Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanol-butanol-ethanol fermentation

Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh(B-593)) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.     

Bacterial community dynamics during <Emphasis Type="Italic">in-situ</Emphasis> bioremediation of petroleum waste sludge in landfarming sites

In-situ bioremediation of petroleum waste sludge in landfarming sites of Motor Oil Hellas (petroleum refinery) was studied by monitoring the changes of the petroleum composition of the waste sludge, as well as the changes in the structure of the microbial community, for a time period of 14 months. The analyses indicated an enhanced degradation of the petroleum hydrocarbons in the landfarming areas. A depletion of n-alkanes of approximately 75–100% was obtained. Marked changes of the microbial communities of the landfarms occurred concomitantly with the degradation of the petroleum hydrocarbons. The results obtained from terminal restriction fragment length polymorphism (T-RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rRNA genes demonstrated that bacteria originating from the refinery waste sludge and newly selected bacteria dominated the soil bacterial community during the period of the highest degradation activity. However, the diversity of the microbial community was decreased with increased degradation of the petroleum hydrocarbons contained in the landfarms. T-RFLP fingerprints of bacteria of the genera Enterobacter and Ochrobactrum were detected in the landfarmed soil over the entire treatment period of 14 months. In contrast, the genus Alcaligenes appeared in significant numbers only within the 10 month old landfarmed soil. Genes encoding catechol 2,3-dioxygenase (subfamily I.2.A) were detected only in DNA of the untreated refinery waste sludge. However, none of the genes known to encode the enzymes alkane hydroxylase AlkB, catechol 2,3-dioxygenase (subfamily I.2.A) and naphthalene dioxygenase nahAc could be detected in DNA of the landfarmed soils.     

Reduction of acetone to isopropanol using producer gas fermenting microbes

Gasification‐fermentation is an emerging technology for the conversion of lignocellulosic materials into biofuels and specialty chemicals. For effective utilization of producer gas by fermenting bacteria, tar compounds produced in the gasification process are often removed by wet scrubbing techniques using acetone. In a preliminary study using biomass generated producer gas scrubbed with acetone, an accumulation of acetone and subsequent isopropanol production was observed. The effect of 2 g/L acetone concentrations in the fermentation media on growth and product distributions was studied with “Clostridium ragsdalei,” also known as Clostridium strain P11 or P11, and Clostridium carboxidivorans P7 or P7. The reduction of acetone to isopropanol was possible with “C. ragsdalei,” but not with P7. In P11 this reaction occurred rapidly when acetone was added in the acidogenic phase, but was 2.5 times slower when added in the solventogenic phase. Acetone at concentrations of 2 g/L did not affect the growth of P7, but ethanol increased by 41% and acetic acid concentrations decreased by 79%. In the fermentations using P11, growth was unaffected and ethanol concentrations increased by 55% when acetone was added in the acidogenic phase. Acetic acid concentrations increased by 19% in both the treatments where acetone was added. Our observations indicate that P11 has a secondary alcohol dehydrogenase that enables it to reduce acetone to isopropanol, while P7 lacks this enzyme. P11 offers an opportunity for biological production of isopropanol from acetone reduction in the presence of gaseous substrates (CO, CO₂, and H₂). Biotechnol. Bioeng. 2011;108: 2330–2338. © 2011 Wiley Periodicals, Inc.     

Induction of aromatic ring: cleavage dioxygenases in <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> strain KB2 in cometabolic systems

Stenotrophomonas maltophilia KB2 is known to produce different enzymes of dioxygenase family. The aim of our studies was to determine activity of these enzymes after induction by benzoic acids in cometabolic systems with nitrophenols. We have shown that under cometabolic conditions KB2 strain degraded 0.25–0.4 mM of nitrophenols after 14 days of incubation. Simultaneously degradation of 3 mM of growth substrate during 1–3 days was observed depending on substrate as well as cometabolite used. From cometabolic systems with nitrophenols as cometabolites and 3,4-dihydroxybenzoate as a growth substrate, dioxygenases with the highest activity of protocatechuate 3,4-dioxygenase were isolated. Activity of catechol 1,2- dioxygenase and protocatechuate 4,5-dioxygenase was not observed. Catechol 2,3-dioxygenase was active only in cultures with 4-nitrophenol. Ability of KB2 strain to induce and synthesize various dioxygenases depending on substrate present in medium makes this strain useful in bioremediation of sites contaminated with different aromatic compounds.     

New Type of Oxygenase Involved in the Metabolism of Propane and Isobutane

Nocardia paraffinicum (Rhodococcus rhodochrous), a hydrocarbon-degrading microorganism, was used in a study of propane and isobutane metabolism. The bacterium was able to utilize propane or isobutane as a sole source of carbon, and oxygen was found to be essential for its metabolism. Gas chromatographic analysis showed that n-propanol was the major compound recovered from the metabolism of propane by resting cells, although trace amounts of isopropanol and acetone were detected. When a mixture of propane and isobutane was used, drastic inhibition (72 to 88%) of hydrocarbon utilization by resting cells occurred. The ratio of hydrocarbon to oxygen consumed was found to be approximately 2:1 during the metabolism of propane or isobutane by resting cells when these substrates were provided individually to the organism. Gas chromatographic-mass spectrometric analysis of products formed from ¹⁸O₂ confirmed that the initial oxidative step in the metabolism of these substrates involved molecular oxygen. The proportion of the alcohol containing ¹⁸O was the same as that of ¹⁸O₂ in the gas mixture. Only a negligible amount of ¹⁸O was detected in the alcohol when H₂¹⁸O was incorporated into the system. The observed 2:1 ratio of hydrocarbon to oxygen consumption suggests that the oxygenase in N. paraffinicum, unlike the conventional mono- or dioxygenases, requires two hydrocarbon-binding sites for each of the oxygen-binding sites and is therefore an intermolecular dioxygenase. The newly described oxygenase, which catalyzes the reaction of two molecules of propane with one molecule of oxygen to yield two molecules of a C₃ alcohol, is proposed as the initial oxidation step of the hydrocarbon substrate.     

