For practical application of autotrophic production of P(3HB) from CO2 by A. eutrophus, the loss of substrate gas utilization efficiency concomitant with the exhaustion of the gas from fermenter, and the potential of explosion of the substrate gas mixture are very serious problems to be solved. A recycled-gas closed-circuit culture system attains high gas utilization efficiency by reusing the exhausted gas. Figure 2 shows the gas-recycling culture system in bench scale. This system could also eliminate the potential for the gas mixture to explode by maintaining the oxygen concentration in the gaseous phase below the lower limit for explosion(6.9 %(v/v)) and introducing several other safety measures. Oxygen consumption by the cells in autotrophic synthesis of P(3HB) is very large as shown in the following equations, the decrease in the driving force for oxygen from the gas phase into the liquid phase then results in the serious decrease in P(3HB) productivity.

Exponential cell growth; 21.36 H2 + 6.21 O2 + 4.09 CO2 + 0.76 Nffi

-» C4.09H7.1 ЗО 1.89N0.76

P(3HB) formation; 33 H2O + 12 O2 + 4 CO2 —> C4H6O2 + ЗО H2O

Hence, a doughnut-shaped agitation system to attain a KLa of 2,970 h_1 was used in the bench plant to compensate for the decrease in oxygen transfer. As a result, cell and P(3HB) concentrations increased to 91.3 gednr3 and 61.9 gedm’3 respectively, under 02-limited condition after 40 h of cultivation (Fig.3). While the 62 concentration in the gas phase was maintained at very low level, the overall productivities of biomass and P(3HB) obtained in this cultivation were 2.28 g*dnr3* h_1 and 1.55 g-dnr^lr1, respectively, which were much higher than those reported for other autotrophic cultivation of hydrogen-oxidizing bacteria (Table I).

General Process. Figure 1 shows a schematic flowsheet of the enzymatic biomass — to-ethanol process. Lignocellulosic biomass consists mainly of cellulose, hemicellulose and lignin. In the production of ethanol the sugars in the hemicellulose and cellulose are converted to ethanol. As a by-product, lignin is formed which can be used as a solid fuel.

In the pretreatment stage the hemicellulose is solubilised into sugars. The cellulose structure is opened up and becomes more accessible to enzymes. Prior to pretreatment, the raw material must be chipped. There are many types of pretreatment methods and steaming has been recognised as one of the most effective for lignocellulosics (12, 23-27). Although companies such as Stake Technology (Stake Technology Ltd, Canada) currently sell equipment for the steam pretreatment of wood, the technology is still considered to be in the development stage (28). Furthermore, the use of catalysts to enhance the cellulose accessibility and the yield of hemicellulose sugars of a number of feedstocks has been recognised (14, 15, 29, 30). If catalysts such as sulphuric acid or sulphur dioxide are used, the corrosive effects of long-term usage on the equipment must be considered. The effect on the quality of the lignin by-product, due to possible sulphonation, must also be taken into account. If a catalyst is employed, the raw material must usually be pre-steamed to expel trapped air from the pores in the material. This can be performed using low-pressure steam.

In the hydrolysis step, the cellulose is hydrolysed by enzymes into glucose. To reduce the cost of concentrating the end-product, a high sugar concentration is desirable, which means a high dry matter content of the cellulosic material entering the hydrolysis stage. However, a high dry matter content in the reactor makes stirring difficult which reduces mass transfer. Fed-batch hydrolysis, i. e. gradual feeding of the material into the hydrolysis vessel, is an option that may be attractive.

Large quantities of enzymes are required for the hydrolysis to be fast due to the low specific activity of the cellulase system. The cellulose-degrading enzymes should preferably be produced on the same raw material as that used for ethanol production. A minor part, about 5%, of the pretreated material is used as a carbon source for some cellulase-producing organism, such as Trichoderma reesei (31-33). Unfortunately, this fungus does not excrete sufficient amounts of one of the enzyme components, (i — glucosidase. As the consumption of P-glucosidase is much lower than that of the other cellulases, this enzyme may either be produced by another organism such as Aspergillus phoenicis (34, 35) or be purchased to avoid increased complexity in the process. Extensive research is necessary to find more effective enzymes which will reduce the hydrolysis time. It is also essential to find inexpensive substrates for enzyme production.

After hydrolysis, the solid residue is separated from the hydrolysate and washed. The residue consists mainly of lignin which can be dried and used as a high-quality solid fuel. Separation can be performed either through filtration in a filter press (36) or by using a decanting centrifuge (18). The sugars in the hydrolysate are then fermented to ethanol.

