Biodiesel Transesterification

## **Chapter 2**

## Direct Transesterification: From Seeds to Biodiesel in One-Step Using Homogeneous and Heterogeneous Catalyst

*Issis Claudette Romero-Ibarra, Araceli Martínez Ponce Escuela, Gabriela Elizabeth Mijangos Zúñiga and Wendy Eridani Medina Muñoz*

## **Abstract**

Biodiesel is a renewable alternative biofuel and is an option to diversify the conventional fossil fuels. Moreover, biodiesel is nontoxic, biodegradable, and biomass renewable diesel fuel and its combustion produces low amount of CO, CO2, hydrocarbon, and particulate matter. It can be produced through transesterification reaction. The most common method is homogeneous transesterification process using basic catalyst as NaOH. However, this route has drawbacks as long timespans, saponification reaction, a large amount of solvent, and a large amount of water to neutralize the methyl esters to eliminate the catalyst. This chapter presents the direct transesterification as a green and sustainable alternative method to improve the benefits of conventional transesterification. The direct transesterification is a one-step process to obtain biodiesel from seed crops in presence of a catalyst. *Jatropha curcas* L. and *Ricinus communis* have been evaluated as non-edible seeds feedstocks. Also, various acid and basic homogeneous and heterogeneous catalysts have been investigated. Results shown that heterogeneous direct transesterification yields ~99% with 5 wt% catalyst in 4 h without n-hexane for oil extraction or water for purify the biodiesel. Heterogeneous direct transesterification is a promising method of obtaining biodiesel as methanol acts as a reactant and as a solvent.

**Keywords:** direct transesterification, homogeneous catalyst, heterogeneous catalyst, biodiesel, *Jatropha curcas* seeds, *Ricinus comunis* seeds

## **1. Introduction**

The production of biofuels from renewable biomass resources is an attractive way to mitigate CO2 emissions and alleviate the shortage of fossil fuels. Biofuels have

emerged as one of the most promising renewable energy sources, offering one alternative to substitute for petroleum-based diesel fuel. Biomass offers an alternative solution, low-cost solution to a drop-in transportation fuel for blending with conventional diesel. The most common biofuels are based on vegetable oil for fatty acid methyl esters (FAMEs) biodiesel. However, the use of edible oils for bio-based fuels production may create controversy. The biodiesel production from edible oil crops has been questioned in several fields, and their negative effects by its high cost and their destruction of soil cultivation are discussed. Recently, agro-industrial waste and sustainably managed non-food feedstocks have been considered as a new generation of fatty acid methyl esters (FAMEs) or advanced biodiesel. For example, non-edible oilseed crops such as *Ricinus communis* oil and *J. curcas L*.. *R. communis* are a new option for biodiesel production because they are not edible, due to their high oil content, easy propagation, resistance, rapid growth, and adoption in wide agroclimatic conditions [1–7], and it provides commercially viable alternative to edible oils. These oils are not suitable for human consumption [8], and therefore, its production does not interfere with the food industry or harvesting lands.

Biodiesel from renewable biomass resources is an attractive way to mitigate CO2 emissions and alleviate the shortage of fossil fuels [4–6, 9, 10]. Biodiesel can be produced *via* esterification or transesterification process. Triglycerides (TG) from vegetable oils and animal fats react with short-chain alcohols and acid or base catalysts to render fatty acid methyl esters (FAMEs) and glycerol as by-product [9, 10]. Every triglyceride molecule reacts with three equivalents of alcohol to produce glycerol and three fatty acid (methyl) ester molecules. Methanol is the most common alcohol due to its low price, high activity, and green chemistry metrics. Nowadays, the biodiesel is produced industrially by homogeneous transesterification due to the high yields, lower cost, and the short reaction time. The most common homogeneous catalysts used are NaOH and KOH as basic catalyst and H2SO4 as an acid catalyst. However, homogeneous acid and base catalysts present many disadvantages such as long timespans, a large amount of solvent, reactors and engine manifolds corrosion, the difficulty of phase separation, their removal from the resulting biodiesel from the reaction, the neutralization and purification of the biofuel at the end of the reaction, and the excessive use of water to removal emulsions and soaps for these processes. The base catalysts cause saponification, a competition reaction, and decrease the yield of the biodiesel obtained. The acid catalysts increase the biodiesel yield, but stable conditions are needed, or the reaction is affected and slows down the transesterification. As by-product, glycerol has significant value to the industry. However, the recovered glycerol is normally impure due to the presence of salts, soaps, monoglycerides, and diglycerides, and the glycerol purification process consequently adds an additional cost [11, 12].

On the other hand, the use of heterogeneous catalysts represents another strategy to obtain biofuels. These catalysts seek to make the production of biodiesel greener, generating less aggressive residues to the environment and reducing manufacturing costs. In transesterification reaction with heterogeneous catalyst, the reagents and the catalyst are in different phases. Usually, the catalyst is solid; meanwhile, the reagents are in liquid phase. The use of heterogeneous catalysts would result in simpler, cheaper separation processes, a reduced water effluent load as well as lower energy consumption and cleaner operating conditions [9].

Furthermore, catalyst would not have to be continuously added and would be easier to reuse. In general, the saponification reaction is not common with heterogeneous catalyst, and the purity of the products increases because the catalyst is more

#### *Direct Transesterification: From Seeds to Biodiesel in One-Step Using Homogeneous… DOI: http://dx.doi.org/10.5772/intechopen.108234*

selective. There would be no neutralization products, enhancing the purity of the glycerol by-product [11, 13]. Several solid base and acid catalysts are explored in transesterification reaction because they offer enhanced process, avoid the quenching steps, and allow continuous operation. Solid acid as heterogeneous catalyst in transesterification reaction of oils into biodiesel renders lower activity, also it is necessary for higher reaction temperatures, and they are lower in comparison with base catalyst [14]. In contrast to solid bases catalysts, solid acids can esterify free fatty acids (FFAs) though to FAME. Some examples of acid heterogeneous catalysts are ZrO2, TiO2, SnO2, zeolites, etc., whereas the basic heterogeneous catalysts most commonly used are CaO, MgO, SrO. The yields obtained with these basic catalysts were 97%, 92%, and 98%, respectively [2, 3, 15, 16]. Some of these catalysts are used to obtain biodiesel by means of used cooking oil, algae, and different oils such as palm oil [13, 15–19]. CaO/TiO2 using canola oil results in a yield of 96.9% [18]. Another alternative is the use of biomass, for example, papaya seed ash as a catalyst, as it contains metal oxides such as K2O, MgO, and CaO having a yield of 95.6% [19]. Besides, it was shown that sodium zirconate (Na2ZrO3) exhibited interesting catalytic properties as a basic heterogeneous catalyst to produce biodiesel *via* a soybean oil transesterification reaction, which led to good purity [20–22]. Furthermore, the alteration of the chemical composition as a possible increment of active basic sites, incorporating cesium to the Na2ZrO3 catalyst by a simple impregnation method, improves the yield of reaction due to its basic character. The catalytic system Cs-sodium zirconate (cesium-impregnated sodium zirconate) was considered as modified in transesterification reaction of soybean oil and jatropha oil for biodiesel production [23]. Results showed outstanding FAME conversion, from soybean oil, of 98.8% using 1 wt% of catalyst in just 15 min. These catalysts can operate at low concentrations and in short reaction times due to its high basicity and low solubility [20, 23]. Recently, other ceramic materials such as sodium silicate (Na2SiO3) [24] and sodium zincsilicate (Na2ZnSiO4) [25] have been evaluated with high yields to produce biodiesel 99% and 98%, respectively.

However, these conventional processes consist of two steps: first, the oil extraction and then transesterification of triglycerides in the presence of the catalyst. Both steps generate drawbacks that promote a more expensive biodiesel production. Therefore, for both environmental and economic reasons, there is increasing interest in new alternatives to produce biofuels. It will face new challenges as a technology to overcome the feasible routes to biofuels. Hence, green chemistry presents a new synthesis route to obtain products from conventional or traditional methods favoring the sustainable process, avoiding hazardous and toxic substances (e.g., hexane or dichloromethane) to extract vegetable oils, and favoring the energy efficiency. As a new alternative, the direct transesterification represents an innovative and *in situ* route to obtain biodiesel from non-edible oils-seed in one step in the presence of homogeneous and heterogeneous catalysts [21, 22].

## **2. Direct transesterification of seeds**

It is well known that biodiesel is conventionally produced by transesterification of vegetable oils using an alcohol and homogeneous or heterogeneous catalysts. The conventional transesterification of oils is based on two steps: 1) the oil extraction and then 2) the transesterification of triglycerides (**Figure 1**) [15, 16, 26, 27]. In the first step, there are several basic methods for obtaining oils, such as chemical extraction, supercritical fluid extraction, steam distillation, mechanical extraction. The second

**Figure 1.** *Conventional and direct transesterification of seed oil.*

step is the transesterification of triglycerides from homogeneous or heterogeneous catalysts. Both catalysts could be acid or basic. For example, biodiesel production through the conventional transesterification from soybean, sunflower, and *Cynara cardunculus* oils (edible oils) and Jatropha and *R. communis* oil (non-edible oils), with methanol or ethanol using acidic (HCl, H2SO4) and basic (NaOH, KOH) homogeneous catalysts, has been reported [5, 26–28].

An alternative to the conventional method is the direct transesterification (**Figure 1**). **Figure 1** shows the comparison between conventional (two steps) and direct transesterification (one step). Direct transesterification is a novel green one-pot synthesis to produce biodiesel from non-edible seeds using homogeneous or heterogeneous catalysis. In this *in situ* one-step process, the extraction and transesterification take place simultaneously in one vessel. The one-step process is more economical and efficient than conventional method [21, 22]. Recently, a few studies have demonstrated that biodiesel production can be achieved from the direct transesterification of seed oils or microalgae biomass with homogeneous catalysts [28–34]. Although both alkaline and acid homogeneous catalysts were suitable for the reaction, most studies use a basic catalyst (sodium hydroxide, potassium hydroxide, or sodium methoxide) due to reduced corrosiveness, lower amount of catalyst, and reaction time. Also, biodiesel production through the homogeneous direct transesterification from edible oils such as soybean, sunflower, and *Cynara cardunculus* seeds, and non-edible oils such as *Jatropha curcas L* and *Ricinus communis* seeds, with methanol or ethanol, has been reported [1, 5, 28, 31]. FAME yields are very high and comparable with conventional homogeneous as well as heterogeneous catalysis (99%).

