Biotechnol. Prog. 1992, 8,316-326
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Activity Regeneration in Continuous Clostridium acetobutylicum Bioconversions of Glucose Kenneth F. Reardon*': and James E. Bailey*9§ Department of Chemical Engineering, California Institute of Technology, Pasadena, California 91125
The regeneration of product-forming activity by periodic nutrient feeding was studied in a continuous immobilized Clostridium acetobutylicum reactor system. Activity levels were increased by feeding ammonia and vitamins during selected intervals of the experiments; these compounds were not otherwise present in the medium. In contrast with experiments utilizing rich, complex media, regeneration with a defined medium resulted in increased rates of product formation far in excess of rates of concomitant cell growth. During one nutrient feeding phase, the increase in the butyric acid production rate was five times that of the cell growth-rate increase, while the analogous ratio for butanol was 18. A hypothesis explaining the patterns of substrate consumption and product formation during the regeneration and deactivation periods has been developed: decreasing levels of exogenous ammonia induce the high-affinity ammonia uptake system, causing the adenylate energy charge of the cells to increase; this in turn leads to the repression of glucose carbon flow through the EMP pathway and to the use of the acid-forming catabolic reactions for more efficient production of ATP. Pulsewise addition of three different vitamins during one regeneration phase yielded interesting results, including a 50 3'% increase in butyric acid production rate that appeared to be connected with the addition of biotin. Finally, enumeration of the different morphological types of immobilized cells provided the first measurements of population dynamics in an immobilized cell system. Changes in the numbers of each cell type are consistent with the sporulation and germination processes of C. acetobutylicum observed with suspended cells.
Introduction The use of viable, nongrowing cells in immobilized cell biocatalysts has several advantages over immobilized growing cells, including cell-free product streams and the potential for higher product:biomass ratios. However, because the nongrowth state is usually achieved by nutrient limitation, loss of the desired conversion activity inevitably occurs. The cause@)of this activity loss might include enzyme inactivation, cofactor leakage, or membrane damage. Many reports have indicated that the activity of nutrient-starved immobilized cells can be regenerated by exposure to growth medium [e.g., Ohlson et al. (1979), Forberg et al. (1983), Cheetham et al. (1985), Kloosterman and Lilly (1985), and Klein and Wagner (1987)l. In most cases, the nutrient medium contained undefined complex compounds such as yeast extract and peptone [e.g., Forberg et al. (1983), Cheetham et al. (1985), and Kloosterman and Lilly (1985)l. However, effects of exposure to defined medium containing the missing nutrient (nitrogen) have been investigated in only a few cases (Briffaud and Engasser, 1979; Inloes et al., 1985; Reardon et al., 1986). The possible physiological mechanisms of activity regeneration by nutrient addition include protein synthesis, activating or relaxing inhibition of enzyme activity, and/or cell growth. Studies with polyacrylamide- or calcium alginate-entrapped Arthrobacter simplex cells indicated that the increase of steroid-A'-dehydrogenase activity by exposure to peptone-glucose solutions was t Current address: Department of Agricultural and Chemical Engineering, Colorado State University, Fort Collins, CO 80523. 5 Current address: Institut fur Biotechnologie, ETH-Honggerberg, CH-8093 Zurich, Switzerland.
primarily due to cell growth, since the presence of benzylpenicillin or chloramphenicol (inhibiting cell wall synthesis and protein synthesis, respectively) prohibited regeneration (Ohlson et al., 1978,1979). Although these results were achieved on the basis of activity increases rather than on the regeneration of lost activity, an investigation by Cheetham et al. (1985) showed that the presence of the same two antibiotics prevented the regeneration of isomaltulose-forming activity in deactivated immobilized Erwinia rhapontici cells. Forberg et al. (1983) developed a nutrient dosing technique for use with Clostridium acetobutylicum entrapped in calcium alginate. The feed to the continuousflow reactor contained no nitrogen or vitamin sources, but it was supplemented with complex nutrients for 15-min periods at intervals ranging from 2 to 12 h. By gradually increasingthe nutrient dosing interval, butanol production was sustained and a lower fraction of feed glucose carbon appeared as biomass. Unfortunately, the number and type of immobilized cells was not reported; microscopic examination of the calcium alginate beads indicated that some sporulation had occurred. Inloes et al. (1985) studied the production of ethanol by immobilized Saccharomyces cereuisiae cells which were alternately fed a complex nutrient medium and a nitrogenfree medium. During periods of nitrogen deficiency, ethanol productivity decreased, amounts of acetaldehyde and glycerol produced relative to ethanol increased, total protein content of the cells decreased, and formation of large intracellular lipid droplets was observed. Exposure to the complex medium regenerated the ethanol-forming activity, but the level of activity achieved decreased with succeeding regeneration periods. The majority of these regeneration studies have utilized
8756-7938/92/3008-0316$03.00/0 0 1992 American Chemical Society and
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complex nutrient compounds, and, with the exception of the investigation just described, little has been reported on the details of the regeneration process (Le., cell physiology and morphology). In this report, several experiments will be discussed that investigate product formation and immobilized cell concentration changes during alternating periods of bioconversion and regeneration in fermentations of immobilized C. acetobutylicum. Regeneration was accomplished by supplying either nitrogen or growth vitamins plus nitrogen to the calcium alginate-immobilized C. acetobutylicum cells, which had been starved for both types of nutrient. The use of an oligosporogenous mutant strain provided additional information on the events occurring during bioconversion and activity regeneration.