Hexoses, mainly glucose, mannose and galactose, are generally fermented by Saccharomyces cerevisiae in continuous fermenters with yeast recycling. The technology and equipment required for glucose fermentation in a biomass-to-ethanol facility are believed to be similar to those used in the sugar — and starch-to-ethanol processes or in the production of ethanol from spent sulphite liquors.

The pentose fermentation step is essentially still at the laboratory stage, although significant advances have been made in understanding the mechanism and in the modification of pentose-fermenting micro-organisms (37-39). None of the wild-type, adapted or engineered strains, have to our knowledge, been able to routinely ferment the pentose-rich, water-soluble stream obtained from steam-pretreated hardwoods or softwoods.

An alternative to separate hydrolysis and fermentation is simultaneous saccharification and fermentation (SSF), i. e. operation of the hydrolysis step in combination with the fermentation step. This operation reduces the end-product inhibition resulting from glucose and cellobiose build-up by continuously converting the glucose to ethanol. Although ethanol and other fermentation products may decrease the activity of individual cellulase enzymes, this inhibition is much weaker than the inhibition caused by equivalent glucose or cellobiose concentrations (40-42). The conditions for running SSF are a compromise between the optimal conditions for cellulose hydrolysis and those for fermentation. It is probably necessary to produce new yeast and enzymes for each SSF batch because of the difficulty in separating the cells and enzymes from the unhydrolysed solid residue (43).

The ethanol in the fermenter is rather diluted and must be recovered by distillation, which is considered to be a mature technology. The experiences from production of alcoholic beverages can be used to design and construct distillation units for recovery of fuel ethanol.

Four major by-product and liquid waste streams are expected from the enzymatic hydrolysis process: a waste stream from the pretreatment unit; spent fungal mycelia from the enzymatic production unit; unhydrolysed cellulose residue containing spent enzyme and lignin; and stillage from the distillation column which contains solubilised non-volatiles from the raw material, carbohydrate- and lignin — degradation products as well as by-products from the fermentation stage. The stillage waste stream is the largest in volume. It has high Biological and Chemical Oxygen Demands, (BOD7 and COD, respectively), and a low pH. It often contains high levels of colouring compounds and has been noted for its high corrosiveness (44). Biological treatment of the stillage stream generally involves anaerobic treatment prior to aerobic treatment and a tertiary treatment to remove colouration. Recycling of liquid streams would minimise the fresh water requirement and lower the amount of waste water produced. However, this recycling leads to an increase in the concentration of various substances in the hydrolysis and fermentation steps (45). To avoid the accumulation of non-volatile inhibitors in the process, the stillage stream could be evaporated prior to recirculation (36). The volatile fraction from the evaporation step is then recycled and the non-volatile residue incinerated. This is described in more detail in the sections ” Recycling of process streams” and ’’Process integration”.

Bench-Scale Unit. In scaling up, the rate-limiting steps must be identified. The rates of all other process steps must be carefully assessed to determine whether they may decrease on scale-up and become new bottlenecks on a larger scale. Since microbial processes are complex, it is important to evaluate how various recycling scenarios will affect the microbial environment in continuous fermentation in a fully integrated process. The final investigations should preferably be performed on a pilot scale, but it is often more cost-effective to evaluate various options and operating conditions first on a bench scale.

At Lund University, a bench-scale unit was set up in 1995 for the development of a process for ethanol production from lignocellulosics based on enzymatic hydrolysis (36). Figure 2 shows a schematic flowsheet of the unit. The different unit operations are not physically connected so the material is passed manually from one step to the next. This makes the unit very suitable for studying various process configurations and also the recycling of process streams.

The pretreatment unit consists of a 10-litre pressure vessel (corresponding to approximately 1 kg of dry wood chips) and a flash tank to collect the pretreated material. An electric steam boiler supplies steam up to a maximum pressure of 30 bar, corresponding to a saturated steam temperature of 235°C. The temperature, pressure, and hold-up time in the reactor are monitored and controlled via a computer.

Figure 1. Production of ethanol using enzymatic hydrolysis.

A laboratory filter press is used to filter and wash the pretreated material. The material is stored in a 100-L stirred tank, slurried with water, then pumped through a filter cloth at a maximum pressure of 10 bar. After filtration, the filter cake is dewatered by applying a pressure of 15-17 bar and then washed under pressure with water. The unit can be used to determine scale-up design data, such as the filter-cake resistance.

