**2. Experimental**

#### **2.1. Experimental setup and analyses**

The leaching experiments were carried out in thermostated Plexiglas columns (length 50 cm, inner diameter 6 cm) at temperature 20.0±0.2°C (Figure 1.) Each column contained 1.4 L (2.2 kg) of soil, which was added into the column and tightened by manual shaking. In the experiments the columns with soil were treated once with 80 cm3 of the diluted solution of bioremediation agent SR-100 (E-Tech, USA), which contained 9.18% of anionic surfactants as MBAS [101]. Every week, 30 cm3 of aerated distilled water was added to the column to moisten the soil and supply the soil with oxygen in order to model natural conditions (rain). After the experiments the content of the columns was divided into four equal fractions by volume (Figure 2A).

The experiments with soil compost were carried out in the same columns (Fig. 1), which contained 1.4 L (1.9 kg) of soil compost. In the experiments the columns with soil compost were treated twice per week with aerated distilled water or 0.02% solution of Tween 80 and the amount of added liquid was calculated by mean annual precipitation in Estonia. After the experiments the content of the columns was divided into three equal fractions by volume (Figure 2B). All experiments continued two months.

First series of the experiments were carried out with contaminated coarse-grained (diameter 2-8 mm) sandy soil from Ämari Airport (northwestern Estonia). The soil was contaminated with jet and diesel fuel as well as lubricating oil. To achieve a higher porosity in the test the polluted soil was mixed with unpolluted sand and the final concentration of the hydrocarbons was about 1000 mg HEM kgˉ1 DS. Another series of experiments was carried out with fine (diameter 0.2-4 mm) natural sandy soil from Kloogaranna beach (northwestern of Estonia). It

was artificially contaminated with used diesel oil and the concentration of hydrocarbons was about 1400 mg HEM kgˉ1 DS. The column with the unpolluted sand was used to compare the

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leaching of hydrocarbons and surfactants.

**Figure 2.** Fractions of soil (A) and soil compost (B)

**Figure 1.** Experimental setup for leaching experiments

Biodegradation and Leaching of Surfactants During Surfactant-Amended Bioremediation of Oil-Polluted Soil http://dx.doi.org/10.5772/56908 129

**Figure 1.** Experimental setup for leaching experiments

by traditional plating techniques and because it is more important to create suitable conditions for the indigenous bacteria than to introduce new species [10]. The germination tests showed that no immediate phytotoxic properties could be attributed to the compost after 12 weeks of

The main objective of the experimental work was to study the behavior of surfactants in oil-polluted soil and soil compost. The sub-objectives for achieving the main goal are the

**a.** To study leaching and biodegradation of anionic surfactants and their influence on the

**b.** To study leaching and biodegradation of non ionic surfactant Tween 80 and its influence on the properties of leachate and on the behavior of petroleum hydrocarbons in soil

The leaching experiments were carried out in thermostated Plexiglas columns (length 50 cm, inner diameter 6 cm) at temperature 20.0±0.2°C (Figure 1.) Each column contained 1.4 L (2.2 kg) of soil, which was added into the column and tightened by manual shaking. In the

bioremediation agent SR-100 (E-Tech, USA), which contained 9.18% of anionic surfactants as

the soil and supply the soil with oxygen in order to model natural conditions (rain). After the experiments the content of the columns was divided into four equal fractions by volume

The experiments with soil compost were carried out in the same columns (Fig. 1), which contained 1.4 L (1.9 kg) of soil compost. In the experiments the columns with soil compost were treated twice per week with aerated distilled water or 0.02% solution of Tween 80 and the amount of added liquid was calculated by mean annual precipitation in Estonia. After the experiments the content of the columns was divided into three equal fractions by volume

First series of the experiments were carried out with contaminated coarse-grained (diameter 2-8 mm) sandy soil from Ämari Airport (northwestern Estonia). The soil was contaminated with jet and diesel fuel as well as lubricating oil. To achieve a higher porosity in the test the polluted soil was mixed with unpolluted sand and the final concentration of the hydrocarbons

(diameter 0.2-4 mm) natural sandy soil from Kloogaranna beach (northwestern of Estonia). It

of aerated distilled water was added to the column to moisten

DS. Another series of experiments was carried out with fine

of the diluted solution of

composting fresh biowaste together with the diesel-contaminated soil (10%) [90].

behavior of petroleum hydrocarbons in oil-polluted sandy soil;

experiments the columns with soil were treated once with 80 cm3

(Figure 2B). All experiments continued two months.

*1.1.5. Objectives of experimental work*

128 Applied Bioremediation - Active and Passive Approaches

following:

compost.

**2. Experimental**

(Figure 2A).

**2.1. Experimental setup and analyses**

MBAS [101]. Every week, 30 cm3

was about 1000 mg HEM kgˉ1

**Figure 2.** Fractions of soil (A) and soil compost (B)

was artificially contaminated with used diesel oil and the concentration of hydrocarbons was about 1400 mg HEM kgˉ1 DS. The column with the unpolluted sand was used to compare the leaching of hydrocarbons and surfactants.

In the latter experiments the coarse-grained soil was mixed with the hydrophobic porous adsorbent (10% v/v) prepared by modifying of the urea-formaldehyde resins. The leachate was collected and the concentrations of the leached surfactants and hydrocarbons were deter‐ mined.

**Time, days**

during the leaching experiments.

surfactants are presented in Table 2.

**Fraction of the column**

the experiments.

**Unpolluted soil Oil-polluted soil Oil-polluted soil with adsorbent**

**Coarse soil Fine soil Coarse soil Fine soil Coarse soil Fine soil**

Biodegradation and Leaching of Surfactants During Surfactant-Amended Bioremediation of Oil-Polluted Soil

 172 292 172 260 552 612 121 176 91.4 142 461 549 95.7 108 85.4 97.5 213 401 51.7 46.6 48.5 2.1 109 254 8.9 2.3 8.4 1.1 64.8 119 8.1 1.5 8.1 0.93 43.7 87

**Table 1.** Concentrations of anionic surfactants (mg MBAS gˉ1) in the upper layer (0-5 cm) of the columns with soil

surfactants by the soaking of the solution of surfactants in the soil.

The columns with unpolluted or polluted coarse-grained soil had a negligible difference in the concentration of surfactants in the upper soil layer (0-5 cm) during the experiment. The higher concentration of surfactants in the upper soil layer in case of a mixture of soil and adsorbent could be explained by the specific higher surface of adsorbent which adsorbed the anionic

For fine soil, the initial concentration of surfactants and its reduction in the upper layer (Table 1) was similar in all soil samples. The surfactants were washed out of the upper layer in the columns of unpolluted and polluted soil during the first 30 days. The concentration of anionic surfactants decreased linearly during the last 40 days of the experiment with the mixture of polluted soil and adsorbent and the surfactants were not completely washed out of the upper layer during the 60 days in the column. The experiments continued for 60 days after which all four fractions of soil samples and leachate were analyzed and the calculated masses of anionic

1 (0-12 cm) 22.7 10.4 8.5 19.6 61.1 74.4 2 (12-24 cm) 52.6 17.1 75.9 75.5 70.7 81.3 3 (24-36 cm) 64.3 93.8 4.4 88.4 79.9 84.8 4 (36-48 cm) 271 617 4.1 351 71.3 97.5 leachate 125 40.4 20.2 50.4 0 0 total 535 779 113 584 283 338 **Reduction (%)** 34.4 4.5 86.2 28.4 65.3 58.6

**Table 2.** The cumulative masses of anionic surfactants (mg MBAS) in soil fractions and leachate of the columns after

**Unpolluted soil Oil-polluted soil Oil-polluted soil with**

**Coarse soil Fine soil Coarse soil Fine soil Coarse soil Fine soil**

**adsorbent**

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131

Soil compost was sampled from the composting windrows (composting facility of Ragn-Sells Ldt, Estonia) and for composting the polluted soil, which consist different petroleum hydrocarbons was mixed with gravel (to enhance the porosity), horse manure (as source of microorganisms), wood and bark chips and peat (as bulking agents). The concentration of hydrocarbons was about 77 g HEM kgˉ1 DS.

#### **2.2. Chemical analysis**

The pH of the leachate and water extracts was measured by a pH-meter (SensION1, Hach, USA and Model 3320, Jenway, UK). Soil pH was determined by extracting the soil samples with 5 volumes of distilled water and measured with a glass electrode. The conductivity (EC) of water extracts was measured by a conductivity meter (Model 4320, Jenway, UK) and the values have been corrected to a constant temperature of 25 °C [102].

Colorimetric methods are quite widely used for the determination of the concentrations of surfactants [103 - 106]. Anionic surfactants form ion pairs with Methylene Blue (MB) and extracted with chloroform. The concentration of anionic surfactants in the soil and leachate was determined by the spectrophotometric method using MB [105]. The chloroform phase with the dissolved colored complex was separated and its absorbance was measured at 654 nm by the spectrophotometer KFK-3 (USSR). The concentration of the anionic surfactants in the leachate was calculated by the calibration curve as methylene blue active substances (MBAS). The concentration of non ionic surfactants was determined as cobalt thiocyanate active substances (CTAS) by method 512C [102]. The surfactant-cobalt complex will partition into methylene chloride from excess aqueous cobalt thiocyanate by a single extraction and measurement of CTAS in the methylene chloride at 620 nm by spectrophotometer (Model 6300, Jenway, UK).