Materials and Methods The culture media, calcium alginate immobilization procedure, experimental apparatus for batch and continuous operations (a gradientless packed-bed bioreactor system), and assay methods have been described in detail previously (Reardon and Bailey, 1989a,b). Experiments 1and 2 utilized C. acetobutylicum ATCC 824, while experiment 3 employed the oligosporogenous strain C. acetobutylicum ATCC 39236. Differences in the culturing and immobilizationprotocols between these two strains are discussed in another report (Reardon and Bailey, 1989b). The regeneration experiments discussed in this report all began with the batch growth, rinsing, and continuous bioconversion procedures, materials, and methods outlined elsewhere (Reardon, 1988; Reardon and Bailey, 1989a). The bioconversion feed in these experiments was a defined nitrogen- and vitamin-free medium (10 g/L glucose for run I, 15 g/L for runs 2 and 3), while the feed during the regeneration phases consisted of the bioconversion feed plus NH4C1 and (sometimes) vitamins. All continuousfermentation media were filter-sterilized.
Experimental Protocols 1. Experiment 1. Following a standard batch growth phase and nutrient rinsing, the immobilized cell reactor operated using the schedule listed in Table I. The pH was maintained a t 5.0 for the entire run. 2. Experiments 2 and 3. Experiment 2 utilized strain ATCC 824, while run 3 used strain ATCC 39236. In both experiments, the dilution rate was 0.22 h-I and the pH was maintained a t 4.5. The changes in feed composition during each experiment are given in Table I. In these runs, the change to regeneration conditions was done in a stepwise manner by injecting a concentrated solution of nutrient(s) into the fermentor vessel; the level of nutrient( 8 ) in the injection solution was calculated to bring the reactor liquid concentrations to that in the feed (at the moment of injection). In experiments 2 and 3, the "vitamin mixture" (Table I) contained (final concentrations) 1mg/ L p-aminobenzoic acid (PABA), 1mg/L thiamin hydrochloride, and 0.05 mg/L D-biotin. During the fourth regeneration period of experiment 3, these vitamins were present in the feed medium a t one-tenth these values. In order to assess the effects of each of these vitamins on immobilized cell product-forming activity, pulses of each were injected into the fermentor so that the concentration of that vitamin in the system at the time of injection would be at the one-tenth of vitamin mix level. That is, PABA was injected at 250 h (to a system concentration of 0.1 mg/L), thiamin hydrochloride was
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Table I. Experimental Schedules Experiment 1 time dilution interval. h rate. h-' feed 0-73 0.22 bioconversion 73-135 0.04 bioconversion 135-148.5 0.21 bioconversion + 2 g/L butyric acid 148.5-165 0.21 bioconversion + 2 g/L butyric acid + 1 g/L casamino acids 165-208 0.21 bioconversion + 2 g/L butyric acid ~~~
~~
Experiment 2 time interval, h 0-101 101-141 141-221 221-261 261-291 291-331
feed bioconversion regeneration (0.6g/L NH4CI) bioconversion regeneration (0.6g/L NH4C1) bioconversion regeneration (0.6g/L NH4C1, vitamin mixture) Experiment 3
time interval, h 0-85 85-100 100-115 115-155 155-165 165-195 195-205 205-235 235-250 250-270 270-285 285-335.5
feed bioconversion regeneration (0.6g/L NHGl) regeneration (0.6g/L NH4C1, vitamin mixture) bioconversion regeneration (0.6g/L NHdCl, vitamin mixture) bioconversion regeneration (0.6g/L NH4C1, l / l O t h vitamin mixture) bioconversion regeneration (0.6g/L NH4C1) regeneration (0.6g/L NH4C1, PABA at 250) regeneration (0.6g/L NHaC1, thiamin at 270) regeneration (0.6g/L NHdCI, biotin at 285)
injected at 270 h (0.1 mg/L), and biotin was injected a t 285 h (0.005 mg/L).