*Direct Transesterification: From Seeds to Biodiesel in One-Step Using Homogeneous… DOI: http://dx.doi.org/10.5772/intechopen.108234*

Recently, heterogeneous direct transesterification represents a new alternative in biodiesel production; due to these, solid catalysts have many advantages over liquid catalyst used in homogeneous direct transesterification. The heterogeneous catalyst exhibits easy separation, lower energy consumption, and cleaner operation. Using solid catalyst could be eliminated the contaminated waste, the formation of soaps, and the emulsification of products that are generated in the homogeneous acid and basic transesterification [21, 22]. It is important to note that the heterogeneous reaction can proceed without polluting or hazardous solvents, for example, without *n*-hexane for oil extraction. Thus, methanol as a reactant acts as a solvent to carry out the reaction and obtain the methyl esters. This important solvent change increased in seven times the greenness of the heterogeneous reaction in comparison with the conventional method (two-step). Besides, the water consumption to purify the biodiesel is not necessary. Direct transesterification shows the environmental benefits related to solvents and energy consumption [21, 22].

## **2.1 Direct transesterification of** *J. curcas* **and** *R. communis* **using homogeneous catalyst**

#### *2.1.1 Seed-oil extraction*

*J. curcas L.* is a plant oil with more than 3500 species. It is native to Mesoamerica, covering northern Mexico and Central America. Jatropha seed contains a high percentage of vegetable oil that can be used in biodiesel production [28, 30, 35]. Another vegetable oil used for biodiesel production is the *R. communis*, which is native to tropical Africa and is considered a highly invasive species in Some Asian and European countries [5, 21, 22]. *J. curcas L.* and *R. communis* seeds oils are suitable for biodiesel production because they are highly available seeds and neither their fruits nor plants are edible. These seeds are widely distributed in several places as a weed in urban and agricultural areas, and therefore, they have a great capacity for adaptation that allows them to be cultivated in all tropical and subtropical regions, although it is typical in semiarid regions [32–35]. Both species are considered toxic plants in the human. For example, jatropha seed contain toxic compounds known as phorbol esters, while in the *R. communis* seed, there is an albumin known as ricin. These toxic compounds can cause diarrhea, rapid breathing, tumor promotion in humans, etc., in high concentrations [36]. The extraction of the oil is carried out by several methods such as chemical extraction, supercritical fluid extraction, steam distillation, mechanical extraction, solvent extraction, CO2 extraction, maceration, enfleurage, among others [30, 37, 38]. Also, seed oils can be obtained through the Soxhlet extraction method [37]. These methods of extraction require an excessive energy consumption so in the most cases the cost of the final product becomes more expensive. In addition, the extraction of the vegetable oil involved the use of toxic solvents and water, which makes the process less environmentally friendly.

**Table 1** shows the results of jatropha and *R. communis* oils extraction from the seed and the shell, using *n*-hexane and methanol as solvents. These seeds were provided by the State of Morelos in Mexico. The oil content of jatropha and *R. communis* seeds corresponded to yields ranging from 48 to 52% and from 50 to 52%, respectively (*entries 1–4*), while the oil content of the shell was only <7% (*entries 5 and 6*). It is important to know that the quantity of the seed oils helps to quantify the amount of biodiesel in the direct transesterification of seeds.


#### **Table 1***.*

Ricinus communis *and jatropha oils extraction.*

The amount of vegetables oils extracted agreed with the values reported in the literature, which ranges from 40 to 60% for jatropha oil [1, 28, 36–39] and ranges from 40 to 56% for *R. communis* oil [39] by the chemical oil extraction method (Soxhlet extraction). Therefore, the percentage of the vegetable oil extraction in both seeds with n-hexane and methanol is similar. Therefore, the methanol is an interesting option to carry out the reactions as a reactant and solvent.

Vegetable oils (triglycerides) contain mainly mixture of triglycerides (TAG), with a different composition of the alkyl chains depending on their origin. **Figure 2** shows the jatropha (**Figure 2A**) and *R. communis* (**Figure 2B**) oil compositions that were calculated according to electrospray ionization mass spectrometry (ESI-MS) analysis [40]. Jatropha

#### **Figure 2.**

*TAG and FFA fingerprints of jatropha (A) and* Ricinus communis *(B) oils obtained by ESI(+)-MS and ESI ()-MS ionization technique.*

*Direct Transesterification: From Seeds to Biodiesel in One-Step Using Homogeneous… DOI: http://dx.doi.org/10.5772/intechopen.108234*

oil mainly contains oleic (O), linoleic (L), and linolenic (Ln) triglycerides. The most abundant [TAG + Na]+ ions are of *m/z* 905 (C57H102O6), 901 (C57H98O6), and 899 (C57H96O6) that correspond to LOO, LLL, and LnLL, respectively (**Figure 2A**). Other triglycerides of *m/z* 907 (C57H104O6), 903 (C57H100O6), and 897 (C57H94O6) are attributed to OOO, OLL, and LnLnL in less amount. Diglyceride as LL (*m/z* 639, C39H68O5) was found. The free fatty acids (FFAs) contained in Jatropha oil were detected by ESI()-MS. **Figure 2A** shows the spectrum of ESI()-MS that displays mainly ions corresponding to the deprotonated molecules [FFA-H] from oleic (*m/z* = 281, C18H34O2), linoleic (*m/ z* = 279, C18H32O2) acids as the most abundant. In the same way, *R. communis* oil contains several lipids such as tri-, di-glycerides, and FFA. The [TAG+Na]+ ion most abundant is the ricinoleic TAG (RRR) with *m/z* = 955 ion. Diglycerides such as RR (m/z, C39:2) and ricinoleic acid (*m/z* = 297, C18:0) were detected (**Figure 2B**).

#### *2.1.2 Conventional and direct transesterification processes*

The production of biodiesel from vegetable oils and fats can be carried out by several routes (pyrolysis, microemulsion, transesterification, etc.). The most used conversion method is from the transesterification of triglycerides in the presence of homogeneous basic catalysts (NaOH and KOH) with methanol. It has been reported that by this synthesis route, the conversion of oil to biodiesel is 99.99%. One of the disadvantages at the industrial level is the recovery of the catalyst and the emulsions and soaps that are obtained during the reaction. Moreover, this conventional process consists of two steps: First, it is necessary extract the vegetable oil from different seeds prior to the transesterification, commonly the oil is extracted with a hazardous solvent or mechanical extraction with high energy consumption and then, in a second step, subsequently transesterification of triglycerides in the presence of the catalyst. These disadvantages promote excessive energy consumption and high operational and biodiesel production costs. Therefore, a new strategy for obtaining biodiesel is proposed. Direct transesterification is a method (*in situ* or one-pot) for transforming seed oils (biomass) to free acid alkyl esters and glycerin, in the presence of a short-chain alcohol and the catalyst. This one-step process is more economical and efficient than conventional method. **Figure 1** shows the comparison between conventional (two steps) and direct transesterification (one step).

**Table 2** shows the conventional and direct transesterification reactions of jatropha and *R. communis* oils using acidic (HCl) (*entries 1 and 9*) and basic (NaOH) homogeneous catalysts.

The Jatropha and *R. communis* oils were transesterified in methanol (CH3OH) using acidic catalyst (HCl/CH3OH, 5 *v/v %*) that corresponds to (HCl/oil, 31 *wt. %*); the oil conversion to fatty acid methyl esters (FAME's) was 98.70% and 99.10%, respectively (*entries 1 and 9*). In the same way, these non-edible oils were transesterified using basic catalyst (NaOH/CH3OH, 5 *wt. %*) that corresponds to (NaOH/oil, 1.20 *wt. %*) with 99.99% of conversion to FAME.

It has been reported that the use of acidic homogeneous catalysts can catalyze esterification and transesterification simultaneously; however, it is not sensitive to the free fatty acids (FFA) content in the oils, the oil conversion to FAME needs high catalyst concentration, long reaction times, high molar ratio of alcohol to oil, and the catalyst separation is difficult in comparison with the base-catalyzed process [41–47]. In **Table 2**, the vegetable oil transesterification using the acidic catalyst (HCl) was conducted by conventional reflux for 6 hours, while for the basic catalyst (NaOH), the reaction remained at 50°C for 3 hours (*entry 10*). Therefore, the direct


*a Conventional transesterification.*

*b Molar ratio of methanol to oil.*

*c Isolated fatty methyl esters, FAME.*

#### **Table 2.**

*Biodiesel from conventional and direct transesterification of jatropha and* Ricinus communis *seed oils using homogeneous catalysts.*

transesterification of seed oils was carried out from the basic homogeneous catalyst, NaOH. The solvent extraction step that is required in the conventional process but not in direct transesterification, and is usually the most capital and running cost-intensive [46]. Few studies have demonstrated the FAME production achieved from the *in situ* transesterification using a homogeneous catalyst, also reactive extraction, of several seed oils such as soybean [30], sunflower [1, 31], Jatropha [22, 28], *R. communis* [21], microalgae (*Schizochytrium limacinum, Chlamydomonas, and Chlorella*) [32, 33], and others. In this method, oil-bearing seeds are ground and then reacted directly with the alcohol and catalyst (basic homogeneous and heterogeneous catalysts), thereby eliminating the timespans and a large amount of solvent for the oil extraction.

In particular, the homogeneous direct transesterification of the jatropha and *R. communis* seed oils in hexane or methanol as solvents, in the presence of NaOH basic catalyst, was studied. **Table 2** shows the results of the jatropha (*entries 3–8*) and *R. communis* (*entries 11–15*) seed oil conversion to free acid methyl esters (FAMEs, biodiesel) to different conditions of reaction (ratio of catalyst to oil, molar ratio of CH3OH to oil, and time). The effect of several factors that are type of solvent, catalyst concentration, temperature, reaction time, methanol-oil ratio, and particle size has been investigated to optimize the direct transesterification of seed oils for achieving maximum oil yield [33, 48–50]. Jatropha and *R. communis* seed oil conversion to FAME from direct transesterification was optimized using the following parameters: NaOH homogeneous catalyst amount, the ratio of methanol/oil, and the effect of reaction, which are described below.

