Carbon dioxide emission in the biosphere is attributed to two sources, one biological and the other industrial. Industrial carbon dioxide emission is mainly from the exhaust gases of industrial processes. Where carbon dioxide occurs in high concentration in such processes, the carbon dioxide in the exhaust gas can be trapped and then recovered for uses as a raw material. However, carbon dioxide released from automobiles, for example, is too diluted to be trapped for recovery. Photosynthesis is the most successful means for the trapping and recovery of car­bon dioxide released to the air from diluted emission sources. A counter-measure against the elevation of carbon dioxide in the atmosphere is to strengthen the path of carbon dioxide recycling through photosynthesis and then efficiently utilize the plant biomass formed as a result.

2-1 Oil Palm

In terms of photosynthesis, tropical areas offer great advantages over tem­perate zones due to high solar radiation. High energy crops native to South East Asia (and Africa and South America) are therefore desirable tools for the counter­measure to carbon dioxide elevation.

Year Atm Oil Preaa fibre Effluent

million ha million ton million ton million kl

(ai fuel 15 t)

Energy recovery 6% 0.8 fbel t/h у

Fig.2. Total biomass production and waste materials in palm oil industry.

Oil palm is one crop which has a very high energy fixation per land area, i. e. 5 t/ha year of oil (4.5xl07 kcal/ha year of energy) can be harvested. However, as shown in Fig.2, oil is only 1/3 of the total biomass of the tree. Hence the total en­ergy fixed by biomass is estimated to be 13.5×107 kcal/ha year, this being equiva­lent to 15 tons of fuel. In the world as a whole, the plantation area for oil palm trees is estimated to be about 4,000,000 ha (equivalent to 12,000,0001 oil/year). Thus, the total biomass fixed in oil palm trees is equivalent to about 30,000,0001 of fuel. As shown in Fig. 2,2/3 of the biomass in the oil palm indus­try is agricultural waste. A number of valuable resources are wasted and dumped in the plantation and oil mills. Among them, potential resources are press fiber and waste liquor. The chemical composition of the carbohydrate component of delignified palm fiber is 56.4% glucose, 36.0% xylose, 5.9% arabinose, and 1.7% mannose (1). Our experiments showed that delignified palm fiber can be digested to form glucose and xylose by commercial cellulases such as Meicellase and Onozuka and the hydrolysate can be subjected to fermentation to produce biofuel and chemicals (1). Our study estimates that 3,000,0001 of glucose and 2,000,000 t of xylose could be recovered from this waste per year.

Another big resource in the palm oil industry is the waste liquor from the oil mill. Two kinds of waste from the crude oil separation process are discharged, sterilized condensate and separator sludge. The mixture contains about 30,000 ppm of BOD and it is very hard to reduce BOD to the regulation limits. Our tech­nology proved that the waste which digested by commercial cellulase can be fer­mented well by CL saccharoperbutylacetonicum N1-4 to produce acetone, bu­tanol and ethanol without any additional medium supplementation (2-3). Extrac­tion of butanol by fatty acid methylester stimulated fermentation rates by release of end product inhibition (4) and the extract was similar to diesel fuel. We are confident that a process using crude palm oil (CPO, the product from the oil mill) methylester will stimulate the fermentation rate to produce biodiesel efficiently. Thus, about 200,0001 of biodiesel could be recovered from the waste by this process (Fig.3).

Gregarious sago palm is limited to tropical areas such as Malaysia (Sarawak), Indonesia, and Papua New Guinea (5). The starch productivity of sago palm is several times greater than that of tapioca (cassava), wheat, and rice, i. e. sago palm has a high efficiency of photosynthesis. Recently, Sarawak, a state of Malaysia, has successfully cultivated a sago plantation on 7,000 ha of land and has developed 85,000 ha of land for planting. The productivity of starch is re­ported to be 25 t/ha y. As shown in Fig. 4, one tree is planted per 100 m2 (10 m x 10 m) land and one shoot from the mother tree will be remained every year. Thus, one grown tree can be harvested from 100 m2 land every year after ten years ini-

tial propagation. In this plantation, 25 tons of starch per hectare can be recovered and this is almost 3 times higher than that of rice. Approximately 1,500,000 ton of starch will be harvested from this plantation per year and the starch could be used for bioconversion. Either 750,000 ton of ethanol or 1,350,000 of lactic acid could be produced by anaerobic bioconversion from this plantation.

Natural rubber is also one of biomass made by recycling carbon dioxide by photosynthesis. Cost saving and increase productivity of natural rubber give a chance to win the competition of natural rubber against synthetic rubber made by petrochemical industry. Recently, our laboratory has developed NRSP (Natural Rubber Serum Powder) which is the spray dried product of the waste from natural rubber industry. The waste is discharged from the centrifugal machine to separate latex. The waste is subjected to digestion by papain, a proteinase, prior to spray drying. It is noted that NRSP contains strong growth promoters for various kind of microorganisms particularly anaerobes such as lactic acid bacteria (6), wide range of Bifidobacterium (7), and Zymomonas (8). Usually, anaerobic microor­ganisms require complex nutritious factors. Bifidobacterium is important intesti­nal microflora and there is a great interest in the possibility of increasing number of this microorganism in intestine by the special growth promoter. It is expected that NRSP can increase the number of Bifidobacterium in intestine to enhance the health of human.

Zymomonas mobilis is capable of producing ethanol with a higher produc­tivity than yeast, but this microorganism requires complex nutritional factors.

This microorganism thus can not grew in simple and economical medium such as that used in industrial ethanol production. By our study, the medium prepared with NRSP with soy bean hydrolysate as a source of complex nutritional factors exhibited the same fermentation capability with Z. mobilis as medium prepared using yeast extract (8).

