**3.2 Ontogenetic changes in δ15N and δ13C values in common minke whales**

**Figure 4** shows the δ15N and δ13C signatures with a focus on calves of MW [40]. MWs are mysticete and opportunistic feeders that change their prey items temporally and regionally; they feed on zooplankton and small fish. MW may strand during their migration [75].

A clear δ15N-enriched peak was found in MW calves (n = 12), including newborn animals at 2.6 and 3.0 m BL. Similar to BWs (**Figure 3**), the increase in δ15N values toward this peak could represent nursing, and the decrease in δ15N values from this peak could represent weaning [40]. The δ15N values of calves fitted to a quadratic function (*p* < 0.05), and this peak calculated by the fitted equation was 4.0 m BL and 13.3‰, suggesting the onset weaning at 4 m BL. In addition, the BL of weaned animals was estimated at 5 m BL from the fitted equation and the average δ15N values of mature animals as 11.9‰, which coincided with the reported BL of weaning [76]. Thus, the weaned BL of MWs (**Figure 4**) and BWs (**Figure 3**) estimated using δ15N profiles in muscle samples coincided with the weaned BL estimated from ecological and morphological studies. We believe that the δ15N value of the muscle from calves of mysticetes reflects the dietary shift quite rapidly with little time lag, because of the brief weaning period [5, 6] and the fast turnover rate of small and growing animals [48, 49].

*Typical Changes in Carbon and Nitrogen Stable Isotope Ratios and Mercury Concentration… DOI: http://dx.doi.org/10.5772/intechopen.103067*

#### **Figure 4.**

*Ontogenetic changes of δ15N and δ13C signatures in muscle of common minke whale stranded in Hokkaido. This figure was reprinted from Endo et al. [40] with permission from Aquatic Mammals.*

No particular pattern was found in δ13C values of MW calves, probably owing to the large variation in δ13C-depleted lipid concentrations in milk [3, 33, 42].

The Hg concentrations in immature (weaned) animals were apparently higher than those in MW calves because of the opportunistic eating of fish. Marked increases in Hg owing to weaning were found in liver samples of MWs (data not shown).

Studies on the ontogenetic changes in δ13C and δ15N values in mysticetes were conducted by Borrell et al. [3] using muscle samples from FWs and by Mitani et al. [53] using baleen plate samples of MWs. However, the study on FWs did not include calf samples, and a clear δ15N-depleted peak related to weaning was not found among the few δ15N-depleted peaks observed in the baleen plate of MWs.


**1. Introduction**

The global prevalence of spinocerebellar ataxia is 1 to 5 per 100,000 but higher in Asian populations while lower in European countries [1]. Ataxia is the loss of neural control over muscular tone and function affecting gait, coordination, eye movement, and speech; swallowing and other voluntary activities are also affected. Spinocerebellar ataxia (SCA) is a large and complex group of hereditary (genetic, familial), progressive, neurodegenerative, and heterogeneous disease that mainly affects the cerebellum. SCA is a subgroup of inherited cerebellar ataxia and is a rare disease. Currently, in excess of 40 distinctive genetic SCAs have been identified. The genetic contributions and impact of SCA is very complex to understand but is critical to diagnosing and classifying the disease which determine treatment approaches [1, 2]. SCA presents clinically with several symptoms related to the neuromuscular systems and is characterized by cerebellar gait and limb ataxia (with dysmetria, dysdiadochokinesia, intention tremor, dysarthria, and nystagmus), which may be

present together with extracerebellar signs, such as ophthalmoplegia, pyramidal signs, movement disorders (including parkinsonism, dystonia, myoclonus, and chorea), dementia, epilepsy, visual disorders (including pigmentary retinopathy), and peripheral neuropathy. These symptoms contribute to significant physiological impact which influences Disability Adjusted Life Years (DALY). One DALY is the equivalent of one healthy life-year lost and contributes to the global disease burden [3, 4]. Individuals diagnosed with SCA may present clinically with self care deficits and experience increased nutrient requirements coupled with limitations in dietary intake. The progressive nature of the disease makes it critical for early nutritional intervention and management to correct, resolve and limit the negative health and nutrition outcomes associated with the disease. Consequently, health care practitioners must collaborate to create the best therapeutic outcome for the client diagnosed with SCA.


**2. Evolution from high silver to low silver solder**

In 2000s, SAC system had been identified as replacement for Sn-Pb eutectic solders in fulfilling the RoHS compliance. Different countries or organizations had chosen different SAC composition as their preferred solder composition. They are Sn3.0Ag0.5Cu, SAC305 (Japan), Sn3.5Ag0.9Cu, SAC3509 (European Union), and Sn3.9Ag0.6Cu, SAC3906 (USA) [3].

In 2000, it was the era of consumer product, which had incorporated many electronic components in its system such as personal computer, television, radio, rice cooker, and windowmounted air conditioner. These products were mostly using through-hole components and/ or big surface mount technology (SMT) components. It implies that the consumption of solder per unit device was huge during that period of time. It explains the reason of mass adoption of SAC305 instead of other Pb-free solutions. It is because the lower silver content of SAC305 as compared to other SAC alloys mentioned in former paragraph. Silver is a precious metal and its inclusion in solder alloy does increase the cost of solder significantly. For example, to produce 1 kg of SAC305, 1 ounce of silver needs to be added. It depends on the market price of silver, assuming USD20 per ounce, the cost of silver in 1 kg of SAC305 is almost 50% of the selling price of 1 kg of SAC305 solder bar. When the silver price surges, for example, in April 2011, when the silver price was USD 40 per ounce, the cost of silver of a SAC305 solder bar was more than 50% of its selling price. For those compositions with higher silver content, the ratio of silver cost against solder bar selling price is usually more than 50% depending on the tin price. It becomes a big burden to solder users. Solder bar is mainly used in wave soldering process. It was a very common process in printed circuit board assembly (PCBA) for those consumer products mentioned above. During the peak of this era, the consumption of solder bar could exceed 2000 kg per month for a single customer who has more than 10 wave soldering lines in production. Another reason of mass adoption of SAC305 instead of other higher Ag bearing SAC alloy is more a political reason. During that time, Japanese consumer product was very popular and common. *National* brand electric rice cooker should be one of the must have electrical appliances in every home. SAC305 was the Japan Electronics and Information Technology Industries Association, JEITA recommended Pb-free alloy too.

Nevertheless, the industries were looking for alternatives for SAC305 very quickly after its introduction to industries, even though with its acceptance as Pb-free alloy in replacing SnPb-6337 solder. In general, there are two major reasons in justifying this direction, cost and drop impact resistance of SAC305 alloy. The users were looking for alternative to mitigate the high cost of SAC305 as mentioned above. This was the time when low SAC alloy started to join the supply chain of solder. **Table 2** lists some of the low SAC alloys available in market offered by different solder suppliers. The introduction of these low SAC alloys was as soon as 2 years after the mass adoption of SAC305 alloy as Pb-free solder in the industries. Nevertheless, SAC305 is still the major adopted alloy in the industries after 10 years of RoHS enactment. It is worth to note that Nihon Superior has introduced Ag-free Pb-free solution since 1999. It has become a main solution in soldering especially in wave soldering and Pb-free hot air solder leveling (HASL) process.

existing alloys to close the gap between the user and supplier of solder material. In the chapters that follow the evolution of Pb-free alloy and the techniques used in enhancing solder

In 2000s, SAC system had been identified as replacement for Sn-Pb eutectic solders in fulfilling the RoHS compliance. Different countries or organizations had chosen different SAC composition as their preferred solder composition. They are Sn3.0Ag0.5Cu, SAC305 (Japan),

In 2000, it was the era of consumer product, which had incorporated many electronic components in its system such as personal computer, television, radio, rice cooker, and windowmounted air conditioner. These products were mostly using through-hole components and/ or big surface mount technology (SMT) components. It implies that the consumption of solder per unit device was huge during that period of time. It explains the reason of mass adoption of SAC305 instead of other Pb-free solutions. It is because the lower silver content of SAC305 as compared to other SAC alloys mentioned in former paragraph. Silver is a precious metal and its inclusion in solder alloy does increase the cost of solder significantly. For example, to produce 1 kg of SAC305, 1 ounce of silver needs to be added. It depends on the market price of silver, assuming USD20 per ounce, the cost of silver in 1 kg of SAC305 is almost 50% of the selling price of 1 kg of SAC305 solder bar. When the silver price surges, for example, in April 2011, when the silver price was USD 40 per ounce, the cost of silver of a SAC305 solder bar was more than 50% of its selling price. For those compositions with higher silver content, the ratio of silver cost against solder bar selling price is usually more than 50% depending on the tin price. It becomes a big burden to solder users. Solder bar is mainly used in wave soldering process. It was a very common process in printed circuit board assembly (PCBA) for those consumer products mentioned above. During the peak of this era, the consumption of solder bar could exceed 2000 kg per month for a single customer who has more than 10 wave soldering lines in production. Another reason of mass adoption of SAC305 instead of other higher Ag bearing SAC alloy is more a political reason. During that time, Japanese consumer product was very popular and common. *National* brand electric rice cooker should be one of the must have electrical appliances in every home. SAC305 was the Japan Electronics and Information Technology Industries Association, JEITA recommended Pb-free alloy too.

Nevertheless, the industries were looking for alternatives for SAC305 very quickly after its introduction to industries, even though with its acceptance as Pb-free alloy in replacing SnPb-6337 solder. In general, there are two major reasons in justifying this direction, cost and drop impact resistance of SAC305 alloy. The users were looking for alternative to mitigate the high cost of SAC305 as mentioned above. This was the time when low SAC alloy started to join the supply chain of solder. **Table 2** lists some of the low SAC alloys available in market offered by different solder suppliers. The introduction of these low SAC alloys was as soon as 2 years after the mass adoption of SAC305 alloy as Pb-free solder in the industries. Nevertheless,

Sn3.5Ag0.9Cu, SAC3509 (European Union), and Sn3.9Ag0.6Cu, SAC3906 (USA) [3].

alloy strength are discussed.

94 Recent Progress in Soldering Materials

**2. Evolution from high silver to low silver solder**

With lower silver content in SAC alloy, some users were hoping the hot tear or shrinkage cavity could be mitigated. This phenomenon which was first believed as a process defect was found when users migrating from SnPb-6337 to SAC305. To date, many users are still confused by this phenomenon even though IPC has classified it as an acceptable phenomenon. **Figure 1** shows the comparison of few low SAC alloys and a eutectic Tin Copper Nickel alloy.

