**7.3 Validation and reliability of the research instruments**

To ensure validity, the questionnaire was hand-delivered to two NS Subject Education Specialists (SES) and one science teacher for critic feedback before it was used for data collection. A Subject education specialist is someone who provides support to teachers with subject content knowledge, practical activities, relevant resources to be used in schools based on their areas of specialization as well as organizing workshops [20]. The study considered the Cronbach alpha test to ensure the reliability of the instrument. In **Table 1**, Part 5 illustrates the outcomes of the Cronbach's alpha test for the quantitative instrument that was used in this study.

Cronbach's alpha for 14 items in the instrument used in this study is shown as 0.703. This is construed as an indication of high reliability and internal consistency in the questionnaire.

#### **7.4 Procedure for data collection**

The structured questionnaire was distributed to 30 science teachers who were requested to complete the task within seven days. After seven days, only 16 had

#### **Table 1.**

*Cronbach's alpha reliability of internal consistency for Likert scale.*

completed. Those who were not ready for submissions were given four more days to respond to the questionnaire which was later collected by the researchers.

#### **7.5 Data analysis**

Both descriptive and inferential statistics were employed in the analysis of the collected data. The research question was answered using science teachers' frequency counts and percentages as follows: promotion of science learning through science content and practical assessment responses: Principles of assessment, those who agreed were 70% and above, while neutral were 30% below; disagree—0%; and no response—0%.

#### **8. Ethical considerations**

Respondents were informed about the significance of the study and its purpose. School principals and science teachers signed the informed consent. Respondents were also informed, as part of their consent that the results of the study would be made available to them on request. Moreover, they were told that their responses would remain anonymous and confidential and their names would not be written even if the findings were published.

#### **9. Results**

**Table 2** illustrates quantitative results on how educators assess science learning and promote science learning.

The data in **Table 2** illustrate how teachers assess SL. The responses were as follows: agree, neutral, disagree, and no response.

#### **9.1 Agree**

Align with cognitive domains 86.7%; Principles of assessment 77%; Investigations 66.7%; Simulation 63.3%; Debates 50%; each among these: Assignment, Experiments, and Examinations 40%; Problem-Solving 37%; Projects 34%; Presentation 23%; Roleplay 17%; Tests 13.3%; and Quiz 7%.

#### **9.2 Neutral**

Presentations 70%; Problem-Solving 60%; Experiments 57%; projects 53%; each among these: Tests and Examinations 50%; Assignments 46,7%; Debates 34%; each among these: Simulations and Investigations 30%; Roleplay 27%; Quiz 23%; Principles of assessment 13%; and Align with cognitive domain 10%.

#### **9.3 Disagree**

Quiz 70%; Roleplay 53%; Tests 13.3%; each among these: Debates 13.3%; Examinations 13%; each among these: Debates and Projects 13%; each among these: Principles of assessment, Assignments, and Examinations 10%; Presentations 7%; Simulations 3.3%; each among these: Problem-Solving and Align with Cognitive domain 3%; each among these: Experiments and Investigations 0%.

*Promotion of Science Learning through Science Content and Practical Assessment DOI: http://dx.doi.org/10.5772/intechopen.105407*

#### **Table 2.**

*Responses on how educators assess SL.*


3.3.2. Relative Absolute Error

The relative absolute error (RAE) is the ratio of the total absolute error produced by a model to the total absolute error of a simple predictor. In this case, the simple predictor is just the average of the target values. The RAE is thus computed as

$$RAE = \frac{\sum\_{i=1}^{n} |P\_i - A\_i|}{\sum\_{i=1}^{n} |\bar{A} - A\_i|} \times 100\% \tag{10}$$

where *Pi* is the predicted value by a model for an instance *i* out of a total number of *n* instances, *Ai* is the target value for the instance *i*, and *A*¯ is the mean of the target values given by

$$\bar{A} = \frac{1}{n} \sum\_{i=1}^{n} A\_i \tag{11}$$

One advantage of the RAE metric compared to the root mean square error (RMSE) described later is that it treats each error equally by ensuring that only the absolute value is considered and not the square of the error. Consequently, systems that are invariant to the effects of outliers can be best evaluated by the RAE instead of the RMSE.


*2.4. Animal Experiment*

The animal experiments were conducted in Beijing Animal Experimental Center. The Animal Ethics Committee of IAR, CAAS (Beijing, China) provided approval for the animal experimentation. The registration number is CAAS-IAR-ER0046. Female BALB/c mice (6-week-old, 16–20 g) were kept in cages, housed in a 12-h light–dark cycle at 20–24 ◦C and a relative humidity of 50 ± 5%, and supplied with filtered pathogen-free air, standard AIN-93 laboratory diet and sterile water. After acclimation for one week, all mice were randomly divided into five groups (*n* = seven per group), and named as CK, OVA, BP, 2E-BP and 3E-BP. The mice administration procedure is shown in Figure 1A. Briefly, mice in all groups were given a standard diet and water. For the CK group, mice were intraperitoneally injected with a saline solution every seven days for 28 days; but for the OVA group (as a positive control), mice were injected intraperitoneally with 0.2 mL of 0.1 mg/mL OVA solution (containing 1% Alum Adjuvant) every seven days for 21 days, and finally injected with 0.2 mL of 0.5 mg/mL OVA solution (containing 1% Alum Adjuvant) on the 28th day; for BP, 2E-BP and 3E-BP groups, mice were injected intraperitoneally with 0.2 mL of 0.5 mg/mL BP, 2E-BP and 3E-BP solution (containing 1% Alum Adjuvant) every seven days for 21 days, respectively, and finally injected with 0.2 mL of 2.5 mg/mL BP, 2E-BP and 3E-BP solution (containing 1% Alum Adjuvant) on the 28th day, respectively. The frequency of scratching behavior was observed and recorded 15 min after final injection. Mice were sacrificed after intraperitoneal injection with an excitation dose of samples for 30 min on the 28th day. The blood was extracted from eyes, and then centrifuged at 3000 rpm for 10 min for serum collection.

**Figure 1.** BALB/c mice administration schedule. (**A**) Scratching frequency of mice in different treatment groups. (**B**) Different letters indicate a significant difference among different groups (*p* < 0.05).


**5. Role of bioactive components**

#### **5.1. Hypertensive aspects**

fractions are centrifuged to separate the insoluble fiber fraction—called rice bran fiber—from the aqueous dispersible fraction—called rice bran soluble. The mixture of both insoluble and soluble extracts is called rice bran balance. Using different technologies, the bran is fully stabilized and the oil is removed. The resultant food‐grade, defatted rice bran is temporarily stored in food grade silos until it can be used in edible applications. Bleaching of the edible oil typically leaves minor flavor and odor compounds that must be removed by steam distillation before the oil is used. Steam distillation is the final step in the processing of edible oil, whereby

We produced two types of rice bran fraction: Driselase® fraction (DF) and ethanol fraction (EF). To process the rice bran, 500 g bran was agitated in 1.0 L of 70% ethanol for 2 h; this yielded two fractions: the solid and filtered fractions. The DF was derived from the solid fraction. Driselase® is a commercial plant cell wall‐degrading enzyme mixture containing cellulase, xylanase and laminarinase; however, it is esterase free. The solid fraction of rice bran was dried at room temperature and then suspended in 10 mM acetate buffer (500 mL) containing Driselase (0.2 mg/L) from *Basidiomycetes* spp. The bran was treated in this manner overnight at 37°C; the suspension was then filtered and finally lyophilized. As a result, Driselase‐treated rice bran had increased quantities of bioactive components that improve glucose and lipid

metabolism in the SHRSPs—a genetic animal model of metabolic syndrome [33, 34].