To develop any chemical or biochemical technology for recycling carbon dioxide to serve as a counter-measure against carbon dioxide elevation in the at­mosphere, it is obvious that the process should not use fossil fuel. In this regard, it is also better to avoid aerobic bioprocesses which are accompanied by carbon di­oxide emission. Aerobic bioprocesses involve the oxidation of organic substrates by oxygen and this is the same principle of fossil fuel combustion. It is therefore preferable that anaerobic bioprocess should be adapted for bioconversion which allows less carbon dioxide emission. Some anaerobic reactions do not involve the production of carbon dioxide, some result in small quantities being produced, but others produce comparatively large amounts during the entire course of the fer­mentation. For example, fermentation involving the metabolic pathway in which pyrvate is converted into acetyl-CoA, with the release of carbon dioxide, inher­ently produce one mole of carbon dioxide for each mole of substrate consumed. Hence, anaerobic fermentation yielding ethanol, butanol, acetone, acetoin, and butanediol are accompanied by the production of carbon dioxide, while lactate formation from pyrvate is not accompanied by the release of carbon dioxide. Thus, lactate fermentation has advantages over other anaerobic fermentation such as ethanol fermentation from the point of view of carbon dioxide release during the biochemical reaction.

In general, anaerobic fermentation has advantages as 1. energy gaining metabolism, 2. less carbon dioxide, 3. accompanying metabolites as electron ac­ceptor, while, it has disadvantages as 1. low cell density, 2. product inhibition, and 3. complex nutrition requirement. These disadvantages are sometimes bottle­neck to attain efficient fermentation.

Lactic acid fermentation has great advantage over other bioconversions. The stoichiometry for homofermentation from hexose can be expressed,

СДА———— * 2C3HA

whilst lactate and acetate are formed from pentose without the release of carbon dioxide.

C5H10O5——— ► C3HA + C2H402

As indicated in the above stoichiometry, neither reaction loses any atoms of car­bohydrate during bioconversion. Thus, bioconversion using lactic acid fermenta­tion does not produce carbon dioxide and does not lose any material. Thus, this is an excellent counter-measure against the global problem of carbon dioxide accu­mulation. Processes by which glucose and xylose are first formed from lignocellu — losic agricultural waste and next converted into lactic acid (from hexose) /or lac­tic acid and acetic acid (from pentose) are an anaerobic bioconversion; and resulting organic acid is transformed into commodity chemicals such as polylac­tate, biodegradable plastics, by synthetic methods. Recent technology involved in polylactate production can blend the various forms of polyester such as PHA to improve the characteristics of the plastics. Our laboratory has developed the fer­mentation technology to produce PHB from lactic acid and acetic acid (9,10) and this PHB could be used for preparing mixtures of polylactate and PHA whose characteristics is better than that of lactate homopolymer.

Lactococcus lactis 10-1 isolated in our laboratory (11) is capable of effi­ciently fermenting xylose up to 50 g/1 initial concentration into acid (12,13). However, the rate of fermentation in xylose medium is slower than in glucose medium but a high dilution rate culture with cell recycling could be applied to stimulate xylose consumption rate overcoming lactate inhibition (14). We have observed an interesting phenomenon that NRSP, the product from natural rubber

waste, changed the molar ratio of the two products (lactic acid/ acetic acid) of lactic acid fermentation using xylose to increase lactic acid production (unpub­lished).

Bioconversion through lactic acid fermentation can therefore contribute to the reduction of the carbon dioxide concentration in atmosphere. We propose a new concept of biochemical industry named "Lactate Industry" (15,16) in which biomass is converted into lactic acid without carbon dioxide release and no ele­mental loss. The resulting lactic acid could be used to produce commodity chemi­cals such as biodegradable plastics. Used plastics would be digested into carbon dioxide by composting and recycled back to biomass by photosynthesis (Fig. 5).

2. Conclusion

Elevated levels of carbon dioxide in the atmosphere is a serious problem that will interfere with life of all organisms on the planet. To solve this crisis and to allow our continued survival, a favorable ecosystem in which all elements are recycled in the biosphere from the organic state to the inorganic state has to be maintained. Excess carbon dioxide is released into atmosphere by the consump­tion of fossil fuel to support our comfortable way of life. Carbon dioxide emission from non biological action can not yet be balanced by the recycling of elemental carbon in the biosphere. However, new technology introduced as a counter­
measure to this environmental problem will inevitably results in other changes to our ecosystem. Such change is sometimes serious problem that all living organisms in the earth never have.

To tackle the global task of reduction of carbon dioxide accumulation through the application of biotechnology, we must assess the other risks that the introduction of the new technology could cause. We have to avoid these dangers we would be suffered by the new technology.

[1] CFPP = Cold-filter plugging point, b) tbs = to be standardized.

The iodine value (IV; see Table III) has been included in the European standards and is based on rapeseed oil as biodiesel feedstock. It is set at IV = 115, which would exclude soybean oil (neat vegetable oils and their methyl esters have nearly identical I Vs) as biodiesel feedstock. The discussion in the previous section, however, shows that

[3] Corresponding author

© 1997 American Chemical Society

The complexity of the catabolic pathway of B. methylotrophicum makes it difficult to extract patterns of metabolic regulation from the experimental data. Metabolic models that calculate the fluxes of carbon, electron, and ATP fluxes through the various branches from the experimental data can aid in this process. However, development of such models requires that the stoichiometry of the individual reactions of the pathway be known. Sufficient information about the CO metabolism by B. methylotrophicum is available (77,74,27) for development of a metabolic model.