*Direct Transesterification: From Seeds to Biodiesel in One-Step Using Homogeneous… DOI: http://dx.doi.org/10.5772/intechopen.108234*

**Figure 3.** *FAME yield for the direct transesterification of jatropha and* Ricinus communis *seed oil* versus *catalyst amount (A) and ratios [CH3OH]/[oil] (B).*

**Figure 3** shows the FAME yield obtained from homogeneous direct transesterification of jatropha and *Ricinus communis* seeds. **Figure 3A** shows the FAME yield *versus* catalyst amount, and **Figure 3B** shows the relation between FAME yield *versus* molar ratio [CH3OH]/[oil].

1.Effect of NaOH amount

**Figure 3A** shows the results of direct transesterification reactions of jatropha and *Ricinus communis* seeds using methanol and NaOH as catalyst with ratio of NaOH/oil = 0.3 *wt. %* to 1.2 *wt.* %. The molar ratio [CH3OH]/[oil] = 16:1, time = 9 hours were maintained constant using hexane as solvent (**Table 2**, *entries 4 and 12*). The catalyst amount affects the yield of the FAME products for both seeds. Low catalyst concentration (NaOH/oil = 0.3% by weight) reached the maximum yield for jatropha and *R. communis* seed oil of 54.53% and 53.00%, respectively. The conversion increased as the amount of catalyst increased from 0.5 *wt. %* to 0.8 *wt. %*, varying for the jatropha seed from 79.5% to 97.7% and for the *R. communis* seed from 88.45 to 98.2%, respectively. Then, the maximum yield reached an equilibrium. The optimum catalyst amount for both seed oils was at 1.2 *wt. %* of catalyst, and the FAME yield was 99.99%. An increment in catalyst amount (2 *wt. %*) does not affect the oil conversion to FAME.

2.Effect of methanol-oil ratio

**Figure 3B** shows the plot of methanol-oil ratio *versus* FAME yield of the direct transesterification of jatropha and *Ricinus communis* using a molar ratio

NaOH/oil = 1.2 *wt.* % (**Table 2**, *entries 5–7, 13,* and *14*). The methanol-to-oil molar ratio varied for jatropha and *R. communis* seed oils within the range of 9:1–65:1. The maximum oil conversion to FAME products (99.99%) in both seed oils was obtained at the methanol-to-oil molar ratio of 16:1. **Table 2** shows the results of these reactions when *n*-hexane is used as solvent (*entries 5, 6,* and *13*). The excess methanol in the direct transesterification (methanolto-oil molar ratio of 37:1 and 65:1) is used as a solvent, and the conversion of FAME was 99.99% (**Table 2**, *entries 7* and *14*).

3. Influence of the reaction time

**Figure 4** shows the kinetic curves of FAME yields *versus* reaction time of the jatropha and *Ricinus communis* seeds. According to the previous results, the optimum catalyst was 1.2 *wt. %* of NaOH and a molar ratio [CH3OH]/ [oil] = 16:1. The oil conversion to FAME jatropha and FAME *R. communis* was increased 65% and 64% from 0.5 hours, respectively. Over the period from 1 to 8 hours, FAME yield from jatropha seed was increased with values ranging from 71 to 98% and for *R. communis* seed was from 68.5% to 98.4%, respectively. The reactions reached equilibrium after 9 hours with the maximum conversion of 99.99% in both cases. The maximum conversion to FAME jatropha and FAME *R. communis* is observed in the <sup>1</sup> H-NMR spectra (**Figure 5**).

**Figure 5** shows the <sup>1</sup> H-NMR spectra of FAME products obtained from jatropha seeds (A) and FAME products obtained from *Ricinus communis* (B). The signal arising in 3.66 ppm region corresponds to the protons of CH3-O-. The signals observed at 5.39–5.30 ppm (A) and 5.59–5.53 ppm to 5.42–5.34 pp. (B) represent the protons of

*Direct Transesterification: From Seeds to Biodiesel in One-Step Using Homogeneous… DOI: http://dx.doi.org/10.5772/intechopen.108234*

**Figure 5.** *1 H-NMR (400 MHz, CDCl3) spectra of jatropha FAME (A) and* Ricinus communis *FAME (B).*

vinyl group (HC=CH) contained in the unsaturated fatty acids. The FAME obtained from jatropha; the spectrum (A) shows an intense signal at 2.79 ppm compared with the spectrum (B) that corresponds to protons of -CH2 group bonded to carbon-carbon double bond in the methyl linoleate chain. The FAME *R. communis* is composed of methyl ricinolate chains. The spectrum (B) shows the signal of the hydroxyl group (-CH-OH) that is observed at 3.65–3.59 ppm.

The composition of the Jatropha and *R. communis* biodiesel products obtained from direct transesterification of seed oil using NaOH/MeOH catalysts was determined from electrospray ionization mass spectrometry (ESI-MS) technique. The biodiesel composition was detected as (FAME + Na]<sup>+</sup> ions. **Figure 6A** shows the chromatogram of fatty acid methyl esters (FAMEs) from the seed of *Jatropha curcas L.* We can see that the oleic- (C18:1, 30.00%) and linolenic (C18:2, 48.30%) methyl esters compounds are the most abundant, respectively. The palmitic- (C16:1), stearic- (C18:0), and eicosanoic methyl esters (m/z = 349, C20:0) signals are shown in low ratio. **Figure 6B** shows the chromatogram of FAMEs from the seed of *R. communis*. The most abundant signal corresponds to the ricinoleic methyl ester (C18:1-OH, 91.50%),

**Figure 6.** *ESI (+)-MS of fatty acid methyl esters (FAME) from (A)* Jatropha curcas *and (B)* Ricinus communis *seeds.*

followed by FAMEs in smaller amounts such as linoleic- (C18:2), oleic- (C18:1), stearic- (C18:0), eicosanoic- (C20:0), and dihydroxystearic (C18:0-(OH)2) methyl esters, respectively.

## **2.2 Direct transesterification of** *J. curcas* **and** *R. communis* **using heterogeneous catalyst**

An enhancement of conventional transesterification is direct transesterification. In this method, to produced biodiesel, the biomass is reacted directly with the shortchain alcohol and the catalyst in one step. To overcome some drawbacks of the homogeneous direct transesterification reaction, heterogeneous catalysts have been used. The heterogeneous *in situ* or direct transesterification is expected to be an effective FAME production process with low cost and minimal environmental impact because of simplifying the production under mild reaction conditions. It is a cheaper method than conventional because the extraction of the triglycerides and the transesterification are done *in situ* at the same time decreasing the time to obtaining

#### *Direct Transesterification: From Seeds to Biodiesel in One-Step Using Homogeneous… DOI: http://dx.doi.org/10.5772/intechopen.108234*

biodiesel and the excessive use of different resources. However, there are few studies were reported with solid heterogeneous catalyst in direct transesterification.

Direct transesterification has been reported using different heterogeneous catalysts. As acid heterogeneous catalyst, the CT 269 ion-exchange resin was reported to obtain biodiesel from microalgae [51]. The optimum conditions of this reaction were 95°C, and mass ratio catalyst/biomass equals to 0.52:1. On the other hand, the heterogeneous basic catalyst reported was LiOH-pumice [52] obtained by acid treatment and wet impregnation. The highest yield obtained was 47% with 20 wt% of catalyst at 80° C in 3 hours of reaction time and a relation methanol/biomass 12 mL/g. The biomass used in this reaction was *Chlorella sp. microalgae*. The same microalgae were used to produce biodiesel using carbon-dot functionalized strontium oxide [53]. The reaction was carried out with microwave radiation with dried microalgae mixed with chloroform, methanol, and 0.3 g of the catalyst. The temperature of the reaction was 60° C, the conversion of the lipids into FAME's was 97 wt%, and the maximum yield was 45.5%. CaO obtained from eggshell waste was evaluated in direct transesterification from *A. obliquus* microalgae. The eggshell needed a previous treatment to obtain the CaO as catalyst and obtained FAMEs. The biomass was mixed with the catalyst and methanol with a ratio 10:1 wt/vol; the temperature of the reaction was 70°C for a period of 1–5 h. About 86.41% was the yield reported using 1.7% (w/w) [54]. Biodiesel was produced from palm kernel by *in situ* transesterification using CaO as catalyst. The biomass was mixed with methanol and CaO. The reaction was carried out at 65° C for 3 hours. The oil content in the biomass was 33.08%. In this case, the size of the catalyst was very important, and the optimal size was <1 mm approximately [55]. In another study, strontium oxide as catalyst was evaluated using castor and jatropha seeds with microwave and ultrasound irradiation. The yield obtained with castor seeds was 57.2% of the total weight, and the conversion into FAMEs was 99.9%. With jatropha seeds, the yield was 41.1% with a conversion of the oil into FAMEs of 99.7% by microwave irradiation. By ultrasound irradiation, the yields were 48.2% and 32.9% from castor and jatropha seed, respectively [32].

Recently, Na2ZrO3 was evaluated as heterogeneous catalyst using *J. curcas L.* and *R. communis* seeds. This ceramic material exhibits interesting catalytic properties as basic catalysts and their low solubility and high stability. For heterogeneous direct transesterification, the seeds were ground and mixed with methanol and the catalyst with a molar ratio 1:65 (oil/methanol) at 65°C. At the first reactions, n-hexane was added to the reaction to favor the vegetable oil extraction. The presence of n-hexane in the reactions using 10 wt% of Na2ZrO3 as catalyst decreases the conversion of FFAs in 76% for *Jatropha Curcas L*. and 66% for *R. communis* seeds [21, 22]. This is caused by the decrement in the contact area between the reaction products caused by the nonpolar solvent despite the increase to 10 wt% of the catalyst. The ideal conditions using only methanol as reactant and solvent were 5 wt% of catalyst at 65° C for 8 hours obtaining conversion to FAME of 99.9% in the first cycle and 72.5% after 5 cycles of reusing the catalyst. **Figure 7** shows the kinetic curves of the FAME yield of *in situ* transesterification reaction of Jatropha seeds. The use of methanol as reactant and solvent in transesterification reaction represents an environmental benefit due to methanol being environmentally friendly, and it can be a replacement for n-hexane or dichloromethane, which are toxic and hazard solvents [21].

