Long chain organic acid bioproduction
09856499 ยท 2018-01-02
Assignee
Inventors
Cpc classification
C12N1/38
CHEMISTRY; METALLURGY
C12P7/40
CHEMISTRY; METALLURGY
International classification
C12N1/38
CHEMISTRY; METALLURGY
Abstract
Method of cell culture, comprising adding a redox active compound with a redox potential of between 0.116 to 0.253 to a culture capable of forming hydrogen via a hydrogenase so that the redox potential is diverted from hydrogen to form a longer chain acids, e.g., butryic acid.
Claims
1. A method of producing increased amounts of butyric acid, said method comprising culturing a Clostridium cell in a medium including sugar and a redox compound with a standard redox potential 0.116 to 0.253 V without using external electrodes to change the redox potential in the medium until said butyric acid is produced, wherein the redox compound is phenazine-1 carboxylic acid (PCA) present at about 50 M; and wherein the amount of the produced butyric acid is increased by at least 30% as compared to without supplementation of the redox compound.
2. The method of claim 1, wherein said Clostridium cells are selected from a group consisting of Clostridium acetobutylicum, Clostridium bifermentans, Clostridium pasterianum, Clostridium sordelii, Clostridium sp., Clostridium tyrobutyricum, and Clostridium kluyveri.
3. The method of claim 1, further comprising adding sugars to said media.
Description
DESCRIPTION OF FIGURES
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DESCRIPTION OF THE INVENTION
(9) We studied TNT degradation by Clostridia, as part of our efforts to develop suitable bioremediation treatments for contaminated sites. However, as a result of the metabolic studies underlying our bioremediation work, we also found that TNT addition to cell culture can affect the metabolite profile of such organisms.
(10) Experiments designed to elucidate the underlying mechanism for this phenomena showed that during TNT degradation, the cells shift metabolism away from hydrogen formation towards reduction of TNT, and the resulting effects on cell redox cofactors generate a higher proportion of butyrate than would otherwise occur as part of the cells effort to reduce excess. Furthermore, these results were fairly consistent across a variety of cell genetic backgounds tested, although only a selection of data is presented herein. It is our hypothesis, that the cells eliminate the excess NADH in the only way they canby making acetoacetyl coA, and thus bypassing the acetylCo, that leads to acetate. Thus, the cells accumulate more butyrate than acetate.
(11) We then tested a number of colored molecules with a range of redox potentials, and found that those molecules with a range of redox potential 0.116 to 0.253 V show similar effects, while dyes outside this range do not. Thus, the method has generally applicability in driving Clostridia and other ferrodoxin hydrogenase containing anaerobes towards the production of butyrate and other C4 and higher products.
(12) Materials:
(13) 2,4,6-trinitrotoluene, 2,6-dinitrotoluene, 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were purchased from ChemService (Westchester, Pa., USA); p-nitrotoluene and 2,4-dinitrotoluene were purchased from Sigma-Aldrich (St. Louis, Mo., USA); 2-hydroxylamino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene were from SRI International (Menlo Park, Calif., USA).
(14) All medium components for Clostridium culture were obtained from Difco (Detroit, Mich., USA) or Sigma-Aldrich (St. Louis, Mo., USA). All Restriction enzymes were obtained from New England Biolabs, Inc. (Beverly, Mass., USA). pCR2.1-TOPO vector from Invitrogen Corporation (Carlsbad, Calif., USA) was used for PCR product cloning. Automated DNA sequencing was performed by LoneStar automated DNA sequencing (LoneStar Laboratories Inc., Houston, Tex.).
(15) Bacterial Strains, Plasmids and Growth Conditions:
(16) All bacteria strains and plasmids are listed in Table 1.
