David E. Leiva-Candia and M. P. Dorado

Abstract The biodiesel industry is gaining interest in the past years due to the depletion of the easily extracted petroleum, the increasing demand to the automotive market, and the environmental damage. It is acknowledged that the main obstacle to biodiesel marketing is the cost of production, which is mostly due to the price of the raw material (usually vegetable oils). In this way, the goal is to provide low-cost raw materials. This may be achieved by feedstocks that do not require arable land, do not depend on growing seasons, and that give added value to waste, helping also to its recycling. In this way, oleaginous organisms may be considered an alternative feedstock for the biodiesel industry, as they meet all the previous requirements. This chapter presents the state of the art and the main characteristics of the oil and bio­diesel provided by macroorganism (insects) and microorganism (bacteria, filamen­tous fungi, and yeasts).

1 Introduction

It is worldwide accepted that biodiesel is an attractive alternative to fossil diesel fuel in terms of exhaust emissions besides its renewable nature (Demirbas 2009). However, the market inclusion of first-generation biodiesel is controversial due to the “food versus fuel” discussion (Pinzi et al. 2009). Moreover, it is not economi­cally viable in the absence of both tax exemption and high petroleum-derived

D. E. Leiva-Candia • M. P. Dorado (H)

Department of Physical Chemistry and Applied Thermodynamics, University of Cordoba, Cordoba, Spain e-mail: pilar. dorado@uco. es

D. E. Leiva-Candia e-mail: z82lecad@uco. es

A. Domingos Padula et al. (eds.), Liquid Biofuels: Emergence, Development and Prospects, Lecture Notes in Energy 27, DOI: 10.1007/978-1-4471-6482-1_11, © Springer-Verlag London 2014

fuel prices (Janda et al. 2012), as a result of the high cost of the raw materials (60-75 % of the total cost of biodiesel) (Dorado et al. 2006; Gui et al. 2008). In this sense, research is focused on new renewable non-edible low-cost raw materi­als that do not need arable land. Second-generation biodiesel, mainly constituted by non-edible oil, waste oil, and animal fat-based biodiesel, partially complies with the above requirements, as in some cases, it requires land to produce the raw materi­als. Third-generation biodiesel uses non-edible oleaginous alternative sources fully independent of climate or availability of land. Among the possibilities, there is a novel source of raw materials composed by macro — and microorganisms that are able to produce oil.

In the category of macroorganisms, insects show a great potential in terms of fat accumulation, in some cases above 25-30 %, especially during the immature stages (larva, pupa, and nymph) (Manzano-Agugliaro et al. 2012). The fat contents of oleaginous insects vary according to the species and location, being Coleoptera and Lepidoptera species the ones that provide the highest amount of fat (Ramos — Elorduy 2008). Insects have shown a high potential to replace oleaginous seeds as raw material for biodiesel production, due to their high food efficiency, high repro­duction rate, and short life cycle (Li et al. 2012). Furthermore, biodiesel derived from insect oil fulfills both ASTM D6751 and EN 14214 standards (Leung et al. 2012; Li et al. 2012).

Microbial oil or single-cell oil proceeds from different oleaginous microorgan­isms, i. e., bacteria, fungi, and microalgae (Li et al. 2008). These microorganisms are able to accumulate intracellular lipids above 20 % of their dry cell weight. Besides, they do not require arable land and allow the recycling of residual bio­mass, as it can be used as a carbon source (Azocar et al. 2010). The accumu­lation of lipids depends on the kind of microorganism, culture conditions, and the relation C/N, as under nitrogen limitation, the accumulation of oil increases. The oleaginous microorganisms are able to consume a variety of carbon sub­strates following different metabolic pathways (Xu et al. 2013). Currently, tech­nologies for the production of microbial oil are still in pilot scale, i. e., Nestea Oil Company uses waste as medium and expects commercial production after 2015 (Neste oil 2012).

