Influence of substrate carbon on the metabolism of Clostridium thermohydrosulfuricum (original) (raw)
2009
Ethanolic fermentation of simple sugars is an important step in the production of bioethanol as a renewable fuel. Significant levels of organic acids, which are generally considered inhibitory to microbial metabolism, could be accumulated during ethanolic fermentation, either as a fermentation product or as a by-product generated from pre-treatment steps. To study the impact of elevated concentrations of organic acids on ethanol production, varying levels of exogenous acetate or lactate were added into cultures of Thermoanaerobacter ethanolicus strain 39E with glucose, xylose or cellobiose as the sole fermentation substrate. Our results found that lactate was in general inhibitory to ethanolic fermentation by strain 39E. However, the addition of acetate showed an unexpected stimulatory effect on ethanolic fermentation of sugars by strain 39E, enhancing ethanol production by up to 394%. Similar stimulatory effects of acetate were also evident in two other ethanologens tested, T. ethanolicus X514, and Clostridium thermocellum ATCC 27405, suggesting the potentially broad occurrence of acetate stimulation of ethanolic fermentation. Analysis of fermentation end product profiles further indicated that the uptake of exogenous acetate as a carbon source might contribute to the improved ethanol yield when 0.1% (w/v) yeast extract was added as a nutrient supplement. In contrast, when yeast extract was omitted, increases in sugar utilization appeared to be the likely cause of higher ethanol yields, suggesting that the characteristics of acetate stimulation were growth condition-dependent. Further understanding of the physiological and metabolic basis of the acetate stimulation effect is warranted for its potential application in improving bioethanol fermentation processes.
Applied Microbiology and Biotechnology, 2006
We have investigated hydrogen (H 2 ) production by the cellulose-degrading anaerobic bacterium, Clostridium thermocellum. In the following experiments, batch-fermentations were carried out with cellobiose at three different substrate concentrations to observe the effects of carbonlimited or carbon-excess conditions on the carbon flow, H 2production, and synthesis of other fermentation end products, such as ethanol and organic acids. Rates of cell growth were unaffected by different substrate concentrations. H 2 , carbon dioxide (CO 2 ), acetate, and ethanol were the main products of fermentation. Other significant end products detected were formate and lactate. In cultures where cell growth was severely limited due to low initial substrate concentrations, hydrogen yields of 1 mol H 2 /mol of glucose were obtained. In the cultures where growth ceased due to carbon depletion, lactate and formate represented a small fraction of the total end products produced, which consisted mainly of H 2 , CO 2 , acetate, and ethanol throughout growth. In cultures with high initial substrate concentrations, cellobiose consumption was incomplete and cell growth was limited by factors other than carbon availability. H 2 -production continued even in stationary phase and H 2 / CO 2 ratios were consistently greater than 1 with a maximum of 1.2 at the stationary phase. A maximum specific H 2 production rate of 14.6 mmol g dry cell −1 h −1 was observed. As cells entered stationary phase, extracellular pyruvate production was observed in high substrate concentration cultures and lactate became a major end product.
Closing the carbon balance for fermentation by Clostridium thermocellum (ATCC 27405)
2012
Our lab and most others have not been able to close a carbon balance for fermentation by the thermophilic, cellulolytic anaerobe, Clostridium thermocellum. We undertook a detailed accounting of product formation in C. thermocellum ATCC 27405. Elemental analysis revealed that for both cellulose (Avicel) and cellobiose, >92% of the substrate carbon utilized could be accounted for in the pellet, supernatant and off-gas when including sampling. However, 11.1% of the original substrate carbon was found in the liquid phase and not in the form of commonly-measured fermentation products-ethanol, acetate, lactate, and formate. Further detailed analysis revealed all the products to be <720 da and have not usually been associated with C. thermocellum fermentation, including malate, pyruvate, uracil, soluble glucans, and extracellular free amino acids. By accounting for these products, 92.9% and 93.2% of the final product carbon was identified during growth on cellobiose and Avicel, respectively.
