**1. Introduction**

410 Biodiesel – Feedstocks and Processing Technologies

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> These days the interest of fuels preparing from sustainable natural resources is continuously increasing due to the rising prices of the fossil fuels and the political instability in the oil producing countries. The fuels manufacturing from local vegetal resources can sustain the every country' prosperity, including rural, agricultural, economically disadvantaged regions. Nowadays only the bioethanol and the biodiesel are already produced at industrial level from sustainable raw materials.

> The biodiesel is manufactured by the chemically catalysed transesterification of the triglycerides from the vegetable oils, rapeseed oil in Europe and soya oil in USA. As the methanol is often used as alcohol reagent, the reaction is consequently named methanolysis. The most applied catalysts are alkalines (especially NaOH) or mineral acids. So the biodiesel represents the methyl esters of the fatty acids from the vegetable oils. The present diesel engines can normally use a mixture of diesel with 5% v/v biodiesel.

> Biodiesel contains virtually no sulfur or aromatics, and use of biodiesel in a conventional diesel engine results in substantial reduction of unburned hydrocarbons, carbon monoxide and particulate matter. The production and use of biodiesel, compared to petroleum diesel, resulted in a 78.5% reduction in carbon dioxide emissions. Moreover, biodiesel has a positive energy balance.

> The chemical transesterification applied at industrial level has important advantages, but also limitations: in spite of the high conversion yields and the short reaction duration, the global transformation is energetically intensive, the glycerol recovery is difficult, the alkaline catalyst must be separated, the wastewaters are to be treated by a rather complex procedure, and both the free fatty acids and water can badly influence the reaction.

> These unfavourable situations can be diminished by performing the enzymatic transesterification on conditions that: (a) the immobilised lipase used as biocatalyst must be as cheap as possible; (b) one can obtain the economic efficiency of the whole biotransformation process similar to that characteristic to the chemical process, these objectives being presented function of the research methodology and results. The comparison between the chemical way and the enzymatic way is presented in the Table 1.

Progress in Vegetable Oils Enzymatic Transesterification to Biodiesel - Case Study 413

The reaction can be realised either in organic solvents, or in solvent-free media (where there are only the substrates' mixture). Normally in organic solvents' systems the lipases can catalyse the biotransformation when the alcohol is added stepwise at the beginning (a "batch" system), by comparison with the free-solvent media, where the alcohol is added

The **key factors affecting the enzymatic transesterification** are presented in the Figure 1.

Fig. 1. Key factors of influence on the enzymatic transesterification (Antczak *et all*, 2009)

the enzyme recycling.

(Fjerbaek *et all*, 2009)

There are two categories of enzymatic biocatalysts: (1) extracellular lipases (i.e. the enzyme has previously been recovered from the cultivation broth and then purified) especially from the microbial producers *Candida rugosa*, *Candida utilis*, *Candida antarctica* and *Pseudomonas cepacia*, generally bacteria and yeasts; (2) intracellular lipases which still remain either inside or attached to the cellular wall; in both cases the enzymes are immobilized directly or together with the whole cell and this use can eliminate downstream operations and assure

The **extracellular lipases** are mostly produced by bacteria and yeasts and the large scale production of these lipases should be economical, fast, easy and efficient. Unfortunately, the cost of specific separation and purification operations is high enough. Still the majority of immobilized lipases that are commercially available are extracellular. The most commonly used is: Novozym 435 which is the lipase from *Candida antarctica*. Meanwhile the bacteria and yeasts can probably form growth associated lipases, in a first stage, linked to the cellular

When preparing the **intracellular lipases** the costly step of purification can be eliminated and this has led to using whole cells as biocatalysts. After the intracellular production of lipases the direct use of fungal cells immobilized within porous biomass support particles as a whole biocatalyst represents an attractive process for bulk production of biodiesel

The main criteria to choose between the two lipase types can be: (a) the bacteria and yeasts strains which biosynthesise extracellular lipases, can be considered as recommended producers based on the cultivation conditions, namely easy to apply and reproducible

membranes, then released into the cultivation medium as extracellular enzymes.

several times for maintaining a certain molar ratio with the oil concentration.


Table 1. Comparison between the alkaline catalysis and the enzymatic method for biodiesel preparation (Bajaj *et all*, 2010)

The now-a-day technological progress regarding the enzymatic transesterification is demonstrated by the realisation of 2 industrial pilots in China (Moore 2008a, 2008b; Uthoff *et all*, 2009) to apply this advanced methodology, though the biodiesel manufacture price still remains higher than the diesel price no matter the transesterification route, due to the raw materials high prices (Bisen *et all*, 2010). Developments to meet the economical framework are needed, including: (a) the introduction of the enzymatic transesterification of plant oils as a part from a comprehensive technology of complete valorisation of the vegetable oil, meaning the application of the bio refinery concept; (b) the increase of the available vegetable oil quantity with limited interference with the vegetable oils' food use; (c) the possible preparation of methanol from natural resources.
