Metabolic pathways and energetics of the acetone-oxidizing,sulfate-reducing bacterium,Desulfobacterium cetonicum

Acetone degradation by cell suspensions of Desulfobacterium cetonicum was CO₂-dependent, indicating initiation by a carboxylation reaction. Degradation of butyrate was not CO₂-dependent, and acetate accumulated at a ratio of 1 mol acetate per mol butyrate degraded. In cultures grown on acetone, no CoA transfer apparently occurred, and no acetate accumulated in the medium. No CoA-ligase activities were detected in cell-free crude extracts. This suggested that the carboxylation of acetone to acetoacetate, and its activation to acetoacetyl-CoA may occur without the formation of free acetoacetate. Acetoacetyl-CoA was thiolytically cleaved to two acetyl-CoA, which were oxidized to CO₂ via the acetyl-CoA/carbon monoxide dehydrogenase pathway. The measured intracellular acyl-CoA ester concentrations allowed the calculation of the free energy changes involved in the conversion of acetone to acetyl-CoA. At in vivo concentrations of reactants and products, the initial steps (carboxylation and activation) must be energy-driven, either by direct coupling to ATP, or coupling to transmembrane gradients. The G of acetone conversion to two acetyl-CoA at the expense of the energetic equivalent of one ATP was calculated to lie very close to 0kJ (mol acetone)^-1. Assimilatory metabolism was by an incomplete citric acid cycle, lacking an activity oxidatively decarboxylating 2-oxoglutarate. The low specific activities of this cycle suggested its probable function in anabolic metabolism. Succinate and glyoxylate were formed from isocitrate by isocitrate lyase. Glyoxylate thus formed was condensed with acetyl-CoA to form malate, functioning as an anaplerotic sequence. A glyoxylate cycle thus operates in this strictly anaerobic bacterium. Phosphoenolpyruvate (PEP) carboxykinase formed PEP from oxaloacetate. No pyruvate kinase activity was detected. PEP presumably served as a precursor for polyglucose formation and other biosyntheses.Abbreviations MV ²⁺ Oxidized methyl viologen - PEP Phosphoenolpyruvate - PHB Poly--hydroxybutyrate     

Effects of N-serve (2-chloro-6-(trichloromethyl)pyridine) formulations on nitrification and on loss of nitrate in sand culture experiments

Summary N-serve (2-chloro-6-(trichloromethyl)pyridine) was tested as an inhibitor of nitrification of ammonium or urea in sand cultures. Nitrification was reduced but not prevented by N-Serve present at between 5 and 20 ppm in solution or by weight of sand. In the presence of root debris and acetone, used in some experiments at 2–4 ml/l of nutrient to convey N-Serve, denitrification was stimulated under the same conditions and resulted in loss of a large proportion of nitrate, probably mainly as gaseous products and some nitrite. These losses were greater when N-serve was also present. There was also conversion of nitrate to an insoluble form in the sand. A smaller proportional loss of nitrate occurred in other treatments in the presence of root debris when N-Serve was added without acetone, either as the commercial formulation 24E or as a solid. Thus, using N-Serve to inhibit nitrification may encourage denitrifying organisms especially in the presence of carbon sources including root debris or acetone. Large decreases of nitrate reductase activity in plants produced by using N-Serve in the presence of ammonium or urea were caused as much by losses of nitrate in the presence of acetone as by prevention of nitrate formation. Other N-Serve treatments (solid or 24E) decreased enzyme induction by between 50 and 90 per cent as a result mainly of reduced nitrification.     

Anaerobic biotechnological approaches for production of liquid energy carriers from biomass

In recent years, increasing attention has been paid to the use of renewable biomass for energy production. Anaerobic biotechnological approaches for production of liquid energy carriers (ethanol and a mixture of acetone, butanol and ethanol) from biomass can be employed to decrease environmental pollution and reduce dependency on fossil fuels. There are two major biological processes that can convert biomass to liquid energy carriers via anaerobic biological breakdown of organic matter: ethanol fermentation and mixed acetone, butanol, ethanol (ABE) fermentation. The specific product formation is determined by substrates and microbial communities available as well as the operating conditions applied. In this review, we evaluate the recent biotechnological approaches employed in ethanol and ABE fermentation. Practical applicability of different technologies is discussed taking into account the microbiology and biochemistry of the processes.     

Simultaneous determination of eight lipid peroxidation degradation products in urine of rats treated with carbon tetrachloride using gas chromatography with electron-capture detection

One of the major processes that occur as a result of radical-induced oxidative stress is lipid peroxidation (LPO). Degradation of lipid peroxides results in various products, including a variety of carbonyl compounds. In the present study eight different lipid degradation products, i.e., formaldehyde, acetaldehyde, acetone, propanal, butanal, pentanal, hexanal and malondialdehyde were identified and measured simultaneously and quantitatively in rat urine after derivatization with O-(2,3,4,5,6-pentafluorbenzyl)hydroxylamine hydrochloride, extraction with heptane and using gas chromatography–electron-capture detection (GC–ECD). The identity of the respective oximes in urine was confirmed by gas chromatography–negative ion chemical ionization mass spectrometry (GC–NCI-MS). Simultaneously measured standard curves were linear for all oxime-products and the detection limits were between 39.0±5.3 (n=9) and 500±23 (n=9) fmol per μl injected sample. Recoveries of all products from urine or water were 73.0±5.2% and higher. In urine of CCl₄-treated rats an increase in all eight lipid degradation products in urine was found 24 h following exposure. ACON showed the most distinct increase, followed by PROPA, BUTA and MDA. It is concluded that the rapid, selective and sensitive analytical method based on GC–ECD presented here is well suited for routine measurement of eight different lipid degradation products. These products appear to be useful as non-invasive biomarkers for in vivo oxidative stress induced in rats by CCl₄.     