The concentration of total petroleum hydrocarbons (abbreviated as TPH) in the solids and leachate was determined gravimetrically as HEM by the USEPA method 1664 [107]. The microbial activity of soil compost samples was characterized through the oxygen uptake over a 7-days period determined by manometric OxiTop system (WTW, Germany) at temperature 20±0.2 °C [108-110].

#### **2.3. Results of column studies**

#### *2.3.1. Sandy soil*

Concentrations of anionic surfactants were measured regularly in the upper layer (0-5 cm) of the soil in the columns [101, 111]. The determined concentrations of anionic surfactants as MBAS in the experiments with polluted sandy soil are presented in Table 1.

Biodegradation and Leaching of Surfactants During Surfactant-Amended Bioremediation of Oil-Polluted Soil http://dx.doi.org/10.5772/56908 131

In the latter experiments the coarse-grained soil was mixed with the hydrophobic porous adsorbent (10% v/v) prepared by modifying of the urea-formaldehyde resins. The leachate was collected and the concentrations of the leached surfactants and hydrocarbons were deter‐

Soil compost was sampled from the composting windrows (composting facility of Ragn-Sells Ldt, Estonia) and for composting the polluted soil, which consist different petroleum hydrocarbons was mixed with gravel (to enhance the porosity), horse manure (as source of microorganisms), wood and bark chips and peat (as bulking agents). The concentration of

The pH of the leachate and water extracts was measured by a pH-meter (SensION1, Hach, USA and Model 3320, Jenway, UK). Soil pH was determined by extracting the soil samples with 5 volumes of distilled water and measured with a glass electrode. The conductivity (EC) of water extracts was measured by a conductivity meter (Model 4320, Jenway, UK) and the

Colorimetric methods are quite widely used for the determination of the concentrations of surfactants [103 - 106]. Anionic surfactants form ion pairs with Methylene Blue (MB) and extracted with chloroform. The concentration of anionic surfactants in the soil and leachate was determined by the spectrophotometric method using MB [105]. The chloroform phase with the dissolved colored complex was separated and its absorbance was measured at 654 nm by the spectrophotometer KFK-3 (USSR). The concentration of the anionic surfactants in the leachate was calculated by the calibration curve as methylene blue active substances (MBAS). The concentration of non ionic surfactants was determined as cobalt thiocyanate active substances (CTAS) by method 512C [102]. The surfactant-cobalt complex will partition into methylene chloride from excess aqueous cobalt thiocyanate by a single extraction and measurement of CTAS in the methylene chloride at 620 nm by spectrophotometer (Model 6300,

The concentration of total petroleum hydrocarbons (abbreviated as TPH) in the solids and leachate was determined gravimetrically as HEM by the USEPA method 1664 [107]. The microbial activity of soil compost samples was characterized through the oxygen uptake over a 7-days period determined by manometric OxiTop system (WTW, Germany) at temperature

Concentrations of anionic surfactants were measured regularly in the upper layer (0-5 cm) of the soil in the columns [101, 111]. The determined concentrations of anionic surfactants as

MBAS in the experiments with polluted sandy soil are presented in Table 1.

DS.

values have been corrected to a constant temperature of 25 °C [102].

mined.

hydrocarbons was about 77 g HEM kgˉ1

130 Applied Bioremediation - Active and Passive Approaches

**2.2. Chemical analysis**

Jenway, UK).

20±0.2 °C [108-110].

*2.3.1. Sandy soil*

**2.3. Results of column studies**

**Table 1.** Concentrations of anionic surfactants (mg MBAS gˉ1) in the upper layer (0-5 cm) of the columns with soil during the leaching experiments.

The columns with unpolluted or polluted coarse-grained soil had a negligible difference in the concentration of surfactants in the upper soil layer (0-5 cm) during the experiment. The higher concentration of surfactants in the upper soil layer in case of a mixture of soil and adsorbent could be explained by the specific higher surface of adsorbent which adsorbed the anionic surfactants by the soaking of the solution of surfactants in the soil.

For fine soil, the initial concentration of surfactants and its reduction in the upper layer (Table 1) was similar in all soil samples. The surfactants were washed out of the upper layer in the columns of unpolluted and polluted soil during the first 30 days. The concentration of anionic surfactants decreased linearly during the last 40 days of the experiment with the mixture of polluted soil and adsorbent and the surfactants were not completely washed out of the upper layer during the 60 days in the column. The experiments continued for 60 days after which all four fractions of soil samples and leachate were analyzed and the calculated masses of anionic surfactants are presented in Table 2.

**Table 2.** The cumulative masses of anionic surfactants (mg MBAS) in soil fractions and leachate of the columns after the experiments.

In the case of unpolluted soil the anionic surfactants were washed mainly deeper than 36 cm and the higher cumulative masses of surfactants indicated the higher level of degradation in the more porous coarse soil. Due to the low porosity of fine soil the amount of oxygen that diffused into the soil was not enough to achieve the aerobic conditions needed for the surfac‐ tants degradation [25].

but porosity of soil was important factor, too. Analysis of leachate showed very low content of hydrocarbons (less than 1% from dry residual) indicating quite active biodegradation of

Biodegradation and Leaching of Surfactants During Surfactant-Amended Bioremediation of Oil-Polluted Soil

The profile of leachate generation is presented in Figure 3 and the generation of leachate had no significant difference by adding of non ionic surfactants Tween 80. The leaching experi‐ ments in the columns with the oil-polluted soil compost continued for 60 days. The measure‐ ments of leachate masses showed that 64% of added water and solution of Tween 80 leached out during the experiments and therefore the non ionic surfactant did not increase the amount of leachate during 2 months [112]. The results of leachate analysis are presented in Table 4.

> 1 6 11 16 21 26 31 36 41 46 51 56 61 66 Time, days

**Figure 3.** Profile of generation of leachate from the column of soil compost. The sampling of leachate is also present‐

(Table 4). It means that the added non ionic surfactant can generate the leaching of petroleum

Similar trend was observed for the non ionic surfactant as well and leachate had more than 3 times higher concentration of non ionic surfactant as CTAS. The solution can be the turning of

Lwater40

LTween48

Lwater51

) content of TPH, but it was

) for the last period through the added Tween 80

LTween64

Lwater68

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133

Lwater23

LTween34

LTween22

In general, the leachate had quite low (below 15 mg HEM Lˉ1

increased significantly (up to 70 mg HEM Lˉ1

hydrocarbon during longer period of treatment.

petroleum hydrocarbons in the soil columns.

*2.3.2. Polluted soil compost*

0

ed.

50

100

150

200

Solution mass, g

250

300

350

400

added water added Tween 80 leachate (water) leachate (Tween 80)

**Table 3.** The cumulative masses of TPH (mg HEM) in soil fractions and leachate of the columns after the experiments.

Addition of porous urea-formaldehyde adsorbent increased the mass of generated leachate (up to 25%) due to the higher porosity of soil but anionic surfactants were no leached out. The residual content of anionic surfactants was about 35-40% from the added surfactants inde‐ pendently on the soil properties and fractions. The analysis of soil fractions indicated no significant accumulation of anionic surfactants in the upper layer of mixture of soil and adsorbent.

The analysis of residual TPH (Table 3) showed that porosity was quite important factor for the biodegradation of hydrocarbons as coarse soil contained less hydrocarbon in comparison with fine soil. Added anionic surfactants accelerated the degradation of petroleum hydrocarbons, but porosity of soil was important factor, too. Analysis of leachate showed very low content of hydrocarbons (less than 1% from dry residual) indicating quite active biodegradation of petroleum hydrocarbons in the soil columns.

#### *2.3.2. Polluted soil compost*

In the case of unpolluted soil the anionic surfactants were washed mainly deeper than 36 cm and the higher cumulative masses of surfactants indicated the higher level of degradation in the more porous coarse soil. Due to the low porosity of fine soil the amount of oxygen that diffused into the soil was not enough to achieve the aerobic conditions needed for the surfac‐

**Initial soil** 1039 1463 882 1245 **Soil + H2O** 1 (0-12 cm) 216 318 154 246 2 (12-24 cm) 220 326 160 251 3 (24-36 cm) 229 340 175 259 4 (36-48 cm) 218 333 169 248 leachate 42 20 18 10 total 925 1337 676 1014 **Reduction (%)** 11.0 8.6 23.4 18.6 **Soil + SR-100** 1 (0-12 cm) 107 186 60 152 2 (12-24 cm) 69 193 53 164 3 (24-36 cm) 82 258 63 144 4 (36-48 cm) 128 183 61 139 leachate 30 59 10 18 total 416 879 247 617 **Reduction (%)** 60.0 39.9 72.0 50.4

**Table 3.** The cumulative masses of TPH (mg HEM) in soil fractions and leachate of the columns after the experiments.

Addition of porous urea-formaldehyde adsorbent increased the mass of generated leachate (up to 25%) due to the higher porosity of soil but anionic surfactants were no leached out. The residual content of anionic surfactants was about 35-40% from the added surfactants inde‐ pendently on the soil properties and fractions. The analysis of soil fractions indicated no significant accumulation of anionic surfactants in the upper layer of mixture of soil and

The analysis of residual TPH (Table 3) showed that porosity was quite important factor for the biodegradation of hydrocarbons as coarse soil contained less hydrocarbon in comparison with fine soil. Added anionic surfactants accelerated the degradation of petroleum hydrocarbons,

**Oil-polluted soil Oil-polluted soil with adsorbent**

**Coarse soil Fine soil Coarse soil Fine soil**

tants degradation [25].