Applied and Environmental Microbiology, 1981
The fermentation of various saccharides derived from cellulosic biomass to ethanol was examined in mono- and cocultures of Clostridium thermocellum strain LQRI and C. thermohydrosulfuricum strain 39E. C. thermohydrosulfuricum fermented glucose, cellobiose, and xylose, but not cellulose or xylan, and yielded ethanol/acetate ratios of >7.0. C. thermocellum fermented a variety of cellulosic substrates, glucose, and cellobiose, but not xylan or xylose, and yielded ethanol/acetate ratios of ∼1.0. At nonlimiting cellulosic substrate concentrations (∼1%), C. thermocellum cellulase hydrolysis products accumulated during monoculture fermentation of Solka Floc cellulose and included glucose, cellobiose, xylose, and xylobiose. A stable coculture that contained nearly equal numbers of C. thermocellum and C. thermohydrosulfuricum was established that fermented a variety of cellulosic substrates, and the ethanol yield observed was twofold higher than in C. thermocellum monoculture fermentation...
Microbiological Research, 2002
Clostridium thermobutyricum produces butyrate as the main fermentation product from glucose, and from yeast extract, which is required for substantial growth. After sequential transfer in the presence of increasing butyrate concentrations, strain JW 171K grew in the presence of up to 350 mM butyrate either at pH 5.5 or at pH 8.0 and at 40°C as well as at 60°C. This result indicated that butyrate-dependent growth inhibition was independent from the concentration of undissociated butyric acid. Increased butyrate concentration decreased the level of tolerated glucose from above 15% to below 10%. At 0.05 and 2.0% (wt/vol) yeast extract, the YGlucose was 30 and 55 g dry weight cells per mole glucose, respectively. YATP values between 18 and 21 g weight cells per mole ATP, obtained after growth in the presence of 2% yeast extract, indicate that the butyrate fermentation under thermophilic growth conditions is as energy efficient as it is under mesophilic conditions. Externally added acetate stimulated the production of butyrate. Supplementedloc = “pre”14C-acetate was converted to butyrate, resulting in the formation of 44% labeled butyrate (i.e. formed fromloc = “pre”14C-acetate) and 56% unlabeled butyrate (formed from glucose and yeast extract). Continuous removal of H2 in batch cultures led to a shift in the fermentation products from more butyrate to the more oxidized and more energy yielding acetate.
Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 1C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h À1 , a biomass yield on glucose of 0.51 g g À1 , and a maximum specific consumption of oxygen of 11.1 mmol g À1 h À1 were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h À1. At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol À1 substrate. At 0.5 h À1 , although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol À1 of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.
End-product induced metabolic shifts in Clostridium thermocellum ATCC 27405
Applied Microbiology and Biotechnology, 2011
When attempting to increase yields of desirable end-products during fermentation, there is the possibility that increased concentrations of one product redirects metabolism towards the synthesis of less desired products. Changes in growth, final end-product concentrations, and activities of enzymes involved in pyruvate catabolism and fermentative end-product formation were studied in Clostridium thermocellum in response to the addition of individual end-products (H 2 , acetate, ethanol, formate, and lactate) to the growth medium. These were added to the growth medium at concentrations ten times greater than those found at the end of growth in cultures grown under carbon-limited conditions using cellobiose (1.1 gl −1 ) as model soluble substrate. Although growth rate and final cell biomass decreased significantly with the addition of all end-products, addition of individual end-products had less pronounced effects on growth. Metabolic shifts, represented by changes in final endproduct concentrations, were observed; H 2 and acetate yields increased in the presence of exogenous ethanol and lactate, while ethanol yields increased in the presence of exogenous hydrogen (H 2 ), acetate, and lactate. Late exponential phase enzyme activity data of enzymes involved in pyruvate catabolism and end-product formation revealed no changes in enzyme levels greater than 2-fold in response to the presence of any given end-product, with the exception of pyruvate:formate lyase (PFL), ferredoxin-dependent hydrog-enase (Fd-H 2 ase), and pyruvate:ferredoxin oxidoreductase (PFO): PFL and Fd-H 2 ase activities increased 2-fold in the presence of ethanol, while PFO activity decreased by 57% in the presence of sodium formate. Changes in enzyme levels did not necessarily correlate with changes in final end-product yields, suggesting that changes in final end-product yields may be governed by thermodynamic considerations rather than levels of enzyme expressed under the conditions tested. We demonstrate that bacterial metabolism may be manipulated in order to selectively improve desired product yields.