**4. Extraction, isolation and identification of the active components in rice**

Different conventional methods are used to extract the bioactive compounds from plant materials. Of these, solvent, supercritical fluid, microwave‐assisted and ultrasonic‐assisted extraction are notable. Microwave‐assisted extraction and ultrasound‐assisted methods are used to detect antioxidant and anticancer bioactive components in the plant extracts [35, 36]. Imsanguan et al. described conventional solvent extraction, with different modifications at different temperatures (32–60°C), using 100 ml hexane at a rotating speed of 200 rpm for 24 h to extract γ‐oryzanol from rice bran [37]. However, this conventional technique does not fully remove toxic solvent residues from the final product; for this reason, Herrero et al. used the prominent technique of supercritical fluid extraction, which offers better extraction and purification of bioactive compounds [38]. Zigoneanu et al. described antioxidant extraction from rice bran oil using microwave‐assisted extraction, which uses electromagnetic radiation

We developed the DF method to identify active components in the rice bran. As already described, the DF was derived from the solid fraction and chromatographed onto a silica gel column. One fraction derived from the methanol eluate was further fractionated using an octadecylsilane (ODS) column. The active fraction was obtained from the methanol/water (20– 70%) eluate and separated by high‐performance liquid chromatography (HPLC) using an ODS column. The BP‐lowering activity of each fraction was examined using a single oral adminis‐ tration to male, 14‐week old SHRSPs; we found that gavage of a certain fraction at 40 mg/kg

any off‐flavor and residual free fatty acids left in the oil are removed.

294 Superfood and Functional Food - An Overview of Their Processing and Utilization

**bran**

in the range of 0.3–300 GHz [39].

The following factors increase the risk of diet‐related disorders such as obesity, cancer and cardiovascular disease (CVD): consumption of fewer plant‐based foods, changing dietary patterns, increased consumption of Westernized food and socioeconomic conditions. High BP, one manifestation of CVD, continues to be a major cause of morbidity and death and one public health strategy is dietary management of high BP. Studies have shown that a 5‐mm Hg decrease in BP is related to a 16% decrease in CVD [45]. The risk factors for CVD are higher plasma cholesterol levels, lower high‐density lipoprotein (HDL) levels and higher low‐density lipoprotein (LDL) levels. Several bioactive compounds from rice bran that have been identified and used may reduce the risk of CVD. For instance, angiotensin‐converting enzyme (ACE) inhibitors reduce BP via the renin‐angiotensin system and FA has plasma ACE‐inhibitory activity. In this regard, Ardiansyah et al. showed that food supplementation using FA reduces high BP by inhibiting plasma ACE activity [5]. Later, the novel compound adenosine, isolated from the DF fraction, was also found to have BP‐lowering activity. Specifically, single‐dose or long‐term orally administered adenosine may reduce BP in the spontaneous hypertension of the SHRSP model [4]. Administered adenosine increases plasma nitric oxide levels, which in turn increase vasodilation. Adenosine also causes potent vasodilation by activating adenosine receptors (A2) on vascular smooth muscle; moreover, it stimulates K+ATP channels, resulting in the hyperpolarization of smooth muscle [4].

The risk of CVD is elevated in conditions of oxidative stress. Urinary 8‐hydroxydeoxyguano‐ sine (8‐OHdG) serves as a sensitive biomarker of oxidative stress resulting in genetic damage. FA significantly reduces urinary 8‐OHdG levels; thus, it can reduce CVD risk factors [4, 5].

Diets containing cholesterol‐lowering phytochemicals and antioxidants can prevent the progression of atherosclerotic lesions. Experiments have demonstrated that γ‐oryzanol possesses potent anti‐atherogenic and antioxidant activity. Furthermore, in a rat model of two‐ kidney, one‐clip renovascular hypertension, Boonla et al. described the vasorelaxant and antihypertensive effects of peptides derived from rice bran protein hydrolysates [46]. **Table 1** describes the anti‐hypertensive roles of various bioactive components of rice bran.

#### **5.2. Metabolic disorder aspects**

Metabolic disorders consist of metabolism‐related diseases, including hyperglycemia, hypercholesterolemia, hypertriglyceridemia and insulin resistance; they accompany type 2 diabetes mellitus, obesity and CVD. Rice bran and its various active components, prevents or ameliorates metabolic disorders. Specifically, a rice bran enzymatic extract‐supplemented diet can prevent the adipose and macrophage changes associated with diet‐induced obesity in mice [54]. In addition, the antihyperlipidemic effects (lower cholesterol and triglyceride levels) of α‐tocopherol have been investigated in F344 rats fed a Western diet [55]. Pigmented rice, which contains anthocyanins and proanthocyanidins concentrated in the bran layer, stimulates glucose uptake by 3T3‐L1 adipocytes—a key function in glucose homeostasis. Specifically, basal glucose uptake is increased two to three fold, while mRNA levels of both GLUT1 and GLUT4 are upregulated [56]. γ‐Oryzanol and FA ester with phytosterols—both of which are abundant in rice bran—prevent high‐fat and high‐fructose diet (HFFD)‐induced metabolic syndrome [57]. In addition, only γ‐oryzanol treatment is more effective than FA in significantly decreasing the liver index and hepatic triglyceride content. Decreased serum C‐reactive protein and IL‐6 levels and increased serum adiponectin concentration confirmed that FA and γ‐ oryzanol can be used as dietary supplements to alleviate the deleterious effects of HFFD [57]. Adenosine, in particular, effectively mitigates metabolic syndrome in SHRSP [50]. Specifically, single‐dose and long‐term oral administration of adenosine improves hyperlipidemia and hyperinsulinemia; it also regulates body weight gain and food intake. Studies have shown that enhanced plasma adiponectin levels alleviate hyperinsulinemia and that dietary adenosine can elevate plasma adiponectin and increase insulin sensitivity. Adenosine administration for 3 weeks downregulates mRNA levels of glucose‐6‐phosphatase, a gene encoding the rate‐ controlling enzyme of hepatic gluconeogenesis. Adenosine also plays an important role in regulating hepatic mRNA expression of genes involved in β‐oxidation, fatty acid synthesis and AMP‐activated protein kinase [4, 50]. In conclusion, various active components of rice bran ameliorate metabolic‐related diseases.

ACE, angiotensin‐converting enzyme; ApoB, apolipoprotein B; BW, body weight; FFA, non‐esterified fatty acid; HDL‐C, high density lipoprotein cholesterol; LDL‐C, low density lipoprotein cholesterol; PL, phospholipid; TC, total cholesterol; TG, triglycerides; VLDL‐C, very low density lipoprotein cholesterol; eNOS, endothelial nitric oxide synthase.

**Table 1.** Summary of bioactive components and their prospective effects on blood pressure and metabolic parameters in different species.

#### **5.3. Anti‐cancer aspects**

patterns, increased consumption of Westernized food and socioeconomic conditions. High BP, one manifestation of CVD, continues to be a major cause of morbidity and death and one public health strategy is dietary management of high BP. Studies have shown that a 5‐mm Hg decrease in BP is related to a 16% decrease in CVD [45]. The risk factors for CVD are higher plasma cholesterol levels, lower high‐density lipoprotein (HDL) levels and higher low‐density lipoprotein (LDL) levels. Several bioactive compounds from rice bran that have been identified and used may reduce the risk of CVD. For instance, angiotensin‐converting enzyme (ACE) inhibitors reduce BP via the renin‐angiotensin system and FA has plasma ACE‐inhibitory activity. In this regard, Ardiansyah et al. showed that food supplementation using FA reduces high BP by inhibiting plasma ACE activity [5]. Later, the novel compound adenosine, isolated from the DF fraction, was also found to have BP‐lowering activity. Specifically, single‐dose or long‐term orally administered adenosine may reduce BP in the spontaneous hypertension of the SHRSP model [4]. Administered adenosine increases plasma nitric oxide levels, which in turn increase vasodilation. Adenosine also causes potent vasodilation by activating adenosine receptors (A2) on vascular smooth muscle; moreover, it stimulates K+ATP channels, resulting

The risk of CVD is elevated in conditions of oxidative stress. Urinary 8‐hydroxydeoxyguano‐ sine (8‐OHdG) serves as a sensitive biomarker of oxidative stress resulting in genetic damage. FA significantly reduces urinary 8‐OHdG levels; thus, it can reduce CVD risk factors [4, 5].