Model Development Following the approach developed by Papoutsakis (22), an equation was written for cell mass production that is balanced for carbon, electrons and ATP. In acetogenic anaerobes, such as B. methylotrophicum, acetyl-CoA is a precursor for cell mass production and links catabolism with anabolism (7). Consequently, cell mass was assumed to be produced from acetyl-CoA, as shown in Equation 1:

Acetyl-CoA + yi NADH2 + y2 ATP -> 2 Cell Mass (1)

Applying an electron balance to Equation 1, the coefficient yi can be estimated to be 0.2 mol NADH^mol Acetyl-CoA. In an alternative approach (17), the value of yi was estimated to be 1.5 mol NADH2/mol Acetyl-CoA from the following stoichiometric equation that was determined from batch growth of B. methylotrophicum on CO (20):

4 CO -» 2.17 C02 + 0.74 CH3COOH + 0.45 Cell mass (2)

This equation balances to within 3% for both carbon and electrons. The elemental composition measured for B. methylotrophicum cells closely matched the average cell formula (CHi. gOo. sNo. s) suggested by Roels (25).

Estimation of y2 required the assumption of a second mechanism of ATP production. When only substrate-level phosphorylation (SLP) is considered, conversion of CO to acetate is an ATP-neutral process (one ATP is consumed by formyl-THF synthase for each ATP produced by acetate kinase), and production of

butyrate, ethanol, and butanol result in net consumption of ATP by SLP. Zeikus et al. proposed that an electron-transport phosphorylation (ETP) mechanism contributes the remaining ATP needed for cell maintenance and growth (7). In this mechanism, electrons generated by CO dehydrogenase are shuttled through two membrane-bound electron carriers. One of these carries both a proton and an electron, and the other carries only electrons. The net result is the ejection of protons from the cell, generating a transmembrane proton gradient. The protons reenter the cell via a proton-translocating ATP synthase that generates ATP. Six moles of electrons are produced by CO dehydrogenase per mole acetyl-CoA produced, so a theoretical maximum of 6 protons could be ejected per acetyl-CoA produced. A conservative value of 2 moles of protons ejected per mole of acetyl — CoA produced was used, along with a standard ratio of 1 mole ATP produced per 3 moles of protons translocated (24), to calculate ATP yields by both SLP and ETP for the growth data given in Equation 2. The net ATP yield for production of 0.74 mol of acetate and the acetyl-CoA used to produce the cell mass was calculated to be 2 mol ATP/mol acetyl-CoA used for cell mass. This value equals the amount of ATP available to convert one mole of acetyl-CoA into cell mass (i. e., y2). This y2 value translates into a Yx/atp value of 26 g cells/mole ATP. By comparison, the accepted value for cell growth on glucose is 10.5 g cells/mole ATP (25).

Reaction equations were written that capture the stoichiometry and structure of the branched pathway. Consecutive reactions that did not involve branch points reactions were lumped together.

Equations 1 and 3-11 were each assigned an unknown rate (or flux) coefficient. An expression for the rate of production of each species (n) was then written from these equations in terms of the unknown flux coefficients and the reaction stoichiometries. The reaction rates for non-secreted intermediates NADH2, [CH3OH], acetyl-CoA, butyryl-CoA, and acetoacetyl-CoA were set equal to zero, based the pseudo-steady-state assumption (22). The r* terms for other compounds (acetate, butyrate, ethanol, butanol) were calculated from experimental measurements of their liquid-phase concentrations (Q) , using the following, unsteady-state conservation equation:

r{ = — j — — DC( (12)

at

where D is the dilution rate. The CO2 production rate was determined from the flow rate and CO2 concentration of the effluent gas stream. For the steady-state experiments, the time derivative was set equal to zero. The resulting set of 10 equations with 10 unknowns was solved using Gaussian Elimination to calculate the flux coefficients.

Application of the Metabolic Model to Experimental Data. Calculation of the pathway fluxes allowed the relative ATP contributions from ETP and SLP to be calculated (77). Even though ATP is a non-secreted intermediate, the pseudo­steady-state assumption does not apply, because ATP can be consumed in a variety of unknown reactions in the сей, including miscellaneous maintenance-energy requirements. The model predicted that, for the steady-state fermentations, there was significant net consumption of ATP due to SLP (-0.12 mol ATP/mol CO). However, this was offset by sufficient production of ATP via ETP (+0.14 mol ATP/mol CO) to result in a small net gain of ATP.

An unsteady-state approach had to be used for the oscillatory fermentations. The time

derivative in Equation 12 was evaluated by graphically differentiating the C* vs. time data. The net ATP yield predicted by the model was slightly negative throughout the experiment. Since the culture could not be sustained under a long­term ATP deficit, this result suggests that there is more ATP produced than is accounted for by the model. The assumed ratio of 2 protons ejected per acetyl — CoA may have been too conservative. Assuming 4 moles of protons ejected per mole of acetyl-CoA produced instead of 2 would give a Yx/atp value of 10 g cells/mol ATP, which closely matches the accepted value for glucose (25). Alternatively, there is evidence (G. J. Shen and J. G. Zeikus, unpublished results) that the electron carrier for the reduction crotonyl-CoA to butyryl-CoA is membrane-bound and may thus participate in ETP.