As with jatropha seeds, the heterogeneous direct transesterification of *Ricinus communis* seed was performed with Na2ZrO3 in the presence of methanol. The yield obtained with optimal conditions (65° C, 5 wt%) was 99.9% just in 4 hours. **Figure 8** discloses the FAME yield *versus* the reaction time. The selectivity between the catalysts

**Figure 7.**

*Kinetic curves of the FAME conversion efficiency of* in-situ *transesterification reaction of Jatropha seeds and methanol heterogeneous catalysts.*

**Figure 8.**

*kinetic curves of the FAME conversion efficiency of* in situ *transesterification reaction of* Ricinus communis *seeds and methanol heterogeneous catalysts.*

reduced the reaction time and increased the yield and conversion. The hydroxyl group, -OH, of the ricinoleic methyl ester interacts with the high surface basicity of the solid catalyst, increasing their catalytic activity (**Figure 5B** shows the 1H-NMR spectra of the FAME *R. communis*, similar spectra). In this reaction *n*-hexane was

*Direct Transesterification: From Seeds to Biodiesel in One-Step Using Homogeneous… DOI: http://dx.doi.org/10.5772/intechopen.108234*

substitute by methanol. The toxicity and environmental hazard decreased. Methanol as solvent increased the greenness of heterogeneous process, and the products obtained show high purity.

In addition, some reusing reactions are being carried out to determine the selectivity and stability of the catalyst. The heterogeneous direct transesterification (onestep process) using Na-based catalyst is a promising alternative for more sustainable, cleaner, and efficient FAME production.

After a few reusing reactions, a reduction in FAME conversions was observed. This may be attributed to the adsorption of organic matters from the biomass and the glycerol on the catalyst surface, which generates a low contact area between the oil and the Na2ZrO3.

In recent years, heterogeneous acid catalysts have been reported in biodiesel production and are an emerging field of research. But before producing competitive biodiesel, it is necessary to improve some aspects of catalyst as the stability of acid sites and the control of the surface properties.

## **3. Conclusions**

Direct transesterification reaction from non-edible crops was successfully evaluated using both homogeneous and heterogeneous catalysts, from *Jatropha curcas L* and *R. communis* seeds, in one step. However, heterogeneous direct transesterification provides a new alternative and promising method to obtain highpurity biofuels. No toxic organic solvent was used to extract the oil from seeds, or no water was used to neutralize the products. Furthermore, methanol acts as reactant and solvent in the *in situ* reaction. This method improved the FAMEs production because the heterogeneous catalyst produces maximum conversion of 99.9% with 5 wt% of catalyst at 65°C. For *R. communis*, the optimal conditions were reached in 4 hours of reaction time due to the -OH group. In addition, the reuse and the stability of the solid catalyst in the direct transesterification reactions with yields of>72.5% in fifth cycle were evaluated. However, it is necessary investigate the optimal condition to increase the yield of FAME by favoring the contact between the biomass and the catalyst. Finally, heterogeneous direct transesterification has potential environmental and energy benefits in comparison with the conventional biodiesel production method.

## **4. Transesterification future research direction**

The processes for the biodiesel production must be carried out in a sustainable way using third- and fourth-generation feedstocks. Currently, our work group is focused on the development of green processes for the preparation of biodiesel through green routes that imply incorporating principles of sustainable chemistry. These clean processes incorporate the principles of a) atomic efficiency, b) use of renewable raw materials, c) preparation of heterogeneous catalysts, d) prevention of waste generation, and e) heterogeneous design reactors.

a. According to the results discussed in this chapter (Section 2), direct transesterification *(in situ)* is a promising alternative for obtaining biodiesel, this method simplifies the conventional process and eliminates the oil extraction stages. With this chemical synthesis, we are reducing the use of solvents and making the process of obtaining biodiesel more efficient and environmentally friendly in short times.


## **Acknowledgements**

Authors are grateful to Centro de Nanociencias y Micro y Nanotecnología del Instituto Politécnico Nacional (IPN) for their assistance in ESI-MS and 1H-NMR analyses. Also, the authors greatly appreciated the support from SECTEI CM-059/ 2021, SIP-IPN 2104 (modulo 20220625) projects and BEIFI scholarship. Finally, A. Martínez Ponce acknowledges the financial support from the DGAPA-UNAM PAPIME PE106522 project.

*Direct Transesterification: From Seeds to Biodiesel in One-Step Using Homogeneous… DOI: http://dx.doi.org/10.5772/intechopen.108234*

## **Author details**

Issis Claudette Romero-Ibarra<sup>1</sup> \*, Araceli Martínez Ponce Escuela<sup>2</sup> , Gabriela Elizabeth Mijangos Zúñiga3 and Wendy Eridani Medina Muñoz3

1 National Polytecnical Institute (Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria en Ingeniería y Tecnologías Avanzadas - UPIITA), Mexico City, Mexico

2 Nacional de Estudios Superiores, Unidad Morelia, Universidad Nacional Autónoma de México, Morelia, Michoacán, Mexico City, Mexico

3 National Polytecnical Institute (Instituto Politécnico Nacional, UPIITA), Mexico City, Mexico

\*Address all correspondence to: iromero@ipn.mx

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Kumar A, Sharma S. An evaluation of multipurpose oil seed crop for industrial uses (Jatropha curcas L.): A review. Industrial Crops and Products. 2008;**28**: 1-10. DOI: 10.1016/j.indcrop.2008. 01.001

[2] Reddy B, Ramesh S, Kumar A, Wani SP, Ortiz R, Ceballos H, et al. Biofuel crops research for energy security and rural development in developing countries. Bioenergy Research. 2008;**1**: 248-258. DOI: 10.1007/s12155-008-9022-x

[3] Joshi G, Rawat DS, Sharma AK, Pandey JK. Microwave enhanced alcoholysis of non-edible (algal, jatropha and pongamia) oils using chemically activated egg shell derived CaO as heterogeneous catalyst. Bioresource Technology. 2016;**219**:487-492. DOI: 10.1016/j.biortech.2016.08.011

[4] Achten WMJ, Mathijs E, Verchot L, Singh VP, Aerts R, Muys B. Jatropha biodiesel fueling sustainability. Biofuels, Bioproducts and Biorefining. 2007;**1**: 283-291. DOI: 10.1002/bbb.39

[5] Hincapié G, Mondragón F, López D. Conventional and in situ transesterification of castor seed oil for biodiesel production. Fuel. 2011;**90**(4): 1618-1623. DOI: 10.1016/J.FUEL.2011. 01.027

[6] Koh MY, Mohd GTI. A review of biodiesel production from Jatropha curcas L. oil. Renewable and Sustainable Energy Reviews. 2011;**15**:2240-2251. DOI: 10.1016/j.rser.2011.02.013

[7] Divakara BN, Upadhyaya HD, Wani SP, Laxmipathi CL. Biology and genetic improvement of Jatropha curcas L.: A review. Applied Energy. 2010;**87**: 732-742. DOI: 10.1016/j.apenergy. 2009.07.013

[8] Kuete V. Physical, hematological, and histopathological signs of toxicity induced by African medicinal plants. In: Kuete V, editor. Toxicological Survey of African Medicinal Plants. Cameroon: Faculty of Science, University of Dschang; 2014. pp. 635-657. DOI: 10.1016/B978-0- 12-800018-2.00022-4

[9] Melero JA, Iglesias J, Morales G. Heterogeneous acid catalysts for biodiesel production: Current status and future challenges. Green Chemistry. 2009;**11**:1285-1308. DOI: 10.1039/ B902086A

[10] Luque R, Lovett JC, Datta B, Clancy J, Campeloa JM, Romero AA. Biodiesel as feasible petrol fuel replacement: Amultidisciplinary overview. Energy & Environmental Science. 2010;**3**:1706-1721. DOI: 10.1039/ C0EE00085J

[11] Marx S. Glycerol-free biodiesel production through transesterification: A review. Fuel Processing Technology. 2016;**151**:139-147. DOI: 10.1016/j. fuproc.2016.05.033

[12] Tan HW, Abdul AR, Aroua MK. Glycerol production and its applications as a raw material: A review. Renewable and Sustainable Energy Reviews. 2013; **27**:118-127. DOI: 10.1016/j.rser.2013. 06.035

[13] Atadashi IM, Aroua MK, Abdul AR, Sulaiman NMN. The effects of catalysts in biodiesel production: A review. Journal of Industrial and Engineering Chemistry. 2013;**19**(1):14-26. DOI: 10.1016/j.jiec.2012.07.009

[14] Lotero E, Liu Y, Lopez D, Suwannakarn K, Bruce D, Goodwin J. Synthesis of biodiesel via acid catalysis. *Direct Transesterification: From Seeds to Biodiesel in One-Step Using Homogeneous… DOI: http://dx.doi.org/10.5772/intechopen.108234*

Industrial & Engineering Chemistry Research. 2005;**44**(14):5353-5363. DOI: 10.1021/ie049157g

[15] Lam MK, Lee KT, Mohamed AR. Homogeneous, heterogeneous, and enzymatic catalysis for transesterification of high free fatty acid oil (waste cooking oil) to biodiesel: A review. Biotechnology Advances. 2010; **28**(4):500-518. DOI: 10.1016/j.biotech adv.2010.03.002

[16] Lee DW, Park YM, Lee KY. Heterogeneous base catalysts for transesterification in biodiesel synthesis. Catal Surv from Asia. 2009; **13**(2):63-77. DOI: 10.1007/s10563-009- 9068-6

[17] Wali Khan I, Naeem A, Farooq M, Ali Z, Saeed T, Perveen F, et al. Biodiesel production by valorizing waste non-edible wild olive oil using heterogeneous base catalyst: Process optimization and cost estimation. Fuel. 2022;**320**:123828. DOI: 10.1016/j. fuel.2022.123828

[18] Abdulkareem A, Nasir N. Biodiesel production from canola oil using Ti*O*2/ CaO as a heterogenous catalyst. Journal of Advanced Research in Fluid. 2022;**93**: 125-137. DOI: 10.37934/ arfmts.93.2.125137

[19] Iskandinata I, Taslim T, Bani O, Purba HLM. Potential of papaya seeds as a heterogenous catalyst in biodiesel synthesis. IOP Conference Series: Earth and Environmental Science. 2022; **912**(2):24-25. DOI: 10.1088/1755-1315/ 912/1/012022

[20] Santiago-Torres N, Romero-Ibarra IC, Pfeiffer H. Sodium zirconate (*Na*2*ZrO*3) as a catalyst in a soybean oil transesterification reaction for biodiesel production. Fuel