Besides the appearance of solder whose difference is easily noticeable, the users were also concerned about the reliability of low SAC especially the intermetallic compound (IMC) formed in between the solder and soldering pad. **Figures 2** and **3** show the IMC of SAC 305, low SAC, SAC1205, and Ag-free Pb-free solder (SnCuNi) on Copper (Cu) pad and Nickel Gold (NiAu) pad, respectively. Both SAC alloys regardless of Ag content were showing columnar growth of IMC and the IMC continued to grow during the heat treatment at 125°C for 500 h. The SnCuNi IMC appeared to be flat and stable even after the heat treatment. Besides the columnar IMC, crack was observed at the Cu pad after heat treatment within the IMC of SAC305 but not at the SAC1205 and SnCuNi alloys. This observation matched the explanation proposed by Nogita et al. in his journal [4]. The Ni addition in Pb-free alloy has successfully suppressed the polymorphic transformation of Cu6 Sn5 , which is the culprit of this IMC crack. There is transformation of hexagonal to monoclinic Cu6 Sn5 and vice versa at temperature of 186°C due to the allotropic attribute of Cu6 Sn5 . This transformation involves volume change of the Cu6 Sn5 IMC, which causes high stress at this layer. The stress has exceeded the strength of this IMC and caused the crack within this layer. Micro crack might have formed within the SAC305 IMC during the solidification process of joint formation and the cracks propagated during the heat treatment due to IMC growth. This has explained why the crack still formed even though the heat treatment temperature was just 125°C, which was far below the polymorphic transformation temperature.

**Table 2.** List of low SAC solder and no Ag solder as alternatives of SAC305.

**Figure 1.** Appearance comparison of different SAC alloys and a eutectic SnCuNi alloy.

**Figure 2.** IMC formed on Cu pad with solder alloy of SnCuNi (SN100C®), SAC305 and SAC1205 (LF35).

Besides the cost concern mentioned earlier, another driving force which triggered the industries to consider low SAC alloy was the drop impact resistance. It is widely accepted that silver addition can effectively reduce the liquidus and increase the yield strength and modulus of SAC alloy; also because of the high strength and high modulus which make the alloy

**Figure 3.** IMC formed on NiAu pad with solder alloy of SnCuNi (SN100C®), SAC305, and SAC1205 (LF35).

readily transfer the stress to the solder joining interface which is the region of IMC [5]. Due to this mechanism, most of the failures found in drop impact resistance test with conventional SAC alloys mostly locate at the IMC region, and most of these failures are early failures. By reducing the Ag content of SAC alloy, the modulus of alloy decreases. It has significantly increased the resistance to the drop impact. The urge of looking for an alloy with high drop impact resistance increased further due to the popularity of portable devices in mid-2000 to late-2000. During that period of time, portable music player such as MP3 player and iPod and handheld game console were very common. The robustness against drop shock was crucial to keep a reasonable reliability of such devices. For example, during the initial implementation of Pb-free soldering, most of the solder ball used in ball grid array, BGA package is SAC305 composition. Many mobile integrated circuits, ICs had migrated to LF35 or SAC1205 BGA solder ball in order to increase the drop test performance of the ICs. However, by only lowering the Ag content of SAC alloy did not give a total solution to meet the expectation of users. Many other microalloying had been deployed by solder manufacturers in order to increase the alloy performance of low SAC alloy such as adding Nickel, Ni, Zinc, Zn and Manganese, Mn into the alloy system. As shown in **Figure 2**, the addition of Ni into LF35 composition has suppressed the polymorphic transformation of Cu6 Sn5 during the solidification. It cannot be achieved by only reducing the Ag content of SAC alloy.

The acceptance of low SAC or SnCuNi as Pb-free solution is also encouraged by the readiness of other material to Pb-free soldering process. One of the major challenges in adopting low SAC or SnCuNi alloy is the high melting point of this alloy. It has approximately 44 and 10°C higher melting point comparing to eutectic SnPb alloy and SAC alloy, respectively. However, the market is now more ready for this Pb-free soldering. The reflow and wave soldering machines are having higher heat capacity to accommodate the high melting point of Pb-free solder, PCB, and electronic components are using higher glass transition (Tg) temperature materials and soldering flux can survive longer in high-temperature reflow. All these

Besides the cost concern mentioned earlier, another driving force which triggered the industries to consider low SAC alloy was the drop impact resistance. It is widely accepted that silver addition can effectively reduce the liquidus and increase the yield strength and modulus of SAC alloy; also because of the high strength and high modulus which make the alloy

**Figure 2.** IMC formed on Cu pad with solder alloy of SnCuNi (SN100C®), SAC305 and SAC1205 (LF35).

**Figure 1.** Appearance comparison of different SAC alloys and a eutectic SnCuNi alloy.

96 Recent Progress in Soldering Materials

improvements have enabled the adoption of low SAC and SnCuNi alloy in soldering. This has also explained the market share of this low SAC and SnCuNi alloys is expanding in recent years. SN100C® has become one of the preferred choices in Pb-free wave soldering process.

In short, there are two driving forces, which have triggered the proliferation of low SAC and SnCuNi alloys for electronic industries. Someone has named the low SAC as second generation of Pb-free alloy. **Figure 4** summarized the evolution of this Pb-free alloy from conventional SAC to low SAC alloy for electronic industries. This transition is very important to support the IoT era. One of the characteristics of IoT is huge in quantity. The high usage of electronic components in IoT era also prompts the usage of solder. The type of solder for this era must be able to fulfill the specific requirement in manufacturability, reliability, toxicity, cost, and availability. The second generation Pb-free alloy should be a better choice for the IoT era.

The following section discusses the development of Pb-free alloy into third generation alloy for high reliability application.

**Figure 4.** Evolution of SAC alloys in Pb-free solution from first generation to second generation.


**2. Related work**

#### **2.1. Chronic obstructive pulmonary disease**

COPD is one condition of COAD and we introduce some studies of COPD disease. The research of the World Health Organization (WHO) and the global initiative for chronic obstructive lung disease revealed that nowadays the COPD is the fourth cause of death in the United States. For the entire world, the COPD can be considered the fifth cause of death [4]. In another study, the COPD can be considered the fourth leading cause of death in the world [5] and is projected to be the fifth by 2020 as a worldwide burden of disease [6].

Generally speaking, chronic disease is gradually rising because of the ageing population and changing habits. The treatment for COPD patients is pulmonary rehabilitation programmes, including when patients are discharged at home [7]. Economic analyses have shown that over 70% of COPD-related health-care expenditures result from emergency room visits and hospital care for exacerbations; this translates to >\$10 billion annually in the Unites States [8]. COPD patients have a lower physical activity level than healthy peers [9]. The reduced level of physical activity also related to an increased risk [10]. Liao et al. [11] proposed a review that concentrated on describing wearable devices for measuring physical activity level in COPD patients. In [12], the authors evaluated a method for detecting an exacerbation onset in COPD patients. They used data collected through a pulse oximeter, permitting an easy way to the cohort of patients composed of elderly people affected by COPD. The study [13] provided a system, which offers an effective platform for the satisfaction of the clinical and the patient's needs in the area of early diagnosis of patient's health status. The portable system aims at the effective management in the health status of the patients who are suffering from COPD.

#### **2.2. Gait analysis**

People increase the respiratory ventilation when they are walking or exercising. However, those people who suffer from chronic obstructive airway disease (COAD) cannot increase their respiratory ventilation quick enough to maintain the exercising behaviour. Consequently, they change their behaviour such as walking slowly so that they can maintain their respiratory ventilation. We can perform gait analysis on COAD patients because their walking behaviours are different with normal people when they are exercising because of their respiratory function. Chronic obstructive pulmonary disease (COPD) is one condition of COAD. Nowadays, there are many chronic diseases in our daily life. COPD is one of them. COPD is a chronic airway disease characterized by progressive going downhill of the breathing functions [2]. One characteristic of COPD disease is the decreasing of forced expiratory volume in 1 s (FEV1) because of the obstructive airway [2]. Depending on their disease severity, they have different walking behaviours. Therefore, gait analysis can be used to judge the COAD patients' airway condition by observing their walking behaviour. However, it is difficult to collect the data of COAD patients without medical staff. Without clinical data, we cannot verify the correctness

By cooperating with Shuang-Ho Hospital in New Taipei, Taiwan, we set up an experiment. We film the side view of the subjects when they are performing a 6-min brisk walking test. By gait analysis, we can extract the features from walking behaviour such as pace distance and walking speed variation. However, in order to obtain that physiological information, we may need to wear sensors or markers on the subjects. Our method does not need to wear any sensors

In gait analysis, it is common to wear markers or sensors to record walking behaviour. In the experiment [1], the subjects need to wear a recorder on the ankle so that it could record the stride interval. In another experiment [3], the subjects also need to place a designed insole with

However, there are some drawbacks of using makers or sensors. Firstly, it is inconvenient and uncomfortable of attaching them on human body and might affect the normal walking. Secondly, some sensors are heavy or hard to use for the elderly. Thirdly, some sensors have the electromagnetic interference that might affect and harm human body. In addition to the sensor problem, it is hard to tell a subject suffering from COAD disease or not by a single experiment. Without a complete examination, it is hard to judge whether the subjects are COAD patients or not. Consequently, we decide to access the respiratory function. By the pulmonary spirometer, we can obtain the tested subjects' respiratory data. Nowadays, the parameters from pulmonary spirometer are the standard to access the respiratory function.

COPD is one condition of COAD and we introduce some studies of COPD disease. The research of the World Health Organization (WHO) and the global initiative for chronic obstructive lung

of our gait analysis algorithm.

2 Advanced Biosignal Processing and Diagnostic Methods

12 sensors into their own shoes.

on the subjects.

**2. Related work**

**2.1. Chronic obstructive pulmonary disease**

Gait analysis plays an important role in accessing human's walking behaviour and it aims to extract biomechanics information. The obvious disease on lower limbs is Parkinson's disorder. In [14], they provided a feasible method image marker to measure gait with little skin move‐ ment. Then, they performed quantitative analysis to extract gait parameter. According to their analysis, the joint angle, rotation angle of lower limbs, stride velocity and stride length have significant difference between Parkinson's disease patients and non-diseased subjects. The study [1] demonstrated that the gait variability in terms of statistical parameters of stride interval such as STC*γ*, would be increased in Parkinson's disorder. In addition to Parkinson's disease, Alzheimer's disease (AD) also can be detected by gait analysis. The study [15] presents an inertial-sensor-based wearable device and its associated stride detection algorithm to analyse gait information for patients with Alzheimer's disease.

The above methods all use markers or sensors to get interest points and then use gait analysis. With the advances in smart phones, Susu Jiang et al. [16] used a smart phone with an acceler‐ ometer and a gyroscope to collect human walking gait data in daily life. However, one smart phone only records one feature data. If we want to acquire many interest point data, testers need to tie many smart phones on the body, which might be inconvenient for walking.

Now, we proposed a gait analysis algorithm without any wearable marker or sensor. In addition, we also collect clinical COAD and control data to verify the correlation between pulmonary spirometer and our gait analysis method.