The hydrolysis unit consists of two tanks with working volumes of 20 litres and 40 litres. The tanks are stirred by propeller-type agitators which have been proven to give efficient mixing of pretreated material up to 10% dry matter (DM). Both vessels are equipped with a water jacket to maintain a constant temperature in the range 30- 90°C. The hydrolysis residue can be filtered in the filter press described above.

The enzyme production and fermentation units consist of fermenters with total volumes of 22 litre and 16 litre, respectively. The fermenters are equipped with sensors for control and data sampling of temperature, pH, dissolved oxygen and anti­foam addition. The outlet gas is analysed for oxygen and carbon dioxide.

The distillation unit consists of a 1.5 m packed column, corresponding to about 15-20 ideal stages, a jacket reboiler and a condenser. The distillation unit can be operated batchwise or continuously. The reflux ratio can be varied between 0.1 and

20.

The evaporator is designed for two different purposes: flash-sterilisation of fermentation broth, or concentration of the non-volatile components in liquids. Heat transfer is achieved via plate heat exchangers using live steam and cold water as heating and cooling media, respectively. The maximum capacity of the evaporator is 16 kg/h evaporated water. The evaporator can be used for the simulation of condensate withdrawal in a multiple-effect evaporator.

Experimental Runs. In the introductory experimental runs, willow, a fast growing energy crop, was used as substrate. The willow was pre-steamed with low-pressure steam (2 bar) prior to impregnation with gaseous sulphur dioxide in a plastic bag. The amount of sulphur dioxide added was approximately 4% of the DM. The impregnated material was then subjected to steam pretreatment at 205-210°C for 5 minutes. The pretreated material was slurried with water to approximately 5% DM, and filtered. After filtration, the filter cake was dewatered to approximately 40% DM.

Hydrolysis was performed using the fibrous material and the filtrate at a dry matter content of 5%, supplemented with Celluclast 2L and Novozym 188 (Novo AS, Denmark), corresponding to 15 FPU/g substrate. Commercial enzyme preparations were used, as the main purpose of the study was to investigate the influence of the recycling of waste streams. The temperature was maintained at 40°C and the pH at 4.8 during the entire hydrolysis time of 90 hours.

Fermentation of the willow hydrolysate was carried out in the 22-litre fermenter containing 16 litres medium at 30°C, pH 5.5, at a stirring speed of 300 rpm. The hydrolysate was supplemented with a rich medium, and inoculated with compressed baker’s yeast, S. cerevisiae, to a final cell concentration (DM) of 6 g/L.

The fermentation broth was stripped in the evaporation unit, prior to distillation, to avoid problems associated with suspended particles in the distillation column. The evaporated fraction was then distilled. The COD, the BODy and the fermentability of the stillage and the evaporation residue were determined.

The enzyme production was run in a parallel study. Enzyme production was performed in the 16-litre fermenter. A modified Mandels medium was used (46), in which the yeast extract and proteose peptone were replaced by dried yeast. A separate batch of willow, pretreated under the same conditions as described in the pretreatment section, was used as substrate for the enzyme production.

Some of the results from the experimental study in the bench-scale unit are described in the section ’’Recycling of process streams”. A more detailed description of the equipment and the experimental procedure can be found in the original publication (36). Similar experiments have also been performed using a mixture of softwoods (47).

Photosynthesis is the fundamental biological process that converts the electromagnetic energy of sunlight into stored chemical energy that supports essentially all life on Earth. In green algae as in higher plants, photosynthesis occurs in a specialized organelle, the chloroplast. Light energy captured by the photosynthetic reaction centers is stored predominately by reduction of C02, using water as the source of electrons. As illustrated in Figure 1, the key components of the photosynthetic apparatus involved in light absorption and energy conversion are embedded in thylakoid membranes inside the chloroplast. They are two chlorophyll (chl)-protein complexes, Photosystem I (PSI) with a reaction center, P700, and Photosystem II (PSII) with another distinct reaction center, P680. According to the current and prevailing concept of oxygenic photosynthe­sis, the Z-scheme, first proposed by Hill and Bendall (2) and now described in many textbooks (5-7), PSII can split water and reduce the plastoquinone (PQ) pool, the cytochrome (Cyt) b/f complex, and plastocyanin (PC), while PSI can reduce ferredoxin (Fd)/nicotinamide adenine dinucleotide phosphate (NADP+) and oxidize PC, the Cyt b/f complex, and the PQ pool. As a result, the electrons derived from water splitting are transferred to Fd/NADP+, which provides the reducing power for reduction of C02 to carbohydrate in the stromal region of the chloroplast by a series of enzymatic reactions collectively called the Calvin cycle. Electron transport in the membrane is coupled with proton transport from the stroma into the lumen, generating a proton gradient across the thylakoid membrane. The proton gradient drives phosphorylation through the coupling factor CF0-CF, to make essential ATP for the reduction of C02. This is the common description of oxygenic photosynthesis.