The potential use of microbial oil as a feedstock for the biodiesel industry is surrounded by a great expectation, as oleaginous microorganisms can be grown in conventional microbial bioreactors, improving the biomass yield and reduc­ing the cost of produced biomass and oil (Vicente et al. 2009). For the reasons mentioned above, this chapter includes the main characteristics and properties of microbial oil, with special focus on the use of waste as substrate and the sub­sequent biodiesel. Microalgae have been removed from this chapter as the sole explanation of the cultivation technology requires a fully dedicated chapter.

The commercial potential for microalgae represents a largely untapped resource, once there is a huge number of algae species. Some microalgae are mainly used to human nutrition, but are suitable for preparation of animal feed supplements. Like a biorefinary, it is possible to produce from biofuel and coproducts (espe­cially glycerin) to pigments and nutraceuticals.

The production of microalgae started in the early 1960s with the culture of Chlorella as a food additive and had expanded in others countries (Japan, USA, India, Israel, and Australia) until 1980s (Brennan and Owende 2010). The oil (tri­glycerides) extract from microalgae Chlorella, produced by dark fermentation, has high nutrient value and protein content, and their omega-3 fatty acid—DHA has been used as an ingredient in infant formulas (Brennan Owende 2010; Benemann 2012). D. salina is exploited for its beta-carotene content. Many strains of cyanobacteria (e. g., Spirulina) have been studied to “produce the neurotoxin b-N-methylamino — L-alanine (BMAA) that is linked to amyoptrophic lateral sclerosis-parkinsonism dementia complex, Lou Gehrig’s disease (ALS), and Alzheimer’s disease” (Brennan and Owende 2010, p. 572). The human consumption of microalgae biomass is restricted to very few species (Chlorella, Spirulina, and Dunaliella species domi­nate the market) due to the strict food safety regulations, commercial factors, market demand, and specific preparation. According to Subhadra and Edwards (2011),

a market survey of global algal producers indicated that more companies are planning to grow algae and extract the O3FA to market to consumers […] an immediate market of 0.2-0.4 million ton can be foreseen for algal based O3FA. A small portion can be further refined for marketing as human nutraceuticals and a significant portion for fortifying the AM produced as a co-product by algal biofuel refineries.

In the end of biodiesel production, it is possible to obtain a significant amount of glycerin that “there is a clear existing market from many industries such as paint and pharmaceuticals.” Some studies “have also shown that glycerin in turn can be effectively utilized to grow more algal biomass, another viable method of using glycerin in algal biofuel industry” (Subhadra and Edwards 2011).

Although the microalgae biomass is being produced essentially to human nutritional products, perhaps it is most attractive as animal feeds (Benemann 2012). Algae are the natural food source of aquaculture species such as mol — lusks, shrimps, and fish. In addition, it assists the stabilization, improvement, and enhancement of the immune systems of this cultures (Brennan and Owende 2010). They possess high protein rate (typical 50 %), high energy content (~20 MJ/kg), high concentrations of astaxanthin (used in salmon feed), and valuable carotenoids (e. g., lutein—used in chicken feed). Microalgae have also a long chain of omega-3 fatty acids to replace fish meal/oil (Benemann 2012).

Due to the many problems associated with the implementation of second-genera­tion biofuels, initiatives are now undertaken to research third-generation biofuels that mainly make use of algal biomass as the feedstock (John et al. 2011). Algal

• Simple and well-known

production methods:

Produced directly from food crops by extracting the oils for use in biodiesel or producing bioethanol through fermentation

• Scalable to smaller production

capacities

• Experience with commercial

production and use in many countries

• Well-recognized feedstocks:

Wheat and sugar are the most widely used feedstock for bioethanol while oil seed rape for use in biodiesel

• Fungibihty with existing

petroleunr-based fuels

• Major issue is ‘fuel versus food’