The mechanism driving the oscillations is believed to be related to metabolic regulation, rather than the CO mass-transfer rate or the liquid flow rate. Both the rate of CO addition and the liquid flow rate were constant throughout the experiments. The mean residence time of the liquid (66 h) was much shorter than the period of the oscillations (about 250 h). Although there were oscillations in both the acetate and butyrate concentrations, these oscillations were out of phase, indicating that the carbon flux was being alternately regulated through the two — carbon and four-carbon pathway branches. The cells obtain twice as much ATP via SLP per carbon equivalent by producing acetate from acetyl-CoA than butyrate. However, acetate production eliminates fewer electrons per carbon equivalent. Thus, the oscillations may have arisen from the cells alternately responding to needs to eliminate electrons and generate ATP. Consistent with this hypothesis, the oscillations in the CO uptake rate are in phase with butyrate production and out of phase with acetate production. More CO has to be consumed when butyrate is produced to maintain an equivalent rate of ATP production via SLP.

Gas Mass-Transfer Issues in Synthesis-Gas Fermentations

Oxygen mass transfer from the gas to the liquid phase is commonly rate-limiting in commercial-scale, aerobic fermentations (26). For this reason, design of fermenters for aerobic applications centers around providing an adequate volumetric mass — transfer coefficient (Кід). In a commercial-scale synthesis-gas bioprocess, providing sufficient mass transfer would be expected to be even more challenging, for two reasons. First, about twice as many moles of gas must be transferred per electron equivalent in the substrate for fermentations based on synthesis gas than those based on glucose. Second, under mass-transfer limiting conditions, the volumetric mass-transfer rate is directly proportional to the gas solubility (18), and the molar solubilities of CO and H2 are only 77% and 65% of that of oxygen, respectively (27).

Mass-transfer-limiting conditions are readily identified in synthesis-gas fermentations by applying an unsteady-state mass balance to the gas uptake rate data. This approach has been used to demonstrate mass-transfer limitations in a variety of bioreactor configurations, including batch (28), stirred tanks (18), airlift fermenters, and trickle-bed reactors (16). Under such conditions, the gas uptake rate is constant. Thus, increases in the concentration or intrinsic reaction rate of the cells will not translate into improved productivity unless comparable increases are made in the gas mass-transfer rate.

Traditionally, gas mass transfer has been enhanced by increasing the power input to the bioreactor, which reduces the average bubble size and hence increases the interfacial area. In the previously described continuous CO fermentations using cell recycle (17), efforts were made to increase interfacial area by rapidly recycling the gas from the headspace, through a frit, and back into the fermentation broth. A high impeller rate was also used to maintain small bubble size. Even then, the highest specific CO gas consumption rate observed was 0.0044 mol/h*g cell. Calculations based on batch data with this organism at lower cell densities (20) have yielded CO consumption rates as high as 0.02 mol/h®g cell for much lower cell densities. These results suggest that, even with a high agitation rate and gas recycle, CO mass transfer was still rate-limiting. Moreover, this approach would not be economically feasible at the commercial scale, because power consumption increases with the impeller diameter to the fifth power and the impeller rate to the third power (29).

Formation and Stability of Microbubble Dispersions. Microbubble aeration has recently been proposed as an energy-efficient approach to enhancing synthesis-gas mass transfer (30). Aficrobubbles are surfactant-stabilized gas bubbles having radii on the order of 25 pm. The surfactant layer provides a surface charge that prevents bubble coalescence by electrical repulsion (57). Microbubble dispersions have colloidal properties and can be pumped, unlike conventional foams that collapse upon pumping. The formation and coalescence properties of microbubbles have been studied (30). The microbubble generator consisted of a 5-cm diameter, stainless-steel disk spinning at 7000 rpm in the vicinity of stationary baffles. The number-averaged diameter was 107 pm for room-temperature microbubbles generated using Triton X-100 at a concentration of twice the critical micelle concentration (30). Modifications of the microbubble generator later reduced the number-averaged bubble diameter to 56 pm.

The rate of drainage of the microbubble dispersion was measured as a function of surfactant concentration and type. This technique gives information on stability and initial gas void fraction. As the concentration of surfactant increased beyond the critical micelle concentration, the stability of the dispersion increased to an asymptotic value that varied with the surfactant used. The initial gas void fraction of the dispersion was virtually unaffected by surfactant concentration, surfactant type, or the addition of sodium chloride. The constant value of the initial gas void fraction approximated the theoretical packing limit for monosized spheres. These results indicated that salts commonly used in growth media should not interfere with microbubble formation and stability.

The power required to generate microbubbles was measured using a Lightnin Labmaster unit capable of simultaneously measuring the impeller rate and power input (Bredwell and Worden, manuscript in preparation). The Power Number of the microbubble generator was measured to be 0.036, and the projected power requirement for microbubble generation for commercial-scale B. methylotrophicum fermentations was calculated to be 0.0081 kW per m3 of fermentation capacity. Compared to a nominal power input for commercial-scale fermentations of 1 kW/m3 (26), these data indicate that power requirements for microbubble production should be low at the commercial scale. Moreover, minimal power input would be required for the bioreactor, because the mass-transfer rate from microbubbles is virtually independent of agitation rate (33). Some power input would be required for liquid-mixing requirements, but this input could be minimized by the use of advanced, axial-flow impellers or a pneumatically mixed bioreactor configuration (e. g., airlift) reactor.