In many green algae, such as Chlamydomonas, there is a hydrogenase that can be induced under anaerobic conditions (8, 9). The hydrogenase can catalyze the reduction of protons to produce H2 using electrons from the reduced Fd as shown in Figure 1. Since protons are also produced by water splitting at PSII, the net result of this Fd/hydrogenase pathway is simultaneous photoevolution of H2 and 02, using water as the substrate and light energy as the driving force.

Under anaerobic conditions and darkness, H2 may be produced by fermentative metabolic degradation of organic reserves such as starch (Figure 1). This fermentative metabolic process has been well studied (70, 77). Since the substrate of the fermenta­tive pathway is generated photosynthetically by reduction of C02, the net result of the

Figure 1. Conventional photosynthetic pathway based on the Z-scheme.

sequential process, photosynthesis and fermentation, is still splitting water to H2 and 02, with C02 as an intermediate.

Cell Immobilization Studies. Cell immobilization can greatly increase the cell density in bioreactors. Immobilized-cell biocatalysts are well suited for energy — efficient bioreactor configurations, such as airlift and fluidized-beds. The suitability of several support matrices for immobilization of Butyribacterium methylotrophicum has been evaluated (34). The mass of cell protein immobilized was measured as a function of time for celite, wood powder, activated carbon, ion exchange resin, molecular sieves, and alumina during batch growth on CO in the presence of the support. The fluidization properties (bed expansion as a function of superficial liquid velocity), rate of product formation, and proportion of two — and four-carbon products were also measured for celite, ion-exchange resin and molecular sieves. All of these latter three supports were deemed satisfactory for immobilized-cell culture of B. methylotrophicum.

Continuous, Cell-Recycle Fermentations. High cell densities can also be achieved in bioreactors using cell recycle. This approach was evaluated during long-term, continuous CO fermentations by B. methylotrophicum, in which a

0. 3 ^im pore size cellulosic membrane was used in a cross-flow mode to achieve total cell recycle (77). Runs were conducted at different pH values, because previous experiments had shown that pH strongly regulates this fermentation (75,79), as evidenced by changes in relative proportions of the products. The experiments were performed with the same dilution rate used during previous continuous runs done without cell recycle (19) to study the effect of cell density on reactor productivity. At pH values of 7.2, 6.8, 6.4, and 6.0, steady-state was achieved. At pH values of 5.75 and 5.5, oscillations in the concentrations of several fermentation products were observed. A viable culture could not be maintained at pH values below 5.5.

Butyribacterium methylotrophicum has perhaps the most versatile metabolic capabilities of known microbes capable of anaerobic CO metabolism. It grows on a wide range of carbon and energy substrates, including 100% CO, H2/CO2, methanol, formate, and glucose (20). When grown on CO, it produces acetate, butyrate, ethanol, and butanol as catabolic products. The direct pathway from CO to butanol is apparently unique to B. methylotrophicum (21).

The reaction stoichiometries for the steady-state runs, balanced for carbon and electrons, are given in Table I. As was evident in similar chemostat experiments done without cell recycle (79), a reduction in pH resulted in the production of less acetate and more butyrate and alcohols. This trend is also evident in the Tables II and III, which show the partitioning of carbon and available electrons in the fermentation products. The fractions of carbon and electrons going into alcohols approximately double between a pH of 6.4 and 6.0. Production of 4- carbon compounds (Le., butyrate and butanol) accounts for over 50% of the total electrons from CO at a fermentation pH of 6.0. Between a pH of 7.2 and 6.0, the partitioning of carbon and electrons to acetate decreases by approximately 35%. However, even at a pH of 6.0, on a molar basis, acetate remained the predominant product. Previously, butyrate was found to be the major product during batch culture with a pH shift from 6.8 to 6.0 at the onset of the stationary phase (75).

At pH values of 5.75 and 5.5, the cultures initially exhibited transient primary butanol production, followed by prolonged oscillations in acetate and butyrate formation. Table IV shows the initial fermentation stoichiometries for these two oscillatory fermentations during the period of primary butanol production. Average product concentrations over the time period of primary butanol production were used to obtain these balances. These initially high butanol production levels, up to 2.7 g/L, were significant in that they demonstrated that B. methylotrophicum is capable of producing butanol as the major product from CO metabolism (21). Butanol accounts for as much as 44% of the total available electrons from the CO feed at a pH of 5.5. The AG0’ of butanol production is sufficiently exergonic to drive ATP synthesis (75). An obvious research challenge is how to control the pathway fluxes so as to sustain high butanol yields. Interpretation of the experimental data with the metabolic model described below can help elucidate the trends in pathway regulations.