132 Applied Bioremediation - Active and Passive Approaches

**Fraction of the column**

adsorbent.

The profile of leachate generation is presented in Figure 3 and the generation of leachate had no significant difference by adding of non ionic surfactants Tween 80. The leaching experi‐ ments in the columns with the oil-polluted soil compost continued for 60 days. The measure‐ ments of leachate masses showed that 64% of added water and solution of Tween 80 leached out during the experiments and therefore the non ionic surfactant did not increase the amount of leachate during 2 months [112]. The results of leachate analysis are presented in Table 4.

**Figure 3.** Profile of generation of leachate from the column of soil compost. The sampling of leachate is also present‐ ed.

In general, the leachate had quite low (below 15 mg HEM Lˉ1 ) content of TPH, but it was increased significantly (up to 70 mg HEM Lˉ1 ) for the last period through the added Tween 80 (Table 4). It means that the added non ionic surfactant can generate the leaching of petroleum hydrocarbon during longer period of treatment.

Similar trend was observed for the non ionic surfactant as well and leachate had more than 3 times higher concentration of non ionic surfactant as CTAS. The solution can be the turning of

*2.3.3. Fluxes of pollutants and surfactants*

reduction of content of TPH was below 25%.

soil and 58.6% for fine one).

specialized soil bacteria [68].

of TPH in soil compost.

**3. Conclusion**

Calculation of mass balance of anionic surfactants showed the highest degradation of anionic surfactants (about 86% of added surfactants) in the column of coarse-grained oilpolluted soil, while only 28.4% of anionic surfactants were degraded in the column of fine polluted soil (Table 2). Quite low degradation of anionic surfactants was determined in the columns of unpolluted soil (34.4% for coarse soil and 4.5% for fine soil) while added adsorbent significantly accelerated the degradation of anionic surfactants (65.3% for coarse

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The highest degradation of TPH (72%) was determined for the mixture of polluted coarse soil with adsorbent (Table 3) treated with SR-100 containing anionic surfactants. The treatment of polluted coarse soil with SR-100 reduced the content of TPH to 60%. In the case of fine soil the reduction of content of TPH was about 20% lower. Without added anionic surfactants the

The cumulative amount of added Tween 80 was 116 mg CTAS and residual amount of non ionic surfactant was 66 mg CTAS and therefore 43% of non ionic surfactant was degrad‐ ed during 60 days of experiments, while the leachate contained 9.7% of surfactant and therefore main part of Tween 80 biodegraded in soil compost during experiments. The other experiment also showed not so good biodegradation of Tween 80 due to its high molecu‐ lar weight [113]. From biodegradability tests, both in liquid and in solid phase, Tween 80 resulted to be, in tested conditions, extremely biodegradable and mineralisable also by not

Calculation of mass balance of TPH showed that 22% of TPH was biodegraded in the column of soil compost treated with water and 23% of TPH was degraded through addition of solution of Tween 80. It means that non ionic surfactant had no accelerating effect on the biodegradation

Surfactants are used to increase the bioavailability of hydrophobic pollutants (hydrocarbons), but the added anionic surfactants were washed out of the upper layer of soil columns during the first 30 days regardless of the type of soil. After the experiments (60 days), the analysis of different soil fractions showed that up to 86% of anionic surfactants had degraded in the column of coarse-grained polluted soil while up to 28% of the added surfactants had degraded in the column of fine sandy soil. Higher amount of anionic surfactants was determined in the leachate of columns of coarse-grained soil and the higher porosity of soil could increase the

Addition of porous urea-formaldehyde adsorbent increased the mass of generated leachate (up to 25%) due to the higher porosity of soil but anionic surfactants were no leached out and the analysis of soil fractions indicated no significant accumulation of anionic surfactants in the

leaching of surfactants due to the lower surface area of coarse-grained soil.

upper layer of mixture of soil and adsorbent.

**Table 4.** Leachate from the column of composted soil

soil compost which was impossible in the columns. The compost windrows were turned at least once per month in the composting facility and therefore the leaching of petroleum hydrocarbons and non ionic surfactant would not occur. After the experiments the fractions of soil compost were analyzed and the results are presented in Table 5.

**Table 5.** Characteristics of fraction of soil compost after leaching experiments in the columns

The analysis of compost fractions showed that both water and solution of Tween 80 reduced the content of petroleum products in the compost mixture about 20%. The reduction of hydrocarbon content was generated through biodegradation as less than 0.01% of hydrocar‐ bons were leached out from the soil columns. It is important to note that addition of surfactant did not increase the leaching of petroleum products [112]. In the column of soil compost which was treated with water the lowest TPH content as well as rate oxygen demand and conductivity were determined for the centre fraction (16-32 cm). In the case of treatment with Tween 80 the rate of oxygen demand was the highest for the lower fraction, but content of TPH was lightly higher for the upper layer. The analyses of soil compost fractions showed that non ionic surfactant was leached into the lower fractions of soil (Table 5) and only up to 10% of non ionic surfactant was leached out from the soil columns during 60 days.

### *2.3.3. Fluxes of pollutants and surfactants*

Calculation of mass balance of anionic surfactants showed the highest degradation of anionic surfactants (about 86% of added surfactants) in the column of coarse-grained oilpolluted soil, while only 28.4% of anionic surfactants were degraded in the column of fine polluted soil (Table 2). Quite low degradation of anionic surfactants was determined in the columns of unpolluted soil (34.4% for coarse soil and 4.5% for fine soil) while added adsorbent significantly accelerated the degradation of anionic surfactants (65.3% for coarse soil and 58.6% for fine one).

The highest degradation of TPH (72%) was determined for the mixture of polluted coarse soil with adsorbent (Table 3) treated with SR-100 containing anionic surfactants. The treatment of polluted coarse soil with SR-100 reduced the content of TPH to 60%. In the case of fine soil the reduction of content of TPH was about 20% lower. Without added anionic surfactants the reduction of content of TPH was below 25%.

The cumulative amount of added Tween 80 was 116 mg CTAS and residual amount of non ionic surfactant was 66 mg CTAS and therefore 43% of non ionic surfactant was degrad‐ ed during 60 days of experiments, while the leachate contained 9.7% of surfactant and therefore main part of Tween 80 biodegraded in soil compost during experiments. The other experiment also showed not so good biodegradation of Tween 80 due to its high molecu‐ lar weight [113]. From biodegradability tests, both in liquid and in solid phase, Tween 80 resulted to be, in tested conditions, extremely biodegradable and mineralisable also by not specialized soil bacteria [68].

Calculation of mass balance of TPH showed that 22% of TPH was biodegraded in the column of soil compost treated with water and 23% of TPH was degraded through addition of solution of Tween 80. It means that non ionic surfactant had no accelerating effect on the biodegradation of TPH in soil compost.


*Review* **Postharvest Ultraviolet Radiation in Fruit and Vegetables: Applications and Factors Modulating Its Efficacy on Bioactive Compounds and Microbial Growth**

**Magalí Darré 1, Ariel Roberto Vicente 1,\*, Luis Cisneros-Zevallos 2 and Francisco Artés-Hernández 3**

**Abstract:** Ultraviolet (UV) radiation has been considered a deleterious agent that living organisms must avoid. However, many of the acclimation changes elicited by UV induce a wide range of positive effects in plant physiology through the elicitation of secondary antioxidant metabolites and natural defenses. Therefore, this fact has changed the original UV conception as a germicide and potentially damaging agent, leading to the concept that it is worthy of application in harvested commodities to take advantage of its beneficial responses. Four decades have already passed since postharvest UV radiation applications began to be studied. During this time, UV treatments have been successfully evaluated for different purposes, including the selection of raw materials, the control of postharvest diseases and human pathogens, the elicitation of nutraceutical compounds, the modulation of ripening and senescence, and the induction of cross-stress tolerance. Besides the microbicide use of UV radiation, the effect that has received most attention is the elicitation of bioactive compounds as a defense mechanism. UV treatments have been shown to induce the accumulation of phytochemicals, including ascorbic acid, carotenoids, glucosinolates, and, more frequently, phenolic compounds. The nature and extent of this elicitation have been reported to depend on several factors, including the product type, maturity, cultivar, UV spectral region, dose, intensity, and radiation exposure pattern. Even though in recent years we have greatly increased our understanding of UV technology, some major issues still need to be addressed. These include defining the operational conditions to maximize UV radiation efficacy, reducing treatment times, and ensuring even radiation exposure, especially under realistic processing conditions. This will make UV treatments move beyond their status as an emerging technology and boost their adoption by industry.