Microbiology, 1987
Thermostable lactate dehydrogenases (EC 1 . 1 . 1 .27) were purified to homogeneity from Clostridium thermohydrosulfuricum cells grown on starch and producing mainly ethanol (LDH,) and from cells grown on sucrose and producing mainly lactic acid (LDHJ, and were found to be distinct isoenzymes. The two enzymes both had native M, values close to 145 x lo3, but slightly different subunits with M , values about 37 x lo3. LDHL dissociated into subunits more readily. The isoelectric points were 5.0 for LDHL and 5-2 for LDHE. The catalytic activity of LDHE had an almost absolute requirement for fructose 1,6-bisphosphate (FBP) at all temperatures (22-fold activation with K I l 2 12 p~-F B P at 65 "C, pH 6.0). LDHL was activated by FBP only at temperatures over 40 "C (5-fold activation with K,12 80 ~M -F B P at 65 "C, pH 6.0). For both enzymes the optimum temperature for pyruvate reduction in the presence of 1 mM-FBP was 70 "C and the pH optimum at 65 "C was sharp and at 5-5-6-0. FBP lowered the apparent K , of LDHL for pyruvate. At 50 p~-F B P both enzymes showed a positive co-operative dependence on NADH.
Diol metabolism and diol dehydratase in Clostridium glycolicum
Archives of Biochemistry and Biophysics, 1986
Levels of the five enzymes involved in the fermentation of 1,2-ethanediol and 12-propanediol in the strictly anaerobic bacterium, Clostridium glycolicum, were investigated. All enzymes with the exception of the first enzyme in the pathway, diol dehydratase, were found to be constitutive, stable to exposure to oxygen, and present in the cytosol. Diol dehydratase was found to be extremely oxygen sensitive and strongly associated with the cell membrane. Treatment with ionic and nonionic detergents, butanol, phospholipase As, or osmotic shock procedures failed to solubilize any diol dehydratase activity. Limited proteolysis using subtilisin released small amounts of activity. Diol dehydratase was found to be specific for 1,2-ethanediol and 1,2-propanediol and required the addition of a reducing agent for maximal activity. The enzyme was strongly inhibited by low concentrations of EDTA, ethylene glycol his@-aminoethyl ether)-N,N,i?',ZV'tetraacetic acid, o-phenanthroline, hydroxylamine, hydroxyurea, and sulfhydryl reagents. Addition of adenosylcobalamin or high levels of intrinsic factor did not affect the reaction rate. Irradiation with light also did not inhibit the enzyme activity. These results suggest that the catalytic mechanism of diol dehydratase from C g&&urn does not involve a cobamide coenzyme. o 1986 Academic POW, IIIC. Growth on 1,2-ethanediol or 1,2-propanediol as the sole carbon and energy source has been investigated in various aerobic and facultatively anaerobic bacteria (l-8). A cobamide coenzyme-dependent diol dehydratase (EC 4.2.1.28), the first enzyme in the fermentative pathway catalyzed by Klebsielh pneumonias, has been purified and studied extensively (9-12). However, there have been no detailed investigations on the metabolism of these diols in a strictly anaerobic organism such as Clostridium gl~colicum. Gaston and Stadtman (13) isolated this anaerobe and showed that it could utilize 1,Zethanediol or 1,Bpropanediol as its source of energy and carbon. They found that C. glgcolicum ferments these diols to equal amounts of their corresponding alcohols and acids. This organism differs in carbon source ' To whom correspondence should be addressed. MATERIALS AND METHODS ~anism and growth, The organism used was from the original strain of C. gZvwZ&m isolated from a stagnant pond near the Chesapeake and Ohio Canal by Gaston and Stadtman (13). It was grown anaerobically at 30 and 35'C on three different carbon sources: l,2-ethanediol(1%),1,2-propanediol (l%),and glucose (4%). In addition to the carbon source, the
World Journal of Microbiology and Biotechnology, 2005
Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H 2 /mol sugar) as much hydrogen as monosaccharides (1.29 mol-H 2 /mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H 2 /mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H 2 /mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.
