic perspective. The main differences highlighted are the office layouts, addition of collaborative areas, and perimeter hallways as opposed to perimeter offices. With this new open-office concept the addition of sound masking and ceiling tiles with higher absorption were critical to the acoustic comfort within the space.

**Table 1.** Comparison of acoustic comfort spatial and surface characteristics between office buildings.

#### *2.2. Measurement Procedure*

The acoustic comfort experienced in an open-plan office can be assessed through the measurement of acoustic parameters such as spatial decay rate of speech, speech transmission index (STI), distraction distance, privacy distance and background noise levels. STI and background noise levels were measured to evaluate the indoor environmental quality (IEQ) of the LEED platinum-certified building described in Section 2.1.

The speech transmission index represents the transmission quality of speech with respect to intelligibility. Background noise levels represent the measured sound pressure level in absence of any occupants within the evaluated space. Both distraction and privacy distances are determined from the STI measurements and represent the distance from the speaker at which the STI falls below 0.50 and 0.20 respectively. ISO 3382-3 [19] provides a detailed description of these acoustic parameters along with the standardized measurement procedure which was used to acquire the data presented.

Speech is essentially the modulation of a band of noise. Measurements of speech transmission index (STI) were conducted using the STIPA (STI for Public Address Systems) method [20]. This method is an alternative to the full STI measurement, which uses a Gaussian noise signal as the carrier and is divided into seven-octave bands from 125–8000 kHz, where each band is modulated by fourteen modulation frequencies in one-third octave bands, from 0.63–12.5 Hz. This results in 98 combinations, which creates a time-consuming measurement. In the STIPA method a total of 12 modulation indices are measured from carrier frequencies in one-third octave bands ranging from 0.63 to 12.5 Hz. The modulation represents the combination of modulation and carrier frequencies used to mimic speech

excitation. A Nor140 (Norsonic sound level meter) was used to measure the STI using the STIPA method at specific distances from the excitation source within the space. The sound level meter was also used to measure the background noise level at various workstations throughout the open office, in accordance with ISO 3382-3.

Workstations within the LEED-certified open office building were evaluated for the speech transmission index and background noise levels with and without sound masking. Ideally, the STI values will drop below 0.50 between 2 and 4 m. The background levels are expected to fall below a noise criteria (NC) level of 40 dB. Noise criteria are single numbers used to define goals for maximum allowable noise in a given space.

Measurements at various workstations allowed for a comprehensive acoustic comfort mapping of distraction distance from the STI. The maps can be used for quick visualization and spatial assessment of the expected acoustic comfort of the open-plan space with regard to distraction and privacy distances.

#### *2.3. Post-Occupancy Evaluation Survey*

The Space Performance Evaluation Questionnaire (SPEQTM) is an online occupant's survey with developed categories and scales representative of the most important issues identified by occupants to impact their comfort, satisfaction, performance, and health, as perceived and experienced in their work environments. The survey semantics and linguistics have been designed based on proven language constructs that represent layperson descriptions of their physical environment. SPEQTM was cross-tested and calibrated in field and lab settings. The survey was peer-reviewed by an expert panel of 20 professional building scientists, psychologists, space planners, architects, and physicians.

The data collected by the questionnaire evaluates 30 pre- and post-occupancy issues in 76 questions classified into seven main categories. All questions contained a skip-logic approach to skip irrelevant information based on the occupant's responses. This made the instrument more effective and reduced respondents' fatigue. The average response time of the questionnaire is 12 min, with a minimum of 8 and a maximum of 20 min. All answers were recorded on a five-point Likert scale, semantic-differential scale, a numerical open scale, or a categorized aggregated scale. The scales allowed for continuous data that was easily analyzed using simple and more complex statistical modeling and regressions. In addition, open-ended responses were encouraged for all questionnaire categories to allow occupants to voice their opinions without restrictions. The questionnaire contained a forced response to a consent form and pre-set reminders for missed question responses. Respondents were allowed to skip questions on the second attempt for most questions, with the ability to skip demographic questions to maintain the respondent's privacy. Neither compensation nor a fee was administered for a respondent to respond to the questionnaire. To facilitate statistical analysis, questionnaire responses were recorded into a numerical scale of one to five, such that five was "strongly agree", three was "neutral", and one was "strongly disagree". Subjective responses from SPEQTM questions were paired with objective acoustical measurements identified in Section 2.2 using timestamps for crosstabulation analyses.

#### **3. Results**

The level of background noise in a space directly impacts speech intelligibility. Background noise level is governed by noise criteria (NC). For an open-office plan with forcedair distribution systems, the ASA S.12.2-2008 [21] recommended noise criteria (NC) curve limit is NC-40 or less. At NC-40 the corresponding sound pressure level range is 46 dBA to 48 dBA.

Background noise levels were measured in the LEED building with and without the sound masking system turned on during occupied hours. The dots in Figure 2 indicate locations on the third floor at which the background levels were measured. The average sound pressure levels in the north wing with and without the sound masking system turned on were 46.6 dBA and 41.1 dBA, respectively. The sound masking system generated white noise in the space through an evenly distributed loudspeaker system located in the ceiling plenum. This increased the average background noise to the desired level to achieve good speech privacy between workstations. In Figure 2, the green dots indicate values that met the design criteria during occupied hours. Red dots indicate values that exceeded 48 dBA due to conversations (red unfilled ovals highlight locations) occurring near the measurement location. However, this value was only exceeded with the noise contribution from the sound masking system.

**Figure 2.** Background noise survey north wing of LEED-certified building with sound masking on (**left**) and off (**right**). The green dots denote values that met the design criteria during occupied hours whereas the red dots denote values exceeding 48 dBA. Design criteria were exceeded in some instances due to nearby conversation, denoted by the red, unfilled oval shapes.

Workstations were evaluated for spatial sound distribution of the STI to assess the distraction distance from the speaker at which the speech transmission index fell below 0.50. A comprehensive acoustic comfort mapping strategy was applied in order to fully characterize the space and provide visuals of spatial performance. STI measurements were conducted in the northeast wing of the building with and without the activation of the sound masking system. Additionally, the impact of speaker height (standing or seated) on distraction distance was assessed.