• Produce negative net energy gains Releasing more carbon in their

production than their feedstock’s capture in their growth

• High-cost feedstocks lead to high-cost

production

• Low land-use efficiency

• Produces sustainable energy but

also can capture and store CCb

• Biomass materials, which have

absorbed CO2 while growing, are converted into fuel using the same processes as second-generation biofuels

• Require nretabohcally engineering

nricroalgae that can capture CCb and synthesize biofuels at the same time

• Technically very cumbersome and

commercially not viable

biomass is derived from both micro — and macroalgae and contains high amount of lipids. Such biomass has high potential as biodiesel precursors as they con­tain up to 70 % of oil on dry-weight basis (Demirbas 2011). However, it should be noted that all species of microalgae are not suitable for biodiesel production. Microalgae require low maintenance and are able to grow in wastewaters, non­potable water or water unsuitable for agricultural purpose, and even in sea water (Alp and Cirak 2012). The biomass can double in less than a day, and its produc­tion can be combined with CO2 from petroleum industries. The main limitation of microalgae-based biofuels is the requirement of large areas for their cultivation or costly photo-bioreactors. Moreover, such large units need to be located near the production unit, which is not feasible in many instances. The major decisions to be taken for setting up a microalgae-based biofuel production facility involve selec­tion of open or closed system and batch or continuous mode of operation. Algal biomass can be easily cultivated in open-culture systems such as lakes and ponds and in closed-culture systems like photo-bioreactors. However, both open-culture and closed-culture systems have their own merits and demerits. The closed-culture systems can be operated in either batch or continuous mode. Although continu­ous mode of operation seems convenient, it suffers from contamination and dif­ficulty in controlling the non-growth-related products. Among the macroalgae, the Laminaria spp. and Ulva spp. are the most important ones from the energy per­spective. On the other hand, there are at least 30,000 known species of microalgae. In brief, the supply chain of algae-derived biofuels includes biomass generation, harvesting, pretreatment, downstream processing, and market.

The overall schematic diagram of the apparatus used in this study is shown in Fig. 1. The experiment was carried out in a Pyrex glass reactor (volume 75 cm3) under atmospheric pressure at an isothermal temperature of either 350, 400, 450, or 500 °C. Nitrogen gas was continuously passed at a flow rate of 30 cc/min to purge the remain­ing air in the reactor to ensure inert atmosphere. In a typical run, 10 g of HDPE sam­ple and 5-10 % by weight of catalyst were blended together before being fed into the reactor. The reactor was heated to 120 °C in 60 min and held for 60 min at 120 °C. The nitrogen flow was then cut off, and the temperature was increased from 120 °C at a heating rate of 30 °C/min up to the desired temperature. The temperature of the polymer was measured with a thermocouple (Type J). The outlet of the reactor was connected to a water-cooling condenser maintaining at 20-25 °C. The gases (Cl-C5) were separated from the liquid oils (C5-C25) and then analyzed using a gas chromato­graph. The products from the reactor were collected over a period of 3-4 h.

In the past few years, biodiesel production from insect oil is gaining interest in the scientific community (Leung et al. 2012; Li et al. 2011a, b). This technology is based on the fact that many insects possess a lipid body rich in monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids (Rumpold and Schluter 2013). MUFA are among the preferred fatty acids for biodiesel production due to their ability to improve the engine behavior under cold weather conditions, besides bio­diesel oxidative stability (Pinzi et al. 2009).

