Influence of substrate carbon on the metabolism of Clostridium thermohydrosulfuricum (original) (raw)
Related papers
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.