STI data is often presented in a table format or an X–Y graphical plot. Figure 3 (WS3154) shows the location, and STI as a function of distance from the speaker at different angles. This dataset illustrates the scenario of a speaker standing versus sitting with sound masking and captures the impact on the STI. The distraction distance target (STI 0.50-0.30 at 2–4 m) was met for the 90-degree orientation with the standing speaker, and for all orientations except the direct (0-degree orientation) and a single 45-degree orientation when the speaker was seated. Given the layout of the workstations relative to the location of the speaker the differences observed with the orientation of the listener to the speaker were expected.

**Figure 3.** Office map depicting location on the third floor, north wing, at which measurements were conducted along with a snapshot of the interior space (**top**). Speech transmission index (STI) as a function of distance at different angles to a sitting speaker (**bottom left**) and a standing speaker (**bottom right**) with sound masking. The red dashed lines indicate a STI target value between 2–4 m.

Tables and line plots have been used extensively in the presentation of STI data. The plots are easy to interpret and present the data. However, this can at times be a challenge for architects, designers, owners, and developers, who are the solution decision-makers and would prefer the most user-friendly presentation of such data. A color-coded visualization of the STI acoustic comfort mapping can provide a quick and intuitive interpretation of the overall acoustic performance of the space. Overlaying the surface plot on a scaled drawing of the space can provide additional insights under various conditions.

To convey the space performance using the approach to STI acoustic comfort mapping, concentric semicircles were placed at 2-m intervals from the simulated speaker in a sitting

position (Figure 4, left). The color map superimposed on the concentric circles convey the change in STI (Figure 4, right), and, by extension, speech intelligibility, at distances away from the speaker. Detailed scenarios are presented in Figure 5 which illustrate the STI measurements for standing and sitting with the sound masking system on and off. The plots, which can be interpreted relatively quickly, show the positive impact when the sound masking system is on and of the lower distraction distance (lower STI) in the seated versus standing positions.

**Figure 4.** Acoustic comfort mapping with STI of north-facing workstation at the LEED building with the speaker in sitting position and sound masking on. (**Left**) Scaled drawing depicting STI measurements at various location and distances. (**Right**) Associated surface plot highlighting areas from good to bad STI rating.

**Figure 5.** Acoustical comfort visualization through STI surface plots of measurements from the north wing workstation area on the third floor of the building. (**a**) Speaker in sitting position with sound masking on. (**b**) Speaker in sitting position with sound masking off. (**c**) Speaker in standing position with sound masking on. (**d**) Speaker in sitting position with sound masking off. The best- and worst-case scenarios for comfort shown in (**a**,**d**) respectively.

#### **4. Discussion**

#### *4.1. Human-Centric Acoustic Comfort*

The human-centric acoustic comfort model conceptualizes both the quality and quantity of the acoustical environment based on the occupant's perspective [22]. In this model, spatial and temporal impacts of comfort are evaluated physically and perceptually. To test the visualization approach proposed to acoustic comfort and its impacts on occupants' satisfaction, the SPEQTM survey was administered to the occupants of the traditional building prior to the move and one year after the move to the new LEED-certified building respectively. Focus groups were employed before the administration of the surveys to ensure that occupants were well habituated in the building and were even in their perception of both positive and negative attributes of the indoor environment. In addition, the after-move survey was administered one-year post-move to limit bias in perceptions between the new and old buildings.

Response to the survey was very high, with 42% of the 800 employees completing the survey prior to the move and 44% of the 800 plus employees completing the questionnaire one year after the move and relocation. Data tabulation and coding were performed on both the physical and human-response data sets. Physical measurements and survey responses were spatially tagged and statistically analyzed using the software Statistical Package for the Social Sciences (SPSS). This analysis ensured that spatial visualizations of physical acoustics metrics, such as STI, were correlated with occupants' perceptions of acoustical comfort and satisfaction responses to the SPEQTM questionnaire.

#### *4.2. Occupants Perspective Analysis*

To examine the correlation between acoustic comfort visualizations and occupants' perceptions, we calculated the mean difference in dissatisfaction and mean difference in satisfaction for the various aspects related to occupant perceptions of acoustical comfort (Table 2) in a traditional and a LEED-certified building. The reference in these calculations is the LEED building. For example, a negative sign (-) in dissatisfaction means a reduction in dissatisfaction responses for that item, hence more improvement. Similarly, a negative sign (-) in satisfaction means a reduction in satisfaction in this item, hence a lack of improvement and vice versa.

**Aspect Mean Difference in Dissatisfaction (%) Mean Difference in Satisfaction**(%) **Total Perceived Difference (%)** overall acoustical satisfaction −19.5 +22.7 42.2 noise from people talking in your area −7.2 +12.0 19.2 noise from people talking in the hallway −17.3 +16.9 34.2 noise from ventilation systems −27.9 +31.2 59.1 noise from equipment −25.3 +30.1 55.4 noise from lights −10.6 +27.3 32.7 noise from outside the building −10.4 +22.3 32.7

**Table 2.** Differences in perceived satisfaction of acoustic comfort attributes inside the LEED-certified building as compared with the traditional building.

Employees responded to five different questions that collectively evaluated their acoustical comfort in the building pre- and post-occupancy between the traditional and LEED buildings. Across all questions, employees reported a higher level of acoustical comfort improvements of 42.2% in the LEED building as compared with the traditional building (Table 2). In addition, acoustical comfort improved across all parameters between 19% and 59%. The most improvement was the perceived reduction in noise of equipment and ventilation systems by 55.4% and 59.1%, respectively. Thanks to improvements in the building envelope and the provision of better daylighting and electric lighting systems, occupants reported an increased satisfaction of 37.9% for noise from lighting system and an increased satisfaction of 32.7% for outside building noise perception. The sound masking

system and improvements in sound absorption material in the LEED building might be related to a perceived satisfaction with distractions of noise from people talking and walking in hallways despite being in an open-plan office. In general occupants perceived improvements in satisfaction of 19.2% for noise distractions due to talking and 34.2% improved satisfaction due to hallway noise as compared with the traditional building.