The amount of lipids and the fatty acid composition of the insect depend not only on the species but also on the diet used to grow it (Manzano-Agugliaro et al. 2012; Belluco et al. 2013) (Table 1). For the selection of suitable insects to produce fats to be used as biodiesel feedstock, the following parameters should be considered: fat content, duration of the life cycle, requirements of space to grow, reproductive capacity, and low-cost feeding (Manzano-Agugliaro et al. 2012). In the search of more economical nourishment, it is important to select insects that are able to consume waste both to produce oil and for recycling purposes. Therefore, the insect Hermetia illucens, also known as black soldier fly (BSF), has been investigated as a source of oil for biodiesel production (Li et al. 2011b; Zheng et al. 2012a) and also for its capability for waste manage­ment (St-Hilaire et al. 2007). Li et al. (2011b) used BSF larvae for the bioconver­sion of diary manure on biodiesel and sugar. Results showed a consumption of 78 % of the initial value of manure (1,248.6 g of fresh manure) in 21 days. They produced 15.8 g of biodiesel and 96.2 g of sugar from 70.8 g dry BSF larvae. Other wastes, i. e., lignocellulosic materials, have been tested. Zheng et al. (2012a) analyzed different mixtures of restaurant solid waste (RSW), rice straw, and Rid-X (bacteria that facilitate the breakdown of the solid organic wastes). Considering a ratio of 7:3 (RSW/rice straw) plus 0.35 % v/v Rid-X, they achieved 35.6 % of biodiesel per dry insect biomass. Animal waste is another residue that may cause health hazards and environmental pollution. From this group, cattle, pig, and chicken manure have been used to grow BSF larvae (Li et al. 2011a). The highest BSF larvae growth (327.6 g) resulted in 98.5 g of crude fat and 91.4 g of biodiesel.

In another study, Chrysomya megacephaly, a necrophagous blowfly, during its larvae development, was fed with restaurant garbage for 5 days and achieved an oil content in a range from 24.40 to 26.29 % (Li et al. 2012). But the most impor­tant finding is the oil acid value, lower than that of most insects and close to that of vegetable oils (Table 2).

Regarding the production of fatty acid methyl esters (FAME) from insect oil, a two-step process has been implemented in most cases: acid esterification (due to the high acidity of the oil) followed by basic transesterification. Reaction param­eters including temperature, amount of catalyst, time, and methanol-to-oil molar ratio were optimized (Table 3). Results showed that insect oil-based biodiesel properties fulfilled the ASTM D6751 and EN 14214 standards in terms of cetane number, density, flash point, water content, (Table 4), although only a few met the European standard methyl esters content (>96.5 %), kinematic viscosity, alcohol content, and both the acid number value and the oxidation stability required by both standards.

As mentioned above, there are four main types of biomass used for liquid biofuels production: oleaginous (triglyceride source), sugary (sucrose to convert into a glu­cose source), starchy (natural polymer to convert into a glucose source), and cellu — losic (a natural polymer converted into a glucose source). Due to the varying nature of these materials and the processes for converting them to different liquid fuels, they all require different analytical profiles and techniques, each one is considered individually below.

1.1 Oleaginous Biomass

The oleaginous biomass has high contents of triglycerides or lipids, esters derived from glycerol and three fatty acids, within their seeds or grains (Fig. 1). Some free fatty acids may also be present. The chemical composition of the fatty acids within the triglycerides can vary, both with respect to the length of the alkyl chains and the degree of unsaturation depending on the biomass sources (Table 1). This composi­tion can also vary due to soil type, tillage, and climate conditions. The free fatty acids and triglycerides are converted to biodiesel by means of a transesterification reac­tion in the presence of a basic or acidic catalyst and an alcohol (Oh et al. 2012). The chemical composition of the oil along with the free fatty acid content affects both the

transesterification process and the properties of the biodiesel formed, and therefore, analysis of these properties are vital for different oleaginous biomass sources.

Triglycerides can represent 10-25 % m/m in vegetable oils (Gunstone 2004). Table 2 shows values of physicochemical properties from some agricultural species used for biodiesel production.

Some methylic and ethylic esters, observed in biodiesel after transesterification process, are as follows:

• Laurate, derived from lauric acid, C12:0, from palm oil;

• Myristate, derived from myristic acid, C16:0, from tallow;

• Palmitate, derived from palmitic acid, C16:0, cottonseed and palm oils;

• Estereate, derived from estearic acid, C18:0, from tallow;

• Linoleate, derived from linoleic acid, C18:2, C18:2, from cottonseed oil.