Processing Technology. 2014;**120**: 34-39. DOI: 10.1016/j.fuproc.2013.11.018

[21] Martínez A, Mijangos G, Romero-Ibarra IC, Hernadez-Altamirano R, Mena V, Gutiérrez S. A novel green onepot synthesis of biodiesel from Ricinus communis seeds by basic heterogeneous catalysis. Journal of Cleaner Production. 2018;**196**:340-349. DOI: 10.1016/j. jclepro.2018.05.241

[22] Martínez A, Mijangos G, Romero-Ibarra IC, Hernadez-Altamirano R, Mena V. In-situ transesterification of Jatropha curcas L. seeds using homogeneous and heterogeneous basic catalysts. Fuel. 2019;**235**:277-287. DOI: 10.1016/j.fuel.2018.07.082

[23] Torres-Rodríguez DA, Romero-Ibarra IC, Ibarra IA, Pfeiffer H. Biodiesel production from soybean and Jatropha oils using cesium impregnated sodium zirconate as a heterogeneous base catalyst. Renewable Energy. 2016;**93**: 323-331. DOI: 10.1016/j. renene.2016.02.061

[24] Mijangos G, Cuautli C, Romero-Ibarra IC, Vazquez-Arenas J, Santolalla-Vargas C, Santes V, et al. Experimental and theoretical analysis revealing the underlying chemistry accounting for the heterogeneous transesterification reaction in *Na*2*SiO*<sup>3</sup> and *Li*2*SiO*<sup>3</sup> catalysts. Renewable Energy. 2022;**184**: 845-856. DOI: 10.1016/j. renene.2021.11.090

[25] Rodríguez-Ramírez R, Romero-Ibarra I, Vazquez-Arenas J. Synthesis of sodium zincsilicate (*Na*2*ZnSiO*4) and heterogeneous catalysis towards biodiesel production via box-behnken design. Fuel. 2020;**280**:118-668. DOI: 10.1016/j.fuel.2020.118668

[26] Leung DYC, Wu X, Leung MKH. A review on biodiesel production using

catalysed transesterification. Applied Energy. 2017;**87**(4):1083-1095. DOI: 10.1016/j.apenergy.2009.10.006

[27] Ma FA, Hanna M. Biodiesel production: A review. Bioresource Technology. 1999;**70**(1):1-15. DOI: 10.1016/S0960-8524(99)00025-5

[28] Shuit SH, Lee KT, Kamaruddin AH, Yusup S. Reactive extraction and in situ esterification of Jatropha Curcas L. seeds for the production of biodiesel. Fuel. 2010;**89**(2):527-530. DOI: 10.1016/J. FUEL.2009.07.011

[29] Liu Y, Tu Q, Knothe G, Lu M. Direct transesterification of spent coffee grounds for biodiesel production. Fuel. 2017;**199**:157-161. DOI: 10.1016/j. fuel.2017.02.094

[30] Kartika IA, Yani M, Ariono D, Evon P, Rigal L. Biodiesel production from jatropha seeds: Solvent extraction and in situ transesterification in a single step. Fuel. 2013;**106**:111-117. DOI: 10.1016/j.fuel.2013.01.021

[31] Harrington KJ, D'Arcy-Evans C. Transesterification in situ of sunflower seed oil. Industrial and Engineering Chemistry Product Research and Development. 1985;**24**(2):314-318. DOI: 10.1021/i300018a027

[32] Koberg M, Cohen M, Ben-Amotz A, Gedanken A. Bio-diesel production directly from the microalgae biomass of Nannochloropsis by microwave and ultrasound radiation. Bioresource Technology. 2011;**102**(5):4265-4269. DOI: 10.1016/j.biortech.2010.12.004

[33] Johnson MB, Wen Z. Production of biodiesel fuel from the microalga Schizochytrium Limacinum by direct transesterification of algal biomass. Energy & Fuels. 2009;**23**(10):5179-5183. DOI: 10.1021/ef900704h

[34] Hidalgo P, Toro C, Ciudad G, Navia R. Advances in direct transesterification of microalgal biomass for biodiesel production. Reviews in Environmental Science and Biotechnology. 2013;**12**:179-199. DOI: 10.1007/s11157-013-9308-0

[35] Dias L, Missio R, Dias D. Antiquity, botany, origin and domestication of Jatropha Curcas (Euphorbiaceae), a plant species with potential for biodiesel production. Genetics and Molecular Research. 2012;**11**(3): 2719-2728. DOI: 10.4238/2012. June.25.6

[36] Doan LG. Ricin: Mechanism of toxicity, clinical manifestations, and vaccine development. A review. Journal of Toxicology and Clinical Toxicology. 2004;**42**(2):201-208. DOI: 10.1081/CLT-120030945

[37] Kyari MZ. Extraction and characterization of seed oil. International Agrophysics. 2008;**22**:139-142

[38] Sayyar S, Abidin ZZ, Yunus R, Muhammad A. Extraction of oil from Jatropha seeds-optimization and kinetics. American Journal of Applied Sciences. 2009;**6**(7):1390-1395. DOI: 10.3844/ajassp.2009.1390.1395

[39] Perdomo FA, Acosta-Osorio AA, Herrera G, Vasco-Leal JF, Mosquera-Artamonov JD, Millan-Malo B, et al. Physicochemical characterization of seven Mexican Ricinus Communis L. Seeds & oil contents. Biomass and Bioenergy. 2013;**48**:17-24. DOI: 10.1016/ j.biombioe.2012.10.020

[40] Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass spectrometry of large biomolecules. Science (80-.). 1989;**246**: 64-71

*Direct Transesterification: From Seeds to Biodiesel in One-Step Using Homogeneous… DOI: http://dx.doi.org/10.5772/intechopen.108234*

[41] Vyas AP, Verma JL, Subrahmanyam NA. Review on FAME production processes. Fuel. 2010;**89**(1): 1-9. DOI: 10.1016/j.fuel.2009.08.014

[42] Mangesh GK, Dalai AK. Waste cooking oil an economical source for biodiesel: A review. Industrial and Engineering Chemistry Research. 2006; **45**(9):2901-2913. DOI: 10.1021/ IE0510526

[43] Fukuda H, Kondo A, Noda H. Biodiesel fuel production by transesterification of oils. Journal of Bioscience and Bioengineering. 2001; **92**(5):405-416. DOI: 10.1016/S1389-1723 (01)80288-7

[44] Aransiola EF, Ojumu TV, Oyekola OO, Madzimbamuto TF, Ikhu-Omoregbe DIO. A review of current technology for biodiesel production: State of the art. Biomass and Bioenergy. 2014;**61**:276-297. DOI: 10.1016/j. biombioe.2013.11.014

[45] Zhang Y, Dubé M, McLean D, Kates M. Biodiesel production from waste cooking oil: 1. Process design and technological assessment. Bioresource Technology. 2003;**89**(1):1-16. DOI: 10.1016/S0960-8524(03)00040-3

[46] Kasim FH, Harvey AP, Zakaria R. Biodiesel production by in situ transesterification. Biofuels. 2010;**1**(2): 355-365. DOI: 10.4155/bfs.10.6

[47] Kildiran G, Yücel SÖ, Türkay S. Insitu alcoholysis of soybean oil. Journal of the American Oil Chemists' Society. 1996;**73**(2):225-228. DOI: 10.1007/ bf02523899

[48] Patil PD, Deng S. Optimization of biodiesel production from edible and non-edible vegetable oils. Fuel. 2009; **88**(7):1302-1306. DOI: 10.1016/ j.fuel.2009.01.016

[49] Talha NS, Sulaiman S. Overview of catalysts in biodiesel production. ARPN Journal of Engineering and Applied Sciences. 2016;**11**(1):439-442

[50] Singh A, Gaurav K. Advancement in catalysts for transesterification in the production of biodiesel: A review. Journal of Biochemical Technology. 2018;**7**(3):1148-1158

[51] Vicente G, Carrero A, Rodríguez R, del Peso GL. Heterogeneous-catalysed direct transformation of microalga biomass into biodiesel-grade FAMEs. Fuel. 2017;**200**:590-598. DOI: 10.1016/j. fuel.2017.04.006

[52] De Luna MDG, Doliente LMT, Ido AL, Chung TW. In situ transesterification of chlorella sp. microalgae using LiOH-pumice catalyst. Journal of Environmental Chemical Engineering. 2017;**5**(3):2830-2835. DOI: 10.1016/j.jece.2017.05.006

[53] Tangy A, Kumar V, Neel Pulidindi I, Kinel-Tahan Y, Yehoshua Y, Gedanken A. In situ transesterification of Chlorella vulgaris using carbon-dot functionalized strontium oxide as a heterogeneous catalyst under microwave irradiation. Energy and Fuels. 2016; **30**(12):10602-10610. DOI: 10.1021/acs. energyfuels.6b02519

[54] Pandit PR, Fulekar MH. Egg shell waste as heterogeneous nanocatalyst for biodiesel production: Optimized by response surface methodology. Journal of Environmental Management. 2017; **198**:319-329. DOI: 10.1016/j.jenvman. 2017.04.100

[55] Tarigan JB, Prakoso HT, Siahaan D, Kaban J. Rapid biodiesel production from palm kernel through in situ transesterification reaction using CaO As catalyst. International Journal of Applied Chemistry. 2017;**13**(3):631-646

## **Chapter 3**

## Biotechnological Interventions for the Production of Glycerol-Free Biodiesel

*Muhammad Saeed, Ghulam Mustafa, Faiz Ahmad Joyia, Aneela Shadab and Aqsa Parvaiz*

## **Abstract**

Advances in plant biotechnology and microbial genetics are speeding up because of the urgent need to provide a steady supply of resources. Growing cost of crude oil is having a negative impact on economies throughout the globe. Just biodiesel and bioethanol have been recognized as viable fossil fuel replacements. Chemical catalysis is primary way to synthesize biodiesel, besides enzymatic and microbial methods also play important role in biodiesel synthesis. These processes may play a significant part in the replacement of petroleum-based diesel in the future. The growth of sustainable, economically feasible biotechnological tools for the synthesis of biodiesel requires strong collaboration among several disciplines. In this age, lipases are the preferred enzymes for producing methyl esters (FAME), which are significant biological objects in biodiesel, from fatty acid esters (FAE) derived from fats and oils. It has also been shown that designed whole-cell microorganisms may directly produce FAE (MicroDiesel). The expensive cost of the biocatalyst continues to be a barrier to current enzymatic procedures, although advancements have recently been achieved, enabling the first synthetic enzymatic biodiesel synthesis. The fabrication of biodiesel which is enzymatic is primarily desirable due to the initial materials (waste frying oils, oils that were having high water content, etc.), where standard interesterification which is chemical is seldom applicable.