This improvement in satisfaction from the survey findings correlates well with the observation and interpretation of Figures 4 and 5. Within the workstation the STI falls within the range of 0.8–1.0 due to the close proximity to the speaker, and as a result, speech is highly intelligible. However, when sitting and the sound masking is on as illustrated in Figure 4, STI values, on average fall under 0.50 in the 4–6 m range. In this range the speech intelligibility has a fair rating, and a fair to poor rating at distances greater than 6 m. This means that occupants will be less distracted due to office conversations at those distances. Figure 5 clearly illustrates the change in distraction distance between standing vs sitting and sound masking on or off. The perceived satisfaction is expected to decrease to a minimum when the sound masking is off and the occupant is standing as the STI, on average, remains above 0.60 at a rage of 2–8 m. In general, the survey indicated occupants were satisfied with STI ranges of 0.2–0.4 outside of their immediate workstation.

#### **5. Conclusions**

Architectural acoustic designs for open-plan office spaces aim at reducing transmission between workstations while creating acoustical privacy within the individual workstation. This approach, however, doesn't usually result in a pleasant or comfortable work environment due to average numerical values that do not reflect a human-centric acoustical experience for the occupants, according to their spatial locations. Through the use of sound-absorbing surfaces, high-performance, noise-reducing interior partitions, and sound-masking systems, a more spatial acoustical comfort approach is feasible. Predicting the success of this approach in the field can be challenging. The use of STI mapping and visualizations is a tool that can help predict and estimate occupants' perceptions prior to occupancy. The procedure outlined in this paper offers a design and spatial visualization tool to help designers and building owners predict and estimate acoustical performance in spaces from the occupants' perspective.

The results show strong correlations between improved acoustic qualities of the retrofitted, high-performance, LEED-certified building in spaces visualized within the STI standards, and overall acoustical satisfaction (Table 2). A comparative analysis of the occupants' attitudes and perceptions of the impact of the building on their comfort levels reveal a significant perceptual change to the impact of acoustical design on the employee overall satisfaction. A comprehensive design and employment of spatial visualization tools to predict and implement metrics of indoor environmental quality (IEQ) might result in improved comfort, satisfaction, and the perception of well-being for the occupants. It is the hope that further integration of building systems concerning IEQ measures and comfort visualizations can better predict acoustical performance in the early design stages and lead to better work environments that are more responsive to occupant's needs and comfort expectations.

**Author Contributions:** All authorshave equally contributed to this manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study procedures involving human subjects, Protocol Number: 04252016.035, was approved by the University of Oregon Institutional Review Board as per 45 CFR Part 46.110. Upon further review, it was determined that this study qualifies for exemption as per the Common Rule regulations of Title 45 CFR 46.101(b)(2) on 28 April 2018.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study as a requirement to proceed with taking the SPEQ questionnaire.

**Data Availability Statement:** Data for this study is stored in an institutional depository protected by the University of Oregon IRB Protocol Number 04252016.035. Access to the data is only granted to key personnel involved in the study and who successfully passed the CITI training module.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**2. Sample preparation**

Sample preparation step is crucial in every analysis where analytes are present at very low concentration levels. In the speciation analysis, there is also another difficulty that should be overcome. An important requirement for reliable speciation is to retain the concentration and structure of the original chemical forms in the sample. In general, aquatic samples such as rain, ground- or surface water, tap and drinking water, seawater, and soil solutions do not require any pretreatment procedures other than filtration through 0.45 μm filter.

Extraction of selenium species from the solid samples with the highest recovery is quite challenging. According to Peachey et al. [14], selection of the extraction method, which provides high extraction efficiency while preventing the integrity of selenium species, is essential for the accurate measurement of its species. The most used method can be divided into three main groups:

Since selenoaminoacids are water-soluble, extraction with hot water is extensively used [14–17]. However, the efficiency of water extraction from yeast was only 10% [17]. To release bounded selenoaminoacids, enzymatic or acidic hydrolysis was necessary [18–20]. The addition of methanesulfonic acid was used for selenomethionine extraction from yeast when heated under reflux [21]. Casiot et al. [22] reported that extraction of selenium species from yeast with water and ethanol led only to 10–20% recoveries of selenium and not allowed to extract

selenomethionine. The addition of pectinolytic enzymes released additional 20% of selenomethionine, while the addition of dodecyl sulfate solution allowed solubilization of a selenoprotein that accounted for 30% of total selenium. On the other hand, using tetramethylammonium hydroxide solubilized the sample completely, but the extracted selenium species were entirely degraded to selenomethionine and inorganic selenium [22]. It should be noticed that this type of extraction strictly depends on the choice of an enzyme, pH of the extraction solution, as well as temperature and time of the extraction. The most commonly used enzymes for this purpose are proteinase K or proteolytic enzymes (protease XIV), which were used for water-insoluble selenium fraction in many complicated matrices [23]. To reduce the extraction time, also ultrasonic hydrolysis can be used as the breakdown of Se-containing proteins (peptide bonds) into selenoaminoacids occurs [24]. The in vitro digestion with gastric juice was also used for selenium extraction from fish samples [25]. This procedure allowed SeMet determination, but the whole process takes few hours.

The sequential procedure developed by Chassaigne et al. [26] consisted of three steps: first, Tris-HCl buffer was used for extraction of water-soluble fractions, then also Tris-HCl with the addition of SDS for solubilization of protein fraction, and, finally, concentrated HNO3 was used for dissolving of the remaining solid residue. A similar three-step procedure was used for extraction of selenium from mushrooms with 89% extraction efficiency [27]. It should be noticed that sequential treatment was also applied for dietary supplements [28, 29].

Cuderman et al. [30] examined different extraction media to identify selenium species in buckwheat. The optimal extraction efficiencies were obtained by hydrolysis with HCl, followed by breaking the cells with liquid nitrogen and then enzymatic hydrolysis with protease XIV. Ammonium acetate [31], sodium hydroxide [32], and enzymatic hydrolysis with pronase E [33] have been proposed for extraction of selenium species from green onion leaves.

#### **3. Total selenium determination**

Determination of the total selenium content is still the first step of its analysis. This procedure requires that organic forms must be transferred into inorganic selenium that is usually achieved with digestion using strong mineral acids or UV irradiation after addition of hydrogen peroxide [12, 34]. For this purpose, fluorometry, electrochemical detectors, atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), mass spectrometry (MS), and neutron activation analysis have been used (**Table 2**).