**Keywords:** UV; UVB; UVC; UV illumination; photochemical treatments; abiotic stress; antioxidants; phytochemicals; quality; food safety

#### **1. Introduction**

Ultraviolet (UV) radiation comprises the region of the electromagnetic spectrum (EM) between visible light and X-rays (100–400 nm) [1]. It was discovered in 1801 by Johann Wilhelm Ritter, who observed that radiation outside the violet end of the visible solar spectrum could decompose silver chloride [2]. Seven decades later, it was discovered that UV light could prevent microbial growth [3].

The UV region is frequently divided into three sub-regions, UVA (315–400 nm), UVB (280–315 nm), and UVC (100—280 nm) (Figure 1), which are used for CIE and ISO standards [4]. Further sub categorization has been performed by some authors to discriminate

**Citation:** Darré, M.; Vicente, A.R.; Cisneros-Zevallos, L.; Artés-Hernández, F. Postharvest Ultraviolet Radiation in Fruit and Vegetables: Applications and Factors Modulating Its Efficacy on Bioactive Compounds and Microbial Growth. *Foods* **2022**, *11*, 653. https://doi.org/ 10.3390/foods11050653

Academic Editor: Filipa V. M. Silva

Received: 27 December 2021 Accepted: 7 February 2022 Published: 23 February 2022

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

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

within the UVC region vacuum UV (100 and 200 nm) with stronger ionizing power, but less penetration [5–7]. Based on the known mechanisms of plant photoreception, the UVA region has been split into short-UVA (315–350 nm) and long-UVA (350–400) [8].

**Figure 1.** Subregions of the UV spectrum relevant for technological applications and plant photoreception.

#### **2. UV Radiation Sources**

Commercially available UV sources include mercury lamps (low- and mediumpressure), pulsed light (PL), and light-emitting diodes (LEDs) [9]. Although the market has become highly dynamic with the improvement of LEDs, it is still dominated by mercury lamps. These sources are based on the excitation of gas discharges and feature several pitfalls, including a relatively high voltage requirement to operate and a substantial amount of heat released [10]. However, one advantage is that, especially with medium-pressure Hg lamps, high output powers can be achieved.

Xenon inert-gas lamps were introduced during the late 1970s in Japan, leading to the development of sterilizing technology, called PureBright® [11]. PL treatments consist in the exposure of fresh produce to polychromatic light (200–1100 nm), including ultraviolet (180–400 nm), visible (400–700 nm), and near-infrared (700–1100 nm) wavelengths, in the form of intense, but short, pulses (1 μs–0.1 s) [12,13].

Light-emitting diodes (LEDs) are based on the junction of two-terminal semiconductors (p-n junction) converting electricity into radiation. Depending on the materials out of which the semiconductors are made, the LEDs emit at different wavelengths [14]. The first LEDs, in the early 1960s, emitted infrared (IR) light. Over the years, it became possible to develop LEDs of shorter wavelengths. UV LEDs have several advantages relative to mercury lamps, including their lack of requirement for warming time, their lack of mercury, their compactness, their robustness (with UV LEDs, no protection against glass breakage is necessary, and mobile use is possible), and their large wavelength diversity (210 nm to 360 nm by varying the aluminum content in the AlGaN quantum wells) [10]. In addition, they have lower electromagnetic interference, are easily adaptable for fast modulation in terms of radiation intensity and pulse duration, present narrow-band emission without spurious peaks, and require low maintenance [15]. Two important advantages of LEDs are their long lifespan (expected lifetimes of many 10,000 s of h) and low heat emission [16]. The Achilles heel of UV LEDs is their relatively low quantum efficiency [17]. However, in recent years, by reducing dislocations and defects and improving semiconductor doping and light extraction, their quantum efficiency has been increased [18].

#### **3. Uses in the Food Industry**

UV technology has been applied in the food industry for many different purposes (Figure 2).

Surface sterilization: One of the most common uses of germicide UVC lamps is as environmental sterilizers in foodstuffs filling equipment, conveyor belts, containers, and working surfaces [19,20]. Sterilizing UV lamps are frequently used for aseptic packing, a technology that is expected to continue growing in the coming years [21,22].

**Figure 2.** UV radiation applications in the food industry.

Fluid disinfection: UV radiation in the C zone has been used for water disinfection since 1909. It has been also applied for juice pasteurization [23]. UVC does not generate undesirable by-products, but on the other hand, it does not provide residual disinfection capacity [24]. It has been applied to reduce chlorine use.

Air treatment: Air disinfection can be achieved through different strategies, ranging from irradiating just the air in the upper region to treating all air, either when the room is empty or during circulation through air-conditioning systems [25]. The fact that UVC relatively low radiation doses (0.1–0.3 kJ m−2 for 2 log cycle reductions) can inactivate human SARS-coronaviruses has increased the recent interest in using UV radiation for air treatment [26–28].

Waste treatment: Another application of UV radiation has been the elimination of undesirable volatile organic compounds (VOCs) in industrial exhausts [29,30]. This has been achieved through advanced oxidation processes combining UV radiation with photocatalysts, such as TiO2 [9,31]. This strategy generates highly oxidative environments, which facilitates the degradation of unwanted molecules [32].

Insect trapping: For a long time, it has been known that UV radiation can attract insects; thus, it is used for trapping purposes [33]. The most common insect light traps use "black-light" fluorescent tubes emitting ultraviolet (UVA) as an insect attractant in both pre and postharvest [34]. Furthermore, insects may be trapped in glued materials or killed in electrically charged grids [35].

#### **4. Uses in Fruits and Vegetables Postharvest**

UV technology may be of interest for the postharvest treatment of fruits and vegetables for many different purposes [36,37] (Figure 3).

Raw material selection: The presence of skin defects or wounding is a main factor affecting consumer acceptability and purchase decisions. Consequently, one of the most intensive activities of packinghouses is to separate fruit with these defects. Normally, this is performed through visual inspection or machine optical sorting when the fruit is illuminated under proper white light [38]. In citrus, the use of UV lamps during initial classification has may facilitate the identification of physical damages. UVA "black light" illuminates fruit, showing that small peel cracks fluoresce intensely, allowing segregation at early classification steps [39]. The conveyor belts transporting the fruit cross these rooms, where the operators must wear protective glasses and gloves.

**Figure 3.** Main uses of postharvest UV treatments for direct application in fruit and vegetables.

Control of spoilage and pathogenic microorganisms: Relying on UVC germicide properties, a large body of research evaluated this technology in fruits and vegetables to control surface microorganisms [40]. Microbial death induced by UVC has been attributed to DNA mutations, including the formation of cyclobutyl-type dimers (pyrimidine dimers) and pyrimidine adducts [41,42]. Furthermore, the overproduction of reactive oxygen species (ROS) induced by UV radiation can oxidize membrane lipids and inhibit critical cellular enzymes [43]. Most enzymes that contain aromatic amino acids are likely to be sensitive to UV radiation to some extent due to their absorption in this region. Due to its higher energy, UVC is the most effective at killing microorganisms [44]. UV radiation is lethal to bacteria, viruses, protozoa, and fungi [45]. Successive studies showed that UV radiation is more efficient for inactivating Gram-negative than Gram-positive bacteria. This effect has been associated with the difference in the cell wall peptidoglycan structure, which can affect radiation penetration [46]. Furthermore, eukaryotic organisms are normally more resistant to UV than bacteria due to their higher cell size, complexity, and genetic redundancy [47]. The relatively high yeast resistance to UV radiation has also been associated with lower DNA pyrimidine content relative to bacteria, which may increase the likelihood of photons being absorbed by other compounds [48].

Several works have studied the impact of postharvest pre-storage single exposure on most common postharvest fungal pathogens, including *Rhizopus*, *Penicillum digitatum*, *P. expansum* and *Penicillum italicum*, *Monilinia* sp., *Botrytis cinerea*, *Colletrichcum* sp. and *Fusarium* sp. among others [49,50], with positive effects with regards to reducing disease incidence and severity. Direct germicide action compromising microbial viability has been frequently reported [51], but less severe effects, such as the reduced germination speed of viable conidia, have also been observed [52]. With regards to human pathogens, the direct UV irradiation of fresh produce reduced the viability of *E. coli*, *Salmonella*, and *Listeria* [53–57]. These studies have so far mostly been conducted on a laboratory or, in the best scenario, pilot scale. A review of the available research suggests that their widespread commercial use requires some important aspects to be solved, especially with regards to the adaptability of this treatment to continuous processing lines (where the treatment duration may require minutes at low irradiances), safety procedures for workers and even radiation exposure, while avoiding mechanical damage, especially to fruits and vegetables. Furthermore, the fact that wet cleaning methods have long been applied to fresh produce could limit the fast adoption of a different technology.