HO

Fig. 2 Chemical structure of sucrose, a disaccharide present in sugarcane (author). The D-glucose moiety is on the left, and the D-fructose moiety is on the right linked by a-|3-D-disac — charide bonds. Author Sflvio Vaz Jr

As stated before, like a refinery, it is still possible to obtain other products in the cultivation of microalgae, such as methane, biohydrogen, and ethanol. Some examples of these possibilities are presented as follows.

Methane. Since early studies on microalgae biofuels, the production of meth­ane biogas by anaerobic digestion of biomass was a main focus (Benemann 2012). This microbial conversion (of organic matter into biogas) produces a mix­ture of methane, CO2, water vapor, small amounts hydrogen sulfide, and some­times hydrogen (Gunaseelan 1997 in Huesemann et. al. 2010). This process has been successfully and economically viable despite the recalcitrance of some algal species to biodegradation and inhibition of the conversion process by ammo­nia released from the biomass. (Benemann 2012; Huesemann et al. 2010). For Huesemann et al. (2010),

Methane generation by anaerobic digestion can be considered to be the default energy conversion process for microalgal biomass, including algal biomass produced during wastewater treatment and for the conversion of residuals remaining after oil extraction or fermentation to produce more valuable liquid fuels.

Hydrogen. There are three main processes to produce hydrogen from microalgae: dark fermentation; photo-fermentation, and biophotolysis. The first involves anaer­obic conversion of reduced substrates from algae, such as starch, glycogen, or glycerol into hydrogen, solvents, and mixed acids. The second, these organic acids “can be converted into hydrogen using nitrogen-fixing photosynthetic bacteria in a process called photofermentation.” The latter, a biophotolysis process uses micro­algae to catalyze the conversion of solar energy and water into hydrogen fuel, with oxygen as a byproduct (Huesemann et al. 2010). Although these mechanisms were successfully proven in laboratory scale, they have not yet been developed as a practical commercial process to produce hydrogen from algae (Huesemann et al. 2010; Ferreira et al. 2013).

Ethanol. On the other hand, ethanol can be generated from two alternative processes: storage carbohydrates (fermented with yeast) and endogenous algal enzymes (Benemann 2012; Huesemann et al. 2010). The main process is “yeast fer­mentation of carbohydrate storage products, such as starch in green algae, glycogen in cyanobacteria, or even glycerol accumulated at high salinities by Dunaliella.” A self-fermentation by endogenous algal enzymes induced in the absence of oxy­gen has been reported for Chlamydomonas. Against the very low ethanol yield from both fermentation, several private companies are now reported to be developing ethanol fermentations.

Electricity and Gasification. The microalgae biomass can be dried and com­busted to generate electricity, but the drying process is fairly expensive even if solar drying is employed. The combustion and thermal process can destroy the nitrogen fertilizer content of the biomass and generate elevated emissions of NOx. In addition, the combustion process competes with coal and wood biomass that are cheaper than microalgae biomass (Huesemann et al. 2010). Although expen­sive, this can be a key factor for algae to achieve energetic balance and improve its sustainability. A lot of research is being carried in new and more effective drying techniques in order to reduce costs.

Oil. The significant quantities of neutral lipids, primarily as triacylglycerols, can be extracted from the biomass (green algae and diatoms) and converted into biodiesel or green diesel as substitutes for petroleum-derived transportation fuels. “Lipid biosynthesis is typically triggered under conditions when cellular growth is limited, such as by a nutrient deficiency, but metabolic energy supply via pho­tosynthesis is not” (Roessler 1990 in Huesemann et al. 2010). Further information on algae biodiesel is presented in the next chapter.