Results A. Experiment 1. The primarygoal of this experiment, which used the sporogenous strain ATCC 824 and was carried out at pH 5, was to show the effects of activity regeneration by a complex nutrient compound (casamino acids). The time courses of product and glucose concentrations are presented in Figure 1;the level of butyric acid in the feed after 135 h is also shown. The first 73-h period (unsupplemented feed, D = 0.2 h-l) resembles the pH 5 bioconversion results observed in similar experiments (Reardon and Bailey, 1989a): early solvent production followed by acid formation (the high initial ethanol levels were due to incomplete rinsing of the ethanol-sterilized pH probe). The change to butyric acidcontaining feed (at 135 h) did not increase the concentration of any other product, but the addition of 1 g/L casaminoacids to the butyrate-supplemented feed at 148.5 h resulted in an immediate, rapid increase in acetic and butyric acid production and glucose consumption. This was followed about 10 h later by rapid increases in the concentration of acetone, butanol, ethanol, and acetoin. The overall metabolic activity of the immobilized cells declined quickly about 5 h after the feed was switched back to the butyric acid-supplemented bioconversion medium (at 165 h). The profile of 590-nm absorbance of liquid samples withdrawn from the reactor during this run is also shown in Figure 1. The rapid increase in A590 to relatively high levels (for an immobilized cell reactor) following the switch to casamino acid medium is due to both immobilized and
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Figure 1. Medium component concentration profiles from Experiment 1. The gray zone represents the period of casamino acid feeding. Upper left: Butyric acid (solid line) and acetic acid (dotted line); the dashed line indicates the level of butyric acid in the feed during the final 70 h. Upper right: Ethanol (solid line) and acetoin (dotted line). Lower left: Butanol (solid line) and acetone (dotted line). Lower right: Glucose (solid line) and Asw (dotted line, right-hand scale).
suspended cell growth and illustrates one of the drawbacks of regeneration by rich nutrient feeding-the loss of the advantages of using viable, nongrowing cells. B. Experiment 2. 1. Substrate and Product Concentration Profiles. In this pH 4.5 experiment, calcium alginate-immobilizedATCC 824 was exposed to three bioconversion phases (see Table I). The concentrations of the catabolic products and glucose are presented in Figure 2, and the nitrogen concentration profile is shown in Figure 3. Initial solvent-forming activity in the first bioconversion phase was succeeded by acid production. At the end of this period, the only products formed a t appreciable levels were acetic and butyric acids. With the step change to nitrogen-supplemented regeneration medium (0.6 g/L NH4C1) a t 101 h, there was an immediate, rapid uptake of nitrogen and glucose, and the concentrations of acetic acid, butyric acid, acetoin, and ethanol began to increase. The response of the immobilized cell biocatalyst to the addition of ammonia in the second regeneration period was similar to that in the first regeneration phase, except that the maximum concentration and the total amount of each product formed were significantly lower than those that occurred as a result of the previous feeding. The presence of both ammonia and the growth vitamins in the feed used during the final regeneration period stimulated high rates of acetic acid, acetoin, ethanol, and butyric acid within a few hours of the initial nutrient step change. Although lactic acid levels had not exceeded 0.4
m M during the preceding regeneration periods, it was produced a t a high rate during this regeneration period, reaching a maximum concentration of 21 mM. 2. Immobilized Cell Concentrations. The concentrations of each C. aceto buty Zicum morphological cell type in the alginate beads during experiment 2 are shown in Figure 4. Profiles are shown for vegetative cells (rodshaped), pre-spore clostridial forms (phase-bright swollen cigar shapes), mature spores (phase-bright ovoids), and germinating spores (phase-dark ovoids) (Gould, 1969). These concentrations were estimated with a microscope counting chamber. The high counts of rod-shaped objects in the first and second bioconversion periods were due to cell debris. C. Experiment 3. 1. Substrate and Product Concentration Profiles. This experiment, conducted at pH 4.5, used the oligosporogenous C. acetobutylicum mutant strain ATCC 39236. The time trajectories of product and glucose concentrations are presented in Figure 5 and the concentrations of nitrogen (as ammonia) during the run are shown in Figure 6. The experiment consisted of four bioconversionphases and five activity regeneration periods (Table I). The concentration profiles during the first bioconversion phase are generally similar to those of experiment 2. Although the overall activity during this time decreased less rapidly than during the bioconversion phase of run 2, solvent-forming activity was still replaced by acid production.
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Figure 2. Medium component concentration profiles from experiment 2. Gray zones represent regeneration periods (see Table I). Upper left: Butyric acid (solid line) and acetic acid (dashed line). Upper right: Ethanol (solid line) and acetoin (dashed line). Lower left: Butanol (solid line) and acetone (dashed line). Lower right: Glucose.