Applied Microbiology and Biotechnology, 2009
The objective of this research was to understand how carbon loading influences hydrogen (H2) synthesis and metabolic flow patterns in the thermophilic, cellulolytic bacterium, Clostridium thermocellum. C. thermocellum was cultivated in batch cultures with high (5 g L−1) and low (1 g L−1) initial concentrations of α-cellulose at 60°C. The growth rate of C. thermocellum was 22% lower (0.15 h−1) in cultures with low-cellulose concentration compared with cultures with high-cellulose concentrations. Although substrate depletion coincided with the end of log-growth in low-cellulose cultures, the prime reason for growth arrest in high-cellulose cultures was not identified. Ethanol, acetate, and formate were the major soluble end-products with concomitant release of H2 and CO2 under both conditions. Lactate appeared during the late log phase in high-carbon cultures when pH dropped below 6.4 and became the major end-product in stationary phase. During the exponential phase of cell growth, significantly higher yields for H2 and acetate (1.90 ± 0.14 and 1.11 ± 0.04 mol/mol glucose equivalent, respectively) were obtained from low-cellulose cultures compared to those from high-cellulose cultures. The maximum specific rate of H2 production, 6.41 ± 0.13 mmol H2/g dry cell/h, obtained during the exponential phase from low-carbon cultures was about 37% higher than that obtained from high-carbon cultures.
Applied Microbiology and Biotechnology, 2017
Fermentation with acetogens can be affected by cultivation gas phase, but to date, there is not enough evidence on that matter for Clostridium thermocellum and Moorella thermoacetica. In this work, the effects of sparged CO 2 as well as sparged and non-sparged N 2 on these microorganisms were studied using glucose and cellobiose as substrates. It was revealed that sparged CO 2 and non-sparged N 2 supported growth and acetic acid production by C. thermocellum and M. thermoacetica, while sparged N 2 inhibited both of the microorganisms. Notably, part of the sparged CO 2 was fermented by the co-culture system and contributed to an overestimation of the products from the actual substrate as well as an erring material balance. The best condition for the co-culture was concluded to be N 2 without sparging. These results demonstrate the importance of cultivation conditions for efficient fermentation by anaerobic clostridia species. Keywords Carbon dioxide (CO 2). Nitrogen (N 2). Gas sparging. Acetic acid fermentation. Clostridium thermocellum. Moorella thermoacetica (Clostridium thermoaceticum)
International Journal of Hydrogen Energy, 2010
This paper reports investigations carried out to determine the optimum culture conditions for the production of hydrogen with a recently isolated strain Clostridium butyricum CWBI1009. The production rates and yields were investigated at 30 °C in a 2.3 l bioreactor operated in batch and sequenced-batch mode using glucose and starch as substrates. In order to study the precise effect of a stable pH on hydrogen production, and the metabolite pathway involved, cultures were conducted with pH controlled at different levels ranging from 4.7 to 7.3 (maximum range of 0.15 pH unit around the pH level). For glucose the maximum yield (1.7 mol H 2 mol-1 glucose) was measured when the pH was maintained at 5.2. The acetate and butyrate yields were 0.35 mol acetate mol-1 glucose and 0.6 mol butyrate mol-1 glucose. For starch a maximum yield of 2.0 mol H 2 mol-1 hexose, and a maximum production rate of 15 mol H 2 mol-1 hexose h-1 were obtained at pH 5.6 when the acetate and butyrate yields were 0.47 mol acetate mol-1 hexose and 0.67 mol butyrate mol-1 hexose.