Wastewater Treatment. The nutrients for the cultivation of microalgae can be obtained from liquid-effluent wastewater (sewer); therefore, besides providing its growth environment, there is the potential possibility of waste effluents treatment (Cantrell et al. 2008). This could be explored by microalgae farms as a source of income in a way that they could provide the treatment of public wastewater and obtain the nutrients the algae need.

In particular, algae has a potential for recycling nutrients recovered from the wastewater (removing N and P), achieving higher level of treatment and gener­ating biomass. Compared to the conventional water treatment, these processes reduce overall greenhouse gas emissions, burning of digester gas derived from anaerobic digestion.

Biomitigation of CO2 emissions. In the majority of microalgae cultivation, carbon dioxide must be fed constantly during daylight hours. Algae biofuel pro­duction can potentially use CO2 in the majority of microalgae cultivation as car­bon dioxide must be fed constantly during daylight hours. Algae facilities can potentially use some of the carbon dioxide that is released in power plants by burning fossil fuels. This CO2 is often available at little or no cost (Chisti 2007). Thus, the fixation of the waste CO2 of other sorts of business could represent another source of income to the algae industry. This sort of fixation is already being made in some large algae companies in a trial basis though there is a lack of public data of the results yet. Although this is a very promising future possibility, and some species have proven capable of using the flue gas as nutrients, there are few species that survive at high concentrations of NOx and SOx present in these gases (Brown 1996). Public policies could also perform a great boost in this area depending on future CO2 cap and trade emissions or sustainability standards as shown in Chap. “Governance of Biodiesel Production Chain: An Analysis of Palm Oil Social Arrangements”.

There are several production routes for advanced liquid biofuels; however, none have yet reached the fully commercial stage. An overview of the biomass-derived biofuels production is shown in Fig. 1. Biomass is produced via photosynthesis, which is then processed either by biochemical or thermochemical routes to make liquid biofuels like bioalcohols, biodiesel, and biosynfuels. The biorefinery con­cept, usually based on either biochemical — or thermochemical routes, is exploited to produce biofuels from single or multiple feedstocks with value-added co­products and heat and power generation (IE A 2008). In fact, the production of high-value chemicals and bulk quantities of low-value biofuels maximizes the return from biomass feedstock, thereby improving the economic performance of advanced biofuels in a similar fashion as do the oil refineries nowadays. There is no single technology as of now that can use any feedstocks for biofuels process­ing; therefore, on-going research at laboratory, pilot, and demonstration plant is warranted. Such initiative will perfect the processes and technologies tailoring them to different feedstocks. At the moment, it is not clear, which feedstocks,

(Electricity)

Fig. 2 A network illustration to show the applications of products from thermochemical and biochemical conversion routes processes, and pathways will yield the minimal-cost biofuels or otherwise have the maximum potential for cost reductions over time. A network diagram to illustrate the application of products from biochemical and thermochemical routes using biomass feedstocks is shown in Fig. 2.

Both gases and liquid oil products from the reactor were analyzed using a Hewlett- Packard GC equipped with a Supelco Plot Q column and a GC/MS, respectively. Similarly, the liquid oils could also be analyzed using a gas chromatograph with a flame ionization detector while the gaseous products were analyzed using gas chromatograph with a thermal conductivity detector. In addition, some of the car­bonaceous compounds that adhered on the cooling glass tube were eliminated using и-hexane and were measured as waxes. The mass of coke deposited on the catalysts after the degradation was determined by weight difference of the catalyst before and reheating the catalysts at 600 °C for 5 h. In addition, the amount of coke deposit on the catalyst could also be calculated by measuring the desorbed amount of carbon dioxide during temperature programmed oxidation of the used catalysts.