**Keywords:** biodiesel, glycerol, sugarcane, biochar, cyanobacteria

## **1. Introduction**

As a result of climate change-causing greenhouse gas emissions, depleting oil reserves, and skyrocketing crude oil prices, the global community is increasingly turning to biological processes and green technologies to create technological compounds and alternative fuels from renewable resources. Advances in plant biotechnology and microbial genetics are speeding up because of the urgent need to provide a steady supply of resources. There is capability in biotechnology to manufacture a large number of these compounds from inexhaustible sources, at present virtually all the main bulk chemicals, with the exception of ethanol, are produced through

the petrochemical approach [1]. Fermentation products typically have substantially smaller production quantities (less than one million tonnes per year) but higher pricing compared to petrochemical-based compounds [2]. However, bio-based processes offer the benefits of using renewable feedstock, producing environmentally friendly emissions, and operating at relatively moderate temperatures and pressures. Although biotechnology has shown its worth in the fabrication of fine chemicals like organic acids, vitamins, and medicinal compounds, there has been a rising push to optimise green technologies, which is slowly shifting the situation. For example, oil which is a fossil fuel much likely to run out first. Unrefined oil is a multi-component mixture that consists of around 50–95% hydrocarbons. Nearly all glasshouse gas releases are caused by the burning of fossil fuels. In addition to lowering pollution levels, cutting down on fossil fuel consumption would significantly decrease the quantity of carbon dioxide generated. Inquisitiveness in the maintainable generation of chemical raw materials and fuels has increased in light of the known scarcity of crude oil compared to the vast availability of biomass. The quantity of biofuel produced is massive and is anticipated to produce exponentially in the coming years. The growing cost of crude oil is having a negative impact on economies throughout the globe, especially those of the industrialised countries that rely heavily on the commodity. Over the last decade, consumers have seen a quadrupling in the price of essential fuels including natural gas, gasoline, and diesel. Even though the United States and Europe are a wide range of users of fossil fuels, these innovations will also help Asia's rapidly expanding economy. As a result, there has been a rise in the study of potential substitutes for fossil fuels. Just biodiesel and bioethanol, however, have been widely recognised as viable fossil fuel replacements. When customers shop around for ways to save their energy bills, innovative technologies and efficiency improvements become hot commodities. Due to the dramatic changes in the cost of crude oil in recent months, various major oil importers have been actively looking for alternatives. More than only the oil shortage and environmental impact, biofuel's significance cannot be overstated. Feedstock and fuels which are liquids derived from resources that can be renewed will allow us to tap into the vast, as-yet-untapped potential of agricultural and forestry waste products. Other significant arguments in favour of the biofuel alternative include its positive effect on worldwide climate, increased security related to the environment and economics, and practically better and improved goods. Environmental difficulty and political will are necessary, but they are not sufficient to usher in a sustainable age, which is nevertheless hampered by economic constraints. Until recently, biotechnology was seen by the chemical industries as an excessively costly high-tech technique that was not suitable for use on a commercial scale. The current research efforts of large chemical firms to create bulk compounds like 1,3-propanediol using biological approaches show that this perspective is gradually changing. Despite government support, bioethanol has become the biggest fermentation product in use today, demonstrating biotechnology's potential for large-scale chemical production.

## **2. Bioethanol from glycerol as raw material in biodiesel production**

Most delegates to the UN Climate Change Conference in Doha, Qatar, in December 2012 committed to even deeper cuts in carbon dioxide emissions and established the latest objectives and restrictions to be executed under the Kyoto Protocol Extension (2012–2020). In addition, the price of a barrel of Brent oil has risen as high as 87.19 Euros in the last year (www.indexmundi.com). As alternatives to fossil fuels, biofuels

#### *Biotechnological Interventions for the Production of Glycerol-Free Biodiesel DOI: http://dx.doi.org/10.5772/intechopen.108895*

like bioethanol and biodiesel also help the environment. As a result, governments in many developed and developing nations are establishing and expanding research and policy initiatives to boost biofuels' production and consumption. EU leaders have decided to prioritise biofuels in their quest to meet their renewable energy goal of 20% of total energy consumption by 2020. The European Commission has set a target of 10% of total fuel consumption in transportation by 2020. In contrast to fossil fuels, biodiesel may be replenished again and over again. It may be made using either animal or plant-based fats or oils. Since the inception of diesel engines in 1893, the potential of utilising vegetable oils as fuel has been recognised. Vegetable oil has the potential to be an operational substitute fuel oil, but its high viscosity prevents its usage in most conventional diesel engines. Several techniques exist for reducing the thickness of vegetable oils. Methods such as diluting, microemulsifying, pyrolysing, and transesterifying are used to lessen the thickness of a substance. Most biodiesel producers employ transesterification, a process that lowers oil viscosity. The chemical process by which oil is changed into its fatty ester is termed transesterification, although it is also known as alcoholysis. Triglycerides (1) are converted into ethyl esters of fatty acids (3) and glycerol (4) by the transesterification process, in which alcohol (ethanol or methanol) (2) is reacted with a catalytic base (1), (4) (**Figure 1**).

Adding a catalyst speeds up and increases the yield of a reaction. Since the reaction might go either way, an excess of alcohol is utilised to tip the scales in favour of the product side of the equilibrium. An oil-splitting catalyst like NaOH or KOH and an alcohol-like methanol or ethanol are needed for the biodiesel process. Glycerol is the primary output. You may recognise this kind of crude glycerine by its dark colour and syrupy consistency. In contrast to petro-diesel, biodiesel is superior in many ways: flash point, sulphur content, biodegradability, and aromatic content [3]. Transesterification of vegetable oils typically employs homogeneous catalysts. Under moderate reaction conditions, base homogeneous catalysts like NaOH and KOH are the most effective. However, the reaction time for using acid homogenous catalysts is much higher. On the other hand, the price of the production of biodiesel is inflated by the need of treating the waste produced by homogeneous catalysts, which is itself difficult to recover. Biodiesel may be made from vegetable oils, and heterogeneous catalysts are a viable option for this process. Researchers are looking at the transesterification activity of several heterogeneous catalysts [4]. Heterogeneous catalysts have many advantages over their homogeneous counterparts, including the fact that they can be reused multiple times without degrading their performance and the ability to use an uninterrupted procedure without the need for additional decontamination steps, not to mention the possibility of being relatively inexpensive.

**Figure 1.** *Transesterification of triglycerides with alcohol.*

#### **Figure 2.**

*The total energy used in 2010 (REP 2011/2022). The US and Brazilian markets account for the bulk of bioethanol sales, but biodiesel use has surged in the EU. At the end of 2010, Spain has more than 47 biodiesel facilities and four bioethanol plants with a combined installed production capacity of over 4 million tep (Per 2011/2020). The biofuel industry is experiencing a period of the technical revolution that is largely influencing the range of materials amenable to use in production processes. As such, the European Industrial Initiative on Bioenergy's implementation strategy prioritises thermochemical and biochemical processes for material conversion. Rapid expansion is predicted for the biodiesel industry. Its popularity will rise steadily over the next decade, helped by new regulations requiring the labelling of some mixtures. From 2011 to 2020, bioethanol use is predicted to roughly treble. The anticipated increase is due in part to the elimination of gas price subsidies and the standardisation of the labelling of gasoline blends. However, fresh environmental and economic questions regarding the feasibility of alternative fuels have been raised due to the increasing demand for biofuel, in particular biodiesel.*

Financial incentives for both producers and consumers are still needed to increase biofuel usage. In this way, many governments subsidise the biofuel industry via tax credits and other measures. In the beginning, the EU subsidised biofuels like bioethanol and biodiesel. The United States government offers tax breaks to domestic producers in the amount of \$0.51 per gallon for bioethanol and \$1.00 per gallon for biodiesel. Presently, Germany incentivizes the use of biodiesel by taxing it at a lower rate than regular fuel. The yearly fabrication and use of biodiesel in Germany exceeds 2.5 billion litres [5]. The usage of biodiesel has spread to other EU nations, often as an additive to petroleum fuel. However, using objectives to stimulate the future production and use of biofuels are significantly more essential than tariffs and subsidies. Spain's Renewable Energy Plan (REP) 2011–2020 sets goals in conformity with the European Parliament's Directive 2009/28/EC on the encouragement of the utilisation of energy from sources which can be renewed. The goal of the REP is to meet the target set by the EU Directive, which calls for the use of renewable sources to account for at least 20% of gross final energy exhaustion by the year 2020. The International Energy Agency (IEA) reports that in 2010, biofuels substituted for 2% of global oil production. The amazing expansion of renewable energy sources over the last several years may be attributed in large part to the government assistance provided under the Renewable Energy Plan 2005–2010 (**Figure 2**).

## **3. Glycerol, from major commodity to waste effluent**

Pure 1,2,3-propanediol (glycerol) is a colourless, odourless, hygroscopic, viscous liquid having the chemical formula OCH2CHOHCH2OH. It is a member of the alcohol family of organic compounds. The glycerol molecule's space-filling model is seen in **Figure 3**.

*Biotechnological Interventions for the Production of Glycerol-Free Biodiesel DOI: http://dx.doi.org/10.5772/intechopen.108895*

**Figure 3.** *Space-filling model for glycerol molecule.*

Glycerol interacts with inorganic and organic acids to produce ethers, aldehydes, esters, and numerous derived molecules because it has one secondary and two primary alcohol groups per molecule. When there are several alcohol groups present, it is easier to make polymers and coatings (polyesters, polyether, and alkyd resins). Glycerol is a versatile chemical that may be used in a wide variety of industries due to its solubility in water, its biocompatibility, its lack of toxicity when applied topically, and its legal status in the food and drug industries. Also, glycerol's non-toxic qualities enable it for a wide variety of applications. It is safe to say that the cosmetics, explosives, food, pharmaceutical, polymer, and printing sectors are not the only ones that put this chemical component to good use (**Figure 4**). In the construction, automotive, and textile sectors, it is used to make gums and resins. In addition to its uses as a stabiliser in ice cream and a softening agent in baked products, mono and diglyceride emulsifiers also include this substance. As an added bonus, glycerol has several applications in the medical and pharmaceutical fields. It has also been put to use as a safe medium for freezing biological cells, which is a relatively new yet crucial.