Diets containing cholesterol‐lowering phytochemicals and antioxidants can prevent the progression of atherosclerotic lesions. Experiments have demonstrated that γ‐oryzanol possesses potent anti‐atherogenic and antioxidant activity. Furthermore, in a rat model of two‐ kidney, one‐clip renovascular hypertension, Boonla et al. described the vasorelaxant and antihypertensive effects of peptides derived from rice bran protein hydrolysates [46]. **Table 1**

Metabolic disorders consist of metabolism‐related diseases, including hyperglycemia, hypercholesterolemia, hypertriglyceridemia and insulin resistance; they accompany type 2 diabetes mellitus, obesity and CVD. Rice bran and its various active components, prevents or ameliorates metabolic disorders. Specifically, a rice bran enzymatic extract‐supplemented diet can prevent the adipose and macrophage changes associated with diet‐induced obesity in mice [54]. In addition, the antihyperlipidemic effects (lower cholesterol and triglyceride levels) of α‐tocopherol have been investigated in F344 rats fed a Western diet [55]. Pigmented rice, which contains anthocyanins and proanthocyanidins concentrated in the bran layer, stimulates glucose uptake by 3T3‐L1 adipocytes—a key function in glucose homeostasis. Specifically, basal glucose uptake is increased two to three fold, while mRNA levels of both GLUT1 and GLUT4 are upregulated [56]. γ‐Oryzanol and FA ester with phytosterols—both of which are abundant in rice bran—prevent high‐fat and high‐fructose diet (HFFD)‐induced metabolic syndrome [57]. In addition, only γ‐oryzanol treatment is more effective than FA in significantly decreasing the liver index and hepatic triglyceride content. Decreased serum C‐reactive protein and IL‐6 levels and increased serum adiponectin concentration confirmed that FA and γ‐

describes the anti‐hypertensive roles of various bioactive components of rice bran.

in the hyperpolarization of smooth muscle [4].

296 Superfood and Functional Food - An Overview of Their Processing and Utilization

**5.2. Metabolic disorder aspects**

Dietary factors have a significant effect on the risk of cancer. Only 5–10% of all cancer is heredity; all other incidents are directly or indirectly correlated with lifestyle and dietary habits. If dietary supplements are used appropriately, they may reduce the incidence of cancers in humans by as much as 30% [58, 59]. Phytic acid extracted from rice bran has anticancer activity against hepatocellular carcinoma (HepG2) cells, wherein apoptotic activity was evaluated by expression analysis of apoptosis‐regulatory genes (i.e., p53, Bcl‐2, Bax, Caspase‐ 3 and ‐9) [60]. Similarly, δ‐tocotrienol (δ‐T3) is reportedly useful as an anticancer agent against human colorectal adenocarcinoma (DLD‐1) cells under both normoxic and hypoxic conditions. *In vivo*, oral administration of rice bran tocotrienol (mainly γ‐T3; 10 mg/mouse/day) signifi‐ cantly inhibited tumor growth in nude mice [61]. Tumor cells produce reactive oxygen species, which damage cellular integrity. Cycloartenyl ferulate, a major component of γ‐oryzanol, successfully inhibits proliferation in the colorectal adenocarcinoma SW480 cell line because of its antioxidant activity [62].

#### **5.4. General health‐promoting aspects**

Rice bran itself has health benefits, while rice bran oil and isolated active components have immune stimulatory effects. Rice bran that is rich in phytosterols, γ‐oryzanol and compounds with antioxidant properties may modulate the immune system. In addition, rice bran has several generalized health‐promoting characteristics. For example, rice bran supplementation enhances gut health by encouraging the growth and colonization of *Lactobacillus rhamnosus* and it provides effective protection against human rotavirus diarrhea in pigs by modulating gut permeability [63].

Long‐term supplementation has a positive impact on survival, cognition and brain mitochon‐ drial function, which may delay Alzheimer's disease [64]. Rice bran supplements can also be used as ergogenic supplements by body builders and athletes [65] and they may mitigate menopausal symptoms such as hot flashes, as well as bone loss in older women who suffer from osteoporosis [66]. Rice bran can be regarded as a source of plant‐derived active com‐ pounds and as an alternative to expensive vitamin sources from animals. For instance, different colored rice bran has micronutrients, including a rich reserve of β‐carotene, which can be converted to vitamin A [67]. **Table 2** describes the role of various bioactive components of rice bran for general health.

**Table 2.** Summary of bioactive components and their prospective effect on generalized health aspects.


**2. Chemical constituents**

The main constituents of coffee are caffeine, tannin, fixed oil, carbohydrates, and proteins. It contains 2–3% caffeine, 3–5% tannins, 13% proteins, and 10–15% fixed oils. In the seeds, caffeine is present as a salt of chlorogenic acid (CGA). Also it contains oil and wax [2].

The following sections will be discussed in detail after acceptance of this short proposal:

• This article will deal on the types of carbohydrate, protein, lipids, and other chemical constituents in detail.

and play a vital role in the formation of the coffee flavor. The various constituents

Soluble carbohydrates Monosaccharides Fructose, glucose, galactose, arabinose (traces)

Insoluble polysaccharides

Polysaccharides Polymers of galactose, mannose, arabinose, glucose

Cellulose Acids and phenols Volatile acids

Lignin Lipids Wax

Proteins

Trigonelline Minerals

Hemicelluloses Polymers of galactose, arabinose, mannose

Nonvolatile aliphatic acids Citric acid, malic acid, quinic acid Chlorogenic acids Mono-, dicaffeoyl- and feruloylquinic acid

Oil Main fatty acids: N Compounds Free amino acids Main amino acids: Glu, Asp, Asp-NH2

Caffeine Traces of theobromine and theophylline

Most of the carbohydrates present, such as cellulose and polysaccharides

Linoleic acid is the predominant fatty acid, followed by palmitic acid.

The lipid fraction appears to be very stable, and its composition is given below.

The volatile acids include formic acids and acetic acids, while nonvolatile acids include lactic, tartaric, pyruvic, and citric acid. Minor constituents include higher fatty acids and malonic, succinic, glutaric, and malic acids. The degradation products of citric acid are itaconic (I), citraconic (II), and mesaconic acids (III), while

consisting of mannose, galactose, and arabinose, are insoluble.

fumaric and maleic acids are degraded products of malic acid:

along with components of coffee are shown in **Table 1**.

Oligosaccharides Sucrose, raffinose, stachyose

**Constituent Components**

*A Detail Chemistry of Coffee and Its Analysis DOI: http://dx.doi.org/10.5772/intechopen.91725*

**3. Carbohydrates**

*Constituents along with components of coffee.*

Lipid composition. Triacylglycerols. Diterpene esters. Diterpenes. Triterpene esters. Triterpenes (sterols). Unidentified compounds.

**4. Lipids**

**Table 1.**

**5. Acids**

**81**

• This article will review on various analytical methods for the estimation of constituents present in coffee.

Coffee is often used as antioxidants, but more importantly coffee is a good source of chromium and magnesium that assist in controlling blood sugar by ensuring proper usage of insulin.

The main chemical ingredients in coffee beans are given below:

The carbohydrate content of green and roasted coffee (Santos) was identified and measured. Green coffee contained about 6–7% of sucrose as soluble sugars and low amount of glucose. The soluble sugars of roasted coffee were sucrose, fructose, and glucose. The experiment was also carried out for the isolation of holocellulose fractions of green and roasted coffee.

The holocellulose of green coffee was hydrolyzed by a novel method consisting of anhydrous sulfuric acid and 10% potassium insoluble hydroxide, which was partially solubilized on roasting and results in the following ratio of sugars:

1 L-arabinose/2D-galactose/2D-glucose/6D-mannose. Out of these sugars, the arabinose was easily acid-hydrolyzed. Other coffee constituent analyzed and determined were caffeine, trigonelline, caffeic acid, chlorogenic acid, isochlorogenic acid, and the 10 amino acids. The free amino acids disappeared in roasting. An analytical method was developed for evaluating caffeine on chromatograms [3].