1.1 Bacteria

The synthesis of intracellular lipids in oleaginous bacteria occurs during the log­arithmic phase and the beginning of the stationary growth phase (Gouda et al. 2008). However, only few species of bacteria can accumulate lipids suitable for biodiesel, as they mainly accumulate polyhydroxy alkanoates (PHA) and poly­hydroxy butyrate (PHB) (Kosa and Ragauskas 2011; Shi et al. 2011). The spe­cies that produce a large amount of lipids are those belonging to Streptomyces, Nocardia, Rhodococcus, and Mycobacterium (Alvarez and Steinbuchel 2002). The amount of triglycerides (TAG) and fatty acid composition differs depending on the species used for fermentation (Table 5). Gouda et al. (2008) tested Rhodococcus opacus and Gordonia sp. using different agroindustrial wastes (molasses, potato infusion, wheat bran, hydrolyzed barley, orange waste, tomato peel waste, arti­choke waste, and Na-gluconate) as carbon sources. Molasses provided the high­est percentage of lipid in cell, 93 and 96 % for R. opacus and Gordonia sp.,

respectively, while carob waste offered the best source for TAG accumulation, being 88.9 and 57.8 mg per liter of medium for R. opacus and Gordonia sp., respectively, and C17:1 the main fatty acid produced (20.7 %) by R. opaccus. When Gordonia sp. consumed molasses, they followed the same trend in terms of the accumulation of lipid in cell mass (96 %). However, the highest accumulation of TAG (57.8 mg/L) was achieved when orange waste was consumed, being C22:0 the predominant fatty acid, in a percentage close to 35 %. Two different strains of bacterium R. opacus, DSM 1069 and PD630, were inoculated in lignocellulosic compounds (4-hydroxybenzoic and vanillic acids) (Kosa and Ragauskas 2012). The experiments showed that both strains can consume these carbon sources and accumulate lipids close to 20 % of their own weight.

With regard to bacterial biodiesel properties and subsequent engine testing, only one analysis has been reported (Wahlen et al. 2012). In this study, the bacte­rium R. opacus was grown in sucrose and biodiesel properties were compared with those from microalgae and yeast oil-based biodiesel. Biodiesel bacterial molecu­lar properties differ considerably with the other biofuels in terms of carbon chain length. The physical properties were similar to other microbial biodiesel, with the exception of the heating value that was lower. When bacterial biodiesel was ran on a diesel engine, it provided the lowest power output, while NOx and HC emissions were higher and lower than other microbial biodiesel, respectively.

Bacteria that accumulate the highest proportion of triglycerides are providing neither sufficient oil yield under industrial conditions nor an economically sound process. For these reasons, genetic engineering is supporting this biotechnology to be considered a viable alternative for the biodiesel industry. Rucker et al. (2013) demonstrated the feasibility of the lipid metabolism of E. coli for TAG accumu­lation, but the yield achieved was below the threshold to be considered a viable source for biodiesel production. Authors propose two metabolic engineering steps, to increase either the supply of phosphatidic acid during late exponential and sta­tionary phase growth or the supply of acyl-CoA.

One of the most interesting uses of bacteria in the production of biodiesel was described by Kalscheuer et al. (2006). In this study, the genetically modified bac­teria E. coli was recombined with two different enzymes from Zymomonas mobilis and Acinetobacter baylyi. The target was to produce fatty acid ethyl esters (FAEE) in vivo, called “microdiesel.” Under fed-batch fermentation using renewable car­bon sources, they achieved a FAEE concentration of 1.28 g L-1, corresponding to a FAEE content of the cells of 26 % of the cellular dry mass. Gordonia sp. KTR9 may be considered among the suitable bacteria for in vivo synthesis of fatty acid ethyl esters from short-chain alcohols. This species has a large num­ber of genes dedicated to both the formation of fatty acids and lipid biosynthesis. Furthermore, it tolerates the addition of more than 4 % methanol, 4 % ethanol, and 2 % propanol in the medium (Eberly et al. 2013).

It may be concluded from above works that biodiesel produced from bacterial oil can be considered as an alternative to first — and second-generation biodiesel. However, more research is needed to both improve bacterial oil yield and provide economically viable substrates.

D. E. Leiva-Candia and M. P. Dorado