It has been known for some time that some anaerobic bacteria can convert carbon monoxide, carbon dioxide and hydrogen into a mixture of ethanol, butanol and acetic acid. The pathway is shown in Fig. 6.15 where acetyl-CoA is produced via the acetogenic pathway (Henstra et al., 2007). Synthesis gas is a mixture of carbon mon­oxide and hydrogen produced by the gasification of coal, biomass and wastes. In the first step the oxidation of carbon monoxide and hydrogen yields protons which are used to reduce carbon dioxide to formate. The formate bonds to tetrahydrofolate (THF), and this complex adds more protons finishing with a methyl-THF complex.

f 128

Steam, power Fig. 6.13. The logen process for producing ethanol from lignocellulose. (Redrawn from Cardona and Sanchez, 2007.)

This complex reacts with carbon monoxide and CoA to produce acetyl-CoA cata­lysed by acetyl-CoA/carbon monoxide dehydrogenase. If insufficient carbon mon­oxide is available it can be produced from carbon dioxide. The production of acetyl — CoA has a negative energy balance which is recovered by the reduction of acetate formed from acetyl-CoA to ethanol. Butanol is formed from acetoacetyl-CoA. The overall balance in the formation of ethanol from syngas with an equimolar mixture of carbon monoxide and hydrogen is two-thirds of the carbon monoxide can be con­verted into ethanol (Rajagopalan et al., 2002).

The bacteria that have been demonstrated to be capable of growth on hydrogen and carbon monoxide include both mesophilic and thermophilic bacteria and Archaea. Examples of the mesophiles are Clostridium autoethanogenum and Eubacterium

129

Fig. 6.14. The NREL system for simultaneous saccharification and co-fermentation (SSCF). (Redrawn from Cardona and Sanchez, 2007.)

methylotrophicum; thermophiles are Moorella thermoacetica, Archaea, and Methanosarcina acetivorans.

Recently the anaerobic fermentation of syngas has been developed from a labora­tory process to a commercial process by Bioengineering Resources Inc. (BRI, 2007). An outline of the process is given in Fig. 6.16. A single module will process 100,000 t of biomass producing 6-8 million gallons of ethanol (US) and 5-6 MW of energy per year. The energy is derived from heat recovered from the gasifier as the gas has to be cooled to around 37°C before it is introduced into the fermenter and the exhaust gases from the fermenter. Distillation gives 95% ethanol and a molecular sieve is used to separate the remaining 5% water. There are no details of whether this process is more efficient and economical than the conversion of lignocellulose biomass into sugar or the Fischer-Tropsch process.

f 130

Acetate I	Acetoacetyl-CoA I
l Ethanol	l Butanol

Fig. 6.15. Reductive acetyl-CoA pathway. THF, tetrahydrofolate. (From Henstra etal., 2007.)

Butanol

Butanol is another alcohol which has been considered as liquid fuel as it has similar properties to ethanol (Table 6.2) but has a higher energy content. Butanol will give a higher mileage and can be mixed at any proportion with petrol. Butanol has been used as an industrial solvent, paint thinner and a component of brake fluids. It is less corrosive than bioethanol and can be transported through existing pipeline whereas ethanol has to be carried in tankers, by rail or on barges. It is also safer as it has a higher flash and boiling point. With all these advantages over ethanol it is perhaps not surprising that a number of ethanol plants have switched to butanol. BP, DuPont and British Sugar’s plant in Wissington is being converted from ethanol to butanol and Virgin Fuels are interested in butanol produced from cellulose.

At present much of the butanol used is produced from petrochemicals but there is a renewable method of producing butanol using microorganisms. The biological produc­tion of butanol was first observed in 1861 when Pasteur isolated a butyric acid produc­ing bacterium. Studies also showed that acetone was also formed and the organism was

Molecular sieve Fig. 6.16. An outline of the production of ethanol from biomass via gasification. (Redrawn from BRI, 2007.)

not able to grow in the presence of air. The industrial production of acetone and butanol by fermentation has a long history that started in 1914. Acetone and butanol were some of the first biotechnological products and the process that developed was one of the largest. Before 1914 acetone was produced by heating (dry distillation) calcium acetate. Calcium acetate was produced by the dry distillation or pyrolysis of wood. The wood distillate contained about 10% acetic acid that was either distilled off into calcium hydroxide to form calcium acetate or directly neutralized with lime. Between 80 and 100 t of wood was required to produce 1 t of acetone. In 1910 Chaim Weizmann had been working in Manchester as part of a group working for Strange and Graham Ltd trying to produce butanol by fermentation. Butanol was needed as it could be used to form butadiene, a precursor of synthetic rubber. At the time natural rubber was in short supply, as Brazil was the only source and they did not allow the export of rubber trees from their country. By 1914 Weizmann and co-workers had isolated an anaero­bic organism which was later named as Clostridium acetobutylicum that produced both acetone and butanol when grown on starch. In 1914, at the start of the First

132

World War, the demand for acetone increased rapidly as acetone was used as a solvent for nitrocellulose in the manufacture of cordite, a smokeless explosive. By 1915 the demand had exceeded supply and the Nobel Company approached Weizmann and the process of biological production of acetone was adopted rapidly. Brewing capacity was commandeered and by 1916 the bioreactor capacity had reached 700 m3.