**4.1. Three-port circulator utilizing ferrite coupled slotline junction**

The first investigated circuit is three-port circulator realized in coupled slotline technology, which is shown in Fig 19(a).

The device is realized as a cascade connection of a T-junction, ferrite coupled slotline (FCSL) junction, and the output section being the transformer from the coupled slotlines to the microstrip lines. The cross section of the FCSL junction is shown in Fig. 19(b). The FCSL junction is a four-layer structure, where the coupled slotlines are realized on a thin laminate situated above the ferrite material. The T-junction and the output section have the same cross section as ferrite section, although instead of ferrite, a dielectric material with relative

**Figure 19.** FCSL circulator: (a) schematic view of the device and (b) cross section of FCSL junction

permittivity *εr* = 9.6 is used. T-junction is realized as a transformer from coplanar line into coupled slotlines. Due to the fact that the coplanar line is fed from the coaxial connector, the junction provides even excitation of FCSL section. On the other hand, the odd mode, which is transmitted from the ferrite section, is totally reflected in the plane of the coaxial connector. The output structure allows for the transmission of the signal from the specific slot of ferrite junction to the corresponding output microstrip port, while the remaining ports of the circulator are isolated.

In order to determine the scattering parameters of the circulator shown in Fig. 19(a), the scattering matrices of the ferrite junction, T-junction, and output section were first calculated. The scattering parameters of the FCSL junction were simulated with the use of our own software based on the methods described in section 2. The feeding circuits (T-junction and output section) were designed with the use of commercial software. The circulator scattering parameters were obtained by cascade connection of the calculated scattering matrices of individual sections. The simulated frequency characteristics of the scattering parameters are shown in Fig 20.

From the obtained results, it can be seen that the investigated configuration has return losses and isolation better than 10 dB over a wide frequency range from 13 to 20.5 GHz. In the considered frequency band, the average transmission losses are about 1 dB in the case of a single pass of the signal through the ferrite section and about 2 dB in the case of double pass of the signal through the ferrite section. In the analysis, the material losses were not taken into consideration, and the resulting level of losses is due to return losses and the lack of a perfect isolation between the ports of circulator.

A photograph of the manufactured prototype of the circulator is shown in Fig. 21. The measured characteristics of its scattering parameters are depicted in Fig. 22.

Measurements were performed in the frequency range from 10 to 21 GHz. The results show that the investigated device operates in a wide frequency band. In the frequency range from 12 to 19 GHz, the average transmission losses are about 2.5 dB, and the level of isolation and return losses is about 10 dB for a single pass of the signal through the ferrite

T-junction FCL = /4 Output section

Ferrite

(a)

perfect isolation between the ports of circulator.

3

permittivity *εr* = 9.6 is used. T-junction is realized as a transformer from coplanar line into coupled slotlines. Due to the fact that the coplanar line is fed from the coaxial connector, the junction provides even excitation of FCSL section. On the other hand, the odd mode, which is transmitted from the ferrite section, is totally reflected in the plane of the coaxial connector. The output structure allows for the transmission of the signal from the specific slot of ferrite junction to the corresponding output microstrip port, while the remaining ports

In order to determine the scattering parameters of the circulator shown in Fig. 19(a), the scattering matrices of the ferrite junction, T-junction, and output section were first calculated. The scattering parameters of the FCSL junction were simulated with the use of our own software based on the methods described in section 2. The feeding circuits (T-junction and output section) were designed with the use of commercial software. The circulator scattering parameters were obtained by cascade connection of the calculated scattering matrices of individual sections. The simulated frequency characteristics of the scattering parameters are

From the obtained results, it can be seen that the investigated configuration has return losses and isolation better than 10 dB over a wide frequency range from 13 to 20.5 GHz. In the considered frequency band, the average transmission losses are about 1 dB in the case of a single pass of the signal through the ferrite section and about 2 dB in the case of double pass of the signal through the ferrite section. In the analysis, the material losses were not taken into consideration, and the resulting level of losses is due to return losses and the lack of a

A photograph of the manufactured prototype of the circulator is shown in Fig. 21. The

Measurements were performed in the frequency range from 10 to 21 GHz. The results show that the investigated device operates in a wide frequency band. In the frequency range from 12 to 19 GHz, the average transmission losses are about 2.5 dB, and the level of isolation and return losses is about 10 dB for a single pass of the signal through the ferrite

measured characteristics of its scattering parameters are depicted in Fig. 22.

**Figure 19.** FCSL circulator: (a) schematic view of the device and (b) cross section of FCSL junction

slot lines

2

microstrip line

microstrip line

**Hi**

(b)

h0

h1 h2 h3

h4

w1 s w2

ferrite

dielectric r3

dielectric r2

dielectric r1

1

coplanar line

136 Advanced Electromagnetic Waves

of the circulator are isolated.

shown in Fig 20.

**Figure 20.** Simulated scattering parameters of FCSL circulator: (a) transmission with isolation and (b) reflection

**Figure 21.** Photograph of the fabricated FCSL circulator: (a) top view and (b) bottom view

section. The best isolation is observed in the frequency range from 12 to 15 GHz and is better than 16 dB. In the case of a double pass of the signal through the ferrite section, the transmission losses are two times higher and are about 5.5 dB in the frequency range from 12 to 19 GHz. The isolation is better than 14 dB in the entire frequency range. In the measured transmission characteristics, small periodically repeating resonances occur, which result from the inaccuracies in the manufacturing process.

### **4.2. Double isolator utilizing microstrip coupled line section**

Another investigated configuration is a double isolator shown in Fig. 23. This arrangement is composed of two interconnected and magnetized in the same direction three-port circulators (see Fig. 2(c)), in which the appropriate ports are terminated by matched loads. The advantage of this configuration is the ability to achieve high isolation. Unfortunately, due to the fact that the device uses two ferrite sections, the losses in the system are twice as high as in the case of isolator with a single section of FCL.

**Figure 22.** Measured scattering parameters of FCSL circulator: (a) transmission with isolation and (b) reflection

**Figure 23.** Double isolator utilizing microstrip ferrite coupled line junction

Utilizing own software, as well as commercial simulator, the double isolator utilizing ferrite junction from Fig. 8 was designed. The simulation results are shown in Fig. 24.

From the obtained results, it can be seen that in the frequency range from 10 to 16 GHz, the isolation is better than 20 dB with return losses better than 10 dB.

The designed structure was manufactured. A photograph of the prototype is shown in Fig. 25. To obtain the double isolator, ports (3) and (4) of the structure were terminated with the matched SMA connectors. The obtained experimental results are presented in Fig. 26.

It can be seen that the device works in the frequency band from 9 to 16 GHz. In the given frequency range the isolation and the average return losses are better than 15 dB. Furthermore, the average transmission losses are 3.5 dB and they change from 3 dB at 9 GHz to 4 dB at 16 GHz. Based on these results, it can be estimated, that the losses for a single pass of the signal through the investigated microstrip ferrite section are about 1.8 dB. The losses are lower by about 1.5 dB in comparison to the results published for a single section configuration in [4].

**Figure 24.** Simulated scattering parameters of double MFCL isolator: (a) transmission with isolation and (b) reflection

**Figure 25.** Photograph of the manufactured double MFCL isolator

11 12 13 14 15 16 17 18 19 20 21 −22

(a)

T-junction

Frequency (GHz)

FCL-section

microstrip line

**Figure 23.** Double isolator utilizing microstrip ferrite coupled line junction

isolation is better than 20 dB with return losses better than 10 dB.

S13 S21 S32 S12 S23 S31

**Figure 22.** Measured scattering parameters of FCSL circulator: (a) transmission with isolation and (b) reflection

**Hi Hi**

Utilizing own software, as well as commercial simulator, the double isolator utilizing ferrite

From the obtained results, it can be seen that in the frequency range from 10 to 16 GHz, the

The designed structure was manufactured. A photograph of the prototype is shown in Fig. 25. To obtain the double isolator, ports (3) and (4) of the structure were terminated with the matched SMA connectors. The obtained experimental results are presented in Fig. 26. It can be seen that the device works in the frequency band from 9 to 16 GHz. In the given frequency range the isolation and the average return losses are better than 15 dB. Furthermore, the average transmission losses are 3.5 dB and they change from 3 dB at 9 GHz to 4 dB at 16 GHz. Based on these results, it can be estimated, that the losses for a single pass of the signal through the investigated microstrip ferrite section are about 1.8 dB. The losses are lower by about 1.5 dB in comparison to the results published for a single section

junction from Fig. 8 was designed. The simulation results are shown in Fig. 24.

−20 −18 −16 −14 −12 −10 −8 −6 −4 −2 0

( = /4) T-junction

FCL-section ( = /4)

Magnitude S (dB)

11 12 13 14 15 16 17 18 19 20 21 −22

(b)

Frequency (GHz)

S11 S22 S33

2

−20 −18 −16 −14 −12 −10 −8 −6 −4 −2 0

1

configuration in [4].

Magnitude S (dB)

138 Advanced Electromagnetic Waves


**3.8 Epidemiology and aetiology**

Isolated aortic stenosis (AS) is more frequent in men and is found in 2% of people 65 years of age and older. The most frequent causes of AS include agerelated calcific degeneration, bicuspid aortic valve, and rheumatic aortic valve. The distribution of these causes diverges across age groups and geographic regions. Age-related degeneration is the commonest cause of AS in elderly patients. In comparison, bicuspid aortic valve calcification accounts for most surgical cases in younger patients (>65) [33].


*Article* **Allelopathic Inhibition by the Bacteria** *Bacillus cereus* **BE23 on Growth and Photosynthesis of the Macroalga** *Ulva prolifera*

**Naicheng Li 1, Jingyao Zhang 1, Xinyu Zhao 2, Pengbin Wang 3, Mengmeng Tong 1,\* and Patricia M. Glibert 4,5**

Received: 27 August 2020; Accepted: 13 September 2020; Published: 16 September 2020

**Abstract:** Bacteria-derived allelopathic effects on microalgae blooms have been studied with an aim to develop algicidal products that may have field applications. However, few such studies have been conducted on macroalgae. Therefore, a series of experiments was conducted to investigate the impacts of different concentrations of cell-free filtrate of the bacteria *Bacillus cereus* BE23 on *Ulva prolifera*. Excessive reactive oxygen species (ROS) were produced when these cells were exposed to high concentrations of filtrate relative to f/2 medium. In such conditions, the antioxidative defense system of the macroalga was activated as shown by activities of the enzymes superoxide dismutase (SOD) and catalase (CAT) and upregulation of the associated genes *up*MnSOD and *up*CAT. High concentrations of filtrate also inhibited growth of *U. prolifera*, and reduced chlorophyll *a* and *b*, the photosynthetic efficiency (*F*v/*F*m), and the electron transport rate (rETR). Non-photochemical quenching (NPQ) was also inhibited, as evidenced by the downregulation of the photoprotective genes *Psb*S and *Lhc*SR. Collectively, this evidence indicates that the alteration of energy dissipation caused excess cellular ROS accumulation that further induced oxidative damage on the photosynthesis apparatus of the D1 protein. The potential allelochemicals were further isolated by five steps of extraction and insolation (solid phase–liquid phase–open column–UPLC–preHPLC) and identified as N-phenethylacetamide, cyclo (L-Pro-L-Val), and cyclo (L-Pro-L-Pro) by HR-ESI-MS and NMR spectra. The diketopiperazines derivative, cyclo (L-Pro-L-Pro), exhibited the highest inhibition on *U. prolifera* and may be a good candidate as an algicidal product for green algae bloom control.