Table I. Effect of pH on steady-state fermentation stoichiometries

pH__________________ Fermentation Stoichiometry___________________________

7.2 4CO —> 2.21C02 + O.4IOCH3COOH + O. IO5C3H7COOH + O. OI9C2H5OH + О. ОЗ2С4Н9ОН

+ 0.387 CELLS

6.8 400 —> 2.25C02 + О. ЗЗ4СН3СООН + 0.124C3H7COOH + O. O25C2H5OH + O. O4OC4H9OH

+ 0.377 CELLS

6.4 4CO —> 2.26C02 + О. ЗІ6СН3СООН + O. I52C3H7COOH + O. OI8C2H5OH + О. ОЗ2С4Н9ОН

+ 0.279 CELLS

6.0 4CO -> 2.32C02 + O.26OCH3COOH + O. M2C3H7COOH + O. O5OC2H5OH + O. O55C4H9OH

+ 0.279 CELLS

Table П. Effect of pH on Carbon Partitioning During Steady-State Fermentations

Table HI. Effect of pH on Available-Electron Partitioning During Steady — State Fermentations

Table IV. Fermentation Stoichiometries During the Initial Period of Oscillatory Fermentations

pH______________________ Fermentation Stoichiometry_____________________

5.75 4CO —> 2.36C02 + 0.126CH3COOH + 0.074СЗН7ССЮН + 0.021C2H50H + 0.115C4H90H

+ 0.595 CELLS

5.5 4CO —> 2.40CO2 + 0.112CH3COOH + 0.049C3H7COOH + 0.029C2H50H + 0.149C4H9OH

+ 0.533 CELLS

At this writing, we are nearing the end of the seventeenth year of funding from DOE’s Biofuels program to pursue and support the development of a biomass-to-ethanol industry in the United States. During this time, the benefits of a sustained level of funding to the program have become abundantly clear (although sometimes only in retrospect). We have taken a technology with roots in antiquity (i. e., fuel ethanol was produced by desperate governments embroiled in World Wars I and II) and brought it into the age of genetic engineering and advanced computer modeling. This was done for a very important reason-commercializable biomass-to-ethanol technology in the 1990s and beyond must be finely tuned and resembles the old fermentation processes in general form only. Herein lies the dilemma: at the surface bioethanol production appears technically simple; however, when considered in view of today’s complex markets (e. g., costs and values for fuels and biomass) and business/environmental requirements, this initial impression fades and the need for technical acuity becomes obvious.

From our perspective, activities that occur from 1997 to the year 2000 will set the stage for the success or failure of the bioethanol industry. The primary driving factor for this opinion is the sudden and substantial increase in interest by the private sector in bioethanol production. To meet this challenge, biomass conversion researchers must address the issues urgently needed by first (alpha) plants. These plants will be sited in locations specifically chosen by investors for optimal access to infrastructure, including biomass and water resources, fermentation and electrical power facilities, by-product markets, and labor availability. The first processing plants will not be chosen for fit to feedstocks and technology. Even the impact of political mandates will overshadow technical preparedness. The biomass conversion research programs must, therefore, be keenly sensitive to the direction of industry and empowered to support it.

A sustained energy future for the United States also requires energetic support of technologies appropriate to longer-term energy feedstocks. Once the initial bioethanol industry has been established, technologies that provide incremental improvements in specific unit operations, as well as those that permit access to entirely new feedstocks, will ensure a robust and secure industry. A valuable lesson can be learned from the current corn-to-ethanol industry, in which the inability to process varied feedstocks (i. e., lignocellulose as well as com and grain) has left processing plants precariously dependent on the volatile corn market. Future success of bioethanol plants lies in tolerance of feedstock diversity. Incremental technical improvements and new "leap-forward" technologies require active and vigorous advanced research programs. Apart from the obvious benefits to the bioethanol industry, and like all applied/basic research programs, these activities provide spinoff technical benefits that enhance the competitiveness of the general U. S. industry. Specific examples could include insights into cellulose and lignin chemistry from feedstock selection and analysis activities, insights into the kinetics of sugar hydrolysis, sugar destruction, and toxin formation from biomass pretreatment activities, insights into the biochemistry of enzymes that act on insoluble polymers from cellulase improvement activities, insights into the control and analysis of metabolic pathways in fermentative microorganisms from ethanologen improvement activities, insights into the microbiology of methanogenesis from waste treatment activities, and insights into animal feed/nutrient requirement methodologies from plant by-product recovery activities. Thus, the DOE-funded biomass-to-ethanol R&D program offers many levels of benefit to U. S. industry and represents a continuing success story for critical research managed by a national laboratory.