Non-toxic Surfactants for Microbubble Formation. The surfactant used to form the microbubbles must be non-toxic to the biocatalysts. The effects of several anionic, cationic and nonionic surfactants on the growth and product formation by B. methylotrophicum were determined in batch culture on CO (Bredwell, et al, submitted). A phosphate-buffered-basal (PBB) medium was used with the addition of 1, 2, or 3 times the critical micelle concentration of the surfactant in the media. The ionic surfactants, cetyl pyridium chloride and sodium dodecyl sulfate, inhibited growth at concentrations lower than the critical micelle concentration. The non­ionic surfactants tested were polyoxyethylene alcohols (Brij surfactants) and polyoxyethylene sorbitan esters (Tween surfactants). These surfactants had little or no effect on the growth rate of the bacteria. Concentrations of Tween 20, Tween 40, and Tween 80 between 0 and 3 times the critical micelle concentration had a negligible effect on the growth rate. The longer chain length surfactants (Brij 56 and Brij 58) appeared to inhibit growth at higher concentrations. Product concentration was measured using gas chromatography to evaluate the effects of the surfactant on the fermentation products. Carbon and electron balances were used to compute the stoichiometric equations. These equations, listed in Table V, show little effect of the Tween surfactants on the stoichiometry. Combined with the growth data, these results suggest that non-ionic Tween surfactants are well suited for making microbubbles for synthesis-gas fermentations.

Table V. Effect of Surfactants on Fermentation Stoichiometries

pH__________________ Fermentation Stoichiometry___________________

Control 4CO —> 2.17C02 + O.4OCH3COOH + O. O65C3H7COOH + O. O76C2H5OH

+ 0.61 CELLS

Tween 20 4CO —> 2.17C02 + O.45CH3COOH + O. O26C3H7COOH + 0.1 ЮС2Н5ОН

+ 0.60 CELLS

Tween 40 4CO —> 2.18C02 + О. З7СН3СООН + O. O82C3H7COOH + O. O68C2H5OH

+ 0.62 CELLS

Tween 80 4CO —> 2.20CO2 + О. З7СН3СООН + O. O78C3H7COOH + O. O96C2H5OH

+ 0.55 CELLS

Mass-Transfer Properties of Microbubbles. The mass-transfer properties of microbubble dispersions were measured using oxygen as the transferred gas (Bredwell and Worden, manuscript in preparation). The experimental system consisted of a 60-cm long column that had four ports along its length. A stream of oxygen microbubbles was combined with a stream of degassed water in a small mixing zone at the bottom of the column. The resulting steady-state oxygen profile across the column was measured using an oxygen minielectrode. An aqueous solution of Tween 20 at twice the critical micelle concentration was used to prepare the oxygen microbubbles. The concentration of surfactant in the degassed water stream varied from 0 to 5 times the critical micelle concentration.

The overall average mass-transfer coefficients based on the liquid phase (KL, av) calculated from the experimental data varied between 0.00002 and 0.0002 m/s. The largest values, which were obtained when the bulk liquid contained no surfactant, are about a factor of 2 greater than the value predicted by the well — known theoretical result that the Sherwood Number (Sh) = 2.0. The lowest values were obtained when the surfactant concentration in the bulk liquid was 5 times the critical micelle concentration. The high Kl>8v values, coupled with the extremely high interfacial areas provided by microbubbles, resulted in Kl2l values up to 1800 h’1, even without mechanical agitation. By comparison, reported Кьа values for synthesis-gas fermentations are 2.1 h’1 for a packed bubble column, 56 h"1 for a trickle-bed bioreactor, and a range of 28 — 101 h’1 for a stirred-tank bioreactor operated with a high impeller rate (76).

Analysis of the microbubble mass-transfer data indicated that a significant fraction of the transferred gas was lost from the microbubbles within seconds. Consequently, to mathematically model the mass-transfer process, an unsteady-
state approach was needed that accounts for phenomena that can often be neglected when using conventional bubbles, including changes in the microbubble size, gas composition, and intrabubble pressure.

Mathematical Model of Microbubble Mass Transfer. An unsteady-state mathematical model has been developed to explore the dynamics of microbubble mass transfer (Worden et al, submitted). The unsteady-state mass balance on the transferred gas component dissolved in the liquid phase is

This equation was derived in terms of a substantial derivative (DQ/Dt) that follows the movement of the gas-liquid interface as the bubble shrinks at a velocity vR. Two different sets of initial conditions for Q were used for the simulations: a pseudo-steady-state profile (PSS) and a gas-free profile (GF). The PSS initial condition assumes the initial concentration profile surrounding the bubble is that given by the steady-state solution to Equation 13 for the initial bubble radius and gas composition. The GF initial condition assumes the the liquid surrounding the microbubble is initially devoid of the transferring gas. The GF profile resulted in much steeper initial concentration gradients and hence more rapid initial gas mass transfer. The unsteady-state mass balances on the transferred gas and total gas in the microbubble, along with the initial conditions, are given below

(15)

t = 0 X=X0 RB=Ro

where X is the mole fraction of the transferred gas in the gas phase, and Di is the mass diffusivity in the liquid phase. The gas and liquid concentrations are related using Henry’s Law. In some cases, an additional film resistance is added at the interface to account for the surfactant shell.

The model was used to calculate the instantaneous mass transfer coefficient (k) at each time, as well as an average value (kav), defined below, for comparison with experimental Kl,.v values:
(17)

where C* is the liquid-phase concentration of the dissolved gas in equilibrium with the gas phase, and C“ is the concentration in the bulk liquid.

The model, which contained no adjustable parameters, was used to predict the mass-transfer rate from a microbubble into an infinite pool of degassed water. The rate of bubble shrinkage, and the corresponding changes in the к and kaV values are shown in Figure 1 for the GF initial condition. The predicted lifetime of an pure-gas microbubble is on the order of seconds in degassed liquid. During bubble shrinkage, к changes significantly, but kaV is approximately constant except for very early in the transfer process. This result validates the use of average mass-transfer coefficients to characterize microbubble mass transfer in experimental systems. The range of kav predicted by the model compares favorably to the steady-state к values predicted by the theoretical model of Waslo and Gal-Or (32). The experimental KL, av values measured in pure water were similar in magnitude to the predicted kav values. However, KL, av values measured in liquid containing high surfactant concentrations were an order of magnitude less. These results suggest that the mass-transfer resistance of the surfactant shell may be controlled by manipulating the properties of the fermentation medium.