**Keywords:** *Ulva prolifera*; *Bacillus* sp.; allelopathy; photosynthetic system; reactive oxygen species (ROS); antioxidative system

#### **1. Introduction**

Allelopathic interactions are considered to be important factors that affect the growth or survival of organisms within the same ecological habit. Allelochemicals are secondary metabolites from plants, algae, or bacteria [1]. They may have positive benefits (positive allelopathy) or may be detrimental (negative allelopathy) [2]. Allelopathy has been considered to be one potential control mechanism for harmful algae blooms (HABs) [3]. The inhibition effects of allelopathic compounds on algae

include destroying the cell structure [4,5], altering production of the reactive oxygen species (ROS) [6], impacting intracellular enzymatic activities [7], or altering the photosynthesis system [8] and related gene expression [9]. External stress can induce the production of ROS, i.e., hydrogen peroxide (H2O2) and superoxide radical (O2 •−), and can induce the regulation of the antioxidative defense or the photoprotection system [10,11].

A number of bacteria-derived algicidal compounds have drawn wide attention as a control for HABs [12–14] and the algicidal compounds belonging to the *Cytophaga-Flavobacterium-Bacteroides* (CFB) phylum have been identified [15]. Among this phylogenetic profile, the genus of *Bacillus* shows promise in controlling HABs, as negative effects have been demonstrated on the diatom *Skeletonema costatum*, the raphidophyte*Heterosigma akashiwo*, the dinoflagellate*Prorocentrum donghaiense*[16], the prymnesiophyte *Phaeocystis globosa* [16,17], and the cyanobacterium *Microcystis aeruginosa* [18]. The potential allelochemicals that have been isolated and identified from *Bacillus* sp. include terpene, steroids, and alkaloids [19,20]. The active compounds and mechanisms remain to be identified due to the species-specific response to algicidal bacteria [21].

The green tides caused by blooms of *Ulva prolifera* have occurred in the Yellow Sea of China since 2007 [22–26]. These massive blooms negatively impact the local communities, aquaculture operations, and tourism, causing great damage to the local ecosystem service and enormous economic loss [27]. The rapid growth of *U. prolifera*, on the other hand, makes it the strongest competitor for nutrients and light [28,29] in the bloom area, thereby driving the great impact on the marine biodiversity and structure of the community [30–32]. There are currently no effective measures to control these blooms.

The *Bacillus* sp.-derived control of HABs is promising, but limited exploration has been undertaken in mitigating the green tides. As a complicating factor, the life stage of thalli has been reported to be an important factor in green tide development [27]. Therefore, a series of experiments were performed to understand the extent to which bacterial allelopathy may be effective in controlling the thalli of *U. prolifera*. Specifically, the following questions were addressed: (1) does the cell-free filtrate of *Bacillus* sp. inhibit the growth of *U. prolifera* and if so, what is the effective dose? (2) What is the mechanism by which negative allelopathy occurs, particularly with respect to the antioxidative defense system and the photosynthetic system II (PSII) response? (3) What are the potential allelochemicals in the filtrate of *Bacillus* sp. that cause negative effects on *U. prolifera*?

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

#### *2.1. Algal Culture and Identification*

Asexual isolates of *Ulva prolifera* were provided by Zhejiang Xiangshan Xuwen Algal Exploitation Company, China, in October 2018. Specimens were subsequently transferred to the laboratory on ice, sterilized with 0.7% potassium iodide (KI) for 5 min, and then rinsed with autoclaved seawater. The pre-sterilized thalli were maintained in sterilized f/2 medium [33], with salinity of 30, temperature of 20 ◦C, and light of 60 μmol·m2·s−1 (12/12 h of light/dark cycle). The media were replaced every 5 days.

To minimize the interference of carry-over epiphytic bacteria in *U. prolifera*, cultures were pretreated before each exposure experiment by antibiotic mixtures of penicillin (100 mg/L), polymixin (0.75 mg/L), and neomycin (0.9 mg/L) for 48 h [34].

The macroalga was identified using the method described in Li et al. [35]. Total DNA was extracted with a commercial Plant DNA Mini Kit (TaKaRa, China). ITS and 5S sequences were amplified by the corresponding PCR primers (Table 1) and the conducted BLAST analyses in the NCBI database.

**Table 1.** Sequences of primer pairs for *Ulva prolifera* analysis.

#### *2.2. Experiment 1: Bacteria-Derived Allelopathic Inhibition on U. prolifera*

#### 2.2.1. Preparation of Cell-Free Filtrate from *Bacillus cereus*

The bacterium strain *Bacillus cereus* BE23 was previously isolated from the mangrove area in Hainan province, China, and maintained in Luria Bertani (LB) broth (peptone 10.0 g/L, yeast extract 5.0 g/L, sea salt 32 g/L, dissolved in dH2O) at 28 ◦C with shaking at 180 rpm/min. The strain was identified by the 16S rDNA gene and 1439 bp sequence that was acquired by PCR amplification. The bacteria were transferred from stock culture, with the initial concentration of 1010/mL, in 500 mL of LB medium. In 5 days, cell density of *Bacillus cereus* BE23 reached approximately 1 × 1012/mL, then cell-free filtrates were prepared by centrifuging 450 mL of the culture and filtering the supernatant through a Millipore™ (Burlington, MA, USA) Membrane Filter, 0.22 μm pore size.

#### 2.2.2. Preparation of the Exposure Treatment

Triplicate intact macroalga thalli (approximately 1.25 g/L) were cultured in bacterial-free conditions with different ratios of *Bacillus cereus* BE23 filtrate to total media (filtrate + seawater, in volumes of 0:1, 1:100, 1:80, 1:60, 1:40, 1:20, and 1:10, hereafter identified as Control, T1:100, T1:80, T1:60, T1:40, T1:20, and T1:10, respectively) to a total of 400 mL each in 500 mL flasks. Then, stock f/2 medium was added to each flask. All final media were at f/2 levels, assuming that no or low nutrients were carried over by the filtrate. The concentration of bacteria cells in each treatment was 2.5 × 109, 1.25 × 1010, 1.65 × 1010, 2.5 × 1010, 5 × 1010, and 1 × 1011, respectively. The control treatment of *U. prolifera* was cultured in f/2 medium only, without a bacterial filtrate. All experiments were conducted in the same culture environment under a light intensity of 60 μmol·m2·s−1, and with a light/dark cycle of 12/12 h, salinity of 30, and temperature of 20 ◦C. The experiments were conducted in 500 mL flasks containing 400 mL of culture medium. Nutrients (equivalent to the nitrogen and phosphate level in f/2 media) were added every 48 h to exclude any effects of nutrient limitation, and pH values were monitored simultaneously. The culture flasks were randomly changed in terms of incubator position every day to balance the effect of illumination. Sterile conditions were used throughout.

Specimens of macroalga were harvested after 192 h (8 days) of exposure for biomass, photosynthesis, and antioxidant analysis.

#### 2.2.3. Growth

The wet weight biomass of the macroalga was determined (±0.0001 g) at 0 and 192 h, respectively. Samples were treated by blotting with 3 layers of filter paper and conditioning for 10 min at room temperature. The relative growth rates (G) were calculated as

$$\mathbf{Gx} = (\mathbf{Wx} - \mathbf{Wc}) / \mathbf{Wc}$$

where Wc is the initial wet weight (g) of thalli and Wx is the fresh thalli wet weight (g) after treatment X.

The inhibition rate (IR) by the bacterium filtrates was calculated as

$$\text{IR} = (\text{Gc} - \text{Gx}) / \text{Gc}$$

where Gx is the relative growth rate (%) of *U. prolifera* after treatment X, and Gc is the relative growth rate (%) after 192 h in control.

#### 2.2.4. The Antioxidant Defense System

Macroalgal samples (0.2~0.3 g wet weight) were homogenized in a bath of liquid nitrogen and extracted with commercial potassium phosphate buffer (pH = 7.2~7.4, Solarbio, China). Then, the extract was centrifuged at 10,000 rpm/min for 10 min yielding material for further analysis of total soluble protein (TSP), H2O2, and the enzymes superoxide dismutase (SOD) and catalase (CAT). Genes associated antioxidant activity, manganese superoxide dismutase (*up*MnSOD) and catalase (*up*CAT), were also quantified.

The TSP content was measured using the Coomassie blue dye binding assay [36]. Fifty microliters of extracts was homogenized with the Coomassie blue dye for 10 min and absorbance was measured at 595 nm. The results of TSP were expressed as g protein per liter (prot·g/L). One hundred microliters was mixed with the reaction reagents and detected at 405 nm. The concentration of ROS was measured as hydrogen peroxide (H2O2) and measured with a commercial assay kit (Jiancheng, Nanjing, China) following the manufacturer's protocols. Concentrations of H2O2 were determined based on the decomposition of H2O2 by peroxidase and the results were expressed as mmol H2O2 per g of TSP (mmol/g prot). The activity of SOD was measured according to the method of Sun et al. [37]. Samples (20 μL) and reaction reagents were mixed in the microliter 96-well flat-bottom plates and put into the plate reader (Tecan, Switzerland) for incubation at 37 ◦C. After 20 min incubation, the mixtures were detected at 450 nm. One unit of SOD was defined as the amount of enzyme required to generate 50% inhibition of reduction of WST-1 [2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]. The activity of CAT was assayed with the method described by Dhindsa et al. [38]. Briefly, a reaction mixture was composed of 50 μL extracts, 15 mM hydrogen peroxide, and 50 mM phosphate buffer. After addition of the enzyme extract, absorbance at 240 nm was recorded for 1 min. One unit of CAT activity is the amount of enzyme necessary to degrade 1 μmol H2O2 per mg of protein per sec.

The antioxidant enzyme coding genes (*up*MnSOD and *up*CAT) were amplified with gene-specific primer pairs (Table 2). RNA extraction and real-time PCR were performed the same as the photosynthetic genes.