In coffee pulp, condensed tannins are the major phenolic compounds, while in the seeds, phenolic compounds exist primarily as a family of esters formed between hydroxycinnamic acids and quinic acid, collectively recognized as chlorogenic acids (CGA). Green coffee seeds contain up to 14% CGA, which are present in high concentrations and have a greater influence for determining the quality of coffee


**4. Conclusions**

In this study, the analysis of the possibility of using hydroponic cultivation for the removal of 15 pharmaceuticals and endocrine disrupting compounds in municipal wastewater treatment plants

is presented. For the first time, three plants: *Cyperus papyrus* (Papyrus), *Lysimachia nemorum* (Yellow pimpernel), and *Euonymus europaeus* (European spindle) were considered. In order to obtain the most reliable data, the investigation was performed using real MWWTP conditions and with the determination of target compounds not only in raw and treated wastewater, but also in plant materials. The determination of target compounds in raw and treated wastewater samples was performed using a previously proposed method [53]; however, for determining target compounds in plant materials, in this study, a new ASE-SPE-GC-MS(SIM) method was developed and validated. The application of a mixture of MeOH:H2O (1:1, *v*/*v*) at 50 ◦C without the acidification of the ASE extract prior to SPE purification was found to be the most optimal ASE-SPE conditions for the isolation of target compounds from plant material. The MQL values of the proposed method were in the range of 0.4 ng/g d.w. to 2 ng/g d.w.; the intermediate precision measurement was in the range of 0.05 to 21.60%; the mean recoveries in the range of 80% to 102%. Among the 15 investigated, 5 analytes were found in Papyrus (*C. Papyrus*); 5 target compounds in Yellow pimpernel (*L. nemorum*), and 4 in the tissue of the European spindle (*E. europaeus*) plant. The highest concentration of all target compounds was observed for *Lysimachia nemorum* (Yellow pimpernel), therefore, taking into account the summary uptake of target pharmaceuticals and ECDs by the tested plants, this species is the best for supporting conventional MWWTPs. The obtained data proved that the elimination efficiency of the investigated compounds from wastewater was in the range of 35.8% to 100%. Thus, the application of constructed wetlands for supporting conventional MWWTPs allowed a significant increase in their removal from the wastewater stream.

Establishing which plants effectively cumulate certain types of compounds is useful for designing effective constructed wetlands. Moreover, in the future, the proposed method for determining pharmaceuticals and EDCs in plant materials could be used for assessing the quality of food of plant origin in the cultivation of which sewage sludge or purified sewage was used.

**Supplementary Materials:** The following are available online. Table S1 Literature data concerning studies of the usefulness of hydroponically cultivated plants for removing target compounds from the sewage stream; Table S2 Chemical structures and physical and chemical properties of selected non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, β-blockers, β-agonists, antidepressant drugs, and estrogen-based hormones; Figure S1 The mass spectra of target compounds with the MS fragments assignation; Figure S2 Activated sludge chamber with a system of constructed wetlands in the investigated Municipal Wastewater Treatment Plant in Sochaczew (Mazowieckie Voivodeship, Poland); Figure S3 Example chromatogram with marked SIM ions for the determined target compounds in real Papyrus (*Cyperus papyrus*) samples; Figure S4 Example chromatogram with marked SIM ions for the determined target compounds in real Yellow pimpernel (*Lysimachia nemorum*) samples; Figure S5 Example chromatogram with marked SIM ions for the determined target compounds in real European spindle (*Euonymus europaeus*) samples.

**Author Contributions:** Conceptualization, D.W., M.C. and J.K.; Formal analysis, M.P.; Funding acquisition, D.W. and P.S.; Methodology, D.W. and M.C.; Project administration, J.K.; Resources, M.P.; Supervision, J.K.; Validation, D.W.; Writing—original draft, D.W.; Writing—review & editing, J.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** Financial support was provided by the Ministry of Science and Higher Education under grant nos. 539-8610-B334-18/19 and DS 531-8616-D593-19-1E.

**Acknowledgments:** The authors are also grateful to the Municipal Wastewater Treatment Plant (MWWTP) in Sochaczew (Mazowieckie Voivodeship, Poland) for very fruitful cooperation and the possibility to obtain the required samples, and to Agata Miecznikowska for her experimental support.

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


**2. Aim of the article**

The major goal of this article is to search, discuss and review the nongenomic rapid actions of THs in mature mammalian CNS. This article aims to begin with observations describing subcellular distribution, and concentrations of THs within the brain and its biochemical and physiologic consequences, specific binding of THs onto the neuronal plasma membrane to examine for specific plasma membrane receptors of THs and correlate the receptor-binding followed by a specific cellular function. Next, the molecular basis of the TH and plasma membrane receptor interaction-mediated signals are evaluated via possible activation of Gprotein signaling pathway, second messenger systems, and subsequent target protein phosphorylation.


**1. Introduction**

In EU directive 2003/30/EC biodiesel is defined as "methyl ester produced from vegetable or animal oil, of diesel quality, to be used as biofuel". The more recent EU directive 2009/28/EC has set the targets of achieving, by 2020, a 20% share of energy from renewable energy sources in the EU's overall energy consumption and a 10% share of energy from renewable sources in each member State's transport energy consumption. In this context special consideration is paid to the role played by the development of a sustainable and responsible biofuels production, with no impact on food chain.

Nowadays most biodiesel is produced through triglycerides transesterification of edible oils with methanol, in the presence of an alkaline catalyst (Lotero et al., 2005). The so obtained product has low viscosity and is a biofuel (fatty methyl ester) that can replace petroleum-based diesel fuel with no need of engine modifications (Suwannakarn et al., 2005). Furthermore, if compared to fossil fuel, the formed ester fuels are non-toxic, safe to handle, and biodegradable (Krawczyk, 1996). Glycerine is also obtained as by-product as shown in Fig. 1.

Fig. 1. Transesterification of a trygliceride.

C. Pirola1, D.C. Boffito1, A. Di Fronzo1, G. Carvoli1, D. Barnabè2, R. Bucchi2 and A. Rispoli2

*1 Università degli Studi di Milano – Dipartimento di Chimica Fisica ed Elettrochimica, Milano, Italy, 2 Agri2000 Soc. Coop., Bologna, Italy.*

Non Edible Oils: Raw Materials for Sustainable Biodiesel 5

The authors present here preliminary results of a three years study about the feasibility of using new oilseed species for biodiesel production in Italy1. The intent is to propose an innovative agronomic solution that may affect the energy balance and the ability to achieve

A considerable amount of studies are available on mainstream and alternative crops for biodiesel feedstock. The authors made a selection of the most promising crops to be introduced in the Mediterranean zone, taking into account that currently the Mediterranean basin comprises not only temperate climate but also slightly-arid lands. Some of these are being effectively tested under the mentioned project as part of a unique rotation program. Among oil crops the Brassicaceae family has an outstanding position. Rapeseed (*Brassica napus*) is the third largest oil crop with 12% of the world plant oil market with best yields when cultivated in cold-temperate regions (Carlsson, 2009). Yet, the large biodiversity of Brassicaceae reveal incipient species, among which *Brassica juncea*, *Brassica nigra*, *Brassica rapa*, *Brassica carinata*, *Sinapis alba*, *Camelina sativa*, *Eruca sativa* ssp*. oleifera*, etc. Besides the potential as raw material for biodiesel, their high content of glucosinolates (GSL) make them able to recover soils made marginal by soil-borne pests as nematodes (e.g. galling nematodes from the *Meloidogyne* genus and cist nematodes from *Heterodera* and *Globodera* genera) (Romero et al., 2009; Curto & Lazzeri, 2006). Many researchers also report weedsuppressive effects of Brassicaceae (e.g. Al-Khatib, 1997; Krishnan, 1998) as well as filtering-

On the other side an unexpected source of oilseed seems to arise from the tobacco culture. In anticipation of changes in tobacco market, selections of new varieties destined for energy production are coming out. Tobacco, as drought resistant species, seems a good option to