Hydride generation coupled to AAS or AFS detectors is specific for Se(IV) determination, where these species are selectively reduced to volatile SeH2, usually by sodium tetrahydroborate in hydrochloric medium. This technique can be applied for the determination of total inorganic selenium (e.g., sum of selenite and selenate) after quantitative reduction. The content of Se(VI) is then obtained by the difference between two determinations. This technique can be fully automatic by connection with flow injection analysis system. The advantage of such system is minimum sample and reagent consumption as well as short time of single run.

Stripeikis et al. [44] determined selenite and selenate in drinking water using fully automatic online separation/pre-concentration system coupled to electrochemical atomic spectrometry. Preconcentration of both selenium forms was carried out onto a microcolumn packed with an anionic resin (Dowex 1X8) that was placed in the robotic arm of the autosampling device. Selenite and selenate were then sequentially eluted with HCl at concentration of 0.1 and 4 mol L−1 ,

*Chromatographic Analysis of Selenium Species DOI: http://dx.doi.org/10.5772/intechopen.87053*

#### **Table 2.**

*Analytical methods for the determination of total selenium.*

respectively. The interference of large amounts of chloride ions during selenium atomization was prevented by using iridium as a permanent chemical modifier.

Kocot et al. [36] proposed a dispersive micro-solid-phase extraction with graphene as a solid adsorbent and ammonium pyrrolidine dithiocarbamate as a chelating agent for Se(IV) in analysis of inorganic selenium by the energy-dispersive X-ray fluorescence spectrometry; the concentration of Se(VI) was calculated as the difference between the concentration of selenite after and before reduction of selenate.

Due to the high selectivity and sensitivity, wide linear range, as well as multielement and multi-isotope detection, inductively coupled plasma mass spectrometry (ICP-MS) is a great tool for selenium analysis. However, some difficulties can be found when conventional ICP-MS is used for this purpose. To avoid the spectra interferences with 80Se isotope (the largest natural abundance of 49.6%), 82Se and 77Se are often monitored. The use of collision/reaction cell, operating with hydrogen gas, lowers argon dimer interferences. This technique was applied by Reyes et al. to determine the selenium in biological reference materials and serum samples [45] and yeast [46].

### **4. Separation techniques in selenium speciation analysis**

The occurrence of selenium at very low concentration levels as well as the dependence of its toxicity on the form in which it occurs resulted in the need for reliable analytical procedures for identification and quantification of its species. The coupling of separation of selenium species with sensitive detection has become a powerful technique for Se speciation. Liquid chromatography operating in different modes is the most used analytical technique for this purpose; however electrophoresis and gas chromatography also were used in selenium analysis. That is why these two methods will be also described in this chapter, which will mainly focus on liquid chromatography.

#### **4.1 Gas chromatography**

Selenium species can be divided into volatile and nonvolatile compounds. The first group of compounds can be directly analyzed using gas chromatography,

for example, dimethylselenide was determined in human breath after ingestion of Se-enriched selenite [47]. For other nonvolatile compounds, derivatization is required.

In the literature many derivatization procedures can be found. Pelaez et al. [48] tested two methods of derivatization of selenomethionine and selenomethionine in selenium supplements. The first method consisted of esterification of the carboxylic acid group using propan-2-ol and then acylation of amino group. The second described procedure used ethanol-ethyl chloroformated for one step derivatization. Then detection using ICP-MS was performed.

In general, many types of detectors were coupled with GC for speciation analysis of selenium. Yang et al. [49] successfully used ICP detection for the determination of selenium in yeast. Organoselenium species in plants were determined using GC-MIP-AES detection [50]. Because of the requirement of the derivatization, GC is not so widely used in selenium speciation analysis in comparison to liquid chromatography.

#### **4.2 Capillary electrophoresis**

Due to the high resolving power, capillary electrophoresis (CE) has a potential to be used in speciation analysis as an alternative or complementary technique to HPLC. CE coupled with ICP-MS was used in the analysis of selenium in yeast extract after SEC separation [51]. The obtained limit of detection for organic and inorganic species was in range 7–18 μg L−1 . The main difficulty in such separation can be big sizes of selenopolypeptides resulting in their slow migration. In such case, the predigestion of selenoprotein fraction is recommended.

It should be highlighted that gel electrophoresis offers better resolution than liquid chromatography in the analysis of high molecular weight selenoproteins, which was used in such analysis by Chassaigne et al. [26], and in Se-enriched yeast analysis as well by Chery et al. in blood sample analysis [52].

#### **4.3 High-performance liquid chromatography**

#### *4.3.1 Size-exclusion chromatography*

Separation in size-exclusion chromatography (SEC) strictly depends on the size of separated analytes. That is why this technique is widely used as a preliminary step to sample purification or to separate selenoproteins from the matrix. Ayouni [53] observed high molecular weight selenium compounds in the extract of dietary supplements after separation by SEC. Conjunction of SEC with ICP-MS was used for analysis of the products of enzymatic digestion of selenoproteins fraction [54, 55]. Size-exclusion chromatography was also used in selenium analysis in human plasma [56] and extracts from rats' internal organs [57].

#### *4.3.2 Ion-exchange chromatography*

Anion-exchange chromatography has been mainly employed in selenium speciation analysis [58–63]. Mobile phases used in this mode usually contain small content of organic modifier (e.g., 2–5% methanol) and buffered salt solution (e.g., acetate, phosphate, and citrate). During the separation process, the equilibria between the charged solute ion and the oppositely charged surface of the stationary phase are established. The separation is achieved based on the differences of the strength of such interaction between analytes. Anion-exchange

#### *Chromatographic Analysis of Selenium Species DOI: http://dx.doi.org/10.5772/intechopen.87053*

chromatography was used in the selenium speciation analysis in garlic, sunflowers, and radish sprouts [64]. In addition to well-known compounds like SeMet or MeSeCys, several unidentified signals were obtained. The application of high-resolution mass spectrometry enabled identification of additional selenocompounds as inorganic metabolites, such as deamino-hydroxy-seleno-homolanthionine, *N*-acetylcysteine-selenomethionine, methylseleno-pentose-hexose, methylselenoglutathione, 2,3-dihydroxy-propionyl selenocysteine-cysteine, methyltio-selenoglutathione, 2,3-dihydroxy propionyl-seleno-lanthionine, and two Se-containing compounds with proposed molecular formula C10H18N2O6Se and C10H13N5O3Se.