Chemical synthesis from petrochemical feedstock or microbial fermentation [7] is both viable methods for glycerol production. In the last 150 years, scientists have learned that glycerol may be made by microorganisms. During World War I, glycerol was manufactured in industries by microbial fermentation. Due to poor glycerol yields and the difficulties of extracting and purifying glycerol from broth, microbial

**Figure 4.**

*Principal glycerine utilisation in industry [6].*

synthesis eventually fell in favour of chemical synthesis from petrochemical feedstocks. At present, roughly 600,000 tonnes of glycerol are generated each year, and most of this comes from the saponification of lipids as a byproduct of soapmaking (**Figure 5**) [7]. The widespread use of detergents in industrialised countries has reduced the relevance of this step [8].

There are a number of ways to convert propylene into glycerol. Chlorinating propylene yields allyl chloride, which is oxidised with hypochlorite to form dichlorohydrin, which interacts with a robust base to yield epichlorohydrin, the most essential step in the epichlorohydrin process. Glycerol is produced once epichlorohydrin is hydrolysed. The oxidation or chlorination of propylene accounted for around 25% of 2001 global glycerol production in the chemical sector [7]. However, environmental concerns and the rising cost of propylene have caused this route's popularity to decline. Glycerol is a byproduct of biodiesel production, therefore the rising popularity of this alternative fuel has led to a slump in the glycerol market and rendered the epichlorohydrin technique for glycerol synthesis unprofitable on a commercial scale. By 2020, it is expected that there would be a glycerol surplus of six times the yearly need. Glycerol, on the other hand, might be used as a substrate in emerging commercial fermentation processes. Bioconversion of glycerol has resulted in a wide variety of useful byproducts. chotIn particular, the microbial synthesis of 1,3-propanediol from glycerol has received a lot of attention because of the diol's various potential uses in the creation of novel polymers. Only around 62–85% (w/w) of the crude stream is glycerol [9, 10]. Fats (soaps), water, methanol (often used in Europe) or ethanol (typically used in the United States) and catalyst leftovers make up the rest (salts). In addition to carbon, hydrogen, and oxygen, raw glycerol also includes trace amounts of elements including calcium, magnesium, phosphorus, and sulphur [10]. Producers of biodiesel employ a variety of feedstocks, each of which contributes a unique range of purity values. Raw glycerol levels were observed to be greatest (76.6%) in waste vegetable oil, with lower levels (62%) being produced by mustard, rapeseed, canola, crambe, and soybean. Raw glycerol is mostly accumulated by biodiesel factories, which create 10 g of glycerol for 100 g of biodiesel produced [11]. The concentration of raw glycerol is also attributable to the bioethanol sector. Common industrial methods provide 4 g of glycerol for every 48 g of ethanol [12]. Pure glycerol quantities are quite large and growing. Glycerol costs around \$1200 per tonne in 2003. In 2006, the price per tonne was approximately \$600 and declining [13]. Glycerine spot prices in Europe ranged from €260 to €350 per tonne in the month of November 2009. Even crude glycerol is no longer worth what it used to. US\$0–\$70 per tonne was the stated price range in 2006 [13]. Raw glycerol is worthless to those who manufacture it on a small basis. In these situations, the crude stream is often transported to a purification plant at the expense of the producers, disposed of in a landfill, or held in containers

**Figure 5.** *Saponification of fats.*

until a solution is sought. Crude glycerine is currently considered a contaminant that must be disposed of at a cost since it is unfit for most glycerol markets. The dumping of polluted waste glycerol from biodiesel production has caused environmental issues and driven down global glycerol market prices.

## **4. Method and approaches for glycerol-free biodiesel production**

## **4.1 Emerging trends of microorganism in the biodiesel production**

A variety of feedstocks (such as lipids, starch, and sugars) that may be utilised for the generation of biofuels are harvested and processed via the photosynthetic activities of plants (including cyanobacteria, algae, trees, grasses, and crops). Biofuels such as methane, ethanol, and diesel are already widely produced using already accepted "first generation" biofuel systems based on crop plants like sugar beet (*Beta vulgaris*), rapeseed (*Brassica napus*), sugarcane (*Saccharum* spp.), wheat (*Triticum* spp.), oil palm (*Elaeis oleifera*), soya beans (*Glycine max*), and corn (*Zea mays***).** Pressure on food supply has resulted in increased worry and has sparked a heated "food versus fuel" debate, as a consequence of an expanding global population and significant droughts in key grain exporting countries (such as Australia). So, scientists are working on a new generation of land-free biofuel technologies. Most significantly, lignocellulosic methods are being developed to transform plant-based cellulose materials into liquid fuels. The most promising "non-food" plant possibilities for these methods include sorghum, miscanthus, camelina, switchgrass (*Panicum virgatum*), and poplar trees (*Populus* spp.). However, these systems can only be effective if scientists discover and implement energy-saving production techniques, such as enzymatic lignin digesting procedures (although chemical digestion techniques are also being investigated). Even if the resulting need for enzymes seems like a manageable obstacle, this technique may eventually add to food versus fuel dilemmas because of the existing reliance on appropriate land, most of which is already forested. Unless only waste products from existing agricultural or forestry systems are utilised, or feedstocks grown on nonarable land can be created, this might lead to a forest versus fuel dilemma.

The effective synthesis of starch, sugars, and oils by many microalgae makes them ideal feedstocks for the manufacture of biofuels such as biodiesel, ethanol, butanol, methane, and hydrogen. These microalgae may be cultivated in salt water. Microalgae may help with carbon capture because they take up carbon dioxide (CO2) during growth from the atmosphere and, in certain circumstances, from industrial sources. To store carbon, the leftover waste biomass from fuel production may be pyrolysed to create a charcoal-like product (Biochar) that is stable over time. Biochar may replace coal as a fuel source, or it can be sold to the public as a soil amendment. While in principle microalgal biofuel systems may solve both the food versus fuel and the prospective forest versus fuel issues, no such system has yet reached commercial viability. In spite of widespread and overwhelming excitement, which Emily Waltz calls "algae ardour," there are firms actively pushing these technologies towards commercial operation, as Waltz revealed recently. Investment in microalgal biofuels has actually increased after the first failure of a start-up rather than decreasing. This investment makes sense in light of recent economic case studies on standalone microalgal biofuel production models and on a model that co-produces high-value products (HVPs). Key economic determinants, such as building costs, biomass productivity, and cost of the dominating output, were found via sensitivity analysis in these models (and its production in the case of high-value co-products) (**Figure 6**).

**Figure 6.** *Conversion of raw material into ethanol by photosynthesis.*

## **4.2 Biotechnological production of biodiesel fuel using biocatalysed transesterification**

Under supercritical circumstances, Saka and Kusdiana found that methanol facilitated the transesterification of triglyceride (TG) into fatty acid methyl ester (FAME). It is well-known that three sequential reversible processes may be used to convert methanol into biodiesel. First, triglycerides are broken down into smaller molecules called diglycerides (DGs), and then the DGs are broken down further into monoglycerides (MGs). The last process is the transformation of MGs into glycerol. In every stage of the reaction, FAME is the end product. Transesterification results in the formation of three FAMEs, as seen in. The biodiesel generation in supercritical MTBE process is quite similar, consisting of three successive reversible reactions. Triglyceride combines with MTBE to form mono tert-butyl ether (DGE), and DGE then undergoes further reactions to form monoglyceride di tert-butyl ether (MGE). The end result of MGE reacting with MTBE is the production of FAME GTBE. Without the need for a catalyst, MTBE can transform TG into FAME. Although the supercritical MTBE approach operates at a higher temperature, its FAME yield is lower than that of the supercritical methanol route under identical reaction conditions. When utilising supercritical MTBE, however, the yield of FAME reaches 95.4 wt% after a 12-minute residence period, which is almost identical to that achieved using the supercritical methanol approach. Surprisingly, the supercritical MTBE approach has a larger FAME yield than the supercritical methyl acetate route. Since MTBE is less polar than methyl acetate, it is more reactive. Thus, at ambient temperature and pressure, MTBE is more miscible with oil than methyl acetate. In this case, MTBE's miscibility helps it get around the mass transfer issue.

## **4.3 Glycerol-free biodiesel using methyl acetate as acyl acceptor with bio-enzymes**

Using enzymes as the biocatalyst might potentially reduce downstream separation costs since they eliminate the requirement for a separate solvent. Novozyme 435 has

*Biotechnological Interventions for the Production of Glycerol-Free Biodiesel DOI: http://dx.doi.org/10.5772/intechopen.108895*

**Figure 7.** *The use of dimethyl carbonate to catalyse the enzymatic transesterification of vegetable oil.*

been used in almost all published reports of enzymes for synthesis utilising dimethyl carbonate as an acyl acceptor, either as a free enzyme in solution or inactive on a poly-acrylic macro-porous substrate. Novozymes 435 includes lipase B from *Candida antarctica*, which is an approximate lipase. The summary of recently published research on the use of dimethyl carbonate to catalyse the enzymatic transesterification of vegetable oil is given here (**Figure 7**, **Table 1**).

## **4.4 Glycerol-free biodiesel using dimethyl carbonate as acyl acceptor with bio-enzymes**

The environmental friendliness, safe for industrial use (low pressure and temperature is needed), low cost of feedstock (glycerol or glucose), and high theoretical molar yield of the biological conversion of glycerol to 1,3-propanediol are making it a more viable option than the chemical production approach. The sustainability of 1,3-propanediol's biotechnological production is enhanced by the fact that it may be made from a renewable resource. Although the fermentation by bacteria that converts glycerol to 1,3-propanediol has been investigated for about 120 years, its biotechnological potential has just been recognised and further study has only begun after 1990 [2]. Multiple microbial families have been documented as producing 1,3-propanediol since 1996. *Klebsiella*, *Enterobacter* [14], *Clostridium butyricum*, and *Klebsiella pneumoniae* stand out as the most promising producers due to their high substrate tolerance, abundant output, and high rates of production. Dual routes ferment glycerol through a dismutation mechanism. One of these routes involves the enzyme glycerol dehydrogenase converting glycerol to dihydroxyacetone, which then


#### **Table 1.**

*Recent research on dimethyl carbonate as an acyl acceptor in non-supercritical biodiesel manufacturing.*

goes through conventional glycolysis to generate pyruvate and may be further broken down into a variety of compounds such as acids and alcohols. The second process requires the reduction of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol through the expenditure of decreasing power NADH2 and the catalytic action of 1,3-propanediol:NAD oxidoreductase. This reduction requires coenzyme B12. Glycerol dehydratase is a critical limiting enzyme in the second metabolic pathway, which converts glycerol to 1,3-propanediol and is essential for cellular redox homeostasis. Theoretically, glycerol's anaerobic fermentation yields its greatest potential when acetate is the sole by-product [15–17]. The creation of waste substances not only limits the quantity of carbon accessible, but may also stifle the development of microbes.