The mechanisms through which UV prevents deterioration exceed radiation germicide properties and involve host-induced physiological responses [58–60]. They include the inhibition of ripening-related genes [61] and the induction of enzymes and compounds, improving tolerance to opportunistic pathogens or other environmental effectors causing oxidative damage. An array of defensive responses has been observed in UV-stressed tissues. It includes the activation of glucanases and chitinases thought to be involved in the degradation of microbial cell walls, the induction of genes related to phenolic compound biosynthesis or oxidation, such as phenylalanine ammonia lyase (PAL), polyphenol oxidases (PPOs), and peroxidases (PODs) [62,63]. Another frequent change reported is the upregulation of gene coding for antioxidative enzymes, such as superoxide dismutases (SODs), ascorbate peroxidases (APXs), and catalases (CATs) [64]. More recently, Rabelo et al. [65] proposed that the UV response is mediated by the generation of oxidative stress as the primary signaling molecule generated through the partial ionization of water and an increase in mitochondrial activity. With regards to the induction of compounds that could contribute to increasing host tolerance to pathogen attack, there have been several reports on metabolites with direct antimicrobial activity (i.e., hydroxycinnamic acid derivatives, 6-methoxymellein, scoparone, scopoletin, rishitin), or reinforcing structural barriers [49,66]. Phenolic compounds showing in many cases antimicrobial and antioxidant properties have been the most frequently identified family of induced antimicrobial compounds.

Enhancement of bioactive compounds: Early on, in 1977, Langcake and Pryce [67] showed that UV exposure induced bioactive compounds in grapes. This raised the interest in using UV treatments to improve fruit and vegetable phytochemical profiles. A literature review shows that the number of publications on bioactive compounds and UV radiation has increased exponentially in the last three decades. Table 1 provides an overview of some relevant studies that used UV radiation on whole and fresh-cut fruit and vegetables, focusing on bioactive compounds. As a rule, the results show that the induction of antioxidants by UV radiation tends to be greatest for phenolic compounds, although increases in other antioxidant metabolites (ascorbic acid, glutathione, carotenoids) have also been reported [65,68]. The subclass of compounds induced is mostly dependent on the species considered [69,70] and even on the cultivar [71]. Increases in phenolic acids, nonanthocyanin flavonoids, anthocyanins, other flavonoids, isoflavones, and stilbenes have been frequently reported [62,72–76]. Furthermore, the subregion of the UV spectra applied may determine the type of metabolite elicited; in carrots, chlorogenic acid and isocoumarin were more inducible by UVB and UVC radiation, whereas ferulic acid was elicited by all UV regions to comparable levels [77,78]. At low irradiances, UVB and UVC regions are considered more inductive of secondary metabolites than UVA and UVB, which still have effects on antioxidants but also initiate several photomorphogenic responses [79,80]. Other factors affecting phytochemical accumulation besides the UV region and dose that have been less studied include the irradiances of the illuminating source and the mode of exposure (pulse number duration, interval between successive irradiations, etc.) [13,81,82]. Recently, UV has been reported as a suitable green strategy to enhance the nutraceutical content of fruit and vegetable beverages [83]. Considering there are receptors for UVB and UVA but no receptors for UVC, it has been proposed that ROS plays a key role in secondary metabolite biosynthesis for all three UV lights [77,78]. However, further studies are needed to confirm the role of oxidative stress through UVB and UVA receptor-mediated responses. Another proposed hypothesis is that UVB and UVC could share the same photoreceptors, since the action spectrum of UVR8 protein (as the main UVB receptor) ranges from 250 to 310 nm [8], including the UVC region; therefore, this UVB receptor could also be activated by UVC. It can explain why UVC produces similar effects to UVB in some cases according to previous research, since the energy provided by shorter wavelengths of UVC may activate the UVR8 protein [84]. Nevertheless, the UVR8 spectrum of action could slightly vary, depending on the metabolic pathway. The action spectrum on PAL induction is closer to the UVB region and thus may explain why UVB or even the combined effect of UVC + B obtained a higher increase in total phenolics than UVC [85].

**Table 1.** Effects of UV radiation on the phytochemical profile of fruit and vegetables and on other quality attributes.

**Table 1.** *Cont.*

120

**Table 1.** *Cont.*

In addition to their effects on phenolics, carotenoids and ascorbic acid, UV treatments have been shown to increase vitamin D content. Exposure to sunlight and dietary foods are the most important ways for humans to obtain vitamin D [118–120]. Lifestyle changes due to the SARS-CoV-2 pandemic have substantially reduced our regular exposure to the sun, resulting in vitamin D deficiency [120]. Furthermore, patients with vitamin D deficiency were five times more likely to be positive for COVID-19 than patients with no deficiency [121]. Mushrooms are rich in ergosterol, a precursor to vitamin D2, which can be converted to vitamin D2 under proper UV exposure. The eliciting capacity of UVA, UVB, and UVC has been tested in different types of edible mushrooms, increases ranging between 25 and 8000% reported [122]. The UVB zone of radiation shows the greatest inductive effect, with other important factors being the radiation dose applied, the product's water content, and the degree of processing [123].

Retardation of ripening and senescence: In some commodities and under proper treatment conditions, UV radiation may delay ripening and senescence [60,124]. These effects could be understood by recognizing that both developmental processes are genetically regulated and require specific transcriptional programs to be induced. Under UV radiation, cells redirect their normal developmental programs to primarily respond to external stressing stimuli [125,126]. Stress acclimation favors the induction of UV-responsive genes at the expense of ripening and or senescence-related genes [61,127,128]. The efficacy of UV treatments to retard ripening is certainly dependent on the UV irradiation schedule used, and to a great extent on the initial ripening state of the commodity [129]. Ripening

progression would be more effectively delayed in fruit treated at early stages. Hundreds of genes are up- and down-regulated in fruit irradiated with UV radiation. Most frequently, up-regulated genes are mainly involved in signal transduction, defense response, and metabolism. Conversely, genes related to cell wall disassembly, photosynthesis, and lipid metabolism are usually suppressed. The retardation of ripening-related changes such as softening may be one important contributor to the improved tolerance to postharvest spoilage pathogens observed in treated products [61,82,130]. In some cases, especially with long-term exposure, UVB has been shown to promote fruit ripening [131].

Induction of cross-stress resistance and synergistic responses: It is currently known that biotic and abiotic stress responses use common signals, pathways, and triggers [40]. This overlap includes common changes in cellular redox status, reactive oxygen species, hormones, protein kinase cascades, and calcium gradients as common elements [131] and helps to explain cross-tolerance phenomena, whereby exposure to one type of stress can improve tolerance to several different types of stress [132]. UV treatments preceding cold storage have been reported to improve the chilling tolerance of sensitive commodities such as peach [133], sweet pepper [134], and tomato [135]. Some of the metabolic changes behind cross-tolerance include the induction of polyamine biosynthesis in stone fruit and an increase in antioxidant enzymes in the case of pepper [64]. Another effect observed from combining stresses is a synergistic response in plant tissues, as in those reported with wounding and UV exposure. This synergism among stresses applied simultaneously is due to the activation of similar signaling molecules and signaling pathways [136]. This has been reported in the biosynthesis of polyphenols [77,78,102] and betalains [137]. In Table 2, the physiological responses could potentially be higher in fresh-cut products compared to whole tissues, where synergistic effects take place due to skin removal in whole produce, being skin-determinant in the response due to the partial blockage of UV penetration [77].

**Table 2.** Advantages and drawbacks of using UV radiation in foods.

Advantages and disadvantages of UV treatments: UV technology provides several advantages over other conventional preservation methods, but also has some important drawbacks (Table 2). UV treatments can be simply applied and are able to inactivate a wide range of pathogenic and spoilage microorganisms while causing negligible changes in nutritional and sensory quality [95,96]. In contrast to other disinfection practices, it does not require water or generate wastes and leaves no residues on treated surfaces and foods [48]. In addition, it is approved with no major restrictions by the European Food Safety Authority (EFSA), the US Food and Drug Administration (FDA), and most other food regulatory agencies. When compared to electron beam or gamma irradiation, UV technology also

offers several advantages (i.e., lower investment required, fewer regulations, no treatment label needed in the products) [138]. Another plus for UV treatments relates to their ability to be relatively simply combined with other preservation techniques in the search for additive or even synergistic effects [70,139]. UV irradiation is considered a very valuable tool within the hurdle technology, an integrated approach aimed in creating safe and stable foods by combining multiple physical, chemical, and/or biological preservation methods [139]. UV treatments have been applied in metabolically active fruit and vegetables not just due to their germicide action but to activate natural defense mechanisms (i.e., phytoalexins, free radical scavengers' antimicrobial and antioxidant enzymes) [140]. Compared to many conventional methods, UV treatments are also energy-efficient and cost-effective [141]. Finally, UV has historically being applied for a wide range of applications and validation data is available.