(17) TABLE-US-00003 TABLE 1 Bacterial strains and plasmids Strain/ Reference/ Plasmid Description.sup.a source.sup.b Strains Clostridium acetobutylicum ATCC 824 Wild type ATCC M5 pSOL1 (Clark et al. 1989) 824(buk.sup.) Disruption of butyrate kinase (Green et al. 1996) gene in 824 824(pta.sup.) Disruption of phosphotrans- (Green et al. 1996) acetylase gene in 824 824(Mutant B) Disruption of SoIR by pO1X (Nair et al. 1999) Escherichia coli DH10 mcrA mcrBC recA1 Str.sup.r NEB Plasmids pSOS 94 ptb promoter, Ap.sup.r MLS.sup.r ColE I, (Tummala et al. repL 2003) pSC12 Thl.sup.r, shuttle vector, ColE I, (Zhao et al. 2003) repL pDHKM Km.sup.r, 3TI methyltransferase (Zhao et al. 2003) in pDHK29 pSOS-del Control plasmid based on pSOS94 This study by deletion of clostridium structural genes pSOS-as-hydA Antisense down-regulation of This study hydA gene pSOS-as-hydE Antisense down-regulation of This study hydE gene pSOS-as-hydF Antisense down-regulation of This study hydF gene pSOS-hydA Overexpression of hydA gene This study .sup.amcrA mcrBC, methylcytosine-specific restriction system abolished; recA1, homologous recombination abolished; Str.sup.r, streptomycin resistant; MLS.sup.r, macrolide-lincosamide- streptogramin B resistant; repL, plM13 gram-positive origin of replication; ColE I, gram- negative origin of replication; Km.sup.r, kanamycin resistant; Ap.sup.r, ampicillin resistant; Str.sup.r, streptomycin resistant; Thl.sup.r, thiamphenicol resistant. .sup.bATCC, American Type Culture Collection, Manassas, VA.; NEB, New England Biolabs, Beverly, Mass.
(18) E. coli cultures were grown aerobically at 37 C. in Luria-Bertani (LB) medium, C. acetobutylicum was grown anaerobically at 37 C. in buffered Clostridium growth medium (CGM) in a Form a Scientific anaerobic chamber (Thermo Form a, Marietta, Ohio) as described previously (Cai and Bennett 2011; Zhao et al. 2005). For E. coli recombinant strains, the medium was supplemented with ampicillin (100 g/mL), chloramphenicol (35 g/mL), kanamycin (50 g/ml), or erythromycin (200 g/mL) as appropriate.
(19) For C. acetobutylicum strains, erythromycin (40 g/mL for solidified agar plate and 100 g/mL for liquid medium) and thiamphenicol (25 g/mL) were used when necessary. For long-term storage, E. coli strains were cultivated and stored as glycerol stocks at 80 C. C. acetobutylicum strains were stored as lyophilized stocks at room temperature or glycerol stocks at 80 C. (Scotcher and Bennett 2005).
(20) Analysis of Fermentation Products:
(21) Cell growth was monitored by A.sub.600 with a Beckman DU-800 spectrophotometer. On fermentor, cell growth was monitored by a cell density sensor. The concentrations of butanol, acetone, ethanol, butyrate, and acetate were determined using gas chromatography with a Hewlett-Packard 5890 Series II instrument (Hewlett-Packard Company, Palo Alto, Calif.) as described previously (Scotcher and Bennett 2005; Zhao et al. 2003; Zhao et al. 2005). Glucose was measured by HPLC as described (Wang et al. 2011).
(22) TNT Assay in C. acetobutylicum Culture:
(23) TNT concentration in the culture medium was followed by a spectrometric protocol modified from Jenkins et al (Jenkins and Walsh 1992; Watrous et al. 2003). Freshly made sodium sulfite solution (0.2 g/mL) in 50 mM pH 8.0 Tris-HCl buffer reacts with TNT solution to form a colored complex with maximum absorption at A.sub.414 that remains stable for hours under assay conditions. A.sub.414 readings increase linearly up to a 500 M TNT concentration.