Cation-exchange chromatography was used to analyze selenium-enriched yeast in a human adsorption study [65]. As a mobile phase, pyridinium formate buffer with 3% of methanol was used. This method was suitable for separation of organic selenium species, however not suitable to separate selenite and selenate.

### *4.3.3 Reversed-phase chromatography*

Both, simple reversed-phase [66–68] and ion-pair (IP) reversed-phase chromatography [15, 28, 69], are widely used for analysis of ionic and neutral selenium species. The mobile phases are aqueous with small amount of polar organic solvent (usually methanol or acetonitrile). Because of their hydrophilicity, selenoaminoacids are not retained onto typical reversed-phase columns. The use of ion-pairing reagents as mobile phase additives allows their separation. The ion-pairing reagent is usually an alkyl sulfonate, an alkyl sulfate, or an alkylammonium salt. Its nonpolar chain interacts with hydrophobic stationary phase (e.g., C8 or C18), while ionizable group is neutralized by oppositely charged analyte. Hexanesulfonic acid has been used as anion-pairing reagent in the speciation analysis of selenium in Brazil nuts, using C8 column for separation [70]. Obtaining separation was satisfied for organic compounds but poor for selenite and selenate. For separation of organic and inorganic forms of selenium, tetrabutylammonium acetate was proposed [71]. Also mixed ion-pairing reagents (butanesulfonic and tetramethylammonium hydroxide) were also used to simultaneously separate inorganic and organic species with satisfactory separation efficiencies [72].

New mobile phase additives are still developed, for example, room temperature ionic liquids [73]. Their mechanism of action is based on bilayer formulation onto stationary phase. It gives the possibility of additional interactions between the analyte and the bed, which significantly affects the retention and shape of the recorded signals. The effects of several imidazolium chlorides on the separation of selenium species mixture was described in details [74]. In all cases, SeMeCys was the first species eluted indicating its weak retention in the column, while the retention time of Se(VI) was increasing with the increase of alkyl chain.

#### *4.3.4 Hydrophilic interaction liquid chromatography*

Hydrophilic interaction liquid chromatography (HILIC) is a complementary technique to reversed-phase mode. The separation mechanism is mainly based on the partition of the analyte between the thin water layer adsorbed onto the stationary surface and the eluent, which contain high content of organic solvent usually acetonitrile. It is known that also other interactions such as hydrogen bonding dipole-dipole interactions and electrostatic forces may play an important role in the retention mechanism in HILIC [75]. The governed retention mechanism strictly depends on the type of used stationary phase and the buffer conditions

(content of organic solvent, concentration of salt, and pH). TSKgel Amide-80 stationary phase with covalently bound carbamoyl groups is frequently used in the analysis of selenium in HILIC mode [76, 77]. According to the characterization of amide stationary phase, the achieved separation is not pH dependent [78]. Also zwitterionic and silica stationary phases have been also used in HILIC separation of selenium [79, 80]. It should be highlighted that in selenium separation in HILIC mode methanol is recommended instead of acetonitrile as a main component of mobile phase [79] which is shown in **Figure 1**.

The use of methanol enhances peak intensity, improve the separation of SeMet and SeMeSeCys, and shorten time of the single run. The best separation conditions

**Figure 1.**

*The chromatogram of selenium compounds obtained on silica column using (A) MeOH and (B) ACN in the mobile phase [79]. Reprinted with permission from Elsevier.*

*Chromatographic Analysis of Selenium Species DOI: http://dx.doi.org/10.5772/intechopen.87053*

have been obtained for silica column and mobile phase consisted of 85% of methanol and 8 mM of ammonium acetate. Using the zwitterionic column (ZIC-HILIC) instead of silica stationary phase resulted in recording of very asymmetric peaks.

The potential of two orthogonal chromatographic modes—RP and HILIC—was examined in the analysis of onion leaf extracts [79]. Higher separation efficiency (mainly for inorganic selenium species) and shorter retention times were obtained when HILIC mode was used (**Figure 2**).

**Figure 2.**

*The chromatographic separation of selenium species present in onion leaf extracts in (A) HILIC mode and (B) RP mode. (A) Atlantis HILIC (silica) column, mobile phase: 85% MeOH and 8 mM CH3COONH4, pH 7. (B) Luna C8 column, mobile phase: 99.5% HCOOH and 0.5% ACN [79]. Reprinted with permission from Elsevier.*


**4.4 Treatment**

Non-operative treatments involve measures for pain relief and frequent visits to the physical therapists. These measures are effective only in few cases, since patients continue to be in a vicious cycle of increasing pain and reduced range of movement. As we know it is usually confirmed only on arthroscopy, it can certainly be treated at the same time. Diduch, in his study, claims 75% success of pain relief after arthroscopic debridement of impinged PCL stump in total knee replacement patients.


**10.1. Stress**

There is a great deal of translational overlap in the research areas where focus on AVP and OXT is most relevant, and this is especially true with the studies on the effects of stress. Exposure to acute and/or chronic stress is often a predictor of depression/anxiety, addiction relapse, and relationship difficulties. It is suggested that the most valuable paradigms for investigating the roles of AVP and OXT in depression, anxiety, or addiction involve exposure to chronic stress. The use of ethologically relevant stressors in animal models, such as social stress, is most likely to produce translationally consistent results (effects in animals which parallel clinical data). Many commonly used chronic stress protocols used in studies of AVP/OXT and depression and anxiety, such as chronic mild stress, do not use stressors associated with human disorders.