The recent interest in *B. carinata* (also known as Ethiopian or Abyssinian mustard) is mainly a result of its high resistance to biotic and abiotic stresses such as drought tolerance. *Brassica carinata,* is an annual crop noted to be highly resistant to many rapeseed pests: blackleg (*Leptosphaeria maculans),* white rust (*Albugo candida), Sclerotinia* sp. and *Phyllotreta cruciferae* (Pan, 2009). According to Razon (2009), *B.carinata*, together with *E. sativa* ssp. *oleifera*, is the most promising oilseed for biodiesel purpose in temperate zones, not just for the yield but also for its adaptability to hard pedo-climatic conditions. It may be used in a crop rotation system with cereals and on low nutrient soils. Best results are achieved sowing on autumn (IENICA, 2004). Harvesting may be done with same equipment used for rapeseed with the advantage that *B. carinata* shows a good resistance to the dehiscence of mature siliquae. The vegetable oil obtained from *B.carinata* is characterized by the presence of erucic acid, making it unsuitable for human consumption. On the other hand, its physico-chemical properties meet the European

1 SUSBIOFUEL project ("Studio di fattibilità per la produzione di biocarburanti da semi oleosi di nuove specie e da sottoprodotti o materiali di scarto" – D.M. 27800/7303/09), financially supported by the

Ministry of Agricultural, Food and Forestry Policies – Italy.

face the shift of some previously fertile into arid lands caused by climate change.

**2. Agronomical aspects**

**2.1.1** *Brassica carinata*

a high level of sustainability in the oilseeds production.

**2.1 Non edible oil crops in the Mediterranean basin**

buffering effects against heavy metals pollution (Palmer et al., 2001).

Refined, low acidity oilseeds (e.g. those derived from sunflower, soybean, rapeseed, tobacco etc.) may be easily converted into biodiesel, but their exploitation significantly raises the production costs, resulting in a biofuel that is not competitive with the petroleum-based diesel (Loreto et al., 2005). Presumably, as the market increases and technology is improved, costs will be driven down. In any case, the raw materials constitute a large portion of the manufacturing cost of biodiesel (up to 80%) (Bender, 1999).

Current oilseeds production systems raise environmental concerns because lands are intensively cultivated requiring high fertilizer and water inputs. These practices, aiming to increase yield, must be reduced or carefully regulated to prevent emissions of greenhouse gases or other environmental impacts. To do this, improved agronomic practices as the use of mixed species or crop rotation undoubtedly play a key role in mitigating negative impacts and enhancing biodiversity. A deep understanding of the microbial diversity of soils, its impacts on nutrient uptake and therefore on yield is crucial for sustainable cropping systems (The Royal Society, 2008).

Energy crops for industrial destination may represent a strategic opportunity in land use and income generation. However, in addition to the environmental aspects, economical concerns exist regarding the subtraction of lands for food cultivation. In a high market tension, it could have major impact on food/feed prices, increasing inequality, especially in developing countries. In addition, increased demand for food can result in the slowdown in biodiesel production due to reduced raw material availability. This was noticed in 2007 with industrial plants exploiting only 50% of their production capacity (Carvoli et al., 2008).

For all these reasons, it is highly desirable to produce biodiesel from crops specifically selected for their high productivity and characterized by low input requirements, or from low-cost feedstock such as waste cooking oil (WCO), animal fats and greases (Canakci et al., 2005; Zhang et. al, 2003).

While edible crops available for biodiesel production are restricted to few species (mainly palm/ soybean in the U.S. and palm/ rapeseed in the E.U.), the intent of using dedicated alternative feedstock opens a wide choice for new species that may be more suitable for specific conditions resulting on high yields.

The high WCO potential is recognized also by the EU directive 2009/28/EC, where waste vegetable or animal oil biodiesel is reported to save about the 88% of greenhouse emissions, a quite high value if compared to biodiesel from common vegetable oils, whose greenhouse emission savings range from 36 to 62%. The main issue posed by such a raw material is the need of its standardization, especially with regard to acidity decrease. Several methods have been proposed to solve this problem. Among them it is worth mentioning, besides the cited alkali refining method, addition of excess catalyst (Ono & Yoshiharu, 1979), extraction with a solvent (Rao et al., 2009), distillation refining process (Xingzhong et al., 2008) and preesterification method (Loreto et al., 2005; Pirola et al., 2010; Bianchi et. al, 2010; Parodi and Martini, 2008). This last seems to be the most attractive approach and has recently received much attention.

In the following paragraphs, the authors expose how it is possible to exploit waste materials or oils derived from crops not addressed to the food as potential raw materials for biodiesel production. Both the agronomic and chemical aspects deriving from the experimental work of the authors will be displayed.

#### **2. Agronomical aspects**

4 Biodiesel – Feedstocks and Processing Technologies

Refined, low acidity oilseeds (e.g. those derived from sunflower, soybean, rapeseed, tobacco etc.) may be easily converted into biodiesel, but their exploitation significantly raises the production costs, resulting in a biofuel that is not competitive with the petroleum-based diesel (Loreto et al., 2005). Presumably, as the market increases and technology is improved, costs will be driven down. In any case, the raw materials constitute a large portion of the

Current oilseeds production systems raise environmental concerns because lands are intensively cultivated requiring high fertilizer and water inputs. These practices, aiming to increase yield, must be reduced or carefully regulated to prevent emissions of greenhouse gases or other environmental impacts. To do this, improved agronomic practices as the use of mixed species or crop rotation undoubtedly play a key role in mitigating negative impacts and enhancing biodiversity. A deep understanding of the microbial diversity of soils, its impacts on nutrient uptake and therefore on yield is crucial for sustainable

Energy crops for industrial destination may represent a strategic opportunity in land use and income generation. However, in addition to the environmental aspects, economical concerns exist regarding the subtraction of lands for food cultivation. In a high market tension, it could have major impact on food/feed prices, increasing inequality, especially in developing countries. In addition, increased demand for food can result in the slowdown in biodiesel production due to reduced raw material availability. This was noticed in 2007 with industrial plants exploiting only 50% of their production capacity (Carvoli et

For all these reasons, it is highly desirable to produce biodiesel from crops specifically selected for their high productivity and characterized by low input requirements, or from low-cost feedstock such as waste cooking oil (WCO), animal fats and greases (Canakci et al.,

While edible crops available for biodiesel production are restricted to few species (mainly palm/ soybean in the U.S. and palm/ rapeseed in the E.U.), the intent of using dedicated alternative feedstock opens a wide choice for new species that may be more suitable for

The high WCO potential is recognized also by the EU directive 2009/28/EC, where waste vegetable or animal oil biodiesel is reported to save about the 88% of greenhouse emissions, a quite high value if compared to biodiesel from common vegetable oils, whose greenhouse emission savings range from 36 to 62%. The main issue posed by such a raw material is the need of its standardization, especially with regard to acidity decrease. Several methods have been proposed to solve this problem. Among them it is worth mentioning, besides the cited alkali refining method, addition of excess catalyst (Ono & Yoshiharu, 1979), extraction with a solvent (Rao et al., 2009), distillation refining process (Xingzhong et al., 2008) and preesterification method (Loreto et al., 2005; Pirola et al., 2010; Bianchi et. al, 2010; Parodi and Martini, 2008). This last seems to be the most attractive approach and has recently received

In the following paragraphs, the authors expose how it is possible to exploit waste materials or oils derived from crops not addressed to the food as potential raw materials for biodiesel production. Both the agronomic and chemical aspects deriving from the experimental work

manufacturing cost of biodiesel (up to 80%) (Bender, 1999).

cropping systems (The Royal Society, 2008).

specific conditions resulting on high yields.

al., 2008).

2005; Zhang et. al, 2003).

much attention.

of the authors will be displayed.

The authors present here preliminary results of a three years study about the feasibility of using new oilseed species for biodiesel production in Italy1. The intent is to propose an innovative agronomic solution that may affect the energy balance and the ability to achieve a high level of sustainability in the oilseeds production.