Figure 1: Predicted Rate of Change of Microbubble Radius and Mass

Transfer Coefficient

As shown in Figure 2, the rate of microbubble mass transfer is predicted to decrease considerably as the initial concentration of transferred gas in the microbubble decreases. The average mass-transfer rate for a microbubble containing 100% transferable gas was predicted to be 14 times that for a microbubble containing only 20% transferable gas (e. g., air). Because synthesis gas consists of about 90% CO and H2 (2), it is well suited for microbubble mass — transfer on this account.

The significance of the studies summarized above can be discussed in the context of increasing bioreactor productivity in synthesis-gas fermentations. Metabolic modeling can be used to calculate the maximum theoretical yields possible from synthesis gas or combined with experimental data to calculate the fluxes of carbon, electron, and ATP through the branched metabolic pathways. Such information is useful in elucidating the patterns of regulation in response to environmental variables (e. g., pH). However, it is unlikely that manipulation of environmental variables alone will be sufficient to achieve the desired yields. Consequently, additional metabolic-engineering approaches will likely be necessary. Examples include isolation of mutants with altered genetic regulation patterns, elimination of enzyme(s) in unwanted pathways, and overexpression of rate-limiting enzymes in desired pathways. Implementation of these strategies will require development of recombinant tools (e. g., plasmids) for the microbes of interest.

Significant increases in productivity can be achieved via reactor engineering, as well, particularly through increasing cell concentration and increasing the rate of synthesis-gas mass transfer. The cell-immobilization studies indicated that B. methylotrophicum readily attaches to a variety of support materials that would be well-suited for industrial fermentations, and that these support materials do not inhibit cell growth or product formation. The cell recycle approach was highly successful. Both cell and product concentrations were increased several-fold over values obtained without cell recycle (17,19). Moreover, even during runs in excess of 1000 h, virtually no membrane fouling was observed.

The experimental and modeling results to date suggest that microbubble dispersions are well-suited for enhancing synthesis-gas fermentations. Extremely high Кьа values have been measured for microbubbles without mechanical agitation. When the surfactant concentration in the bulk liquid was low, these coefficients approached the theoretical values, suggesting that the mass-transfer resistance of the surfactant shell can be maintained at low levels. The power — consumption rate to produce the microbubbles is projected to be quite low. Because the microbubbles would be produced in a relatively small vessel and pumped to the bioreactor, only the minimal amount of power input required to maintain sufficient mixing would be required in the bioreactor. Consequently, energy-efficient, pneumatically mixed configurations, such as the airlift could be used. Several surfactants have been identified that do not interfere with the growth and product formation yet form high-quality microbubbles. The dynamic microbubble model has been used to evaluate the influence of bubble shrinkage, surfactant-shell resistance, and changes in gas pressure and composition on the mass-transfer efficiency of microbubbles and to help interpret experimental results. Experiments are currently underway in our laboratory to evaluate the suitability of microbubble mass transfer in long-term synthesis-gas fermentations.

Although this paper has focused primarily on issues related to bioreactor productivity, there are also important separations issues related to synthesis-gas fermentations. First, the products are currently produced in relatively low concentrations, so cost-effective methods to separate them from dilute fermentation broths are needed. Second, the acids and alcohols produced in synthesis-gas fermentations become inhibitory as they accumulate. The effects of these products on the growth and stationary-phase product formation in B. methylotrophicum have been measured (77). Cell growth was found to be inhibited at alchohol concentrations on the order of 5 g/L, even though the stationary-phase CO metabolism was unaffected by such levels. Simultaneous fermentation and separation approaches would be expected to be useful in this situation, such as pervaporation membranes, which are selective for alcohols in their transport properties. Flux through the membrane is facilitated by the use of a vacuum, whereby components that diffuse through the membrane are immediately removed by evaporation. The resulting vapor, which is enriched in the alcohols, would then be condensed prior to further purification steps (e. g., distillation). Third, is will likely be necessary to recover and reuse the surfactant when microbubbles are used. However, it may be possible to rely on the surfactants that are naturally produced in the fermentation to form the microbubbles. This approach has been demonstrated in bench-scale yeast fermentations (33).

This paper has identified several engineering issues that currently limit the commercial prospects of synthesis-gas fermentations and has summarized recent research that addresses these issues. Such research, combined with complementary biocatalyst-development efforts, may make bioconversion of biomass-derived synthesis gas into fuels and chemicals a commercial reality.

Ayaaki Ishizaki

Department of Food Science and Technology, Faculty of Agriculture,
Kyushu University, Hokozaki, Higashi-ku, Fukuoka 812-81, Japan

1. Introduction

Today, we are faced with serious global environmental problems. Among them, counter-measure against the elevation of carbon dioxide in the atmosphere must be an urgent tasks. Microbial technology can contribute to the solution of this problem. First, we must note that the composition of the atmosphere over the globe has undergone drastic changes in the past 4.5 billion years of the earth’s his­tory which has resulted in the development and diversification of living organisms and changes in the biota of the earth. However, these changes have been occurred in a very limited part of the earth i. e. biosphere in which the atmosphere and oceans are included. These changes have been accompanied by changes in the flora and fauna as well. The ecosystem of the globe and the environment influ­ence each other in the recycling of atoms in the biosphere to renew the composi­tion of the air and to establish new equilibrium from time to time. However, the global problem of the environment today is a new subject which never arose in the past history of the earth and is caused by new technologies developed to con­trol nature and to ensure a comfortable life. The speed of these developments have been very rapid indeed. Therefore, there has not been enough time for hu­mans to adapt the environment brought by these technologies to suit. Such drastic change has never occurred in the past history of earth and this is the first experi­ence for all living organisms, thus the problem is becoming critical and difficult to solve.