Behavioral Roles of Oxytocin and Vasopressin 65

The animal and human data on OXT support the hypothesis that this peptide hormone is also a valid target for novel maternal mood disorder treatments. An interesting implication in this area is that synthetic OXT is already commonly used to induce labor, yet little is known about how this treatment may affect maternal behavior and/or offspring. OXT or OXT antagonists may also be effective in treating melancholic depression and seasonal affective disorder. There are also interesting non-pharmaceutical interventions which can manipulate OXT levels, such as physical touch and modified birthing practices and procedures (cesarean sections and induced labor vs. natural childbirth). Greater collaboration between animal and clinical researchers will accelerate the development of safe and effective AVP and OXT targeted treatments for depression and anxiety disorders, including postpartum depression, seasonal affective disorder, and PTSD. Projects that involve consistent interactions between animal and clinical researchers throughout the developmental process will be most effective. Another potential therapeutic application of AVP and OXT is in relationship counseling. Both of these hormones are likely to be involved in the mechanisms of establishing and maintaining the social bond necessary for a strong and stable relationship. AVP and OXT targeted treatments may be effective in treating the adverse effects of chronic social conflict, or the effects of other chronic stressors, especially in

Both affiliative behavior and addiction are mediated through similar central reward pathways. Central OXT pathways are also altered by addiction. Endogenous OXT activity is suppressed by chronic drug use, and elevated brain OXT levels may attenuate the negative effects of withdrawal [211]. There is preliminary evidence that exogenous OXT is capable of inhibiting stimulant and alcohol self administration and it may prevent stress and priming induced relapse [212]. As with autism, OXT centered treatments may be a useful adjunct to behavioral cognitive techniques. For example, intranasal OXT may augment the positive

Levels of AVP mRNA increase in the amygdala during early withdrawal from cocaine [214], and the blockade of V1b receptors can block reinstatement in rodents [215]. In a rodent model of ethanol dependence, a V1b antagonist decreases excessive levels of ethanol self administration [216]. There is further evidence that AVP secretion is attenuated in response to social stress in the sons of alcohol dependent fathers, but it is unclear how these results relate to the risk of developing an addiction [217]. While data from humans is lacking, the involvement of AVP in the etiology of stress related depression and anxiety suggests that this hormone may be implicated in the long term effects of addiction and the mechanisms mediating relapse. V1b antagonism may be a productive translational target for not only

While current translational efforts with OXT and autism acknowledge that the effectiveness of intranasal OXT treatments may only be relevant to social behavior deficits, the animal

effects of extinction training for addiction [213] and/or reduce rates of relapse.

drug dependence, but addiction associated depression and anxiety as well.

combination with behavioral cognitive therapy.

**10.3. Addiction**

**10.4. Autism**

While the role of AVP in the endocrine stress response has been studied in detail at the animal level, the effects of stress on OXT are not as well known. Integrative investigations which include both AVP and OXT may indentify novel interactions between these behaviorally potent peptides. The most promising translational area may be PTSD. There is already evidence that male PTSD patients have high plasma AVP, aggression, depression and anxiety levels and similar behavioral effects have been associated with elevated AVP in animals. While it is difficult to separate the changes in depression and anxiety from impairments in social behavior, an increased focus on OXT in PTSD studies may provide insight on the social deficits in PTSD patients. Social bonds are often negatively impacted by exposure to chronic stress, and these bonds can have a positive buffering effect on the negative effects of chronic stress.

An indication of the potential value of social support can be seen in the cultural comparison of postpartum depression prevalence. Societies that have high levels of social support for mothers have low rates of depression, and cultures with low levels of support have much higher rates [208]. There is evidence that social support has protective effects in stress related mood disorders, and understanding the role of AVP and OXT in the positive effects of social support may help maximize the value of social support focused interventions.


*Edited by Everlon Cid Rigobelo*

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4. Hydrogen treatment of rutile TiO2

#### 4.1. Photocatalytic activity of reduced TiO2

TiO2 is often considered as a nonstoichiometric oxygen-deficient compound. According to Eq. (3), a small amount of electrons would be naturally doped. However, the n-type conductivity may be decreased by calcination in air at high temperature owing to the strong oxidation. To confirm the effect of electron density on the fast recombination observed in deactivated TiO2, we performed H2 reduction treatment to the rutile TiO2 particles calcined at 1100�C [8]. The TiO2 samples were reduced by H2 at 500 or 700�C, increasing both the density of longlived charge carriers and photocatalytic activity (Figure 3). These results suggest that the density of oxygen vacancies and/or electrons is an important factor determining the photocatalytic activity.

Figure 3. (A) Time course of photocatalytic H2 evolution from an aqueous ethanol solution with Pt, and (B) transient IR absorption triggered by a 355-nm laser pulse in the presence of methanol: (a) TiO2 calcined in air at 1100�C, (b) TiO2 reduced with H2 at 500�C after calcination at 1100�C, and (c) TiO2 reduced with H2 at 700�C after calcination at 1100�C.

H2 reduction treatment has received extensive attention for improving the photocatalytic activity of anatase TiO2 nanostructures since the recent report of the visible-light sensitivity of "hydrogenated black TiO2" with defect disorders [9]. H2 reduction treatment has also been reported to improve the PEC activity of TiO2 nanowire arrays [10]. The H2 treatment creates a high density of oxygen vacancies and electrons as shown in Eq. (1). We investigated the effects of H2 treatment temperature on the photocatalytic activity of rutile TiO2 particles to discuss the role of oxygen vacancies, Ti3+ ions, and conduction band electrons [11]. The photocatalytic activity was examined using an O2 evolution from an aqueous solution of 50 mmol L�1 AgNO3 as a sacrificial electron acceptor (4Ag+ + 2H2O ! 4Ag0 + O2 + 4H+ ). Calcination above 900�C decreased the photocatalytic activity of rutile TiO2 particles probably owing to strong oxidation, but its initial activity was restored by H2 treatment at above 500�C. The photocatalytic activity of reduced TiO2 was hardly changed after recalcination in air at 300�C, but significantly decreased after recalcination at 500�C.

#### 4.2. ESR and UV-vis-NIR spectroscopy

calcination temperature of the sample increased, suggesting the more recombination of photoexcited carriers. The rutile TiO2 particles calcined at high temperature showed the less density of long-lived photoexcited electrons, which resulted in the low photocatalytic activity. The reason for the deactivation at high temperature calcination is attributed to fast charge carrier

Figure 2. Transient IR spectroscopy setup and the transient absorption at 2000 cm1 triggered by a 355-nm laser pulse with 6-ns duration in the presence of methanol of the rutile TiO2 calcined in air at different temperatures: 500C, 700C,

Figure 1. Effect of calcination temperature on (a) the rate of photocatalytic H2 evolution from an aqueous solution of 50 vol% ethanol with H2PtCl6 (2.0 wt% as Pt) under UV irradiation from light emitting diode (peak wavelength 380 nm) and

TiO2 is often considered as a nonstoichiometric oxygen-deficient compound. According to Eq. (3), a small amount of electrons would be naturally doped. However, the n-type conductivity may be decreased by calcination in air at high temperature owing to the strong oxidation. To confirm the effect of electron density on the fast recombination observed in deactivated

recombination.