#### **2.1 Non edible oil crops in the Mediterranean basin**

A considerable amount of studies are available on mainstream and alternative crops for biodiesel feedstock. The authors made a selection of the most promising crops to be introduced in the Mediterranean zone, taking into account that currently the Mediterranean basin comprises not only temperate climate but also slightly-arid lands. Some of these are being effectively tested under the mentioned project as part of a unique rotation program.

Among oil crops the Brassicaceae family has an outstanding position. Rapeseed (*Brassica napus*) is the third largest oil crop with 12% of the world plant oil market with best yields when cultivated in cold-temperate regions (Carlsson, 2009). Yet, the large biodiversity of Brassicaceae reveal incipient species, among which *Brassica juncea*, *Brassica nigra*, *Brassica rapa*, *Brassica carinata*, *Sinapis alba*, *Camelina sativa*, *Eruca sativa* ssp*. oleifera*, etc. Besides the potential as raw material for biodiesel, their high content of glucosinolates (GSL) make them able to recover soils made marginal by soil-borne pests as nematodes (e.g. galling nematodes from the *Meloidogyne* genus and cist nematodes from *Heterodera* and *Globodera* genera) (Romero et al., 2009; Curto & Lazzeri, 2006). Many researchers also report weedsuppressive effects of Brassicaceae (e.g. Al-Khatib, 1997; Krishnan, 1998) as well as filteringbuffering effects against heavy metals pollution (Palmer et al., 2001).

On the other side an unexpected source of oilseed seems to arise from the tobacco culture. In anticipation of changes in tobacco market, selections of new varieties destined for energy production are coming out. Tobacco, as drought resistant species, seems a good option to face the shift of some previously fertile into arid lands caused by climate change.

#### **2.1.1** *Brassica carinata*

The recent interest in *B. carinata* (also known as Ethiopian or Abyssinian mustard) is mainly a result of its high resistance to biotic and abiotic stresses such as drought tolerance. *Brassica carinata,* is an annual crop noted to be highly resistant to many rapeseed pests: blackleg (*Leptosphaeria maculans),* white rust (*Albugo candida), Sclerotinia* sp. and *Phyllotreta cruciferae* (Pan, 2009). According to Razon (2009), *B.carinata*, together with *E. sativa* ssp. *oleifera*, is the most promising oilseed for biodiesel purpose in temperate zones, not just for the yield but also for its adaptability to hard pedo-climatic conditions. It may be used in a crop rotation system with cereals and on low nutrient soils. Best results are achieved sowing on autumn (IENICA, 2004). Harvesting may be done with same equipment used for rapeseed with the advantage that *B. carinata* shows a good resistance to the dehiscence of mature siliquae. The vegetable oil obtained from *B.carinata* is characterized by the presence of erucic acid, making it unsuitable for human consumption. On the other hand, its physico-chemical properties meet the European

1 SUSBIOFUEL project ("Studio di fattibilità per la produzione di biocarburanti da semi oleosi di nuove specie e da sottoprodotti o materiali di scarto" – D.M. 27800/7303/09), financially supported by the Ministry of Agricultural, Food and Forestry Policies – Italy.

Non Edible Oils: Raw Materials for Sustainable Biodiesel 7

*brasiliensis*) and physic nut (*Jatropha curcas*). Among non-edible oils, the one extracted from castor bean is the most used for a wide variety of industrial purposes. Its oil is primarily of economic interest having cosmetic, medical and chemical applications. The presence of a high proportion of ricin oleic acid makes it suitable for the production of high-quality lubricants (Sanzone & Sortino, 2010). The use of castor oil is particularly supported in Brazil, with attempts to extract the ethyl esters using ethanol from sugarcane fermentation (although less reactive than methanol), making it a complete natural and renewable product (Pinto et al., 2005). Albeit the actual productivity is not very high, between 600 and 1,000 kg seeds/ha year, this value could triplicate with genetic improvement (Holanda, 2004). With the recent report on the draft genome sequence of castor bean revealing some key genes involved in oil synthesis (Chan et al., 2010), this possibility becomes even more palpable. In addition to this, the ease with which it can be cultivated in unfavorable environments contributes to its appeal as a raw material for sustainable biodiesel. In agreement to this, a two years field experiment conducted in south Italy using local ecotypes yielded around 2.3 t/ha of seeds, with up to 38% oil content, a quite high number for the dry conditions of the region (Sanzone & Sortino, 2010). The main limitation is the hand harvest, the current practice in the biggest producer countries as India, Brazil and China. However mechanization of harvesting is recently available for the collection of dwarf hybrid plants

Among the species of interest for the production of biodiesel, the cardoon or artichoke thistle (*C. cardunculus*) is an important resource to be exploited, particularly in light of its adaptability to different soils. *Cinara cardunculus* is a perennial herbaceous species belonging to the family Asteraceae. Its deep root system allows the plant to extract water and nutrients from very deep soil zones revealing a plant with a small demand for fertilization and extremely resistant to drought. This characteristic makes it suitable to be grown on dry marginal or abandoned lands in the Mediterranean basin. Production reaches 30-35 t/ha per year, with about 2 tons of seeds; the seeds contain up to 25 % oil, with a similar composition to sunflower oil (Pasqualino, 2006). Recently, studies have been conducted within the EU project "Biocard - Global Process to Improve *C. cardunculus*". In the framework of this project, a research on the harvesting procedures, i.e. a crucial point of the cultivation of the thistle has also been conducted. As an example, a combine prototype designed to separate and thresh the capitula and to drop the biomass proved to be feasible, with a good

The rationale of this proposal consists in the use of non-edible crops on soils no longer suitable for food production due to infestation by nematodes. The authors tested the possibility to rescue marginal soil fertility in consequence of the cultivation and the green manure of a naturally biocidal crop (*B. juncea* and *B. carinata*). Thanks to this practice the soil could be quickly good enough to produce oilseeds with satisfying yields for industrial destination. Furthermore a reduction in inputs of fertilizers is also expected due to preservation of organic matter content of soil. This practice offers the possibility to rescue soils availability for food production. Indeed, after some cycles of this rotation, the pest

(Clixoo, 2010).

**2.1.5** *Cynara cardunculus*

cost/working capacity relation (Pari et al., 2008).

**2.2 A new proposal for biodiesel production**

specifications defined for biodiesel destination by the normative EN 14214:2002. Beyond its oil production capabilities, it was pointed out that the *B. carinata*'s lignocellulosic biomass can also be used to generate power and especially heat (Gasola et al., 2007), revealing an even greater potential.

#### **2.1.2** *Brassica juncea*

*Brassica juncea* (also known as wild mustard or Indian mustard) varieties are grown for edible leaves or for condiment mustard only in some countries, while its use as an oilseed crop is increasingly growing. Canadian plant breeders have developed *B. juncea* cultivars with canola characteristics (Potts et al., 1999). As a result, canola varieties of *B. napus* and canola-type *B. juncea* have similar compositional characteristics. The key differences between *B. napus* and canola-type *B. juncea* lie in their agronomic characteristics. *Brassica juncea* tolerates high temperatures and drought better than *B. napus*, and thus it is better suited for the warmer, drier climates as the Upper Plains of the U.S. or the Mediterranean area. Green manure of *B.juncea* is a current practice in some countries (e.g. Italy and U.S.) making use of the GSL-Myrosinase system as a natural biofumigant. At the same time, this practice supplies organic matter to soil. To make the most of its biocidal activity against soilborne pests and diseases, the mulching and incorporation to soil must be done at flowering time (Curto & Lazzeri, 2006).