**Table 2.** Sequences of primer pairs in *Ulva prolifera* for real-time PCR.

#### 2.2.5. Photosynthesis System

Concentrations of chlorophyll *a* (Chl *a*) and *b* (Chl *b*) were determined according to Zhao et al. [39]. Macroalgae (0.2 g) were grounded in liquid nitrogen and extracted in 90% *v*/*v*) acetone buffer (5 mL) for 12 h. Then, the mixture was centrifuged at 4 ◦C, 10,000 rpm/min for 10 min. The supernatant was collected for chlorophyll analyses, and optical densities were measured with an ultraviolet–visible

spectrophotometer (HITACHI, U2900, Japan) at 663 and 645 nm wavelength. Concentrations of Chl *a* and *b* were then calculated as follows, and reported as units of mg/g fresh weight (mg/g FW):

Chl *a* = 12.7 OD663 − 2.69 OD645

$$\text{Chl } b = 22.9 \text{ OD645} - 4.68 \text{ OD663}$$

Parameters associated the photosynthesis system II (PSII) were measured using an Imaging-PAM (Walz, Germany). These parameters included the effective quantum yield (Y(II)), non-photochemical quenching (NPQ), relative electron transport rate (rETR), and photochemical quenching (qP). The actinic light was set to be similar to the cultivation light (56 μmol·m−2·s−1). Subsamples of *U. prolifera* were dark-acclimated for 20 min prior to all measurements. All parameters were calculated according to the relationships in Table 3.

**Table 3.** Fluorescence parameters calculated from PAM in *Ulva prolifera* after exposure.

Four genes were selected for characterization: *Psb*S, *Lhc*SR, *Psb*A, and *Psb*D. *Psb*S and *Lhc*SR are associated with photoprotection and non-photochemical quenching (NPQ). *Psb*A and *Psb*D are indicators of the D1 and D2 protein of the PSII apparatus, respectively. The tubulin gene was deployed as a housekeeping gene to standardize the expression variations of target genes [39].

These genes were amplified with gene-specific primer pairs (Table 2). Samples of *U. prolifera* were quickly frozen in liquid nitrogen and stored at −80 ◦C until RNA extraction. Total RNA was extracted by a commercial MiniBEST Plant Total RNA Extraction Kit (TaKaRa, Dalian, China) and the reverse transcripts cDNA were analyzed using a Prime Script™ II 1st stand cDNA Synthesis kit (TaKaRa, Dalian, China). Real-time PCR was performed using the "TB GreenTM Fast qPCR Mix" kit (TaKaRa, Dalian, China). The amplification program of real-time PCR was set at 94 ◦C for 30 s, following 40 cycles of 94 ◦C for 5 s and 60 ◦C for 10 s in Light Cycler® 480 System (Roche, Germany). Dissociation curve analysis of the amplification products was carried out to verify the single PCR production at the end of each thermal program.

#### *2.3. Experiment 2: Isolation and Identification the Potential Allelopathic Compounds from Cell-Free Filtrate of Bacillus cereus BE23*

#### 2.3.1. Step 1: Solid Phase and Liquid Phase Extraction of Potential Allelopathic Compounds

Cell-free filtrate (10 L; approximately 1 × 1016 bacteria cells) of the *Bacillus cereus* BE23 culture was collected after 5 days of growth by centrifuging at 10,000 rpm/min for 10 min and filtering with a 0.22 μm membrane. The filtrate was eluted by solid phase extraction (SPE) with the resin Diaion® HP20 (particle size of 20–60 mesh) and the remaining residuals were rinsed off by methanol. After resuspending the residuals in Milli-Q water, they were used for liquid phase extraction (LPE). Three extracting agents, cyclohexane, ethyl acetate, and 1-butanol, were considered as selection agents for different polarity fragments. Sub-residuals of LPE were extracted from each agent 3 times and concentrated in a rotary evaporator (IKA, RV8V, Germany) in a 30~40 ◦C water bath (Figure 1). The sub-residuals were identified as cyclohexane (Ech), ethyl acetate (Eea), and 1-butanol seriatim (Ebs). These sub-residuals, Ech, Eea, and Ebs, were weighted with an electron balance (±0.0001 g), dissolved in 20 mL dimethyl sulfoxide (DMSO), and stored at 4 ◦C for further bioassay experimentation.

**Figure 1.** Isolation and bioassay program for potential allelopathic compounds from crude extraction of cell-free filtrate of *Bacillus cereus* BE23.

The first *U. prolifera* bioassay experiment was performed in 6-well plates by filling them with macroalgae (approximately 0.05 g) and crude extraction (5 mg/L) or DMSO (control) in 10 mL f/2 medium. Each treatment was conducted in triplicate for 192 h under the same environmental conditions as the primary *U. prolifera* culture. Growth and inhibition rates were used to determine the potential allelopathic activities in each treatment (Supplementary Figure S2). Of the three extracting agents, extractions in cyclohexane (Ech) and in ethyl acetate (Eea) had an inhibition effect (Supplementary Figure S2), therefore, these extractions were used for further investigation.

#### 2.3.2. Step 2: Open Column Chromatography to Select the Potential Allelopathic Compounds

To further purify the potential allelopathic compounds, Ech and Eea were eluted through an open silica gel column chromatography (170 × 30 mm in dimension and with a silica particle size of 200–300 mesh), respectively, and the eluents from each mobile phase were collected. As for extractions in cyclohexane (Ech), the mobile phase was cyclohexane and ethyl acetate with ratios of 200:1, 100:1, 50:1, 25:1, 10:1, 5:1, and 0:1 (hereafter named as Ech1, Ech2, etc.). For extraction in ethyl acetate (Eea), the mobile phase was dichloromethane and methanol with ratios of 50:1(Eea1), 25:1(Eea2), 10:1(Eea3), 5:1(Eea4), 2:1(Eea5), 1:1(Eea6), and 0:1(Eea7), respectively.

Then, a second bioassay was performed in 6-well plates by adding 0.05 g of *U. prolifera* (wet weight) and the corresponding extracted compounds (5 mg/L) in 10 mL of f/2 medium. Each treatment was conducted in triplicate for 192 h under the same environmental conditions as the primary *U. prolifera* culture. The extractions with significant inhibition, Ech5, Eea2, and Eea3 (Supplementary Figure S3), were collected for further detection.

2.3.3. Step 3: Ultra- and High-Performance Liquid Chromatography to Select the Potential Allelopathic Compounds

The bioactive fractions were collected separately and analyzed by analytical ultra-performance liquid chromatography (UPLC, ultimate 3000, Thermo Fisher Scientific, USA) with a C18 column (250 × 4.6 mm, 5 μm, Agilent, China) at a flow rate of 1 mL/min and the UV detection at 210 nm. The mobile phase was methanol or acetonitrile/water (10/90, v/v) −100% methanol with an elution time of 35 min. The dominant components (highest peaks), including 5 components from Ech5, 7 components from Eea2, and 8 components from Eea3, were chosen and the optimal UPLC conditions were retrieved for a further preparative step.

The fractions were then purified and collected by preparative high-performance liquid chromatography (HPLC, Shimadzu, AP20, Japan) with a C18 column (250 × 21.2 mm, 5 μm, NanoMicro, China) at a flow rate of 10 mL/min for different times up to 35 min for Ech5, Eea2, and Eea3, separately, using the recorded optimized mobile phase (Figure 1).

The third bioassay was conducted with the 20 components. Three compounds, Ech5-4, Eea2-5, and Eea3-2, were collected at 23.52, 13.43, and 16.25 min in each extraction run (Supplementary Figure S4).

#### 2.3.4. Structure Identification

The three potential allelochemicals, Ech5-4, Eea2-5, and Eea3-2, were preliminarily analyzed by an Agilent 6230 time-of-flight liquid chromatography–mass spectrometer (TOF LC-MS) (Agilent, CA, USA) to determine the molecular weight. Then, structures were identified by a pulse Fourier transform nuclear magnetic resonance spectroscope (NMR, 600 MHz, JNM-ECZR, JEOL, Japan). Deutero methanol or deutero dimethyl sulfoxide solutions containing trimethylsilyl were used as reference substances and acted as solvents to record 1H and 13C NMR spectra. All chemical shifts were exhibited as relative values.

#### *2.4. Statistical Analysis*

All data were presented as mean ± standard error and were analyzed by one-way ANOVA with a significant level of 0.05 (Sigma plot 12.5, Systat Software Inc., London, UK). A phylogenetic tree was constructed using the neighbor-joining algorithm with the MEGA 7.0 program. Relative gene expression levels were analyzed following the 2−ΔΔCt method.

#### **3. Results**

#### *3.1. Identification of Macroalga and Bacteria*

The 5S sequence of the macroalga, 418 bp, was 100% identical to*Ulva prolifera* (GenBankID:HM584772.1) and the ITS sequence, 614 bp, was 99% identical to *U. prolifera* (GenBankID:KF130870.1). Thus, the macroalga deployed in the present study was identified as *U. prolifera*.

The 16S rDNA sequence of the bacterial strain BE23 (GenBank accession number: MN814015) was 100% identical, with few genetic distance differences, to that of *Bacillus cereus* strain ATCC14597 (Supplementary Figure S1). Thus, bacterial strain BE23 was identified as *Bacillus cereus*.

#### *3.2. Inhibition on the Growth of U. prolifera*

To simplify the treatment and response analysis of *U. prolifera*, two major treatment groups of *B. cereus* filtrates were classified. They are herein separated as high-concentration (HC), i.e., the T1:10 and T1:20 treatments, and low-concentration (LC), i.e., the T1:40, T1:60, T1:80, and T1:100 treatments.

Cell-free filtrates of *Bacillus cereus* BE23 were used as the source of the allelopathic compounds tested on *U. prolifera*. These cell-free filtrates induced growth of *U. prolifera* at LC, i.e., T1:100~T1:40 (ANOVA, *p* < 0.05), with growth rates of 105% ± 11% on average (n = 12), but inhibited growth at HC treatments (T1:20 and T1:10), with inhibition rates of 67% and 75%, respectively (Figure 2). Values of pH were monitored during the exposure in all treatments (Supplementary Table S1) and variation of the pH value was within the optimal range for *U. prolifera* growth [40].

**Figure 2.** Relative growth rates and inhibition rates of *Ulva prolifera* under the exposure of different amounts of cell-free filtrate of *Bacillus cereus* BE23. T1:100, and T1:80~T1:10 indicate the treatments of volume ratio of cell-free filtrate of *Bacillus cereus* BE23 to f/2 medium. Values are means ± SD (n = 3). \* indicates a significant difference (*p* < 0.05) and \*\* indicates a significant difference (*p* < 0.001) compared to control.

#### *3.3. Response of Antioxidant System of U. prolifera*

A significant amount of H2O2 (ANOVA, *p* < 0.001) was produced in the HC treatments, ranging from 38.21 to 50.33 mmol/gprot (Figure 3) after 192 h of exposure. The production of ROS was associated with changes in activities of SOD (ANOVA, *p* < 0.05) and CAT (ANOVA, *p* < 0.001), with concentrations of T1:40 eliciting a response in SOD activity (Figure 4a) but only the highest dosage, T1:10, elicited a response in CAT (Figure 4b). The antioxidant enzyme genes, *up*CAT and *up*MnSOD, were upregulated gradually in response to the increased dosage of cell-free extracts (Figure 4a,b), indicating the initiation of the antioxidant defense system under the stress of the filtrate of *Bacillus cereus* BE23.