900C, and 1100C.

106 Titanium Dioxide

4. Hydrogen treatment of rutile TiO2

(b) BET specific surface area of rutile TiO2 particles.

4.1. Photocatalytic activity of reduced TiO2

Electron spin resonance (ESR) is active for paramagnetic species such as Ti3+ ions (electron trapped in titanium lattice site) and O•� radicals (hole trapped in oxygen lattice site). Figure 4A shows ESR spectra of the H2-reduced TiO2 samples [11]. The TiO2 calcined at 1100�C exhibited signals with g = 2.061 and 2.045. Kumar et al. have reported radical formation (g1 = 2.026, g2 = 2.017, and g3 = 2.008) on rutile TiO2 by calcination at 750�C and assigned the signals to trapped holes on the surface (Ti4+O2�Ti4+O•� radicals) [12]. It has been reported that strong oxidation of TiO2 facilitates the transformation of n-type oxygen-deficient TiO2�x to p-type metal-deficient Ti1�yO2 [13]. The high temperature calcination of TiO2 may be considered as acceptor doping to create titanium vacancy (V0000Ti) and hole (h• ) according to Eq. (10). The hole would be trapped in the oxygen lattice site as an O•� radical.

$$\text{CO}\_2 \rightarrow 2\text{O}\_\text{O}^\times + V^{\prime\prime\prime}\_{\text{Ti}} + 4h^\bullet \tag{10}$$

The signals attributable to O•� radicals (trapped hole) disappeared after H2 treatment at 400�C, indicating that strongly oxidized Ti1�yO2 was reduced to neutral TiO2 by the mild H2 treatment. The ESR spectrum of TiO2 reduced at 500�C exhibited a sharp signal at g = 2.002 assigned to electrons trapped in oxygen vacancies and an intense signal at g = 1.974 assigned to Ti3+ ions (electron trapped in Ti lattice site) in rutile. This indicates that H2 treatment at 500�C further reduced the TiO2 to oxygen-deficient TiO2�x. The signal at g = 1.974 was significantly broadened when the H2 treatment temperature was increased to 700�C, indicating the high density of Ti3+ ions. The TiO2 reduced at 700�C was assumed to be deeply doped n-type TiO2.

The color of the TiO2 samples changed from white to a pale ash color after H2 reduction treatment [11]. Figure 4B shows diffuse reflectance UV-vis-NIR spectra of the TiO2 samples. The onset of intense photoabsorption originating from interband transitions was located at ca. 415 nm corresponding to the bandgap of rutile, 3.0 eV. The spectrum of TiO2 reduced at 500 and 700�C exhibited a broad absorption located in the visible and NIR region, which can be assigned to the transition of electrons in shallow traps and the conduction band. From the NIR absorption, the density of electrons in the TiO2 reduced at 500�C was higher than that in the TiO2 reduced at 700�C. Therefore, it is concluded that H2 reduction at higher temperature increased the density of electrons in the conduction band.

#### 4.3. X-ray photoelectron spectroscopy (XPS)

Figure 5 shows X-ray photoelectron spectra of the strongly oxidized TiO2 and reduced TiO2 [11]. There was no significant change in the Ti 2p spectra. The binding energy of 458.6 eV for Ti 2p3/2 was similar to the literature value for Ti4+ in TiO2. This indicates that the amount of Ti3+ ions on the surface of reduced TiO2 was too small to analyze by XPS. In contrast, there was a

Figure 4. (A) ESR spectra and (B) diffuse reflectance UV-vis-NIR spectra of H2-reduced TiO2 samples: (a) TiO2 calcined in air at 1100�C, and (b–d) TiO2 treated with H2 after calcination at 1100�C. The H2 treatment temperatures are (b) 400�C, (c) 500�C, and (d) 700�C.

Figure 5. Ti 2p and O 1s X-ray photoelectron spectra of (a) TiO2 calcined in air at 1100�C, and (b) TiO2 treated with H2 at 700�C after calcination at 1100�C.

significant difference observed in the O 1s spectra. The peak at 529.8 eV was assigned to lattice oxygen of TiO2, and the shoulder peak at 531.5–532.0 eV was assigned to surface hydroxyl groups. Unexpectedly, the area assigned to the hydroxyl group was higher in intensity for reduced TiO2 than that of oxidized TiO2. This is because the oxygen vacancies at the top surface became filled by reaction with H2O in the air. It is reported that oxygen vacancies induce dissociation of water molecules and form two hydroxyl groups via H+ transfer to a neighboring lattice oxygen according to Eq. (11) [14]. Therefore, oxygen vacancies are not considered to be present on the surface of reduced TiO2 under ambient conditions. This suggests that oxygen vacancies are not a catalytic site in reduced TiO2 photocatalysts.

$$\text{V}\_{\text{O}}^{\bullet \text{\textbullet}} + \text{H}\_{2}\text{O} + \text{O}\_{\text{O}}^{\times} \rightarrow 2(\text{OH})\_{\text{O}}^{\bullet} \tag{11}$$

#### 4.4. PEC and electrochemical properties

as acceptor doping to create titanium vacancy (V0000Ti) and hole (h•

O2 ! 2O �

The signals attributable to O•� radicals (trapped hole) disappeared after H2 treatment at 400�C, indicating that strongly oxidized Ti1�yO2 was reduced to neutral TiO2 by the mild H2 treatment. The ESR spectrum of TiO2 reduced at 500�C exhibited a sharp signal at g = 2.002 assigned to electrons trapped in oxygen vacancies and an intense signal at g = 1.974 assigned to Ti3+ ions (electron trapped in Ti lattice site) in rutile. This indicates that H2 treatment at 500�C further reduced the TiO2 to oxygen-deficient TiO2�x. The signal at g = 1.974 was significantly broadened when the H2 treatment temperature was increased to 700�C, indicating the high density of Ti3+ ions. The TiO2 reduced at 700�C was assumed to be deeply doped n-type TiO2.