Acknowledgments

This work was funded by the Biochemical Conversion Element within the Biofuels Systems Program of the Office of Fuels Development of the U. S. Department of Energy.

Figure 2 shows the hydrogenation rate of four ketones: 2-propanone (acetone), 2-butanone, 2-pentanone, and 3-pentanone (18). From typical VFA compositions in the fermentor, these four ketones comprise about 90% of the expected product. The hydrogenation was performed at 1 atm total pressure and near-ambient temperature. The reaction rates are expressed as moles of alcohol product per minute per gram of Raney nickel catalyst. The right-most data points of each figure indicate the hydrogenation rate of pure ketone. The data points to the left indicate the hydrogenation rate of ketone mixed with 2-propanol, the dominant product in the MixAlco Process.

Comparing the hydrogenation rates of the four ketones at 40°C (the only temperature that is common to all), they are all very similar and differ at most by a factor of two. At a catalyst concentration of 50 g Raney nickel/L, a temperature of 40°C, and a total pressure of 1 atm, the time required to hydrogenate 99% of

Table IV. Thermal Conversion of VFA Salts to Ketones Calcium Acetate* Calcium Propionate* Calcium Butyrate* Activation energy (kJ/mol) 642.6 ± 28.0 b2327 ±162 c691.7 ± 73.3 386.5 ±15.8 Frequency factor (min1) 109.4 ±0.4 b414.7 ± 0.4 c121.1+0.2 66.12 ±0.22 Time required for 90% conversion at 440°C (min) 0.817 0.0283 0.915 Yield (g actual liquid/g theoretical liquid) 93.1 ±0.9 87.5+0.7 94.4 ±0.9 Maximum temperature studied (°С) 455 475 508 2-Propanone (acetone) (%) 91.5 ±4.3 Isophorone (%) 2.78 ±2.6 2,4-Dimethyl phenol (%) 3-Pentanone (%) 0.30 ±0.63 96.9 ±0.9 2-Butanone (%) 1.06 ±0.37 5-Methyl-2-hexanone (%) 4-Heptanone (%) 0.49 ±0.13 94.9 ±3.46 2-Pentanone (%) 2.84 ±1.7 3-Heptanone (%) 0.44 ±0.07 3-Hexanone (%) 0.39 ±0.26 Other (%) 5.43 ±1.71 1.57 ±0.85 1.44 ±1.79 a Error band represent ± two standard deviations. b398°C and below ‘above 398°C

2-propanone is 4.4 h. The reaction rate increases linearly with hydrogen pressure (18), so the reaction time could be reduced to under one hour using a moderate pressure. Alternatively, the catalyst concentration could be increased.

The hydrogen would likely be derived from reformed natural gas, an abundant domestic energy source. In large-scale production, hydrogen costs about the same as gasoline per unit of energy (Appleby, J., Texas A&M University, personal communication, 1996), so there are no economic penalties associated with its use. The ketone may be viewed as a hydrogen carrier which avoids the need for high — pressure tanks to store gaseous hydrogen.

Although not strictly related to the production of succinate by anaerobic microorganisms, the downstream processing of this compound ultimately is of paramount importance for any industrial process, and could indeed influence the selection of microorganism. A few processes have been studied for the recovery of succinate from fermentation media, and each of these has its unique advantages and disadvantages. In general, processes developed heretofor have focused on the characteristic of charge that distinguishes succinate from many other components in fermentation media.

One method to recover succinate is by extraction, and recent articles describe extraction of various organic acids (139-146). Extraction processes for succinate and other negatively charged solutes usually involve the transfer of the solute into an organic phase by the use of a positively charged extractant, such as long-chain tertiary amines. The selectivity of separation is very good with amine extractants, because they have favorable equilibration chemistry with deprotonated acids (140,141). A potential problem is water coextraction (142), which reduces the extraction yield of the acid. Of course, the succinate-amine complex in an envisioned extraction process must still be reextracted (stripped) from the organic phase, a process which might, for example, involve a strong base. An advantage of the process is that extraction is a mature process, with numerous designs and devices available to implement the process. A potential problem is the toxicity of the amine extractant if the extraction is carried out in situ. If a water-soluble volatile tertiary amine such as trimethylamine is used to back-extract the succinate, the solution may be partially evaporated to produce the acid product in crystalline form (143).