**Figure 3.** H2O2 content of *Ulva prolifera* under the exposure of different amounts of cell-free filtrate of *Bacillus cereus* BE23. T1:100, and T1:80~T1:10 indicate the treatments of volume ratio of cell-free filtrate of *Bacillus cereus* BE23 relative to f/2 medium. Values are means ± SD (n = 3). \* indicates a significant difference (*p* < 0.05) and \*\* indicates a significant difference (*p* < 0.001) compared to control.

**Figure 4.** (**a**) Superoxide dismutase (SOD) activity and relative gene expression of manganese superoxide dismutase (*up*MnSOD), and (**b**) catalase (CAT) activity and catalase gene expression (*up*CAT) of *Ulva prolifera* under the exposure of different amounts of cell-free filtrate of *Bacillus cereus* BE23. T1:100, and T1:80~T1:10 indicate the treatments of volume ratio of cell-free filtrate of *Bacillus cereus* BE23 relative to f/2 medium. Values are means ± SD (n = 3). \* indicates a significant difference (*p* < 0.05) and \*\* indicates a significant difference (*p* < 0.001) compared to control.

#### *3.4. Response of PSII System of U. prolifera*

To investigate the effects of the *Bacillus cereus* BE23 filtrate on the photosynthetic pigments of the macroalga, Chl *a* and *b* contents were quantified (Figure 5a). No significant changes of either Chl *a* or *b* were observed in the LC treatments, but significant decreases were observed (ANOVA, *p* < 0.001) in the HC exposures, from 0.41 to ~0.13 mg/g FW for Chl *a*, and from 0.57 to ~0.24 mg/g FW for Chl *b* (Figure 5a).

**Figure 5.** (**a**) The chlorophyll *a* and *b* content, and (**b**) the maximum quantum yields of PSII (*F*v/*F*m) of *Ulva prolifera* under the exposure of different amounts of cell-free filtrate of *Bacillus cereus* BE23. Values are means ± SD (n = 3). \*\* indicates a significant difference (*p* < 0.001) compared to control.

The photosynthetic response of *U. prolifera* under the stress of cell-free filtrate of *B. cereus* BE23 was significant (Figure 5b, Figure 6, Figure 7). The maximum photochemical quantum yields of PSII (*F*v/*F*m) were reduced in the HC treatments, from 0.80 to ~0.29 (n = 6, Figure 5b). Accordingly, values of Y(II), the effective quantum yield of PSII, were significantly downregulated (ANOVA, *p* < 0.001), from 0.22 to 0.15 in the HC treatments (Figure 6a). Similar responses were found in the relative electron transport rates (rETR), coincident with a sharp reduction in photochemical quenching (qP) (Figure 6b). A significant enhancement of NPQ activity (Figure 6b) (ANOVA, *p* < 0.001) was recorded in the LC treatments, from 0.18 to 0.44. However, high doses of the filtrate of *Bacillus cereus* BE23 induced a downregulation of NPQ (ANOVA, *p* < 0.001), indicating photoinhibition damage.

**Figure 6.** Photosynthetic system II parameters of *Ulva prolifera* under the exposure of different amounts of cell-free filtrate of *Bacillus cereus* BE23: (**a**) quantum yield (Y(II)) and relative electron transport rate (rETR), and (**b**) non-photochemical quenching (NPQ) and photochemical (qP). T1:100, and T1:80~T1:10 indicate the volume ratio of cell-free filtrate of *Bacillus cereus* BE23 relative to f/2 medium in the different treatments. Values are means ± SD (n = 3). \*\* indicates a significant difference (*p* < 0.001) compared to control.

The expression of the two assayed photoprotection-related genes, *Psb*S and *Lhc*SR, varied in response to cell-free filtrate exposure (Figure 7a). The relative expressions of both genes increased with the bacterial filtrate dosage from 1:100 (T1:100) to 1:40 (T1:40) but were significantly downregulated in the HC treatments (T1:20 and T1:10). The highest *Psb*S and *Lhc*SR were in treatments of T1:40, reaching 2.66 and 5.29 times that of the control, and the lowest value was in the T1:10 treatment, at 0.75 and 0.72 of the control (Figure 7a). The response of *Psb*A and *Psb*D was not as clear, but a substantial degradation of *Psb*A was observed in the HC treatment, with a value of 0.59 of the control in T1:10 (Figure 7b).

**Figure 7.** Relative expression of the genes (**a**) *Psb*S and *Lhc*SR, and (**b**) *Psb*A and *Psb*D of *Ulva prolifera* under the exposure of different amounts of cell-free filtrate of *Bacillus cereus* BE23. T1:100, and T1:80~T1:10 indicate the treatments of volume ratio of cell-free filtrate of *Bacillus cereus* BE23 relative to f/2 medium. Values are means ± SD (n = 3).

#### *3.5. Identification of Allelochemicals from Bacillus cereus BE23 Filtrate*

To isolate the bioactive compounds, five steps of extraction and insolation (solid phase–liquid phase–open column–UPLC–preHPLC) were conducted. After each isolation, the separated groups were tested for bioactivity (Figures S2–S4). Three bioactive compounds in the cell-free filtrates of *Bacillus cereus* BE23 were identified by high-resolution mass spectrometric data and NMR spectroscopic analysis. The molecular formula C10H13NO of compound Ech5-4 was deduced from its ion at m/z 164.1072 [M+H]+ (Supplementary Figure S5a, calculated for C10H14NO, 164.1075) and its 13C data. The 13C-NMR spectrum (600 MHz, DMSO-d6) of Ech5-4 displayed signals at δC 169.5 (C=O), 140.0 (C,

C-1), 129.1 (CH, C-3, C-5), 128.8 (CH, C-2, C-6), 126.5 (CH, C-4), 40.7 (CH2, C-7), 35.7 (CH2, C-8), and 23.09 (CH3) (Supplementary Figure S5b,c). The 1H-NMR signals were observed at δH 7.92 (1H, brs, NH), 7.27–7.30 (2H, t, J = 8.0 Hz, Ar-H), 7.18–7.20 (3H, m, Ar-H), 3.22–3.26 (2H, m, H-7), 2.69 (2H, t, J = 7.5 Hz, H-8), and 1.78 (3H, s, -CH3). Based on these data and the comparison with the reported data [41], compound Ech5-4 was identified as N-phenethylacetamide (Figure 8a).

**Figure 8.** Structures of the compounds Ech5-4 (**a**), Eea2-5 (**b**), and Eea3-2 (**c**) isolated from the crude extract of *Bacillus cereus* BE23 filtrate.

The molecular formula of C10H14N2O2 for compound Eea2-5 was determined based on its m/z 217.0953 [M+Na]+ (Supplementary Figure S6a, calculated for C10H14N2NaO2, 217.0953). The 13C and 1H NMR spectra of Eea2-5 showed signals for the functional groups of carbonyl (δC 168.1), methine (δC 61.2; δH 4.34, 1H, t, J = 9.0 Hz), and methelene (δC 45.7, 28.2, 23.7; δH 3.45–3.53, 2H, m, 2.25–2.30, 1H, m, 1.99–2.09, 2H, m, 1.91–1.97, 1H, m,) (Supplementary Figure S6b,c). These data and comparison with the reference data [42] indicated that compound Eea2-5 was cyclo (L-Pro-L-Pro) (Figure 8b).

The compound Eea3-2 has the molecular formula of C10H10N2O2 deduced from its m/z 219.1103 [M+Na] (Supplementary Figure S7a, calculated for C10H10N2NaO2, 219.1109). The 13C-NMR spectrum (600 MHz, Methanol-d4) of Eea3-2 exhibited 10 carbon signals, resonating at δC172.8 (C, C-1), 167.8 (C, C-6), 61.8 (CH, C-7), 60.3 (CH, C-2), 46.4 (CH2, C-5), 30.1 (CH, C-8), 29.8 (CH2, C-3), 23.5 (CH2, C-4), 19.1a (CH3, C-10), and 16.9 (CH3, C-9). The 1H NMR spectrum displayed signals at δH 4.20 (1H, t, J = 8.6 Hz, H-2), 4.05 (1H, br t, H-7), 3.56 (1H, m, H-5a), 3.48 (1H, m, H-5b), 2.48 (1H, m, H-3a), 2.31 (1H, m, H-8), 2.02 (1H, m, H-3b), 1.91–1.96 (2H, m, H-4), 1.08b (3H, d, J = 7.3 Hz, H-9), and 0.95b (3H, d, J = 7.3 Hz, H-10). Thus, the compound Eea3-2 was identified as cyclo (L-Pro-L-Val) (Figure 8c) [43].

#### **4. Discussion**

Bacteria-derived interactions play important roles in species distribution and abundance [44], succession of algal blooms [45], and biomass control of microorganisms [46] and macroalgae [47]. Such allelopathic interactions consist of two pathways, direct (bacterial and algal cell contact) and indirect (release of natural products) [12,32]. The present study demonstrated the potential mechanisms of allelopathic stress on *U. prolifera* by products of *B. cereus* BE23 in indirect ways.

The low dosage (i.e., T1:100~T1:40) of *B. cereus* BE23 filtrate promoted the growth of *U. prolifera*, whereas the high dosage (T1:20 and T1:10) inhibited biomass production (Figure 2). The response of the macroalgae in the LC treatments may have resulted from a hormesis effect [48] and adaption to the low concentrations of allelochemicals [49]. The upregulation of physiological activity of *U. prolifera* (Figures 4–6) in the LC treatments contributed to the growth-promotive effect. Meanwhile, the nutrients, including the inorganic nutrient from f/2 + artificial seawater and the nutrient carrying over by the *B. cereus* BE23 filtrate (4~40 mL), contributed to the growth of macroalga. Inorganic nitrogen, i.e., nitrate or ammonium, has been reported to be rapidly taken up by *Ulva* [28], and within 192 h, the addition of inorganic nutrient of f/2 medium was calculated to be sufficient to the thalli of *U. prolifera* [50,51]. The carried-over inorganic nutrient was low (less than 10%), therefore, the effects of nutrients in *B. cereus* BE23 filtrate were minimal to the growth of *Ulva* in the present study.