The color of the TiO2 samples changed from white to a pale ash color after H2 reduction treatment [11]. Figure 4B shows diffuse reflectance UV-vis-NIR spectra of the TiO2 samples. The onset of intense photoabsorption originating from interband transitions was located at ca. 415 nm corresponding to the bandgap of rutile, 3.0 eV. The spectrum of TiO2 reduced at 500 and 700�C exhibited a broad absorption located in the visible and NIR region, which can be assigned to the transition of electrons in shallow traps and the conduction band. From the NIR absorption, the density of electrons in the TiO2 reduced at 500�C was higher than that in the TiO2 reduced at 700�C. Therefore, it is concluded that H2 reduction at higher temperature

Figure 5 shows X-ray photoelectron spectra of the strongly oxidized TiO2 and reduced TiO2 [11]. There was no significant change in the Ti 2p spectra. The binding energy of 458.6 eV for Ti 2p3/2 was similar to the literature value for Ti4+ in TiO2. This indicates that the amount of Ti3+ ions on the surface of reduced TiO2 was too small to analyze by XPS. In contrast, there was a

Figure 4. (A) ESR spectra and (B) diffuse reflectance UV-vis-NIR spectra of H2-reduced TiO2 samples: (a) TiO2 calcined in air at 1100�C, and (b–d) TiO2 treated with H2 after calcination at 1100�C. The H2 treatment temperatures are (b) 400�C, (c)

hole would be trapped in the oxygen lattice site as an O•� radical.

108 Titanium Dioxide

increased the density of electrons in the conduction band.

4.3. X-ray photoelectron spectroscopy (XPS)

500�C, and (d) 700�C.

) according to Eq. (10). The

O þ V0000Ti þ 4h• ð10Þ

Thermally oxidized TiO2 films were prepared by a simple calcination of a Ti sheet at 900�C [11]. Figure 6A shows PEC properties evaluated in dilute sulfuric acid (0.1 mol L�1 H2SO4, pH = 1) under UV irradiation (wavelength >330 nm). The thermally oxidized TiO2 film was inactive for

Figure 6. Effect of H2 treatment temperature on (A) PEC voltammetry curves in 0.1 mol L�1 H2SO4 (pH = 1) and (B) sheet resistance and donor density of thermally oxidized TiO2 films.

PEC water oxidation. The photocurrent was also negligible for the TiO2 films treated with H2 at 400C. However, anodic photocurrent was observed at applied potentials larger than +0.1 V versus Ag-AgCl after H2 treatment at higher temperature, suggesting the formation of longlived holes in the reduced TiO2 films.

Figure 6B shows the relationship between sheet resistance and donor density of the thermally oxidized TiO2 films after H2 treatment [11]. The sheet resistance was measured using a fourpoint probe. The donor densities were evaluated by Mott-Schottky analysis of the capacitance of the space charge layer. The Mott-Schottky plots of the TiO2 films showed a positive slope of n-type conductivity. The resistance of the thermally oxidized TiO2 films was high owing to their low donor density. H2 treatment at above 450C greatly reduced the sheet resistance and increased the donor density. H2 treatment at 600C increased the donor density by 2–3 orders of magnitude. The enhanced PEC and photocatalytic properties are because of the increase of n-type conductivity by H2 treatment.


*2.5. ß-Galactosidase-Measurements*

The ß-galactosidase activity of transcriptional fusions was measured by the hydrolysis of O-nitrophenyl- ß-D-galactopyranoside (ONPG) (Serva, Heidelberg, Germany) and expressed as Miller Units, as described in [36].

## *2.6. Di*ff*erential RNA-seq*

RNA isolation, TEX (Terminator EXonuclease) treatment, library construction and sequencing, read mapping, and transcription start site prediction have been described in detail in [37]. The RNA-seq data sets are available at the NCBI Gene Expression Omnibus database under accession number GSE71844.

#### *2.7. RNA Isolation and Quantitative RT-PCR*

Twenty milliliters of *R. sphaeroides* cells were harvested by centrifugation when an OD660 of 0.5 was reached. Total RNA for quantitative RT-PCR was isolated by using the peqGOLDTriFast kit (Peqlab, Erlangen, Germany), as described by the manufacturer. Remaining traces of DNA were removed by the TURBO DNaseI (Invitrogen, Schwerte, Germany). To further confirm the absence of DNA, PCR was performed targeting *gloB* (RSP\_0799) with the primers listed in Table S2. Quantitative RT-PCR was performed in a Bio-Rad CFX96 Real-Time system, as described in our previous study [20]. The reference gene *rpoZ* encoding the ω-subunit of RNA polymerase of *R. sphaeroides* was used to normalize the mRNA expression levels [38] according to the formula given by Pfaffl [39]. Primers are listed in Table S2.

#### *2.8. Analysis of isc-suf Operon Synteny*

The synteny analysis was performed by applying the EDGAR software [40].

Purification of the IscR, Irr, and OxyR proteins and electrophoretic mobility shift assays with dsDNA and protein.

*E. coli* M15 (pREP4/pQE2.4.1oxyR) was used for the overexpression of His-tagged OxyR protein, and the purification of OxyR was performed, as described earlier [41]. Isolation of His-tagged Irr from *E. coli* M15 (pREP4/pQE2.4.1*irr*) is described in [30], and the purification of His-tagged IscR from *E. coli* M15 (pREP4/pQE2.4.1*iscR*) in [31]. Binding of the proteins to the promoter regions of the *isc-suf* operon was determined by an electrophoretic mobility shift assay, as described previously [30,38]. DNA fragments containing the promoter regions of the *isc-suf* operon were produced by PCR. Primers used in the PCR are listed in Table S2. The final volume of the binding reactions was 15–20 μL. The reaction mixtures contained varying amounts of protein, ~3–7 fmol γ-32PATP labeled DNA fragment (2000 cpm, Hartmann Analytik, Braunschweig, Germany), salmon sperm DNA (1 μg), and binding buffer, as described elsewhere [30,42]. After the binding reactions were incubated for 30 min at room temperature or 4 ◦C, the samples were loaded onto a 4% (*w*/*v*) polyacrylamide gel in 0.5×TBE buffer (22 mM Tris-HCl, 22 mM boric acid, 0.5 mM EDTA, pH 8.3), and the electrophoresis was performed at 130–180 V for 2–5 h at room temperature or at 4 ◦C. Irr and IscR were isolated and tested under non-reducing conditions. OxyR was purified and tested under reducing and non-reducing conditions with the same outcome.