(24) Single C. acetobutylicum colonies from freshly grown plates were inoculated into buffered CGM medium with appropriate antibiotics and incubated overnight as seed cultures. An appropriate amount from seed culture was used for the subculture to yield a starting A.sub.600 of 0.1. The culture was incubated at 37 C. and growth (A.sub.600) measured periodically. TNT degradation rate was assayed during early-exponential phase (A.sub.600 0.3), mid-exponential phase (A.sub.600 1.0) and stationary phase (24 hour culture with A.sub.600 varied) by diluting the culture at different times into fresh medium containing 400 M TNT. At different time points, 250 L samples were taken and immediately reacted with 750 L sodium sulfite solution by vortex; the samples were further centrifuged at 14000 rpm for 10 minutes and the supernatant was used for the TNT spectrophotometric assay. The TNT degradation rate .sub.max for different strains were compared by calculating the rate of TNT decrease during the first 40 minutes.
(25) In order to determine the effect of culture dilution on TNT degradation and confirm the linear range of the measurements, five C. acetobutylicum cultures were prepared to ultimately yield 10, 20, 30, 40, and 50 dilutions respectively. For each dilution sample, three subcultures were made. The appropriate amounts of C. acetobutylicum (A.sub.600 0.3) and CGM/TNT mixture, for a total volume of 10 mL, were transferred anaerobically into 15 mL culture tubes. The tubes were immediately vortexed, and a 0.25 mL sample of each tube was added to 0.75 mL assay buffer. The tubes were left to incubate at 37 C., and 0.25 mL samples were again taken at 20 minutes and 40 minutes. All operations were conducted in the anaerobic chamber, and all solutions were pre-equilibrated in the chamber for twenty-four hours. Once the three time samples were collected and centrifuged at 13000 rpm for 10 minutes, the A.sub.414 absorbance of each supernatant was measured, and the dilution factors were considered to calculate the specific TNT degradation rates. Since the time of the initial rate measurement was short after the dilution compared to a typical lag phase recovery of growth rate the measured rates are considered indicative of the metabolism of the cell at the time points used.
(26) The Colorimetric TNT Assay Protocol:
(27) A simplified Jenkins TNT assay protocol was adapted in our experiments. Major nitro-compounds and TNT metabolites do not interfere with our TNT assay, in which TNT showed an absorption peak at 414 nm, while other partially reduced species did not. The other nitro compounds showed absorption peaks around 350 nm with a much lower intensity than the peak at 414 nm for TNT (data not shown). The measured TNT concentration in the culture declined with time during the assay, and the rate of decrease had diminished considerably after 1 hr.
(28) It was found that sample dilution affected the calculated specific TNT degradation rate. Samples of Clostridial cultures diluted in higher volumes of the CGM/TNT mixture exhibited increasing levels of specific TNT degradation rate up to a dilution factor of 40, and further dilution incurred no additional effect on the specific rate of transformation. Therefore the maximal specific TNT degradation rate as measured by this method was used in the figures and tables.
(29) TNT Degradation Rate is Relatively Stable:
(30) Strains of C. acetobutylicum were compared for their rate of TNT degradation. The strains analyzed were the wild type ATCC 824; strain M5 (Clark et al. 1989; Stim-Herndon et al. 1996), a strain that has lost the pSOL plasmid and does not produce butanol; strain 824(pta.sup.), with a disrupted phosphotransacetylase gene, pta, which produces a lower level of acetate during the early phase of culture; strain, 824(buk.sup.) with a disrupted butyrate kinase gene, buk, which produces butyrate at a lower level during the early phase of culture (Green et al. 1996); and 824 (Mutant B) a strain that has a disruption in the gene upstream of the solvent operon and has increased butanol formation (Nair et al. 1999).
(31) These strains could reveal differences in the TNT transformation rate correlated with the metabolite pattern of the cells, especially in the early log phase. Since the metabolic mutations affected the products of early metabolism and this change in metabolic pattern could in turn affect the relative proportion of hydrogen produced during the early log phase. For example, the difference in acetate versus butyrate production can result in a different level of hydrogen formation and potentially alter the availability of reductant for TNT transformation. Also since some of these strains, the buk.sup. and mutant H and mutant B strains, have been shown previously to enter the solvent producing phase earlier than the wild type parent, it was anticipated they may have lower ability to transform TNT in the early or mid log phase samples as they would be physiologically more like the early solvent producing state.