At the end of the First World War the demand for acetone reduced but butanol was still in demand as a solvent for the nitrocellulose paints used in the rapidly developing motor industry. Acetone was also being used as a solvent in the production of aircraft dopes and for the production of textiles and isoprene. Only certain Clostridia are cap­able of producing reasonable levels of acetone and butanol and C. acetobutylicum has been the one most studied and used in industrial processes. C. acetobutylicum is a gram-positive anaerobic spore-forming rod 0.6-0.9 pm wide and 2.4-4.7 pm long. It is motile and will ferment arabinose, galactinol, fructose, galactose, glucose, glycogen, lactose, maltose, mannose, salicin, starch, sucrose, trehalose and xylose. The optimum growth temperature is 37°C. As the bacterium will form spores readily when the nutri­ents are exhausted it can be easily maintained as spores mixed with sterile soil. Loss of solvent-forming potential is a common problem with C. acetobutylicum cultures but heat treatment restores solvent-forming ability. The concentration of substrate normally used was 6.0-6.5% and the maximum yield of solvent formed was 37% of the substrate used. However, in practice the yields are around 30% with a ratio of buta- nol/acetone/ethanol of 6:3:1 with small amounts of hydrogen and carbon dioxide being formed as well. Thus 100 t of substrate will yield about 22 t of butanol. The yields depend upon a number of factors including the strain of microorganism, temperature, pH and substrate. In the 1930s, a bacterium C. saccharobutylicum was isolated which when grown on sucrose formed acetone and butanol only.

During the exponential phase little solvent is produced but butyric and acetic acids were formed causing the pH of the medium to drop from 6.0 to below 5.5 (Fig. 6.17). In the stationary phase the accumulation of acetone, butanol and ethanol

Fig. 6.17. The anaerobic production of acetone and butanol.

proceeds rapidly at the expense of the acids and therefore the pH rises. In culture C. acetobutylicum can be in three states: acidogenic where acetic and butyric acids are formed at neutral pH; solventogenic where acetone, butanol and ethanol are formed at low pH; and alcohologenic where butanol and ethanol are formed but no acetone at neutral pH, so that it is important to monitor or maintain pH.

Although molasses-based fermentations were more economical than the original starch substrate the expansion of the petrochemical industry from 1945 onwards meant that by the 1960s the process had ceased to be used. The reasons for the decline of the acetone/butanol process were:

• Low yield of solvents (30-35% of substrate).

• Low solvent concentration in medium due to the toxicity of butanol and ethanol at 20-25 g/l.

• Phage sensitivity.

• Autolysin-induced autolysis in stationary phase.

• Cost of distillation.

• Production of considerable amounts of waste.

• High cost of molasses.

• Petrochemical production was cheaper.

However, since the late 1990s the process has been reevaluated in the light of modern developments in genetic manipulation and waste treatment and the sudden increase in oil prices in 1973 (Durre, 1998). The reasons for the possible re-introduction are:

• The process uses renewable substrates.

• Butanol can replace ethanol as a liquid fuel.

• The newer strains can grow on waste starch and whey and metabolic engineering is being attempted so that it can be grown on cellulose.

• The waste can now be treated anaerobically forming biogas.

• The process may be able to operate at 60°C so that the solvents can be removed as they are formed.

• Solvent may be recovered during fermentation using reverse osmosis, perstrac — tion, pervaporation, membrane evaporation, liquid-liquid extraction, adsorption and gas stripping (Durre, 1998). Any process that avoids distillation will be con­siderably cheaper and able to compete with fossil fuels.

It will be interesting to see how ethanol and butanol develop as liquid fuels in the EU and UK as in the short term much of the ethanol will have to be imported from Brazil.

Conclusions

At present the infrastructure is in place to use liquid fuels and therefore the replace­ment or addition to petrol will be ethanol at least in the short term. The main prob­lem with ethanol is that when it is produced from starch considerable processing is required, which means a substantial input of energy. The most economical method is to make ethanol using sugar from sugarcane as in Brazil. However, the quantity of sugar needed to supply the volumes needed to replace ethanol may start to com­promise the rainforest in Brazil as more and more land is use to grow sugarcane. The

f 134

drivers for ethanol use are the directives in the EU and the USA to include ethanol in all petrol sold. If the amount of ethanol added to petrol is increased the increase in sugar and starch crops used for ethanol production may compromise food crops.

It is therefore important that ethanol production from the more abundant ligno — cellulose becomes industrial. Lignocellulose requires treatment and enzymatic degrad­ation before it can be converted into ethanol. This processing requires energy and increases costs and these need to be reduced before lignocellulose ethanol can com­pete. It may be that lignocellulose will not be able to compete with Fischer-Tropsch fuels from biomass and wastes. Butanol may also supersede ethanol as the liquid fuel of choice.