A general stress response in algae is the production of ROS [52,53] and it can be produced in response to abiotic and allelopathic stresses [54–56]. Here, ROS was produced in response to BE23 cell-free filtrates (Figure 3). The source of ROS may include two main pathways: the intrinsic oxidization by allelochemicals, and inactivation of the electron transport in the PSII systems. The production of ROS is also a signal of the pressure from the excitation energy collected by the PSII light-harvesting complex [57,58]. To regulate the extra ROS, algae have a series of antioxidant defense mechanisms, including the ability to vary antioxidant enzymes or genes. Variations in activities of the enzymes SOD and CAT are important in alleviating oxidative damage [59,60]. In general, SOD scavenges the cellular ROS first, catalyzing O2•− to H2O2. Then, the CAT enzyme decomposes H2O2 to O2 and H2O [61]. MnSOD, one of the total SODs, was selected as the representative enzyme; it is mostly detected in the cytosol and thylakoid membrane [62].

Here, a small amount of ROS (H2O2) was produced in the LC treatments, i.e., T1:60 and T1:40, but no significant variation was observed in the quantum efficiency of photosynthesis (*F*v/*F*m), indicating *U. prolifera* may activate photoprotection to defend against such allelopathic stress. However, a significant increase in ROS concentration (ANOVA, *p* < 0.001) was recorded in the HC treatments, accompanied by the decline in rETR, indicating normal electron transport in PSII was disturbed and excess energy likely contributed to the ROS generation in HC treatments. High production of ROS induced oxidative stress in the algae and finally inhibited the photosynthesis systems. To moderate the oxidative damage, *U. prolifera* upregulated the activity of SOD and CAT, supported herein by the gene expression level of *up*MnSOD and *up*CAT in the LC treatments (Figure 5). Similar responses have been noted in *Cylindrospermopsis raciborskii* under hyper-salinity or light-stress conditions [63,64], and linoleic acid stress [65]. The upregulation of the transcript levels of FeSOD and CAT genes in *U. prolifera* have also been reported in response to salicylic acid and hyper-temperature [66]. In the present study, however, the enhanced CAT activities were not sufficient to scavenge the sudden increased H2O2 and this likely caused extensive oxidative stress in this macroalga.

External stresses, including allelopathic stressors, can alter the algal energy flux of PSII by reducing the photosynthetic efficiency [67–69], and by enhancing non-photochemical quenching (NPQ) [65]. The maximum quantum yield (*F*v/*F*m) is an effective indicator of the efficiency of photochemical stress. In *Ulva* sp., changes in *F*v/*F*m have been observed when the algae are exposed to internal or external stresses [70] such as light [71], desiccation [72], salinity [73], and allelopathy [50].

Significant declines in *F*v/*F*m (Figure 5b), growth rate (Figure 2), and Chl *a* and *b* (Figure 5a) were shown after 192 h exposure to high concentrations of *B. cereus* BE23 filtrate, suggesting disruption of the PSII reaction centers' (RCs) complexes [67] including the electron transport chain [74]. Reduced rETR and Y(II) indicate a reduction in the electron transport rate and CO2 assimilative capacity [75]. Therefore, one mechanism by which *U. prolifera* responds to allelopathic stress is a lowering of the photosynthetic performance, which directly impacts carbon fixation and therefore the growth rate [76]. The significant decreases in the Chl *a* and *b* concentrations in the HC treatments may also be considered as an adaptive strategy which decreases the absorption of photons, thereby leading to less ROS production [67].

The NPQ pathways are photoprotective mechanisms for phototrophs [77]. In the present study, no significant variation in *F*v/*F*m (Figure 5b) or rETR (Figure 5a) was observed in the LC treatments; however, a significant increase in NPQ was recorded as the concentrations of the LC treatments increased, namely T1:40 and T1:60. Under the HC treatments, a substantial decrease in NPQ was observed, indicating that allelopathic stress may hinder the operation of photoprotective mechanisms, and thus the macroalgae dissipated excess energy through non-regulated pathways [78]. At high levels of bacterial filtrate, *U. prolifera* was unable to self-protect against photodamage [39]. The significant decrease in qP in the treatments with high concentrations of filtrate indicated a high level of energy dissipation and potential damage to the PSII reaction centers. Thus, the decrease in the efficiency of PSII was associated with a simultaneous decrease in the photochemical and non-photochemical pathways in the HC treatments, reflecting a complete disruption of normal energy pathways.

Previous studies have suggested that *Ulva* sp. can modulate NPQ levels by adjusting the copy number of *Lhc*SR or *Psb*S and regulation of the xanthophyll cycle [79,80]. It thus appears that low levels of exposure to *B. cereus* BE23 filtrate induced an upregulation of *Lhc*SR and *Psb*S in *U. prolifera* and activated the photoprotection mechanism that enables the self-regulation of external allelopathic stress

without loss of electron transfer efficiency of photosynthesis and growth. An upregulated transcript level of both selected genes and a triggering of *Lhc*SR-dependent NPQ was also previously reported in *Ulva* sp. [80]. High amounts of filtrate, in contrast, inhibited the photosynthetic efficiency and the capability of self-regulation of *U. prolifera*, as evidenced by the downregulation of *F*v/*F*m, qP, and NPQ activity, and finally the inhibition of growth. Therefore, the low value of NPQ was a result of the loss of the photoprotection of *U. prolifera* and a failure of self-regulation under allelopathic stress [81].

Allelopathic damage to the PSII systems is also suggested by the responses of the genes located in the D1-D2 protein [54,82]. *Psb*A and *Psb*D, encoding the D1 and D2 subunits of the PSII complex, constitute the heterodimeric photochemical reaction center [80]. Here, no clear variation in *Psb*A and *Psb*D gene expression was observed after 192 h exposure in the LC treatments (Figure 7b), suggesting the excess absorbed electrons (Figure 4a) were dissipated by the upregulated NPQ, together with the upregulation of *Lhc*SR and *Psb*S transcript levels (Figure 7a). In contrast, clear downregulation of *Psb*A expression levels was recorded in the HC treatments, suggesting that the *B. cereus* BE23 filtrate suppressed *Psb*A expression and may have blocked the elector transport on the PSII receptor side from QA to QB [81].

In summary, the inhibition effect on the PSII of *Ulva* due to bacteria-derived stress may go through two main steps: (1) the inhibition of the electron transport chain, and (2) the deleterious effects on PSII RCs' complexes [83,84]. In the present study, the upregulated expression of *Psb*S and *Lhc*SR under LC levels of cell-free filtrate might indicate the successful regulation of stress via regulated NPQ [85,86], but failure in the HC treatments. The depletion of the transcript pools of *Lhc*SR and *Psb*S contributed directly to the decrease in NPQ activity and likely inactivated the PSII RCs' complexes. Downregulation of Chl *a* and *b* corresponded to the downregulation of *Psb*A expression levels, suggesting the BE23 filtrate degraded the absorption of light energy and blocked the electron transport on the PSII receptor side [65,80]. Surplus electrons exceeded the electron transport chain capacity of *U. prolifera* and induced additional ROS production (Figure 3) that, in turn, damaged the PSII systems [16]. Together, these data clearly document the photooxidative stress in *U. prolifera* upon allelopahtic stress in HC treatments.

Using ESI and NMR, three potential allelopathic chemicals were isolated and identified from the cell-free filtrate of *B. cereus* BE23. The chemical cyclo (L-Pro-L-Pro) (Figure 8b), extracted from Eea2, displayed the largest inhibitory effect on *U. prolifera* (Supplementary Figure S6), and has previously been shown to yield a strong algicidal effect on *Microcystis aeruginosa* [55] and *Phaeocystis globosa* [54] by inhibiting the operation of the photosynthesis and antioxidant systems of target algae. In the present study, the diketopiperazine derivatives decreased the gene expression of *Psb*A [54,87], directly impacting the PSII electron acceptor sides, resulting in the failure of the photosynthetic process. Given that cyclo (L-Pro-L-Pro) is easily biodegradable [88], it may be a good candidate as an environmentally friendly algicide for green algae bloom control.

#### **5. Conclusions**

The high concentration of the cell-free filtrate of *B. cereus* BE23 (approximately 1 × 1011/mL) yielded significant inhibition of growth of *U. prolifera* via degradation of the photosynthetic system as shown by changes in biomass accumulation, photosynthetic responses, gene regulation, and enzyme activities. The potential allelopathic compounds inhibited growth by means of reduction of *F*v/*F*m, rETR, and NPQ, resulting in *U. prolifera*'s failure to dissipate the excess energy through regulated NPQ pathways. This alteration of energy dissipation caused excess cellular ROS accumulation and the antioxidative defense system was generated. This ROS production also inhibited the PSII reaction center apparatus. The potential allelochemicals were further isolated and identified as N-phenethylacetamide, cyclo (L-Pro-L-Val), and cyclo (L-Pro-L-Pro). The diketopiperazines derivative, cyclo (L-Pro-L-Pro), exhibited the highest inhibition effect on *U. prolifera* and further study on its potential as an algicidal product for green algae bloom control is warranted.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2077-1312/8/9/718/s1, Figure S1. Phylogenetic tree of *Bacillus cereus* BE23. Figure S2. Relative growth rates and inhibition rates of

*J. Mar. Sci. Eng.* **2020**, *8*, 718

*Ulva prolifera* of the first bioassay test. Figure S3. Relative growth rates and inhibition rates of *Ulva prolifera* in the second bioassay test. Figure S4. Relative growth rates and inhibition rates of *Ulva prolifera* in the third bioassay test. Figure S5. High-resolution electrospray ionization mass spectrometry (HRESIMS) spectrum (**a**), 13C NMR spectrum (**b**), and 1H NMR spectrum (**c**) of compound Ech5-4. Figure S6. High-resolution electrospray ionization mass spectrometry (HRESIMS) spectrum (**a**), 13C NMR spectrum (**b**), and 1H NMR spectrum (**c**) of compound Eea2-5. Figure S7. High-resolution electrospray ionization mass spectrometry (HRESIMS) spectrum (**a**), 13C NMR spectrum (**b**), and 1H NMR spectrum (**c**) of compound Eea3-2. Table S1. Changes of pH values with culture time in exposed experiments.

**Author Contributions:** Conceptualization, N.L. and M.T.; methodology, X.Z. and N.L.; software, N.L.; validation, N.L., J.Z., X.Z., P.W., P.M.G. and M.T.; formal analysis, M.T. and P.M.G.; investigation, N.L., J.Z. and X.Z.; resources, M.T.; data curation, N.L. and J.Z.; writing—original draft preparation, N.L.; writing—review and editing, M.T., P.M.G. and P.W.; visualization, M.T.; supervision, M.T.; project administration, M.T.; funding acquisition, M.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by a National Key R&D Program of China NO. 2016YFC1402104, Key Laboratory of Integrated Marine Monitoring and Applied Technologies for Harmful Algal Blooms, Ministry of Natural Resources of the People's Republic of China (MNR), MATHAB201803 and Funding for Tang Scholar to M.T.

**Acknowledgments:** The authors are grateful to Zhizhen Zhang of Zhejiang University for helping identify the natural products, and Min Wu for providing the bacteria *Bacillus cereus* BE23 strain.