(32) Samples from cultures of each C. acetobutylicum strain (Table 1) were diluted in order to measure the initial rate of the TNT transformation of the specific culture. To measure the TNT degradation rate during different growth phases samples were analyzed from cultures at A.sub.600 0.3 (for early exponential phase), A.sub.600 1.0 (for mid-exponential phase) and 24-hour culture (for stationary phase culture, solventgenic phase).
(33) The initial rate of TNT degradation in each culture is shown in
(34) The maximal specific TNT degradation rate may be limited by hydrogenase levels or by other factors such as the glucose uptake rate, growth rate, supply of reductant (Fd.sub.reduced) or other components. To investigate the effects of these other factors, analyses were done on the medium before and during the time the TNT was being degraded.
(35) In order to quantitate the glucose metabolism during the TNT transformation, the metabolites were analyzed from parental C. acetobutylicum ATCC824 cultures degrading TNT at the high rates presented in
(36) Glucose uptake rates in three of the culture experiments were found by measurement of the acetate and butyrate concentrations before and after the TNT degradation period. The correlation to glucose utilization used the stoichiometric amount of acetate and butyrate produced by Clostridia cultures (A.sub.600=0.3) within an hour of TNT addition. Since the stoichiometric ratio of metabolites produced to glucose consumed is 2:1 for acetate and 1:1 for butyrate, the molar increase observed in these two products during the first hour was used to determine the amount of glucose metabolism to acids during the period of TNT transformation. Ethanol and butanol were measured and discounted from the analysis as negligible. These measurements gave glucose uptake rates of 22, 26 and 34 mM/Hour/A.sub.600.
(37) In two of the experiments, the glucose uptake by Clostridia culture (A.sub.600=0.3) was analyzed by HPLC directly. For each experiment, the glucose concentration values were measured both before and after the hour-long run with TNT. The rate of glucose consumption was directly determined from the concentration difference showing values of 17 and 14 mM/h/A.sub.600. The ratios of TNT to glucose metabolism in these TNT studies ranged from 0.45 to 1.0. So it appeared, a mole of TNT was reduced for about every 1-2 moles of metabolized glucose.
(38) These relatively high ratios suggest that TNT can be reduced competitively against regular cellular metabolism and that when operating at the maximal rate, TNT reduction is not a minor factor in the cells metabolic activities and can represent a significant distortion and sink for the cell's reducing power. Thus, rather than being a minor side reaction of the cells metabolism, the TNT is an effective in vivo reactant with the redox carriers/enzymes such as reduced ferredoxin-hydrogenase, and it can affect the cells metabolism during this time.
(39) The reaction with TNT not only can use a significant proportion of the cells reducing power, but in doing this it affects the overall pattern of metabolism and is in turn affected by competitors which influence the redox situation, e.g. the more effective diversion of redox to solvent producing redox pathways later in the culture. This perspective would place TNT among the group of dyes that can be reduced by Clostridium (Rao and Mutharasan 1987), and thus this work could have broader implications in the general control/understanding of redox regulation and response in these anaerobes.
(40) In the case of TNT, the reduction is irreversible and continues beyond an initial reduction step (Hughes et al. 1998a; Hughes et al. 1998b), while for many dyes the reaction is reversible and the reduced dye can transfer electrons to another carrier or redox reaction and affect the metabolite pattern.
(41) We found TNT addition alters the redox potential of the culture. Addition of TNT to cultures in exponential growth resulted in a dose dependent spike in the redox potential (not shown). This initial spike was followed by a persistent increase in the redox potential of the cultures receiving TNT when compared to the redox level of the control culture. The relative increase in redox potential lasted for the remainder of exponential growth, indicating that TNT addition results in a change to the culture's redox status for three hours.