#### **2.1.3** *Nicotiana tabacum*

The tobacco (*N. tabacum*) is an annual herbaceous plant belonging to the Solanaceae family, widespread in North and South America, commonly grown for the collection of leaves. The seeds are very small (up to 10,000/g) and contain 36 to 39% of oil having a high percentage of linoleic acid (Giannelos et al., 2002). Currently, the common varieties directed to leaf production reach the modest order of 1 to 1.2 t seeds/ha (Patel, 1998, as cited in Usta, 2005) as a result of selection to reduce the amount of seed produced. Recently researchers were able to over express, through genetic engineering, genes responsible for the oil production in the leaves (Andrianov et al., 2010). However, the seeds potential for oil production is much higher. In this sense, another recent outcome on tobacco improvement is a variety that can at least triple seed (up to 5 t/ha) and oil production. The energy tobacco varieties exist both in the non GMO and the GMO version for resistance factors against herbicides and insects (Fogher, 2008). Its high oil yield makes it very competitive in front of mainstream oil crops as rapeseed, sunflower and soybean. The remaining meal revealed to be relevant for combustion or to be used as a protein source for livestock. Tests with pigs demonstrated its palatability to animals, a good conversion rate and therefore its equivalence to the soybean meal (Fogher, 2002). In addition, the presence of consolidate agricultural practices and know-how make clear the advantage of using a well-known species as tobacco as alternative feedstock for biodiesel. The research on Energy Tobacco has also found new economies for the transplant management as well as direct sowing techniques are currently under test. Combineharvesters for the harvest of the whole inflorescences are available.

#### **2.1.4** *Ricinus communis*

*Ricinus communis* (castor bean) is an oilseed crop that belongs to the Euphorbiaceae family, which includes other energy crops as cassava (*Manihot esculenta*), rubber tree (*Hevea*

specifications defined for biodiesel destination by the normative EN 14214:2002. Beyond its oil production capabilities, it was pointed out that the *B. carinata*'s lignocellulosic biomass can also be used to generate power and especially heat (Gasola et al., 2007),

*Brassica juncea* (also known as wild mustard or Indian mustard) varieties are grown for edible leaves or for condiment mustard only in some countries, while its use as an oilseed crop is increasingly growing. Canadian plant breeders have developed *B. juncea* cultivars with canola characteristics (Potts et al., 1999). As a result, canola varieties of *B. napus* and canola-type *B. juncea* have similar compositional characteristics. The key differences between *B. napus* and canola-type *B. juncea* lie in their agronomic characteristics. *Brassica juncea* tolerates high temperatures and drought better than *B. napus*, and thus it is better suited for the warmer, drier climates as the Upper Plains of the U.S. or the Mediterranean area. Green manure of *B.juncea* is a current practice in some countries (e.g. Italy and U.S.) making use of the GSL-Myrosinase system as a natural biofumigant. At the same time, this practice supplies organic matter to soil. To make the most of its biocidal activity against soilborne pests and diseases, the mulching and incorporation to soil must be done at flowering

The tobacco (*N. tabacum*) is an annual herbaceous plant belonging to the Solanaceae family, widespread in North and South America, commonly grown for the collection of leaves. The seeds are very small (up to 10,000/g) and contain 36 to 39% of oil having a high percentage of linoleic acid (Giannelos et al., 2002). Currently, the common varieties directed to leaf production reach the modest order of 1 to 1.2 t seeds/ha (Patel, 1998, as cited in Usta, 2005) as a result of selection to reduce the amount of seed produced. Recently researchers were able to over express, through genetic engineering, genes responsible for the oil production in the leaves (Andrianov et al., 2010). However, the seeds potential for oil production is much higher. In this sense, another recent outcome on tobacco improvement is a variety that can at least triple seed (up to 5 t/ha) and oil production. The energy tobacco varieties exist both in the non GMO and the GMO version for resistance factors against herbicides and insects (Fogher, 2008). Its high oil yield makes it very competitive in front of mainstream oil crops as rapeseed, sunflower and soybean. The remaining meal revealed to be relevant for combustion or to be used as a protein source for livestock. Tests with pigs demonstrated its palatability to animals, a good conversion rate and therefore its equivalence to the soybean meal (Fogher, 2002). In addition, the presence of consolidate agricultural practices and know-how make clear the advantage of using a well-known species as tobacco as alternative feedstock for biodiesel. The research on Energy Tobacco has also found new economies for the transplant management as well as direct sowing techniques are currently under test. Combine-

*Ricinus communis* (castor bean) is an oilseed crop that belongs to the Euphorbiaceae family, which includes other energy crops as cassava (*Manihot esculenta*), rubber tree (*Hevea*

harvesters for the harvest of the whole inflorescences are available.

revealing an even greater potential.

**2.1.2** *Brassica juncea*

time (Curto & Lazzeri, 2006).

**2.1.3** *Nicotiana tabacum*

**2.1.4** *Ricinus communis*

*brasiliensis*) and physic nut (*Jatropha curcas*). Among non-edible oils, the one extracted from castor bean is the most used for a wide variety of industrial purposes. Its oil is primarily of economic interest having cosmetic, medical and chemical applications. The presence of a high proportion of ricin oleic acid makes it suitable for the production of high-quality lubricants (Sanzone & Sortino, 2010). The use of castor oil is particularly supported in Brazil, with attempts to extract the ethyl esters using ethanol from sugarcane fermentation (although less reactive than methanol), making it a complete natural and renewable product (Pinto et al., 2005). Albeit the actual productivity is not very high, between 600 and 1,000 kg seeds/ha year, this value could triplicate with genetic improvement (Holanda, 2004). With the recent report on the draft genome sequence of castor bean revealing some key genes involved in oil synthesis (Chan et al., 2010), this possibility becomes even more palpable. In addition to this, the ease with which it can be cultivated in unfavorable environments contributes to its appeal as a raw material for sustainable biodiesel. In agreement to this, a two years field experiment conducted in south Italy using local ecotypes yielded around 2.3 t/ha of seeds, with up to 38% oil content, a quite high number for the dry conditions of the region (Sanzone & Sortino, 2010). The main limitation is the hand harvest, the current practice in the biggest producer countries as India, Brazil and China. However mechanization of harvesting is recently available for the collection of dwarf hybrid plants (Clixoo, 2010).

#### **2.1.5** *Cynara cardunculus*

Among the species of interest for the production of biodiesel, the cardoon or artichoke thistle (*C. cardunculus*) is an important resource to be exploited, particularly in light of its adaptability to different soils. *Cinara cardunculus* is a perennial herbaceous species belonging to the family Asteraceae. Its deep root system allows the plant to extract water and nutrients from very deep soil zones revealing a plant with a small demand for fertilization and extremely resistant to drought. This characteristic makes it suitable to be grown on dry marginal or abandoned lands in the Mediterranean basin. Production reaches 30-35 t/ha per year, with about 2 tons of seeds; the seeds contain up to 25 % oil, with a similar composition to sunflower oil (Pasqualino, 2006). Recently, studies have been conducted within the EU project "Biocard - Global Process to Improve *C. cardunculus*". In the framework of this project, a research on the harvesting procedures, i.e. a crucial point of the cultivation of the thistle has also been conducted. As an example, a combine prototype designed to separate and thresh the capitula and to drop the biomass proved to be feasible, with a good cost/working capacity relation (Pari et al., 2008).

#### **2.2 A new proposal for biodiesel production**

The rationale of this proposal consists in the use of non-edible crops on soils no longer suitable for food production due to infestation by nematodes. The authors tested the possibility to rescue marginal soil fertility in consequence of the cultivation and the green manure of a naturally biocidal crop (*B. juncea* and *B. carinata*). Thanks to this practice the soil could be quickly good enough to produce oilseeds with satisfying yields for industrial destination. Furthermore a reduction in inputs of fertilizers is also expected due to preservation of organic matter content of soil. This practice offers the possibility to rescue soils availability for food production. Indeed, after some cycles of this rotation, the pest

Non Edible Oils: Raw Materials for Sustainable Biodiesel 9

In table 2 the mean percentage increasing of biomass of *B. napus*, *H. annus*, *G. max*, and

*N. tabacum* % 21 *B. napus* % 15 *H. annus* % 26 *G. max* % 28 Table 2. Increasing of biomass of oilseed crops produced after green manuring of B. *juncea.*

**3. Chemical aspects: Standardization of the raw materials and biodiesel**

parameters for starting oil that can affect the quality of the final biodiesel.

