Biological background

1.1.1 World energy crisis

When we concern about the problem in our life, we’ll find that the most important source will be exhausted and unable to regenerate in a few years. Because the resources are versatile, it is used as primary energy source.

World energy dependence is mainly depends on limited resource such as coal, oil or natural gas. With the economic progression, the energy demands are increasing in developed country and under-development country. According to the report from International Energy Agency in 2007, the demands are drastically increasing in recent ten years and will keep surging in the following ten to twenty years, see Figure1.1 (World Energy Outlook, 2007).

Owing to mass demands and the finite resource, the supply of energy falls short of demands.

Figure 1.2 shows the different fuel prices divided by oil prices in relation to the time scale, which implied that oil price is increasing with time in every way (World Energy Outlook, 2007). It is evident that shortage of this limited natural resource results in energy crisis in the world. Fortunately, there are renewable energy generated from nuclear, hydro and biomass.

Figure 1.3 shows the renewable energy constitutes about 80% of bio-energy, which was energy derived from biomass (World Energy Outlook, 2003). Thus the bio-energy may provide a way to lessen the world energy crisis.

Figure 1.1 World primary energy demand in the reference scenario. (International Energy Agency, 2007)

Figure 1.2 Assumed ratio of natural gas and implied relation of coal prices to oil prices to oil prices in the reference scenario.

Figure 1.3 Ratio of renewable energy from different sources.

1.1.2 Glycerol for biofuels generation

Biofuels like biodiesel and bioethanol become a biological solution for generating renewable energy, which convert animal or vegetable oil to useful chemical compound biodiesel. The chemical structure and the procedure of biodiesel synthesis are shown at Figure 1.4. During the biodiesel production, byproduct glycerol also produced. The glycerol produced from biodiesel production became competitive compared with those generated from general glycerol factory.

Glycerol is commonly called glycerine or glycerin which was used as materials for flexible foams, serves as humectants, and as a thickening agent in liqueurs. Since 2004, the price of glycerol was cut down dramatically for biodiesel expand vigorously that shown in Figure 1.5. Overproduction of glycerol changed the strategy of glycerol factory such as P&G, Uniqema, Dow Chemical and Cognis. Therefore, glycerol was developed for additional role like sources of hydrogen gas [1] or convert to ethanol [2] for saving the energy crisis.

Previous study showed a lot of research works have been done in application of cellulose as carbon source to biofuels. But only a few recent efforts have focus on glycerol as microbial carbon source. Compare with cellulose, glycerol economizes is not only the work of degradation into small molecular but also cost of operation that shown in Figure 1.6. [3]

Because of its availability, low prices, and high degree of reduction[2], glycerol become a good resource from biodiesel waste. The biofuels second generation aims to improve the efficiency of renewable energy production.

Figure 1.4 Biodiesel production and its byproduct glycerol.

Figure 1.5 US biodiesel production and its impact on crude glycerol prices [2]

Figure 1.6 Comparison of ethanol production from corn-derived sugars[2]

1.1.3 Microbes utilize glycerol

The microbes which using glycerol as carbon source in anaerobic condition have some characteristics in gene coding enzymes that utilizing glycerol[4]. That showed the capability of glycerol fermentation are related with 1,3-propanediol dehydrogenase and glycerol dehydratase. But the pathway of glycerol utilize not only use there two reaction that shown in Table 1.1, but also have two path that convert glycerol to glycolysis intermediates for growth biomass and produce fermentation product that shown in Table1.2.they can convert glycerol to 1,3-propanediol, but the yield of 1,3-propanediol from glycerol is not 100%.becuase of NADH and NAD concentration spend affected the reactions are reduction or oxidation . Therefore, glycerol passed through different reactions to achieve chemical and redox potential balance.

Contrast of their pathogenicity and application shown in Table 1.3, Klebsiella pneumoniae, Clostridium butyricum, Citrobacter freundii and Enterobacter gergoviae possess

intense pathogenicity, Lactobacillus reuteri is better for producing the antibiotic. Because of them, the safely and more feasible for gene modify microbe, Escherichia coli, that can ferment glycerol in special condition is suitable to be a biofuels synthesizer and recombinant host.

Table 1.1 Capability of glycerol fermentation and distribution of glyDH and 1,3PD-DH in enterbacterial species.

Glycerol fermentation 1,3-PD dehydrogenase Glycerol dehydratase Citrobacter braakii

Citrobacter farmeri Citrobacter freundii Citrobacter werlamanii

YES YES YES

Enterobacter gergoviae YES YES YES

Klebsiella pneumoniae YES YES YES

Clostridium pasteurianum YES YES YES

Table 1.2 Glycerol utilization pathway reactions.

Glycerol utilization pathway reactions Aerobic Anaerobic(have electron acceptor)

Fermentation glycerol→sn-glycerol-3-p→DHAP→glycolytic

intermediates

YES YES NO

glycerol→DHA→DHAP→glycolytic intermediates NO NO YES

glycerol→3-HPA→DHAP→,3-propanediol YES YES YES

Table 1.3 Microbial pathogenicity and applications which can ferment glycerol.

Species Pathogenicity Applications

Klebsiella pneumoniae Pulmonary disease, enteric pathogenicity, nasal mucosa atrophy, and rhinoscleroma

Lactose fermenting, facultative anaerobic

Clostridium butyricum Botulism, tetanus and gas gangrene Toxic chemicals and detergents

Lactobacillus reuteri Anti-microbial agent

Citrobacter freundii In clinical specimens as an opportunistic or secondary pathogen

Ability to convert tryptophan to indole, ferment lactose, and utilize malonate

Enterobacter gergoviae Nosocomial (hospital-acquired) urinary tract infections