(42) We also observed addition of TNT resulted in a decrease of hydrogen production by cultures of C. acetobutylicum ATCC 824. TNT can act as an alternative electron acceptor for the hydrogenase enzyme, so the effect of TNT addition on H.sub.2 production in cultures during exponential growth was determined by measuring the H.sub.2/CO.sub.2 ratio. The data (not shown) shows that there was a sharp reduction in the H.sub.2/CO.sub.2 ratio following the addition of TNT, which was due to a decrease in H.sub.2 production since CO.sub.2 production remained relatively constant. This provides further evidence that TNT can compete with the natural electron acceptors of the hydrogenase enzyme in actively growing cells and indicates that TNT reduction could alter the metabolic output of the cells.
(43) We also noted that when TNT was added, and the reaction proceeded over a period of time, a different metabolic pattern was seenmore butyrate and less acetate were observed in a variety of Clostridia cultures studied.
(44) We measured the acid metabolites formed during the time after TNT addition. A culture of C. acetobutylicum grown under the conditions where the TNT degradation rate was analyzed was processed and subjected to gas chromatography to determine the metabolites present in the pre-TNT added culture and in the culture 1 hour after the TNT addition, a time when the starting TNT had been depleted. The culture continued to grow during this time and the metabolism of glucose continued during the 1 hour period and seemed unchanged in rate as judged by the total production of acids during this time. However, analysis of the metabolites formed over the period during TNT transformation showed a significant change in the proportions of acetate and butyrate, with a large amount of butyrate being produced in comparison to very little acetate (Table 3).
(45) In the study presented in
(46) If one considers the total reductant available and that some of the redox goes to reduce the TNT, initially it might seem that the loss of this reducing power would lessen the amount of butyrate formed, since formation of butyrate from pyruvate requires more reduction than formation of acetate. However if we examine the process of reduction to form hydrogen and butyrate we see a difference in cofactor used. In the formation of hydrogen, the cofactor is reduced ferredoxin generated by the pyruvate:ferredoxin oxidoreductase in the conversion of pyruvate to acetyl-CoA, providing one reduced Fd per pyruvate consumed and for each CO.sub.2 released, and this is supplemented to some degree by conversion of some of the NADH formed in glycolysis via a NADH-ferredoxin reductase.
(47) In the early growth stage we are studying there is no formation of the reduced solvents, ethanol and butanol and the enzymes forming these compounds are not induced under this growth condition. The normal ratio of acetate to butyrate 1:1 then gives a value of 1.3-1.4 for the H.sub.2/CO.sub.2 ratio and a use of more Fd than would be generated by the pyruvate:ferredoxin reductase indicating use of some NADH to form reduced Fd for the extra hydrogen formation (Table 3). In this circumstance, the shift to more butyrate formation during the period of TNT reduction by the ferredoxin-Fe hydrogenase in our anaerobic chamber experiments, can be considered as a result of a difference in the cofactor balance.
(48) With TNT it appeared that blockage of the usual hydrogenase acceptance of reductant from reduced ferredoxin was lessened, and thus the NADH-ferredoxin reductase reaction did not proceed as well as normal. This situation then resulted in more NADH being available in the cell and since the NADH must be recycled, it was consumed by conversion of acetyl-CoA to butyrate.
(49) The cell cannot reduce acetyl-CoA directly due to a lack of aldehyde-alcohol dehydrogenase (e.g. AdhE), because alcohol forming genes and proteins are not expressed during this early growth phase. Thus, there are few options available to recycle the NADH, and it appeared the NADH was recycled to NAD+ through conversion of the acetyl-CoA to acetoacetyl-CoA, beta-hydroxylbutyryl-CoA, crotonyl-CoA and butyryl-CoA. This route was already operating to a good extent in the cell since almost equivalent molar amounts of acetate and butyrate were formed during the normal acid phase culture and the normal proportion of acids yielded a hydrogen to carbon dioxide ratio of approximately 1.3-1.4 in the pre TNT culture (not shown) and which is consistent with previous literature where uncontrolled pH batch early phase cultures were studied (Husemann and Papoutsakis 1990; Zhao et al. 2003).