Another recovery method is adsorption, whereby an ion exchange resin adsorbs the negatively charged ions from the fermentation media (145,146). To avoid clogging the adsorption bed with cells, a membrane separation is required. By the appropriate selection of ion exchange resin, the succinate along with other organic acids can very effectively be removed from the fermentation media. The next step in the process is removal of the succinate from the ion exchange column and regeneration of the ion exchange material. This process may require the use of both a strong base to strip the succinate and a strong acid both to convert the succinate into succinic acid and to regenerate the column. Alternatively, the acid can be back — extracted into a trimethylamine solution, as described in the previous paragraph.

A related, general extractive means to recover carboxylic acids from fermentation media is through the use of liquid membranes (147), and an example is the recovery of lactic acid (148). In supported liquid membranes, a porous polymeric membrane impregnated with the extractant is situated between the feed and the stripping solution. Often additional compounds such as surfactants are added to enhance the transport rate of the extracted species. Liquid membranes can suffer from instability, relatively high cost and comigration of other components from the fermentation media.

A unique method to recover succinic acid is by electrodialysis. This process has been the subject of patents (84,85,149) and a review article (150). Transportation of other mobile ions (and cells if the media is not ultrafiltered) in the fermentation media such as sodium, potassium, chloride, phosphate, sulfate, etc. can pose a problem for the efficiency of the electrodialysis. Essentially the method utilizes electric charge first by "desalting" electrodialysis to concentrate sodium succinate, and then by "water-splitting" electrodialysis to remove preferentially sodium and hydroxide ions from the salt stream yielding a precipitated succinic acid product. When electrodialysis was applied to a succinate fermentation by A. succiniproducens, a solid product of 99.9% purity was obtained (150).

The production of ethanol from lignocellulose offers many potential opportunities for

So

Table III. Comparison of SSF Results for Different Substrates and Biocatalysts

Os ЧО

Strain P2 — Enzyme Recycle

I

Figure 6. Comparison of ethanol production from waste office paper (MWOP) by K. oxytoca strain P2 and yeast. More detail concerning fermentation conditions and references are provided in Table III.

synergy with other types of manufacturing processes. Perhaps the simplest of these would involve a combination of electricity with boilers fired from the residues after hemicellulose hydrolysis and ethanol production from hemicellulose syrups. This process could reduce heavy metal contamination of boiler feeds derived from municipal landfill waste (metals precipitated with the gypsum) and upgrade thermal value. Low grade steam could be used for ethanol purification and other processes.

Cane sugar production plants could also benefit from increased ethanol yields by fermenting hemicellulose syrups, analogous to the conversion of bagasse to furfural plant in south Florida. Although bagasse is burned to power sugar refining, excess bagasse typically accumulates. Hemicellulose could be stripped by dilute acid hydrolysis and fermented to ethanol, leaving sufficient residue as a boiler fuel for both processes.

Ethanol production from grain and cane sugar offer an extremely attractive opportunity for synergy. Spent yeasts from these processes could be recycled as a nutrient source for hemicellulose or cellulose fermentations. Com fiber residues, com cobs, com stover, bagasse or other lignocellulosic residues could serve as a feedstock for the biomass to ethanol process. Again, undigested residues could be burned to provide the energy.

Acknowledgments

This research was supported in part by the Florida Agricultural Experimental Station (publication number R-05323) and by grants from the U. S. Department of Agriculture, National Research Initiative (95-37308-1843) and the U. S. Department of Energy, Office of Basic Energy Science (DE-FG02-96ER20222).

Besides favorable economics and environmental and health benefits, the development of reliable standards, which will instill confidence in biodiesel users, engine manufacturers, and other parties, is a milestone in facilitating commercialization (6). Austria (ONORM С 1190) and Germany (DIN V 51606) have established similar standards for neat biodiesel. In the United States, an ASTM standard was suggested (9). Table V gives the German standard and Table VI lists the proposed ASTM standard. The standards contain specifications particular to biodiesel (for example, glycerol quantitation) which are not given for conventional DF.

a) This specification is the process of being evaluated by ASTM. A considerable amount of experience exists in the U. S. with a 20 percent blend of biodiesel with 80 percent petroleum-based diesel. Although biodiesel can be used in the pure form, use of blends of over 20 percent biodiesel should be evaluated on a case-by-case basis until further experience is available.