The amount of the elemental carbon existing in the crust of the earth is great but this carbon is inactive since they are contained in the crust such that no chemical reactions occur resulting in no emission of carbon dioxide from the crust into the atmosphere. Thus, carbon dioxide which influences environment is restricted to the elemental carbon recycling in the biosphere. Since there is about 300 ppm of carbon dioxide in the air so the total amount of elemental carbon containing in the atmosphere is about 700 Gt (gigatons). On the land, heterotro­phic organisms including humans release about 50 Gt of elemental carbon (in the form of carbon dioxide) per year into air. If the same amount of elemental carbon

© 1997 American Chemical Society

was fixed into organic materials by autotrophic organisms, a constant concentra­tion of carbon dioxide would be maintained in the atmosphere. In the oceans, bal­ance would be maintained by exchange elemental carbon between autotrophs such as sea weeds and heterotrophs such as fish, resulting in a constant concentra­tion of dissolved carbon dioxide and bicarbonate in the mixing layer of ocean.

The amount of elemental carbon thus recycled between inorganic (carbon diox­ide) and organic matter both on the land and in the sea is a very small amount compared to the total amount of elemental carbon existing in the globe. However, the amount of elemental carbon recycling in the biosphere, 50 Gt, is big enough to influence the carbon dioxide concentration in air because the size of carbon di­oxide pool in biosphere is only 700 Gt.

The approximately 5 Gt of elemental carbon released from the combustion of fossil fuels is considerably large when compared to the amount of elemental carbon recycling in the biosphere, 50 Gt. It is therefore reasonable to attribute ele­vating levels of carbon dioxide concentration in the air to carbon dioxide emis­sions from combustion of fossil fuels. In order to prevent such increase of carbon dioxide concentration in the air, numbers of autotrophic organisms must increase so as to fix 5 Gt of elemental carbon into organic materials.

In the case of nitrogen, the effect of increased artificial nitrogen fixation (synthetic ammonia and urea) to the environment has not been observed. To date, the amount of nitrogen recycling through artificial fixation has reached 30 Gt per year which is almost equivalent to the amount of biologically fixed nitrogen (by
nitrogen fixing bacteria such as Rhizobium). The luck of influence of the rapid growth of artificially fixed nitrogen on the environment of the globe is due to the huge nitrogen pool size in the biosphere, where about 3,800,000 Gt of elemental nitrogen is presented.

Cell Mass. A spectrophotometer (Coleman model 55, Perkin-Elmer, Maywood, IL) was used to measure the absorbance of the samples at a wavelength of 600 nm that is in the visible region. Samples were diluted as required to assure absorbences of less than 0.5. In this region the calibration curve was linear with a slope of 0.65 g dry weight per unit absorbance.

HPLC. Hitachi HPLC (Hitachi Ltd., Tokyo, Japan) with RI detector was used to analyze the concentrations of glucose, xylose, xylitol, ethanol and glycerol. A BioRad HPX-87H Ion-Exclusion column was used. The mobile phase was 0.005M H2S04 at a flow rate of 0.4 mL/min.

Glucose Analyzer. YSI 2700 Select Biochemistry Analyzer (YSI Inc., Yellow Springs, OH) equipped with glucose membrane was used for rapid analysis of glucose concentration in the fermentation media.

The conversion of component TGs to simple alkyl esters (transesterification) with various alcohols reduces the high viscosity of oils and fats (see also Figure 1). Base catalysis of the transesterification with reagents such as sodium hydroxide is preferred over acid catalysis because the former is more rapid (74). Transesterification is a reversible reaction. The transesterification of soybean oil with methanol or 1-butanol proceeded with pseudo-first order or second order kinetics, depending on the molar ratio of alcohol to soybean oil (30:1 pseudo-first order, 6:1 second order; NaOBu catalyst) while the reverse reaction was second order (75).

Methyl esters are the most “popular” esters for several reasons. One reason is the low price of methanol compared to other alcohols. Generally, esters have significantly lower viscosities than the parent oils and fats (Tables III and IV). Accordingly, they improve the injection process and ensure better atomization of the fuel in the combustion chamber. The effect of the possible polymerization reaction is also decreased. The advantages of alkyl esters were noted early in studies on the use of sunflower oil and its esters as DF (29-31). Another advantage of the esters is possibly more benign emissions, for example, with the removal of glycerol (which is separated from the esters) the formation of undesirable acrolein may be avoided, as discussed above. These reasons as well as ease and rapidity of the process are responsible for the popularity of the transesterification method for reducing the viscosity-related problems of vegetable oils. The popularity of methyl esters has contributed to the term “biodiesel” now usually referring to vegetable oil esters and not neat vegetable oils.

In the early studies on sunflower esters, no transesterification method was reported (29-31). Another early study used H2S04 as the transesterification catalyst (76). It was then shown, however, that in homogeneous catalysis, alkali catalysis is a much more rapid process than acid catalysis in the transesterification reaction (74, 77). At 32°C, transesterification was 99% complete in 4 h when using an alkaline catalyst (NaOH or NaOMe). At 60 °С and a molar ratio alcohokoil of at least 6:1 and with fully refined oils, the reaction was complete in 1 h to give methyl, ethyl, or butyl esters. The reaction parameters investigated were molar ratio of alcohol to vegetable oil, type of catalyst (alkaline vs. acidic), temperature, reaction time, degree of refinement of the vegetable oil, and effect of the presence of moisture and free fatty acid. Although the crude oils could be transesterified, ester yields were reduced because of gums and extraneous material present in the crude oils.