(50) The pathway for acetyl-CoA to butyryl-CoA allowed consumption of 2 NADH per molecule of butyrate formed and relieved the cofactor imbalance between ferredoxin and NADH since NADH is the reductant used in this pathway. The redistribution observed also points to the relative ability of TNT to both be reduced by hydrogenase and inhibit the reaction of hydrogenase by serving as an alternative to ferredoxin. In this way the addition of TNT effectively removes a portion of the normal role of hydrogenase while it acts as a competing substrate and leads to a build-up and redistribution of the substrate for the hydrogenase (i.e. reduced ferredoxin) as shown in
(51) The detailed redox calculations shown in Table 3 analyzes the redistribution of reductant from production of hydrogen to more production of butyrate during the time when TNT was present and being reduced. The level of TNT present (400 M) would not consume much of the reductant available directly in this experiment, and the amount would be expected to be 1 mM or less based on the early reduction steps of TNT under these conditions as analyzed previously (Ahmad and Hughes 2000; Khan et al. 1997; Padda et al. 2000).
(52) Calculations of the observed reductant consumed in the formation of acids either before or during TNT transformation and the resulting reducing equivalents available for formation of hydrogen give values of 1.30 for the hydrogen/CO.sub.2 ratio before TNT addition and 1.05-1.12 during TNT degradation. These values are very close to the values measured experimentally (not shown), before TNT addition of 1.36 and during TNT degradation of 1.18.
(53) If the data are presented in a form where the fraction of total reductant formed from glucose metabolism, i.e. reduced ferredoxin and NADH, that goes to either formation of hydrogen or butyrate is calculated, we can analyze the proportion of reductant that is consumed in butyrate formation in the culture during the one hour period before TNT addition and it is 35% of the total reductant. If the same calculation is done on the culture during the one hour period of TNT degradation a proportion of 44-47% of total reductant that goes into formation of butyrate is determined.
(54) The variation of hydrogenase activity observed fits with the finding of Watrous et al. (Watrous 2003) and was shown in our other work, not presented here, that hydA antisense lowered the expression and activity of hydrogenase and TNT reduction activity. On the other hand, increasing the amount of hydrogenase through overexpression affected the rate of TNT transformation in cells to only a small degree, less than two fold, while the expression of the hydA gene as determined by Rt-PCR was elevated by 50 fold. These results suggest the limitation on reducing capacity seems to be at least partially due to the availability of reduced ferredoxin to transfer reducing equivalents to hydrogenase or TNT.
(55) The results also fits with the observation shown in
(56) These findings of the effect of competition of other factors and intracellular cofactor pathways on TNT reduction pave the way for more detailed investigation of the effects these parameters play in defining the rate of TNT degradation in the natural environment. This is particularly interesting in light of the analysis of natural mobile soluble electron carriers in native soil ecosystems where molecules such as quinones, phenazines, and humic acids exist or are made and released by a variety of soil organisms.
(57) The presence of these compounds that could act as competitors or inhibitors of reduced ferredoxin reactions with hydrogenase and TNT could also influence the availability of reduced ferredoxin and allow the shuttling of reductant to other pathways in the cell, e.g. those using NADH and limiting the availability of reduced ferredoxin. This feature would mimic the situation in the later phase cultures where a lower rate of TNT degradation is observed.
(58) Thus, to define the real world effectiveness of various microbial communities to degrade TNT, not only do we need to consider the major groups of direct degraders such as Clostridia and their substrates (sugars, and polymeric substrates such as cellulose) in the environment that enable growth but we also need to consider the community population and small redox molecules in the soil which may generate e-carrier competitors to the reaction with TNT and cell redox processes. The presence of these other natural electron carriers may play a role in limiting the performance of Clostridial TNT degradation in contaminated sites.