b) Or equivalent ASTM testing method.

c) Austrian (Christina Plank) update of USDA test method (author’s note: refers to Refs. 97 and 104).

this is not without problems (55). Biodiesel from vegetable oils with high amounts of saturates (low I Vs) will have a higher CN while the low-temperature properties are poor. Biodiesel from vegetable oils with high amounts of unsaturates (high I Vs) will have low CN while the low-temperature properties are better. Thus, CN and low-temperature properties run counter to each other and this must affect I Vs for biodiesel standards. Another argument against inclusion of the IV in biodiesel standards is the observation that different fatty acid compositions give identical IVs (e. g., neat methyl oleate has the same IV as a 1:1 mixture of methyl stearate and methyl linoleate). The IV also does not take into consideration structural factors of fatty compounds as discussed above where the CNs depend on double bond position, etc. Furthermore, once in place, the IV will hinder further research and development. It is possible that plants with desirable high- cetane fatty acid profile can be genetically engineered and bred (for example, substituting A6 unsaturated Cl8:1 acids for A9 unsaturated ones) or that combustion­improving additives are developed which are highly effective even for high degrees of unsaturation. It was suggested that it appears better to limit the amount of higher unsaturated fatty acids (e. g. linolenic acid) than to limit the degree of unsaturation by means of the IV {34). Note that soybean oil, rapeseed oil, and canola oil (low-erucic rapeseed oil) have very similar 18:3 fatty acid content (Table II), which is the most problematic in the formation of engine deposits through polymerization. However, linseed oil methyl ester (high 18:3 content and IV) satisfactorily completed 1000 hours of testing in a DI engine while neat linseed oil caused the engine to fail {35 and references therein). These observations make the IV even more debatable.

Since most esters have higher CNs than neat vegetable oils and conventional DF, the esters could accommodate higher CNs than the minimum of 40 given in the ASTM standard for conventional DF. For example, the lowest reported CN for methyl soyate is 46.2 (see Table IV).

The German biodiesel standard includes the so-called Cold-Filter Plugging Point (CFPP) that pertains to the low-temperature flow properties of biodiesel. This low — temperature property test is used in Europe, South America, and the Pacific rim. In North America, a more stringent test, the Low-Temperature Flow Test (LTFT), is used and specified by ASTM D4539. Although the LTFT is more useful in evaluating low — temperature flow properties, ASTM requires only specification of cloud point for certification.

The use of a fermenter with very high KLa is not practical in industrial-scale fermentation process because power consumption for agitation will be uneconomically large. Hence, we developed a new culture method using a

conventional-type fermenter under low O2 conditions. In this method, heterotrophic cultivation using fructose as carbon source was first carried out for exponential cell growth. After the fructose in the medium was exhausted, the culture broth was centrifuged. The harvested cells were suspended in sterilized mineral medium and autotrophic cultivation for P(3HB) accumulation was performed by feeding a substrate gas mixture in which the O2 concentration was below 6.9%(second culture stage). This method is referred as a two-stage culture method. The cell and P(3HB) concentrations were 26.3 g«dnr3 and 21.6 gednr3, respectively after 40 h of autotrophic cultivation. The average productivity of P(3HB) in the autotrophic stage was about 0.56 gedm-3»h_1. According to previous reports (9-20) on the fermentative production of P(3HB), the productivity of P(3HB) was 0.08 — 4.00 g-dm-3»^1 in heterotrophic cultures and 0.04 -1.54 g*dm*3»h_1 in autotrophic cultures. Although the KLa of the fermenter used in this experiment was 340 h_1 and the O2

СНз OW CH2 о

1 11 1

о-сн-ст —c 41 о — си — cm — c

‘n4 yn

PoIy-(D-3-hydroxybtyrate-co-D-3-hydroxyvalerate)

Figure 1 Chemical structure of poly-D-hydroxybutyric acid.

1,02 cylinder; 2, H2 cylinder;

З, CO2 cylinder; 4, gas reservoir;

5, differential-pressure type flowmeter; 6, DO controller;

7, alkali feeding pump;

8, pH controller; 9, NH3 solution;

10, fermentor; 11, circulating pump;

12, mixing pump;

13, internal pressure sensor;

14, saline replacement;

15, flowmeter

I

concentration in the gas mixture was very low, a relatively high P(3HB) production rate was obtained. In this culture method, CO2 is evolved from fructose during the heterotrophic stage. However, the two-stage culture works as a CO2 absorption system because the amount of CO2 consumed during the autotrophic stage is about twice that of the heterotrophic stage.