Oil characterization before proceeding with the standardization of the raw material is a very important issue. Some properties remain in fact unchanged from the starting material to the finished biodiesel, or they are anyway predetermined. It is so important to check that the values of such chemical and physical oil properties are in range with those required by the standard regulations (see Table 3). The experimental procedures to get the values of such properties are also standardized and are indicated in the regulations. The following are

High sulphur and phosphorous content in the fuels cause greater engine wear and in particular shorten the life of the catalyst. Biodiesel derived from soybean, rapeseed, sunflower and tobacco oils are known to contain virtually no sulphur (Radich, 2004;

The authors have nevertheless found that the oil obtained from *B.juncea* seeds may contain high concentrations of sulphur due to the presence in the plant's tissues of glucosinolates,

Soybean, sunflower, peanut and rapeseed oils contain a high proportion of linoleic fatty acids, so affecting the properties of the derived ester with a low melting point and cetane number. Quantitative determination of linoleic acid methyl ester is accomplished by gas chromatography with the use of an internal standard after the substrate has been transesterificated and allows also the quantification of the other acid methyl esters (Environment Australia, 2003). The super-critical chromatography is another useful

**Crop Unit Biomass increasing**

**Element Unit Value Standard Test Method**  sulphur mg/kg 112 UNI EN 20846:2005 nitrogen % (mass) 0,35 ASTM D5291-09 phosphorus mg/kg < 4 UNI EN 14107:2003

Table 1. Nitrogen, sulphur and phosphor content in *B. juncea* oil.

**production**

**3.1 Oil characterization**

Zhiyuan et al., 2008).

**Sulfur and phosphorous content:**

the molecules responsible for the biofumigation effect. **Linoleic acid methyl ester, iodine value and viscosity**

analytical technique, suitable for the direct analysis of the oils.

*N*. *tabacum* produced after green manuring of *B. juncea* is summarized.

control and the progressive increase of organic matter should make the soil eligible again for quality productions.


**3.1 Effects of climate change on indices and climate zonification**

### *3.1.1 Uribia, La Guajira*

This study was carried out in Uribia, located in La Guajira, which corresponds to a desert area according to the Lang's Index (LI) and with little or no surplus water according to the Thornthwaite climate classification. It is an approximation of what may happen to the abovementioned classifications under climate change scenarios.

As shown in **Table 2**, this study considered two models and two scenarios. The HadGem2-ES model was the optimistic scenario for the RCP 4.5 scenario in 2050 and the GDFL-CM3 model was the pessimistic scenario for the RCP 8.5 scenario in 2070.

A hydrological balance was made both for the baseline (1976–2050) and for the previously mentioned scenarios, based on the meteorological data measured at the Nazareth station and its corresponding future change scenarios due to the influence of climate change. **Table 3** shows the annual average values for each variable considered in the water balance.

The climate change models were reviewed, obtaining an increase of 1.7°C and a 2.4% decrease in precipitation for the optimistic scenario, as well as an increase of 3.7°C and an 11% decrease in precipitation for the pessimistic scenario. This will have a direct effect on the provisional crops, as soil moisture will be reduced.

As an example, **Table 4** summarizes the changes in the variables resulting from the water balance and the implications that these changes may have on the water requirements of the corn, bean, and melon crops in the study area.

**Water layer**

**77**

Def (mm/year) Pcp (mm/year) ETo (mm/year)

ETa (mm/year)

Water Water Water *The bold values highlight the percentage change.*

**Table 4.** *Summary of water balance and*

*requirements*

 *for the different scenarios and periods [14].*

requirements

 (mm/year)

—Melon

 223.9

 403.8

 179.9

requirements

 (mm/year)

—Bean

 195.9

 363.1

 167.2

requirements

 (mm/year)

—Corn

 257.5

 426.9

 169.4

**Baseline**

**Value Difference**

1060.6

510.2

1570.8

510.2

 446.2

 2227.7

 656.9

64

**12.5**

**65.8** **85.3** **80.3**

394.1

 170.2

356.6

 160.7

426.2

 168.7

445.2

65

**12.7**

**65.5** **82.0** **76.0**

412.5

 188.6

370.2

 174.3

433.7

 176.2

446.2

64

**12.5**

*Effects of Climate Change on Water Resources, Indices, and Related Activities in Colombia*

**68.4**

**89.0**

**84.2**

 446.2

 1781.5

 720.9 64.0

**12.5**

**41.8**

2261.3

 690.5

445.2

65

**12.7**

**44**

2317.0

 746.1

446.2

64.0

**12.5**

**47.5**

**68.0**

1816.1

 755.5

**71.2**

1870.8

 810.1

**76.4**

 **(mm) Change (%) Value Difference**

**GDFL-ESM2G**

 **(GD) 4.52050**

**GDFL-ESM2G**

 **(HD) 6.02070**

 **(mm) Change (%) Value Difference**

**GDFL-ESM2G**

 **(HE) 8.52070**

*DOI: http://dx.doi.org/10.5772/intechopen.90652*

 **(mm) Change (%)**

**Table 3.**

*Meteorological variables of Nazareth station [6].*

## *Effects of Climate Change on Water Resources, Indices, and Related Activities in Colombia DOI: http://dx.doi.org/10.5772/intechopen.90652*

document. Given its broad temporal spectrum, **Table 2** shows the different scenar-

The studies are located in different areas of the country, with different characteristics. The map illustrated in **Figure 1** shows their exact location

**3. Effects of climate change on water resources, indices, and related**

**3.1 Effects of climate change on indices and climate zonification**

This section introduces the studies carried out in different areas of the country, using different methodologies, which are explained in depth in each of the investigations. Additionally, it sets out the possible effects of climate change on the water

This study was carried out in Uribia, located in La Guajira, which corresponds to a desert area according to the Lang's Index (LI) and with little or no surplus water according to the Thornthwaite climate classification. It is an approximation of what may happen to the abovementioned classifications under climate change scenarios. As shown in **Table 2**, this study considered two models and two scenarios. The HadGem2-ES model was the optimistic scenario for the RCP 4.5 scenario in 2050 and the GDFL-CM3 model was the pessimistic scenario for the RCP 8.5 scenario

A hydrological balance was made both for the baseline (1976–2050) and for the previously mentioned scenarios, based on the meteorological data measured at the Nazareth station and its corresponding future change scenarios due to the influence of climate change. **Table 3** shows the annual average values for each variable

The climate change models were reviewed, obtaining an increase of 1.7°C and a 2.4% decrease in precipitation for the optimistic scenario, as well as an increase of 3.7°C and an 11% decrease in precipitation for the pessimistic scenario. This will have a direct effect on the provisional crops, as soil moisture will be reduced.

As an example, **Table 4** summarizes the changes in the variables resulting from the water balance and the implications that these changes may have on the water

**Variable Baseline value** Maximum temperature (°C) 32.5 Average temperature (°C) 27.3 Minimum temperature (°C) 22.0 Precipitation (mm) 510.2 Evaporation (mm) 2044.5 Evapotranspiration (mm) 1521.1

requirements of the corn, bean, and melon crops in the study area.

ios over different periods of time.

**activities in Colombia**

resources in each location.

*3.1.1 Uribia, La Guajira*

considered in the water balance.

*Meteorological variables of Nazareth station [6].*

in 2070.

**Table 3.**

**76**

within Colombia.

*Resources of Water*

Once the water balance has been carried out with the new weather conditions, it is clear that none of the classifications mentioned at the beginning of this section have changed (Lang and Thornthwaite). However, the conditions of such classification are exacerbated since there is a reduction in the Moisture Index (Im), which may have a negative implication, both ecologically and socially, due to the lower availability and access to drinking water.

This suggests that there is a growing need to investigate this area in order to develop an adequate plan that may include water harvesting projects or efficient crop irrigation systems that take into account the future demands and projected precipitation deficits, to serve as a climate change adaptation strategy.


**3.2 Concept for the adaptive playout of CQs with CQPs**

As shown in the use cases in [50], a CQP is a profile that is assigned to an individual learner. CQPs describe each student's personal learning goals (LG) (target profile) and the current state of attested CQs (actual profile) [50].

As shown in **Figure 2**, the LMS Moodle is required. The architecture of Moodle consists of six components [15], which are the core, the subsystems, the plugins, the