However, there are several limitations of UV technology which prevent its wide use in the food industry. It has a low penetration power, especially in solids and turbid media [142]. Moreover, the fact that it does not provide residuality may be a drawback when long-lasting effects are desired. To properly express the UV germicide properties direct and even exposure is required [143]. This may be challenging in some cases under commercial operations. Another difficulty could be to adapt UV treatments to continue processing lines, especially if long exposure times or low irradiances are required. Considering the moments in which UV could be used, UV treatments are defeated compared to ionizing irradiation, since they cannot be applied after packing [144]. UV radiation could be also harmful to operators if direct exposure occurs, but proper safety procedures and personal protective equipment can easily prevent such risk.

Factors determining UV treatment efficacy: In the last two decades, more than 500 publications have tested postharvest UV illumination strategies in fresh horticultural produce. These studies have been useful in identifying the main factors determining the efficacy of the technology on both the commodity and the treatment sides (Table 3).

**Table 3.** Factors affecting the efficacy of postharvest UV treatments in fruit and vegetables.

The factor that has received the most attention is the type of commodity. So far, most commercially relevant fresh fruit and vegetable (>100 products) have been tested. In general, positive results have been found in one or more of the effects outlined in the previous section, depending on the species considered. In a few cases, some damages, mostly related to tissue discoloration, have also been reported [145,146]. The outcome of UV treatments has also been reported to be dependent on the cultivar [71,147]. A third relevant factor with regards to UV irradiation efficacy is the product-ripening stage. As a rule, the treatment of unripe fruit may lead to stronger phenotypes regarding ripening delay than fruit at advanced maturity stages. The ripening stage not only affects the impact of UV irradiation on ripening changes, but also its ability to control postharvest decay. In fresh-cut bell peppers, UV radiation was more effective at reducing soft rot in red ripe fruit [148,149]. The degree of commodity processing also has a substantial impact on quality maintenance and antioxidant elicitation [102,114]. This could be due to the increased surface during processing, which results in the exposure of a greater area of the product. However, the induction of synergic effects induced by simultaneous UV and wounding stresses has been suggested to be involved as well [70,100]. Finally, the nature of the interphase between UV radiation and the product can also have a relevant effect. For instance, surface wetting would be expected to reduce UV penetration in vegetable tissues. Another effect may be the impact of interphases on microorganism arrangements. Heterogeneous microorganism distributions in liquid droplets can lead to preferential concentration in the outer layer at the liquid–air interface, which may protect the cells inside the droplet from UV bactericidal action [55].

Regarding treatment, the subregion of the UV spectra employed has a large influence on the kind of responses obtained [150]. For instance, changes in nutraceutical compounds have been reported for all three zones, but the greatest impact on disease control is clearly observed for UVC [151]. The second relevant process variable is the radiation dose (energy per unit area). A broad range of doses has been explored (i.e., 0.1–50 kJ m−2 for UVC region). However, a single study tried more than two or three doses and complete optimization studies are currently lacking. Working with strawberries, Cote et al. [152] showed that the radiation intensity (energy per unit time and unit area) is another key process factor determining the efficacy of treatments. Strikingly, this variable has been overlooked in several studies that do not report the radiation intensity of the illuminant used. Subsequent work by Darré et al. [81] reported that both radiation dose and intensity should be considered simultaneously when optimizing UV treatments. Low UVB doses (5 kJ m−2) and intensities delay chlorophyll degradation and may be useful to complement refrigeration. Instead, high-intensity UVB exposure may be better suited for the freezing industry as a pre-treatment to increase the antioxidant capacity prior to further processing.

The irradiation pattern is another aspect to be considered when selecting a proper treatment schedule. This has been relatively well studied for pulsed treatments with xenon lamps. Bauer et al. [153] found that UV germicidal efficacy against *Bacillus* spores was a function of pulsed radiation parameter, with shorter pulses and lower frequencies being more effective. With regards to treatments with conventional single-UV radiation sources, almost no attention has been paid to the relevance of the radiation exposure pattern. Ortiz-Araque et al. [82] showed that at the same total dose (4 kJ m−2) and intensity (36 W m−2), fractionated (two-step and five-step) treatments were much more effective at controlling softening and decay than single pre-storage irradiation. The fractionation of the treatments during storage delayed pectin debranching and delayed the solubilization of polyuronides [130].

Although treatment uniformity and ensuring that all food surfaces are exposed to UVC light may be one of most problematic causes in commercial settings, there are very few studies attempting to overcome this limitation. Finally, some studies have reported that light exposure after treatment may affect the outcome of UV treatments. This may be due to photo repair mechanisms that may be activated in the presence of light [11,154,155]. It is not clear whether this effect would be significant in products. It is important to state that eventually, all these factors and their combinations result in the generation of signal molecules, such as ROS, and ROS levels determine the molecular and physiological response of stress-challenged produce, either staying in homeostasis, going through hormesis, or even responding to extreme stress [136].

#### **5. Concluding Remarks**

After microbial control, the elicitation of bioactive compounds is the aspect that has received the most attention with regards to the use of postharvest UV treatments in fresh produce. So far, several works have reported that appropriate exposure to UV radiation may stimulate the biosynthesis of phenolic compounds and, to a lesser extent, of ascorbic acid, carotenoids, and/or glucosinolates. Recent studies have identified the most relevant factors determining the nature and extent of such changes on both the commodity and process sides. These include the species, cultivar, ripening stage, degree of processing, radiationproduct interphase, radiation wavelength, dose, intensity, exposure pattern (i.e., pulse frequency, duration), and illumination regime after treatment.

Despite the significant advances achieved, some important limitations remain. These has likely slowed down the adoption of UV technology by industrial stakeholders. For fresh products, the main challenges remaining are reducing the treatment times to facilitate their compatibility with continuous processing lines and increasing the treatment uniformity for large produce volumes without causing mechanical damage to products. Solving these challenges would likely help UV irradiation to move beyond the stage of emerging technology and translate all the knowledge accumulated about its application into production.

**Funding:** This research was funded by the Agencia Nacional de Promoción Científica y Tecnológica, Argentina grant number PICT 3679-2018.

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

#### **References**


**5. Outlook for clinical translation: what lies ahead in the field of connectomics and epilepsy?**

The vast amount of data generated by neuroimaging makes it a good candidate for computational methodological analysis. This has shed light on epilepsy as a disease of brain networks. While connectome-derived data have been instrumental thus far in improving and expanding our understanding of the pathophysiology of seizure propagation in difficult-to-treat epilepsy. The generation of personalized connectome is clearly the next step forward in presurgical epilepsy workup in the era of personalized medicine. In doing so our understanding of the brain network will be further expanded benefiting not only epilepsy patients but other neurological and neurosurgical patients alike.

## **6. Conclusions**

Improving noninvasive localization is paramount in the surgical treatment of refractory epilepsies. MRI postprocessing techniques allow for more accurate identification of epileptogenic abnormalities, and in doing so they have the potential to increase the diagnostic yield, reduce the need for invasive electrophysiological investigations, and improve epilepsy surgical outcome in years to come. Patient centered connectome-based lesion-symptom mapping will be the future for epilepsy management where therapeutic options can be tailored in order to enable best informed management for both clinicians and patients in their lifelong journey with this disease.

*Neuroimaging for Epilepsy Diagnosis and Management DOI: http://dx.doi.org/10.5772/intechopen.112356*


*Supporting open minds since 2005*

Frailty and Sarcopenia - Recent Evidence and New Perspectives http://dx.doi.org/10.5772/intechopen.95708 Edited by Grazia D'Onofrio and Julianna Cseri

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Charles Lambert, Norihide Fukushima, Jia-Ping Wu, Tiago Fernandes, Bruno Rocha de Avila Pelozin, Luis Felipe Rodrigues, Edilamar Menezes De Oliveira, Carlos Sáez, Sara García-Isidoro, Grazia D'Onofrio

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**3.12 Calcium (Ca)**

The majority of calcium (99 %) in organism is stored in bones and teeth. Calcium is important for activation of numerous enzymes and hormones. The calcium collaborates in processes of blood coagulation, nerves stimulation and muscles contraction. In blood serum approximately 55% of Ca is in ionised form and this is biologically active. The part of ionised Ca depends from blood pH, by decrease of pH the part of ionised Ca increase. A part of Ca in serum is bound to the albumins (40%) and smaller part (5%) to organic acids (Jazbec, 1990; Kraft, 1999b). The most of Ca is absorbed in small intestine.