A Dual Fermentation Process for the Production of Aliphatic Acids
Production of aliphatic acids by fermentation has traditionally been attempted using one of two processes. Propionic and lactic acids have been produced as their calcium salts by fermentation of carbohydrate in the presence of calcium carbonate. After removal of bacterial cells, the undissociated acid was then recovered by acidification of the fermentation broth with sulfuric acid; this acidification resulted in formation of large quantities of gypsum which was separated from the product acid and discarded.
Journal of microbiology and biotechnology, 2016
Lactobacillus brevis ATCC 14869 exhibited a carbon catabolite de-repressed (CCR) phenotype which has ability to consume fermentable sugar simultaneously with glucose. To evaluate this unusual phenotype under harsh conditions during fermentation, the effect of lactic acid and hydrogen ion concentrations on L. brevis ATCC 14869 were examined. Kinetic equations describing the relationship between specific cell growth rate and lactic acid or hydrogen ion concentration has been reduced. The change of substrate utilization and product formation according to lactic acid and hydrogen ion concentration in the media were quantitatively described. Moreover; utilization of other compounds were also observed along with hydrogen ion and lactic acid concentration simultaneously. It has been found that substrate preference changes significantly regarding to utilization of compounds in media. That could result into formation of two-carbon products. In particular, acetic acid present in the media as ...
Clostridiurn thermXm?tiCUrn Was examined On a Variety Of substrates and mixtures of substrates. Nondiauxic growth was noted for both strains on combinations of carbohydrates, organic acids, or a carbohydrate and an organic acid. The mutant strain was able to grow on DL-lactate as sole energy source. The parent strain would not grow on lactate as sole energy source but consumed lactate Neither strain would grow on formate as sole energy source, but both consumed formate when presented with a second fermentable substrate.
Biomass and Bioenergy, 2013
Sixteen combinations of seven growth nutrients, namely a-cellulose, yeast extract (YE), urea, CaCl 2 2H2O,MgCl22H 2 O, MgCl 2 2H2O,MgCl26H 2 O, FeSO 4 $6H 2 O and vitamins, were studied to improve direct cellulose fermentation by Clostridium thermocellum DSM 1237 under carbon-excess conditions. Varied nutrient compositions improved cellulose fermentation conditions for C. thermocellum and displayed two major types of effects: a general growth enhancement effect and a carbon-flux shifting effect. Different concentrations of the four most influential nutrients (YE, a-cellulose, CaCl 2 and MgCl 2) resulted in enhanced yields of ethanol or H 2. High ethanol yields, high ethanol to acetate (E/A) ratios, and low acetate and H 2 yields were obtained when YE, a-cellulose and CaCl 2 were present at high concentrations, in combination with low concentrations of magnesium. Vitamins were identified as a relatively less influential nutrient, but high concentrations of vitamins supported enhanced yields of acetate and H 2. High urea and YE in combination with low MgCl 2 concentrations enhanced cellulose utilization per unit mass of cells and cell-specific yields of both ethanol and H 2. Volumetric yields for ethanol and H 2 were improved by 2.3-fold (76.5 mM) and 2.04fold (71.22 mmol per liter), respectively, compared with the basic combination. The highest hydrogen yield (1.64 mol/mol glucose) was obtained in the combination with the lowest ethanol yields while the lowest hydrogen yielding combination had the highest ethanol yield of 1.36 mol/mole glucose, representing 68% of the theoretical maximum for ethanol. The culture conditions determined by this study can be optimized further for enhanced production of either ethanol or H 2 by direct cellulose fermentation.