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

#### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

#### *Article*


**1. Introduction**

The transportation sector is one of the key contributors to greenhouse gas emissions that are potentially affecting global warming. A great majority of emissions caused by the transportation sector originate from passenger vehicles and trucks, due to exhaust fumes. The combustion of fossil fuels in engines is thus one of the most important contributors to atmospheric greenhouse gases. Traffic delays, radiative forcing, and rolling resistance are factors, which have a significant impact on the fuel consumption of vehicles and trucks. However, also the management of road infrastructure is directly related with

environmental impacts, due to raw material consumption and energy needs during the construction and maintenance of the road, as well as during end-of-life activities [1–3].

European Green Deal introduced several proposals for reducing net greenhouse gas emissions by at least 55% by 2030, compared to the 1990 level. One of the proposals refers to providing efficient, safe, and environmentally sustainable transport [4]. Within this frame, winter road maintenance (WRM) plays an important role. The main activity of WRM agencies is reducing ice and snow from roadways, which is of crucial importance to provide safe driving conditions for traffic and smooth mobility. In countries with cold and humid winters, snowstorms may cause problems in the mobility of road traffic resulting in congestions and delays. In such conditions, fuel economy of vehicles is deteriorated, and consequently emissions increase. From this point of view, ensuring snow- and ice-free road is of great importance to achieve targets set by European Green Deal. However, winter road maintenance also yields a significant amount of greenhouse gases and other emissions, especially in cold regions, with a relatively high frequency of such activities [5]. Moreover, salt and other deicers, which are gritted on the road, pose a negative impact on groundwater and freshwater quality, and consequently also on biodiversity and human health [5, 6]. This represents a serious environmental problem taking into account that significant amounts of road salt and other chemicals are used to remove ice and snow accumulated on the road or to prevent icing and snow compaction on the roads [5]. WRM agencies are under pressure to improve not only the effectiveness and efficiency of their activities but also to optimize the activities from the aspect of sustainability. To improve environmental sustainability, special attention regarding the application of materials, strategies, and equipment is required [7]. Application of best practices from other studies can be a pathway toward achieving environmental sustainability in the field of winter road maintenance.

Several authors addressed the problem of environmental sustainability of winter road maintenance and the number of such studies is growing in recent years. Cui et al. [7] provided a framework for assessing sustainability in the field of winter road maintenance with salt as a road deicer. Adequate selection of deicers (road salt, agro-based, and complex chlorides/minerals-based products) is of crucial importance for successful implementation of winter road maintenance. Decision on selection of deicers has been typically taken based on their cost and effectiveness. However, the environmental impacts of salt or other chemicals used for deicing should also represent an important aspect when deciding about different deicing alternatives. Environmental impacts can be direct, due to release of chemicals into natural environment (soil, surface water), or indirect. The latter refers to the repair of damage (mostly related with corrosion) that deicers cause on vehicles and road infrastructure. Repair of such damage is associated with environmental impacts as well [7].

Environmental impacts related with winter road maintenance in Norway were evaluated in a study by Vignisdottir et al. [5]. They took into account the production and transportation of road salt (deicer) and vehicles for winter road maintenance and the operation of the winter road maintenance (use of the vehicles for plowing and salt spreading, associated with fuel and salt consumption). Data on quantities of road salt used for deicing and data on fuel consumption of WRM vehicles were gathered from public reports, so the results of LCA reflect realistic conditions. The study showed that emissions related to winter road maintenance in Norway contribute around 1% of the total emissions from road transportation in Norway. Such relatively high contribution can be explained by two facts. The first one is that Norway is the leading country in the use of electric and hybrid electric vehicles, which cause zero- and

#### *Pathway toward Sustainable Winter Road Maintenance (Case Study) DOI: http://dx.doi.org/10.5772/intechopen.110764*

low emissions. The second fact is that most part of Norway has a cold and relatively humid climate. In such regions, winter road maintenance is extensive.

Vignisdottir et al. [8] provided a comprehensive review of 35 scientific papers dedicated to the evaluation of environmental impacts and effects of winter road maintenance. Based on this review, some research gaps were emphasized. In most of studies, only local environmental effects of deicers were addressed. While rare studies provide a holistic overview on environmental impacts related with winter road maintenance operation methods or material selection.

The goal of this study is to compare the environmental impacts of two scenarios related to the operation of winter road maintenance. Life Cycle Assessment (LCA) method was applied to conduct such a comparison. In baseline scenario, the agency responsible for WRM does not use a road-weather information system, meaning that it does not have accurate information about the exact timing of the beginning of the snowstorm event. Winter road maintenance operations start only when snow began accumulating on the road.

In alternative scenario, the WRM agency does use road-weather information system. In such a case, it obtains reliable information on timing of snowfall event and if snow or ice will accumulate on the road. Based on such detailed weather forecast, the WRM agency can take preventive measures, and if necessary, start gritting the road just before the snowfall event. In such conditions, the agency can optimize the consumption of road salt required for anti-icing and/or deicing. The purpose of this study is to benchmark the environmental impacts of baseline WRM operation scenario versus alternative scenario (preventive WRM operation scenario).


**Author details**

Dragana Rakašević1 \* and Dragana Gabrić2,3

1 Department of Periodontology and Oral Medicine, Faculty of Dental Medicine, University of Belgrade, Belgrade, Serbia

2 Department of Oral Surgery, School of Dental Medicine, University of Zagreb, Croatia

3 Clinical Hospital Center Zagreb, University Dental Clinic, Zagreb, Croatia

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Effect of Implant Surface Design and Their Decontamination Methods in Peri-Implantitis… DOI: http://dx.doi.org/10.5772/intechopen.99753*


Contents

Preface

Maternal and child health is an important public health issue because we have the opportunity to end preventable deaths among all women, children, and adolescents and to greatly improve their health and well-being. Far too many women, infants, and children worldwide have little or no access to essential, quality health services and education, clean air and water, and adequate sanitation and nutrition. Factors influencing the development of diseases in women and children are many, depending on genetic factors, the way the child is born, the dietary habits of the mother during pregnancy, the use of antibiotics during pregnancy, and so on. Health depends on climate, lifestyle, diet, education, and ethnic and racial divisions, among other factors. Are we educated enough about health? How much do we know about how to be healthy? Is health a decision of our free will with less financial investment and more knowledge? Is health the art of living in harmony with nature or the disease of a

**Dr. Miljana Z. Jovandaric and Sandra Babic**

Clinical Centre of Serbia,

Beograd, Serbia

modern society burdened with financial power?


Wireless Power Transfer in Electrical Systems

**6**

*Wireless Power Transfer – Recent Development, Applications and New Perspectives*

[1] Z. Zhang, H. Pang, A. Georgiadis, and C. Cecati, "*Wireless Power Transfer - An Overview*", IEEE Transactions on Industrial Electronics, vol. 66, no. 2,

[2] P. Pinho, "*Recent Wireless Power Transfer Technologies*", Published by IntechOpen, London, United Kingdom,

[3] A. Triviño-Cabrera, J. M. González-González, and J. A. Aguado, "*Wireless Power Transfer for Electric Vehicles: Foundations and Design Approach*", Power Systems book series, Published

[4] K. Jin, and W. Zhou, "*Wireless Laser Power Transmission: A Review of Recent Progress*", IEEE Transactions on Power Electronics, vol. 34, no. 4, pp. 3842-

[5] K. A. Kalwar, M. Aamir, and S. Mekhilef, "*Inductively Coupled Power Transfer (ICPT) for Electric Vehicle Charging – A Review*", Renewable and Sustainable Energy Reviews, vol. 47,

[6] Z. Zhang, K. T. Chau, C. Qiu, and C. Liu, "*Energy Encryption for Wireless Power Transfer*", IEEE Transactions on Power Electronics, vol. 30, no. 9,

pp. 1044-1058, 2019.

**References**

by Springer, Cham, 2020.

2020.

3859, 2019.

pp. 462-475, 2015.

pp. 5237-5246, 2015.

**9**

amplifier

**1. Introduction**

**Chapter 2**

**Abstract**

*Himanshu Dehra*

Developments in Wireless Power

This chapter presents state-of-the-art and major developments in wireless power transfer using solar energy. The brief state-of-the-art is presented for solar photovoltaic technologies which can be combined with wireless power transfer (WPT) to interact with the ambient solar energy. The main purpose of the solar photovoltaic system is to distribute the collected electrical energy in various small-scale power applications wirelessly. These recent developments give technology based on how to transmit electrical power without any wires, with a small-scale by using solar energy. The power can also be transferred wirelessly through an inductive coupling as an antenna. With this wireless electricity we can charge and make wireless electricity as an input source to electronic equipment such as cellphone, MP3 Player etc. In harvesting energy, technologies of ambient solar radiation like solar photovoltaic, kinetic, thermal or electro-magnetic (EM) energy can be used to recharge the batteries. Radio frequency (RF) harvesting technologies are also popular as they are enormously available in the atmosphere. The energy converted to useful DC energy which can be used to charge electrical devices which need low power consumption. The chapter has also presented a parallel plate photovoltaic amplifier connected to a potentiometer as a Resistance-Capacitance (RC) circuit power amplifier. The effect of inductance and resulting power transfer has been theoretically determined in the RC amplifier circuit. The electrical and thermal properties and measurements from a parallel plate photovoltaic amplifier were collected to analyze the unbalanced power transfer and inductance in a nonlinear RC circuit amplifier using equivalent transfer functions. The concept of Wireless Information and Power Transfer using Electromagnetic and Radio Waves of Solar Energy Spectrum is also briefly outlined.

**Keywords:** wireless power transfer, solar energy, energy harvesting, photovoltaics,

Wireless power transfer (WPT) is defined as the transmission of electrical power without wires through various methods and technologies using time-varying electric, magnetic, or electromagnetic fields. The development of various technologies for wireless power transfer is being taken widely across the power electronics domains. There are applications which include microwaves, solar cells, lasers, and electro-magnetic waves' resonance in wireless power transfer. With wireless power transfer, the electrical devices are continuously charged without the use of power cord. The three types of wireless power transfer systems can be described by microwaves, resonance, and solar cells. From the power source to a receiver in an electrical

Transfer Using Solar Energy


*Edited by Yasemin Yıldız and Aynur Manzak*

In addition to theoretical information, this book provides information on cobalt compounds and nanoparticles made at selected universities and research centers around the world.This book includes information on new cobalt approaches, nanostructured cobalt, and new catalysts in some reactions and biological systems and technologies.I deeply appreciate the authors' great contributions to this work.We hope that the book will shed light on new horizons for scientists, engineers, and students.

Published in London, UK © 2019 IntechOpen © ABGlavin / iStock

Cobalt Compounds and Applications

Cobalt Compounds

and Applications

*Edited by Yasemin Yıldız and Aynur Manzak*