(59) The above studies showed the amount of TNT degraded contributed a substantial proportion of the metabolic redox available during the consumption of glucose during the culture. Table 4 shows the that a considerable of the cells available redox can go to TNT reduction under optimal conditions and that the reaction studied can be a major contributor to total redox partitioning. This shows it is not just a minor side reaction unconnected to main metabolism.
(60) TABLE-US-00004 TABLE 4 Exp. Exp. Exp. Exp. Exp. Exp. Exp. 1 2 3 4 5 6 7 TNT deg. 15.2 15.2 15.2 15.2 15.2 2.0 2.0 Rate (mM/Hour/ A.sub.600) Glucose 22.0 26.2 33.9 16.9 14.3 4.7 3.9 uptake rate (mM/Hour/ A.sub.600) TNT rate/ 0.691 0.580 0.448 0.899 1.06 0.43 0.51 Glucose rate
(61) Glucose uptake rates for experiments 1-3 were found by analyzing the amount of acetate and butyrate produced by Clostridia cultures (A=0.3) within an hour of TNT addition. Since the stoichiometric ratio of solvent produced to glucose consumed is 2:1 for acetate and 1:1 for butyrate, the molar increase observed in these two products during the first hour was used to estimate glucose metabolism. Other solvents produced were discounted as negligible.
(62) Experiments 4-5 were a pair of identical TNT assays where the glucose uptake by Clostridia culture (A=0.3) was analyzed by HPLC. For each experiment, the glucose concentration values were measured both before and after the hour-long run with TNT. The rate of glucose consumption was directly determined from the concentration difference.
(63) Experiments 6-7 were taken from outside literature and were conducted in the absence of TNT. Information from profiles of glucose uptake by C. acetobutylicum cultures (A=1.0) was extrapolated to estimate the consumption rate in the conditions used in our own experiments. The ratios of TNT to glucose metabolism in our TNT assays ranged from 0.448 to 1.06, which is comparable to the range extrapolated from the other papers' data, 0.43-0.51. In all of the experiments, a mole of TNT was reduced for about every 1-2 moles of metabolized glucose. These relatively high ratios confirm that TNT can be reduced competitively against regular cellular metabolism.
(64) Analysis of the metabolites formed over the period when TNT is being transformed (Table 5) shows a significant change in the proportions of acetate and butyrate.
(65) TABLE-US-00005 TABLE 5 Metabolites of C. acetobutylicum cultures before and after TNT addition Ethanol Acetone Acetate Butanol Butyrate Strain mM mM mM mM mM C. acetobutylicum 0.23 0.07 3.76 0 4.31 824 pre TNT C. acetobutylicum 0.29 0.03 5.39 0 10.11 824 1 hr post TNT C. acetobutylicum 0.06 0 1.63 0 5.8 824 during TNT C. acetobutylicum 0.10 0.02 3.71 0 4.32 M5 pre TNT C. acetobutylicum 0 0 4.53 0 11.37 M5 1 hr post TNT C. acetobutylicum 0 0 0.82 0 7.05 M5 during TNT
(66) The above results show that TNT in the culture broth significantly increased the amount of butyrate formed in a wide variety of genetic backgrounds. This suggested to us that electron carriers could be used to affect the profile of metabolites produced in culture, driving production towards e.g., longer acids.
(67) In order to investigate the role of other electron carriers we examined a variety of electron carrier dyes with different redox potentials on the pattern of metabolites of the Clostridium acetobutylicum cultures. The effects of various redox dyes on the pattern of acids was studied, and the results shown in
(68) With 50 uM phenazine-1-carboxylic acid (PCA) (redox potential 0.113) cultures showed a much higher ratio of butyrate to acetate in
(69) These results show that addition of a redox compound with the redox potential 0.116 to 0.253 V produces more longer chain organic acid molecules and fewer short chain organic acid molecules, and thus can be used in cultures of same to improve production levels. Redox compounds outside this range lack the effect. The redox potential and effect of various dyes is summarized in
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