Since acetic acid is necessary for the NADH2 process, the generation of 1,3-propanediol is reduced in tandem with the formation of all these other byproducts, especially ethanol and butanol. The bioconversion of glycerol to 1,3-propanediol has gained popularity since glycerol might sometimes be a surplus product. In the past 10 years, a lot of work has gone into making this procedure more efficient and raising the yield response. Research into genetically engineered strains for 1,3-propanediol synthesis has been conducted in an effort to boost the efficiency of naturally occurring producers during glycerol fermentations. Another issue with genetically modified microorganisms is that it is difficult to conduct fermentations on an industrial level with these

strains since they are so sensitive and fragile. If petroleum supplies are depleted, biotechnology might be the key to continuing 1,3-propanediol manufacturing [18].

## **4.5 Bioconversion of glycerol by** *C. butyricum*

The age of *Clostridium*, a kind of bacterium, is estimated to be about 2700 million years. This organism predated Earth's oxygen atmosphere, which is why *Clostridium* species are oxygen-sensitive. For almost 60 years, scientists have known that anaerobic bacteria, including *Clostridium*, may ferment glycerol into 1,3-propanediol. All member microorganisms in this genus have rod-like shape that is Gram-positive, moderately big, heterotrophic, endospore producing, and motile. Some are psychotropic or thermopholic, but mesophilia is the norm for the vast majority. *Clostridium* thrives in anaerobic settings rich in organic resources. Clostridia bacteria are present everywhere in the environment and may be discovered in a diversity of habitats, including soils, feed, aquatic sediments, and the digestive systems of humans and animals. They can remain alive for extended periods of time in hostile environments because of their spore-forming abilities. As long as they are kept in a medium that provides them with food and the right temperature and humidity, a single bacterial strain may proliferate. Among the roughly 100 species that make up the genus *Clostridium* are both common, free-living bacteria, and serious diseases. *Clostridium* produces a wide variety of extracellular enzymes, which contribute to their robust metabolic activity. Sugars may be fermented by

**Figure 8.** *Pathway of conversion of biomass into biodiesel.*

these bacteria, leading to the production of hydrogen and organic molecules such as organic acids (particularly acetic acids and butyric), acetone, and butanol. Clostridium produces offensive-smelling breakdown products during the metabolism of amino and fatty acids [19]. Additionally, Clostridia can break down a variety of hazardous compounds and create chiral products, both of which need much effort to get by chemical synthesis. There are several distinct enzymes for breaking down starch and hemicellulose, and they have been found in many non-identical types of bacteria. *Clostridium thermocellum* is a prototypical cellulolytic *Clostridium*, producing a multienzyme cellulase complex that can break down cellulose, hemicellulose, and starch [20]. Using clostridial toxins and spores to treat human illness is a great advancement. Dystonias, involuntary muscular problems, pain, and other neurological conditions are treated with botulinum neurotoxin. Therapeutics are being developed to be delivered to tumours using Clostridia spore systems [21]. Clostridia have very basic nutritional needs. Typically, a complex nitrogen supply is necessary for optimal growth and solvent synthesis. There is a good deal of potential commercial interest in the non-pathogenic Clostridia. *C. butyricum* produces 1,3-PD and other byproducts during the fermentation of glycerol. *C. butyricum*'s anaerobic fermentation metabolic pathways produced by-products. These byproducts have a negative impact on *C. butyricum* development because they deplete the available carbon source (**Figure 8**).

## **5. Conclusion**

Advances in plant biotechnology and microbial genetics are speeding up because of the urgent need to provide a steady supply of resources. The growing cost of crude oil is having a negative impact on economies throughout the globe. Cutting down on fossil fuel consumption would significantly decrease the quantity of carbon dioxide generated. Just biodiesel and bioethanol have been recognised as viable fossil fuel replacements. Biofuel's significance cannot be overstated.

Feedstock and fuels which are liquids derived from resources that can be renewed will allow us to tap into the vast, as-yet-untapped potential of agricultural and forestry waste products. Bioethanol has become the biggest fermentation product in use today, demonstrating biotechnology's potential for large-scale chemical production. Environmental difficulty and political will are necessary but not sufficient to usher in a sustainable age, hampered by economic constraints. The price of a barrel of Brent oil has risen as high as 87.19 Euros in the last year. Biofuels like bioethanol and biodiesel also help the environment. EU leaders have decided to prioritise biofuels in their quest to meet their renewable energy goal of 20% of total energy consumption by 2020. Biodiesel is superior to petro-diesel in many ways: flash point, sulphur content, biodegradability, and aromatic content. Transesterification of vegetable oils typically employs homogeneous catalysts, but heterogeneous ones are a viable option for this process.

Financial incentives for both producers and consumers are still needed to increase biofuel usage. The yearly fabrication and use of biodiesel in Germany exceed 2.5 billion litres. The biofuel industry is experiencing a period of technical revolution that is largely influencing the range of materials amenable to use in production processes. The US and Brazilian markets account for the bulk of bioethanol sales, but biodiesel use has surged in the EU. Biodiesel use is predicted to rise steadily over the next decade, helped by new regulations requiring the labelling of some mixtures.

## *Biotechnological Interventions for the Production of Glycerol-Free Biodiesel DOI: http://dx.doi.org/10.5772/intechopen.108895*

Glycerol is a colourless, odourless, hygroscopic, and viscous liquid with the chemical formula OCH2CHOHCH2OH. It is a versatile chemical that may be used in a wide variety of industries due to its solubility in water and its lack of toxicity when applied topically. In the construction, automotive, and textile sectors, it is used to make gums and resins.

## **Author details**

Muhammad Saeed1 , Ghulam Mustafa1 , Faiz Ahmad Joyia1 , Aneela Shadab1 and Aqsa Parvaiz2 \*

1 Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad, Pakistan

2 Department of Agricultural Biochemistry and Biotechnology, The Women University Multan, Multan, Pakistan

\*Address all correspondence to: aqsapervaiz333@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Wilke D. Chemicals from biotechnology: Molecular plant genetics will challenge the chemical and the fermentation industry. Applied Microbiology and Biotechnology. 1999;**52**:135-145

[2] Zeng A-P, Biebl H. Bulk chemicals from biotechnology: The case of 1,3-propanediol production and the new trends. Advances in Biochemical Engineering/ Biotechnology. 2002;**74**:239-259

[3] Bala BK. Studies on biodiesels from transformation of vegetable oils for diesel engines. Energy Education Science and Technology. 2005;**15**:1-45

[4] Yan S, DiMaggio C, Mohan S, Kim M, Salley SO, Simon Ng KY. Advancements in heterogeneous catalysis for biodiesel synthesis. Topics in Catalysis. 2010;**53**:721-736

[5] Silva GP, Mack M, Contiero J. Glycerol: A promising and abundant carbon source for industrial microbiology. Biotechnology Advances. 2009;**27**(1):30-39

[6] Mota C, Silva C, Gonçalves V. Gliceroquímica: Novos produtos e processos a partir da glicerina de produção de biodiesel. Quimica Nova - QUIM NOVA. 2009;**32**. DOI: 10.1590/ S0100-40422009000300008

[7] Wang Z-X, Zhuge J, Fang H, Prior BA. Glycerol production by microbial fermentation: A review. Biotechnology Advances. 2001;**19**:201-223

[8] Agarwal GP. Glycerol. Advances in Biochemical Engineering/Biotechnology. 1990;**41**:95-128

[9] Mu Y, Xiu ZL, Zhang DJ. A combined bioprocess of biodiesel production by lipase with microbial production of

1,3-propanediol by *Klebsiella pneumoniae*. Biochemical Engineering Journal. 2008;**40**:537-541

[10] Thompson JC, He BB. Characterization of crude glycerol from biodiesel production from multiple feedstocks. Applied Engineering in Agriculture. 2006;**22**(2):261-265

[11] Barbirato F, Himmi EH, Conte T, Bories A. 1,3-propanediol production by fermentation: An interesting way to valorize glycerin from the ester and ethanol industries. Industrial Crops and Products. 1998;**7**:281-289

[12] Yazdani S, Gonzalez R. Anaerobic fermentation of glycerol: A path to economic viability for the biofuels industry. Biotechnology. 2007;**18**:213-219

[13] Miller-Klein Associates. Impact of biodiesel production on the glycerol market. 2006. Available from: www.hgca. com

[14] Zhu M, Lawman PD, Cameron DC. Improving 1, 3-propanediol production from glycerol in a metabolically engineered *Escherichia coli* by reducing accumulation of snglycerol-3-phosphate. Biotechnology Progress. 2002;**18**:694-699

[15] Chotani G, Dodge T, Hsu A, Kumar M, LaDuca R, Trimbur D. The commercial production of chemicals using pathway engineering. Biochimica et Biophysica Acta. 2000;**1543**:434-455

[16] Saxena RK, Anand P, Saran S, Isar J. Microbial production of 1,3-propanediol: Recent developments and emerging opportunities. Biotechnology Advances. 2009;**27**:895-913

[17] Zeng A-P. Pathway and kinetic analysis of 1,3-propanediol production *Biotechnological Interventions for the Production of Glycerol-Free Biodiesel DOI: http://dx.doi.org/10.5772/intechopen.108895*

from glycerol fermentation by *Clostridium butyricum*. Bioprocess Engineering. 1996;**14**:169-175

[18] Cho M-H, Joen SI, Pyo S-H, Mun S, Kim J-H. A novel separation and purification process for 1,3-propanediol. Process Biochemistry. 2006;**41**(3):739-744

[19] Buckel W. On the road to bioremediation of 'dioxin'. Chemistry & Biology. 2005;**12**:723-724

[20] Mitchell WJ. Physiology of carbohydrate to solvent conversion by Clostridia. Advances in Microbial Physiology. 1997;**39**:31-130

[21] Johnson EA. Clostridial toxins as therapeutic agents: Benefits of nature's most toxic proteins. Annual Review of Microbiology. 1999;**53**:551-575