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A significant amount of nitrogen was consumed during the early hours of the first regeneration phase, but only very small changes occurred in the acetic acid, butyric acid, and glucose concentrations. Since very little change was noticed during the first regeneration period with NH4C1-supplementedfeed, the second regeneration phase used a feed medium that contained both NHdCl(O.6 g/L) and the vitamin mix described earlier. Shortly after the vitamin mixture was added to the feed, the acid product concentrations increased rapidly. The levels of all of these products decreased during this second bioconversion period until all product concentrations were nearly zero, except those of acetic and butyric acids, which had plateaued a t intermediate values. As before, the change to nitrogen- and vitamin-
supplemented feed at the start of the third regeneration phase (hour 155) initiated rapid increases in the concentration of acetic acid, butyric acid, and glucose, followed shortly by increased acetoin, ethanol, and suspended cell levels. All of these changes were reversed 5-10 h after the end of nitrogen feeding, near the time that the ammonia levels reached zero. Virtually the same patterns occurred as a result of the fourth nitrogen/vitamin feeding (starting at hour 195),although less nitrogen was consumed (Figure 6). Vitamin mix concentrations in the feed during this fourth regeneration interval were one-tenth their value in the third regeneration phase. In the final regeneration phase, each of the three vitamins was injected individually into the bioreactor system at 15-20-h intervals. A goal during this period was to investigate the effects of the individual components of the vitamin mixture. The glucose, immobilized vegetative cell, and product concentration profiles during this period are presented in Figure 7. During the 315-318-h period, the feed to the system was stopped inadvertently; this can be seen as a very slight perturbation in the concentration trajectories. Lactic acid was not produced in this experiment until the end of the second regeneration period. In general, the times a t which the lactic acid concentration increased and the relative levels reached were similar to those of butanol. This indicates that lactic acid may be formed as a means of consuming intracellular reducing equivalents. 2. Immobilized Cell Concentrations. The concentration profiles for the different immobilized cell morphological types observed during this run are shown in Figure 8. The low frequency of sporulation during nitrogen and vitamin starvation indicates that this strain, ATCC 39236, is oligosporogenic (Fitz-James and Young, 1969).
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Figure 4. Immobilized cell concentrations from experiment 2, strain ATCC 824. Gray zones represent regeneration periods. Upper left: Rod-shaped vegetative cells. Upper right: Clostridial forms. Lower left: Mature spores. Lower right: Germinating spores.
No trajectory for clostridial forms is shown because very few were observed. Analysis and Discussion Although the strategy of exposing preparations of immobilized cells that have lost activity to complete nutrient media has been employed in many cases (Ohlson et al, 1979; F6rberg et al., 1983; Cheetham et al., 1985; Kloosterman and Lilly, 1985; Inloes et al., 1985), the procedure and its effects on the immobilized microorganisms have not been analyzed in any detail. Longer term nutrient-dosing experiments in which the feed to the immobilized cell bioreactor alternately consisted of nutrient-deficient and nutrient-sufficient media have been reported (Fbrberg et al., 1983; Inloes et al., 1985), but the media used to regenerate the biocatalyst activity always contained complex undefined nutrient compounds such as yeast extract and/or peptone. Accurate assessment and control of activity regeneration require the use of defined regeneration conditions. A. Rich Nutrient Feeding. Experiment 1was performed to illustrate the effects of regeneration with complex nutrient medium. The metabolic activity of the immobilized cells increased sharply upon exposure to the casamino acid-supplemented medium. However, a comparison of the concentration profiles from this experiment (Figure 1) with the product levels during the first regeneration period of run 2 (Figure 2) shows that while nearly the same product concentrations were achieved, the release of suspended cells was much higher in the
casamino acids feeding case (implying higher rates of cell growth) (Reardon, 1988). Thus, extensive cell growth is not necessary to achieve this degree of regeneration. In the present case, cell growth observed in regeneration with complex medium likely parallels regeneration of specific cell biocatalytic activity. B. Rates of Production and Consumption. Volumetric rates of product formation and glucose consumption during experiments 2 (ATCC 824) and 3 (ATCC 39236) were calculated using the concentration data in Figures 2 and 5 and the time derivatives of those data, obtained by cubic spline curve fitting, in the unsteady-state CSTR mass balance (Reardon and Bailey, 1989b). Cubic splines were used to provide smooth, differentiable curves through the data points since standard polynomial or exponential functions were insufficient (Wold, 1974). In these experiments, the changes in the slope of a concentrationtime profile were usually much smaller than the dilution term of the CSTR mass balance, resulting in volumetric rate trajectories that resemble the corresponding concentration versus time plots (Reardon, 1988). Therefore, these rates are not presented here, and the concentration trajectories may be employed to discern trends in corresponding volumetric rates. Profiles of the rates of ammonia assimilation, determined as described above, are presented in Figure 9. Interesting changes in the uptake rates can be seen during the regeneration periods. At the start of each nitrogen feeding phase, the rate of ammonia uptake was very high but soon decreased rapidly. After a few hours, the
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