Besides sodium hydroxide and sodium methoxide, potassium hydroxide is another common transesterification catalyst. Both NaOH and KOH were used in early work on the transesterification of rapeseed oil (78). Recent work on producing biodiesel (suitable for waste frying oils) employed KOH. With the reaction conducted at ambient pressure and temperature, conversion rates of 80 to 90% were achieved within 5 minutes, even when stoichiometric amounts of methanol were employed (79). In two steps, the ester yields are 99%. It was concluded that even a free fatty acid content of up to 3% in the feedstock did not affect the process negatively and phosphatides up to 300 ppm phosphorus were acceptable. The resulting methyl ester met the quality requirements for Austrian and European biodiesel without further treatment. In a study similar to previous work on the transesterification of soybean oil (74, 77), it was concluded that KOH is preferable to NaOH in the transesterification of safflower oil of Turkish origin (80). The optimal conditions were given as 1 wt-% KOH at 69±1 °С with a 7:1 alcohol: vegetable oil molar ratio to give 97.7% methyl ester yield in 18 minutes.

Patents. Most patents dealing with transesterification emphasize the engineering improvement of the process. Using patented procedures, a transesterification process permitting the recovery of all byproducts such as glycerol and fatty acids has been described (81). The use of alkaline catalysts is also preferred on the technical scale, as is documented by patents using sodium hydroxide, sodium methoxide, and potassium hydroxide (82-85). Different esters of C9.24 fatty acids were prepared with A^Q — or Fe203- containing catalysts (86). A sulfonated ion exchange catalyst was preferred as catalyst in the esterification of free fatty acids (87).

Other procedures. Besides the methods discussed here, other catalysts have been applied in transesterification reactions (88). Some recently studied variations of the above methods as applied to biodiesel preparation are briefly discussed here.

Methyl and ethyl esters of palm and coconut oils were produced by alcoholysis of raw or refined oils using boiler ashes, H2S04 and KOH as catalysts (89). Fuel yields > 90% were obtained using alcohols with low moisture content and EtOH-H20 azeotrope.

Instead of using the extracted oil as starting material for transesterification, sunflower seed oils were transesterified in situ using macerated seeds with methanol in the presence of H2S04 (90). Higher yields were obtained than from transesterification of the extracted oils. Moisture in the seeds reduced the yield of methyl esters. The cloud points of the in situ prepared esters appear slightly lower than those prepared by conventional methods.

Another study (97) reported the synthesis of methyl or ethyl esters with 90% yield by reacting palm and coconut oil from the press cake and oil mill and refinery waste with MeOH or EtOH in the presence of easily available catalysts such as ashes of the waste of these two oilseeds (fibers, shell, husk), lime, zeolites, etc. Similarly, it was reported that the methanolysis of vegetable oils is catalyzed by ashes from the combustion of plant wastes such as coconut shells or fibers of a palm tree that contain K2C03 or Na2C03 as catalyst (92). Thus the methanolysis of palm oil by refluxing 2 h with MeOH in the presence of coconut shell ash gave 96-98% methyl esters containing only 0.8-1.0% soap. The ethanolysis of vegetable oils over the readily accessible ash catalysts gave lower yields and less pure esters than the methanolysis.

Several catalysts (CaO, K2C03, Na2C03, Fe^, MeONa, NaA102, Zn, Cu, Sn, Pb, ZnO, and Dowex 2X8 (anion exchange resin)) were tested (mainly at 60-63 °С) for catalytic activity in the transesterification of low-erucic rapeseed oil with MeOH (93). The best catalyst was CaO on MgO. At 200°C and 68 atm, the anion exchange resin produced substantial amounts of fatty methyl esters and straight-chain hydrocarbons.

An enzymatic transesterification method utilizing lipases and methanol, ethanol, 2-propanol, and 2-methyl-1-propanol as alcohols gave alkyl esters of fatty acids (94, 95). This method eliminates product isolation and waste disposal problems.

Analysis of Transesterification Products. Hardly any chemical reaction, including transesterification, ever proceeds to completion. Therefore, the transesterified product, biodiesel, contains other materials. There are unreacted TGs and residual alcohol present as well as partially reacted mono — and diglycerides and glycerol co-product.

Glyceride mixtures were analyzed by TLC / FID (thin-layer chromatography / flame ionization detection) (96), which was also used in the studies on the variables affecting the yields of fatty esters from transesterified vegetable oils (74). Analysis of reaction mixtures by capillary GC determined esters, triglycerides, diglycerides and monoglycerides in one run (97). Free glycerol was determined in transesterified vegetable oils (98) Besides analyzing esters for sterols (99-101), which are often minor components in vegetable oils, and different glycerides (102-103), recently the previous GC method (97) was extended to include analysis of glycerol in one GC run (104). In both papers (97, 104), the hydroxy groups of the glycerides and glycerol were derivatized by silylation with Af-methyl-Af-trimethylsilyltrifluoroacetamide. A simultaneous analysis of methanol and glycerol was recently reported (105).

Other authors, using GC to determine the conversion of TGs to methyl esters, gave a correlation between the bound glycerol content determined by TLC/FID and the acyl conversion determined by GC (106). Glycerol has also been detected by high- performance liquid chromatography (HPLC) using pulsed amperometric detection, which offers the advantage of being more sensitive than refractometry and also suitable for detection of small amounts of glycerol for which GC may not be suitable (107).

Recently, an alternative method for determining the methyl ester content based on viscosity measurements, which agreed well with GC determinations, was reported (108). The method is reportedly more rapid than GC and therefore especially suitable for process control.