In newborn calves the mean serum concentration of Ca was 3.35 ± 0.27 mmol/L. Six hours after birth the Ca level decreased to 2.41 ± 0.18 mmol/L and in the next days and weeks almost did not change (Bostedt & Schramel, 1982). Kurz and Willett (1991) observed decrease of Ca concentration in the first 24 hours of life. In the first two months of life the concentration of Ca was around 2.7 mmol/L and did not change a lot. After the 3rd month the Ca concentration started to decrease and at the age of 6 months it was 2.53 ± 0.10 mmol/L (Bouda & Jagoš, 1984). Similar dynamics was established in other study in calves which were fed with milk replacer, only they have slightly higher Ca concentration at the age of 5 days (3.02 ± 0.2 mmol/L) and from the age of 15 days to 2 months it was 2.8 ± 0.1 mmol/L (Steinhardt & Thielscher, 2000d). In sucker calves of Simmental breed the decrease of Ca concentration from birth to the age of 28 days was established (2.6 mmol/L), later the concentration almost did not change to the age of 84 days (Egli & Blum, 1998). Mohri et al. (2007) established a decrease of Ca concentration in the first two weeks of life, later the Ca concentration slowly increased.

### **3.13 Potassium (K)**

In ruminants the potassium is absorbed from rumen and small intestine and is excreted over the kidney and with faeces. The K is main cation in milk (36 mmol/L). High concentrations of K in organism have citotoxical effect. Potassium is the main intracellular ion, its concentration in the cells is 25 to 30 times higher than in plasma (Ward, 1966), 96-98 % of total amount of K is in the cells (Wirth, 1999). Potassium is important for making electrical potential for transport of nervous impulses and for maintenance of muscle tonus. Hypokalemia increase the membrane potential and cause hyper polarisation block, what influence on lower muscle tonus and paralysis (Carlson, 1997). Potassium is important for regulation of acid-base balance in the body. The forage of ruminants usually contains enough potassium; more often comes to the surplus than to potassium deficiency in the meal. Tucker et al. (1991) and Weil et al. (1988) established association between potassium amount in the meal and concentration of K in calves' serum, and between mean daily weight gains. The mean K concentration in plasma of 8 weeks old calves was from 5.13 mmol/L, at 0.35% K in the meal, to 5.60 mmol/L at 0.53 % K in the dry matter of the meal. The authors claimed that the measuring of K concentration in serum and control of daily weight gain is a good indicator of supply with this element in

concentrations are on the level of adult animals (1.6-2.3 mmol/L). The colostrum intake did not influence the iP concentration in calves' serum (Steinhardt et al., 1993). Kurz and Willett (1991) established decrease of iP concentration in first 24 hours of age. In sucker calves the increase of iP concentration in the first 14 days was established later it remained stable. The values were all the time higher as in adult animals (Egli & Blum, 1998). At the age of 60 days the concentration of iP in calves' serum was 2.6 mmol/L (Steinhardt & Thielscher, 2000d).

The majority of calcium (99 %) in organism is stored in bones and teeth. Calcium is important for activation of numerous enzymes and hormones. The calcium collaborates in processes of blood coagulation, nerves stimulation and muscles contraction. In blood serum approximately 55% of Ca is in ionised form and this is biologically active. The part of ionised Ca depends from blood pH, by decrease of pH the part of ionised Ca increase. A part of Ca in serum is bound to the albumins (40%) and smaller part (5%) to organic acids

In newborn calves the mean serum concentration of Ca was 3.35 ± 0.27 mmol/L. Six hours after birth the Ca level decreased to 2.41 ± 0.18 mmol/L and in the next days and weeks almost did not change (Bostedt & Schramel, 1982). Kurz and Willett (1991) observed decrease of Ca concentration in the first 24 hours of life. In the first two months of life the concentration of Ca was around 2.7 mmol/L and did not change a lot. After the 3rd month the Ca concentration started to decrease and at the age of 6 months it was 2.53 ± 0.10 mmol/L (Bouda & Jagoš, 1984). Similar dynamics was established in other study in calves which were fed with milk replacer, only they have slightly higher Ca concentration at the age of 5 days (3.02 ± 0.2 mmol/L) and from the age of 15 days to 2 months it was 2.8 ± 0.1 mmol/L (Steinhardt & Thielscher, 2000d). In sucker calves of Simmental breed the decrease of Ca concentration from birth to the age of 28 days was established (2.6 mmol/L), later the concentration almost did not change to the age of 84 days (Egli & Blum, 1998). Mohri et al. (2007) established a decrease of Ca concentration in the first two weeks of life, later the Ca concentration slowly increased.

In ruminants the potassium is absorbed from rumen and small intestine and is excreted over the kidney and with faeces. The K is main cation in milk (36 mmol/L). High concentrations of K in organism have citotoxical effect. Potassium is the main intracellular ion, its concentration in the cells is 25 to 30 times higher than in plasma (Ward, 1966), 96-98 % of total amount of K is in the cells (Wirth, 1999). Potassium is important for making electrical potential for transport of nervous impulses and for maintenance of muscle tonus. Hypokalemia increase the membrane potential and cause hyper polarisation block, what influence on lower muscle tonus and paralysis (Carlson, 1997). Potassium is important for regulation of acid-base balance in the body. The forage of ruminants usually contains enough potassium; more often comes to the surplus than to potassium deficiency in the meal. Tucker et al. (1991) and Weil et al. (1988) established association between potassium amount in the meal and concentration of K in calves' serum, and between mean daily weight gains. The mean K concentration in plasma of 8 weeks old calves was from 5.13 mmol/L, at 0.35% K in the meal, to 5.60 mmol/L at 0.53 % K in the dry matter of the meal. The authors claimed that the measuring of K concentration in serum and control of daily weight gain is a good indicator of supply with this element in

(Jazbec, 1990; Kraft, 1999b). The most of Ca is absorbed in small intestine.

**3.12 Calcium (Ca)**

**3.13 Potassium (K)**

calves. In calves with diarrhoea comes to hyperkalemia which is connected with grade of acidosis (Maach et al., 1992).

The colostrum intake influence on increase of K concentration in calves' serum, what is most likely, the consequence of higher amount of this mineral in the colostrum (Steinhardt et al., 1993). In the age from 1 week to 2-3 months the K concentration in calves' serum almost did not change and was around 5 mmol/L (Maach et al. 1991). Bouda and Jagoš (1984) measured slightly higher values, around 5.4 ± 0.4 mmol/L, at the age of 6 month K concentration slightly decreased to 4.7 ± 0.4 mmol/L. Reece (1980) Established a decrease of K concentration from the 1st week of age when it was 7.2 mmol/L to the age of 15 weeks when it fell to 4.4 mmol/L.

#### **3.14 Sodium (Na)**

The sodium is the most important cation in extracellular fluid, where it is responsible for maintenance of osmotic pressure. Together with chlorine (Cl) collaborates in metabolism of water and regulation of acid-base balance in the organism (Jazbec, 1990). In ruminants a big part of Na which comes in to digestive tract originates from saliva (rotation in the organism). The rumen could contain up to 50% of whole amount of Na which is available for the organism. From the body is excreted with the urine, faeces and milk (Underwood & Suttle, 2001). At diarrhoeas the calves lose higher amounts of Na. Maach et al. (1992) established significantly lower concentration of Na in serum of calves with acute diarrhoea (131.2 ± 7.2 mmol/L) in comparison to healthy calves of same age (140.0 ± 9.9 mmol/L). In newborn calves after colostrum intake the Na concentration increased what was attributed to absorption from the colostrum (Steinhardt et al., 1993). But Maach et al. (1991) established higher concentration of Na before colostrum intake when it was 145.7 ± 3.7 mmol/L as then after (137.8 ± 6.8 mmol/L), later it almost did not change. The concentration of Na did not change a lot in the first three months, it was about 145 mmol/L, and at the age of 6 months it was slightly lower, about 136.6 ± 5.1 mmol/L (Bouda & Jagoš, 1984). Reece (1980) established higher concentration of Na in serum of calves which received milk replacer in comparison to the calves which were fed with milk.

#### **3.15 Chlorine (Cl)**

Chlorine is the most important anion in the extracellular fluid, inside the cells there is only 12% of total amount in organism. Together with Na it is responsible for maintenance of osmotic pressure in extracellular fluid. Chlorine is absorbed in small intestine, and is excreted mainly through the kidney (Wirth, 1999). The calves with acute diarrhoea, in which the volume of faeces can increase for 40 times, with the fluid lose also electrolytes. In such calves significantly lower concentration of Cl was established (95.6 ± 6.9 mmol/L) in comparison to healthy calves (103.3 ± 6.9 mmol/L) (Maach et al., 1992).

In calves immediately after birth higher concentration of Cl was established (107.3 ± 12.3 mmol/L), then it decreased to the 7th day of age to 95.9 ± 6.6 mmol/L, later it increased slightly to 102.3 ± 6.2 mmol/L at the age of 2 months (Maach et al., 1991). In suckler calves the concentration of Cl increased with age from 98.0 mmol/L in the 1st week of age to 102.4 mmol/L at the age of 14 weeks (Reece, 1984). In calves which were fed with limited amounts of milk the increase of Cl concentration was established to the age of 5 weeks, later it decreased slightly and oscillated between 97.7 and 99.3 mmol/L (Reece, 1980).