Production of acetone,butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping

We examined the effect of gas-stripping on the in situ removal of acetone, butanol, and ethanol (ABE) from batch reactor fermentation broth. The mutant strain (Clostridium beijerinckii BA101) was not affected adversely by gas stripping. The presence of cells in the fermentation broth affected the selectivities of ABE. A considerable improvement in the productivity and yield was recorded in this work in comparison with the non-integrated process. In an integrated process of ABE fermentation-recovery using C. beijerinckii BA101, ABE productivities and yield were improved up to 200 and 118%, respectively, as compared to control batch fermentation data. In a batch reactor C. beijerinckii BA101 utilized 45.4 g glucose l^–1 and produced 17.7 g total ABE l^–1, while in the integrated process it utilized 161.7 g glucose l^–1 and produced total ABE of 75.9 g l^–1. In the integrated process, acids were completely converted to solvents when compared to the non-integrated process (batch fermentation) which contained residual acids at the end of fermentation. In situ removal of ABE by gas stripping has been reported to be one of the most important techniques of solvent removal. During these studies we were able to maintain the ABE concentration in the fermentation broth below toxic levels.     

Recent progress on industrial fermentative production of acetone–butanol–ethanol by Clostridium acetobutylicum in China

China is one of the few countries, which maintained the fermentative acetone–butanol–ethanol (ABE) production for several decades. Until the end of the last century, the ABE fermentation from grain was operated in a few industrial scale plants. Due to the strong competition from the petrochemical industries, the fermentative ABE production lost its position in the 1990s, when all the solvent fermentation plants in China were closed. Under the current circumstances of concern about energy limitations and environmental pollution, new opportunities have emerged for the traditional ABE fermentation industry since it could again be potentially competitive with chemical synthesis. From 2006, several ABE fermentation plants in China have resumed production. The total solvent (acetone, butanol, and ethanol) production capacity from ten plants reached 210,000 tons, and the total solvent production is expected to be extended to 1,000,000 tons (based on the available data as of Sept. 2008). This article reviews current work in strain development, the continuous fermentation process, solvent recovery, and economic evaluation of ABE process in China. Challenges for an economically competitive ABE process in the future are also discussed.     

Acetone-butanol fermentation and its variants

Recent intensive research on the acetone-butanol-ethanol and the isopropanol-butanol-ethanol fermentation has increased the basic understanding of these processes substantially. Metabolic investigations on Clostridium acetobutylicum, and Clostridium beijerinkii show that enzyme activities necessary for solvent production are induced only in solvent-producing cells. Although produced, or added, acetic and butyric acid have significant effects on the metabolic activities, the transition from acid to solvent production cannot as yet be fully explained. Based on studies in continuous cultures, the kinetics of product formation can be described. Knowledge of the mechanism of butanol toxicity is accumulating but no dramatic increase in butanol tolerance has so far been obtained. Successful results, approaching the limitations determined by biological and technological possibilities, have been obtained in batch and continuous cultures, and in continuous processes based on immobilized cells. Continuous processes are superior to batch cultures in respect of their productivity.     

The central metabolic pathway from acetyl-CoA to butyryl-CoA in Clostridium acetobutylicum

Abstract: The pathway from acetyl-CoA to butyryl-CoA serves as a major carbon metabolism channel in Clostridium acetobutylicum and other butyrate-forming clostridia, and the steps are similar to those involved in fatty acid metabolism. Recent findings are discussed, reviewing the isolation and characterization of the enzymes of the pathway, and the analyses of metabolic intermediate levels and possible points of regulation of enzyme activity by CoA compounds. DNA analyses have identified the genes for two thiolase proteins, and an apparent operon encoding five proteins involved in the conversion of acetoacetyl-CoA to butyryl-CoA. These five proteins are β-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase and the α and β subunits of an electron transfer flavoprotein.     

Acetone production with metabolically engineered strains of Acetobacterium woodii

Expected depletion of oil and fossil resources urges the development of new alternative routes for the production of bulk chemicals and fuels beyond petroleum resources. In this study, the clostridial acetone pathway was used for the formation of acetone in the acetogenic bacterium Acetobacterium woodii. The acetone production operon (APO) containing the genes thlA (encoding thiolase A), ctfA/ctfB (encoding CoA transferase), and adc (encoding acetoacetate decarboxylase) from Clostridium acetobutylicum were cloned under the control of the thlA promoter into four vectors having different replicons for Gram-positives (pIP404, pBP1, pCB102, and pCD6). Stable replication was observed for all constructs. A. woodii [pJIR_act_thlA] achieved the maximal acetone concentration under autotrophic conditions (15.2±3.4 mM). Promoter sequences of the genes ackA from A. woodii and pta-ack from C. ljungdahlii were determined by primer extension (PEX) and cloned upstream of the APO. The highest acetone production in recombinant A. woodii cells was achieved using the promoters P_thlA and P_pta-ack. Batch fermentations using A. woodii [pMTL84151_act_thlA] in a bioreactor revealed that acetate concentration had an effect on the acetone production, due to the high K_m value of the CoA transferase. In order to establish consistent acetate concentration within the bioreactor and to increase biomass, a continuous fermentation process for A. woodii was developed. Thus, acetone productivity of the strain A. woodii [pMTL84151_act_thlA] was increased from 1.2 mg L⁻¹ h⁻¹ in bottle fermentation to 26.4 mg L⁻¹ h⁻¹ in continuous gas fermentation.