Values of Blood Variables in Calves 313

CK U/L

168.8 ± 211 2

99.6 ± 69.7 1

121.9 ± 48.4 24

106.3 ± 71.8 16,20,24

113.3 ± 69,9 16,24

106.9 ± 46.9 16,20,24

1,2,3,16,20,24 140.4 ± 70.5 216.2 ± 97.7

1,2,3,4,6,20,24 154.5 ± 103.1 196.9 ± 67.9

181.9 ± 94.2 2,4,5,6

166.5 ± 96.3 2,4,6

177.0 ± 115.5 2,3,4,5,6

> Creatinin μmol/L

113.76 ± 36.20 3,4,5,6,8,12,16,20,24

> 116.53 ± 19.41 3,4,5,6,8,12,16, 20,24

100.79 ± 21.66 1,2,5,6,8,12,16,20,24

93.55 ± 16.66 1,2,8,12,16,20,24

89.35 ± 16.67 1,2,3,12,16,20

87.89 ± 15.67 1,2,3,12,16,20

Urea mmol/L

3.64 ± 1.33 2,3

5.90 ± 2.01 1,4,5,6,8,12,16, 20,24

5.12 ± 3.42 1,4,5,6,8,12

4.01 ± 1.21 2,3

3.85 ± 1.34 2,3

3.79 ± 1.26 2,3

ALP U/L

262.2 ± 185 2,3,4,5,12,16,20,24

143.1 ± 61.3 1,6,8

132.8 ± 57.0 1,5,6,8,12

157.8 ± 79.7 1,8

189.5 ± 93.4 1,3

208.1 ± 104.3 2,3

2,3,4,20

1,4

175.6 ± 65.2 1

158.9 ± 55.4 1,8

162.4 ± 58.5 1

> TSP g/L

52.09 ± 7.09 8,12,16,20,24

52.92 ± 5.48 8,12,16,20,24

52.71 ± 4.85 8,12,16,20,24

52.41 ± 3.91 8,12,16,20,24

53.77 ± 4.47 8,12,16,20,24

54.95 ± 5.21 12,16,20,24

GGT U/L

329.8 ± 358.1 2,3,4,5,6,8,12,16,2024

> 79.8 ± 72.1 1

> 45.1 ± 34.5 1

> 30.5 ± 18.3 1

> 24.6 ± 11.2 1

20.2 ± 7.7 1

16.1 ± 3.5 1

13.4 ± 3.0 1

13.0 ± 2.6 1

13.9 ± 3.4 1

14.2 ± 4.9 1

> Alb g/L

26.35 ± 2.10 2,3,4,5,6,8,12,1620,24

28.94 ± 2.79 1,4,5,6,8,12,16, 20,24

30.24 ± 2.48 1,16,20,24

30.99 ± 2.53 1,2,20,24

31.67 ± 2.48 1,2

31.79 ± 2.91 1,2

LDH U/L

483.2 ± 108.8 8,12,16,20,24

451.3 ± 105.2 5,6,8,12,16,20,24

459.8 ± 88.9 5,6,8,12,16,20,24

503.1 ± 160.9 12,16,20,24

532.9 ± 125,9 2,3,16,20,24

544.9 ± 115.3 2,3,12,16,20,24

575.8 ± 122.4

633.6 ± 100.9

671.1 ± 102.7 1,2,3,4,5,6,8

> 722.6.,3 1,2,3,4,5,6,8,12

770.7 ± 125.5 1,2,3,4,5,6,8,12

The age group differs significantly (P<0.05) from age groups in superscript

Fe μmol/L

16.91 ± 10.95 8,12,16,20,24

14.52 ± 7.16 5,8,12,16,20,24

16.61 ± 7.88 8,12,16,20,24

20.97 ± 12.16 12,16,20,24

22.84 ± 13.47 2,12,16,20,24

20.93 ± 11.81 12,16,20,24

Age (weeks) n AST U/L

3,12,16,20,24

8,12,16,20,24

1,8,12,16,20,24

8,12,16,20,24

12,16,20,24

12,16,20,24

2,3,4,12,16,20,24

1,2,3,4,5,6,8

1,2,3,4,5,6,8

1,2,3,4,5,6,8

1,2,3,4,5,6,8

Bilirubin μmol/L

2,3,4,5,6,8,12,1620,24

1,12,16,20,24

1

1

1

1

Table 2. Activity of enzymes in calves (Ježek, 2007)

1 64 38.7 ± 16.1

2 64 32.7 ± 11.1

3 59 31.5 ± 6.2

4 62 33.3 ± 11.0

5 63 33.9 ± 12.1

6 63 35.7 ± 7.4

8 64 40.3 ± 10.2

12 61 47.6 ± 9.2

16 61 48.4 ± 8.1

20 56 50.2 ± 10.3

24 39 53.6 ± 10.1

1 64 16.08 ± 13.16

2 64 8.23 ± 3.41

3 59 6.99 ± 1.94

4 62 6.96 ± 1.52

5 63 6.88 ± 2.11

6 63 6.79 ± 3.05

Age (weeks) n


*Contemporary Challenges and Future Strategies to Mitigate Social Inequality in Urban Housing… DOI: http://dx.doi.org/10.5772/intechopen.107999*

regulating (refugee) migration, combatting pandemic or coding capital are instances of attempts of re-territorialization. However, any attempts to conflate territorial with topological geographies must be wary of fetishizing either of these spaces.

A predominantly territorial, containerized imagination of spatial organization nurtures competition among cities and municipalities in attracting (high-income) households because their public budgets rely strongly on taxes redistributed on the number of primary residents. Demographically shrinking municipalities thus lose income and attention, and they are simultaneously enforced to preserve the still existing infrastructure from dereliction. Demographically growing municipalities must locate new housing blocks and infrastructure, and will yield new income by reallocating taxes after some time. Due to the high attractiveness of most Austrian large cities, suburban municipalities benefit from this competition of people. This is, however, not always and necessarily the case, as medium-sized cities like Eisenerz in Styria illustrate. Eisenerz, a town of 3800 inhabitants in 2020, lost more than 9100 people over the past 70 years. On the other hand, the population of some neighboring municipalities likes, for example, Trofaiach grew remarkably during the same period, partly from households that moved from Eisenerz [17].

The competition between municipalities is geographically volatile since real estate investment strategies have to cope with changing households' lifestyle preferences [18], transforming regional economic and labor markets, and staging mainstream aspirations of where to live the most satisfying life [19]. The main phases had been urbanization, suburbanization, counter-urbanization, and re-urbanization. Gentrification is perhaps the most prominent phenomenon of the current reurbanization of middle- and upper-class households [20, 21]. Due to an accelerating number of heat days and tropical nights or other climate-change effects [22], and because of experienced challenges of corona measures, a renaissance of suburbanization might likely happen in the near future.

A containerized spatial organization of housing (markets) relates closely to the problem of "methodological nationalism", which equates society with the nation state's territory. The methodology construes internal homogeneity and external exclusion to secure own political and economic interests. This principle, in other words, legitimates global inequality [23]. Its principle idea can be scaled down to the local level and applied methodologically to the administrative units of municipalities. Complementing this unilateral orientation with a relational spatial organization would mitigate housing poverty because it relieves the comparative competition between territories.


**2. Catheter associated urinary tract infection**

In addition to being the most common bacterial infection, UTIs are also the most common type of hospital acquired infections (HAI). HAIs can be defined as a localized or systemic condition resulting from an adverse reaction to the presence of an infectious agent or toxin, which occurs in a patient in a health care setting and was not present or incubating at the time of admission [64, 66]. UTIs account for 30% of all HAI [77]. Of these 30% infections, 80% of them are estimated to be catheter-associated [89]. According to the CDC, CAUTIs are defined as an UTI in a patient who had an indwelling urinary catheter in place at the time of or within 48 hours prior to infection onset. CAUTI can lead to complications such as cystitis, pyelonephritis, gramnegative bacteremia, prostatitis, epididymitis, endocarditis, vertebral osteomyelitis, septic arthritis, endophthalmitis and meningitis [20]. Additionally CAUTIs also result in prolonged hospital stay, increased cost and mortality [77]. An estimated 15-25% of hospitalized patients will have a urinary catheter at some point during their hospital stay [175]. Obstruction of indwelling catheters can lead to sepsis, even resulting in mortality [174]. Each year around 13,000 deaths are attributed to UTIs in the United States [77]. The cost associated with CAUTI episodes is about \$750-\$1000 per infection, and the estimated total cost in the United States ranges from \$340-\$450 million annually [132].

Millions of transurethral, suprapubic and nephrostomy catheters or urethral stents are used in patients every year. These devices overcome several host defenses and enable bacterial entry at a rate of 3 to 10% (cumulative rate) per day, which leads to bacteriuria in patients after a month [8]. In intubated patients, bacteria frequently ascend from the urethral meatus into the bladder between the mucosal and catheter surfaces. In certain cases, bacteria may ascend through the drainage system due to contamination of the drainage bag or disruption of the tubing junction. The presence of a device enables the persistence of the etiologic organism in the urinary tract. Several studies have demonstrated that bacteria exist as biofilms on these devices [53]. Formation of a biofilm and incrustation with calcium and magnesium struvites has a significant role in the pathogenesis and treatment of catheter-associated infections.