Metabolic engineering of Clostridium acetobutylicum for the enhanced production of isopropanol‐butanol‐ethanol fuel mixture

Butanol is considered as a superior biofuel, which is conventionally produced by clostridial acetone‐butanol‐ethanol (ABE) fermentation. Among ABE, only butanol and ethanol can be used as fuel alternatives. Coproduction of acetone thus causes lower yield of fuel alcohols. Thus, this study aimed at developing an improved Clostridium acetobutylicum strain possessing enhanced fuel alcohol production capability. For this, we previously developed a hyper ABE producing BKM19 strain was further engineered to convert acetone into isopropanol. The BKM19 strain was transformed with the plasmid pIPA100 containing the sadh (primary/secondary alcohol dehydrogenase) and hydG (putative electron transfer protein) genes from the Clostridium beijerinckii NRRL B593 cloned under the control of the thiolase promoter. The resulting BKM19 (pIPA100) strain produced 27.9 g/l isopropanol‐butanol‐ethanol (IBE) as a fuel alcohols with negligible amount of acetone (0.4 g/l) from 97.8 g/l glucose in lab‐scale (2 l) batch fermentation. Thus, this metabolically engineered strain was able to produce 99% of total solvent produced as fuel alcohols. The scalability and stability of BKM19 (pIPA100) were evaluated at 200 l pilot‐scale fermentation, which showed that the fuel alcohol yield could be improved to 0.37 g/g as compared to 0.29 g/g obtained at lab‐scale fermentation, while attaining a similar titer. To the best of our knowledge, this is the highest titer of IBE achieved and the first report on the large scale fermentation of C. acetobutylicum for IBE production. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1083–1088, 2013     

Bioremediation via In Situ Electrotransformation

Bioremediation of polluted sites relies on bacteria to degrade or transform contaminants into less noxious chemicals. To do so, bacteria require genes that encode the degradation enzymes and the capacity to properly express them, which may be lacking in indigenous bacteria. To increase the ability of indigenous bacteria to bioremediate a contaminated site, this research proposes the use of electrotransformation to facilitate bacterial uptake of exogenous degradation genes. As a proof of concept, a lindane degradation gene (linA) located on a broad host-spectrum expression plasmid (pBLN) was introduced into soil bacteria by electroporation both in vitro, in liquid media, and in situ, in soil. In both cases, the electrotransformed bacteria displayed an increase in lindane degradation and an increase in the linA gene copy number. The use of in situ electrotransformation could improve pollutant degradation rates and could provide another tool for bioremediation.     

Selective bio-oxidation of propane to acetone using methane-oxidizing <Emphasis Type="Italic">Methylomonas</Emphasis> sp. DH-1

Propane is the major component of liquefied petroleum gas (LPG). Nowadays, the use of LPG is decreasing, and thus utilization of propane as a chemical feedstock is in need of development. An efficient biological conversion of propane to acetone using a methanotrophic whole cell as the biocatalyst was proposed and investigated. A bio-oxidation pathway of propane to acetone in Methylomonas sp. DH-1 was analyzed by gene expression profiling via RNA sequencing. Propane was oxidized to 2-propanol by particulate methane monooxygenase and subsequently to acetone by methanol dehydrogenases. Methylomonas sp. DH-1 was deficient in acetone-converting enzymes and thus accumulated acetone in the absence of any enzyme inhibition. The maximum accumulation, average productivity and specific productivity of acetone were 16.62 mM, 0.678 mM/h and 0.141 mmol/g cell/h, respectively, under the optimized conditions. Our study demonstrates a novel method for the bioconversion of propane to acetone using methanotrophs under mild reaction condition.     

Bioremediation techniques–classification based on site of application: principles,advantages, limitations and prospects

Environmental pollution has been on the rise in the past few decades owing to increased human activities on energy reservoirs, unsafe agricultural practices and rapid industrialization. Amongst the pollutants that are of environmental and public health concerns due to their toxicities are: heavy metals, nuclear wastes, pesticides, green house gases, and hydrocarbons. Remediation of polluted sites using microbial process (bioremediation) has proven effective and reliable due to its eco-friendly features. Bioremediation can either be carried out ex situ or in situ, depending on several factors, which include but not limited to cost, site characteristics, type and concentration of pollutants. Generally, ex situ techniques apparently are more expensive compared to in situ techniques as a result of additional cost attributable to excavation. However, cost of on-site installation of equipment, and inability to effectively visualize and control the subsurface of polluted sites are of major concerns when carrying out in situ bioremediation. Therefore, choosing appropriate bioremediation technique, which will effectively reduce pollutant concentrations to an innocuous state, is crucial for a successful bioremediation project. Furthermore, the two major approaches to enhance bioremediation are biostimulation and bioaugmentation provided that environmental factors, which determine the success of bioremediation, are maintained at optimal range. This review provides more insight into the two major bioremediation techniques, their principles, advantages, limitations and prospects.     

Pervaporation performance of a composite bacterial cellulose membrane: dehydration of binary aqueous–organic mixtures

Summary Acetobacter xylinum (Gluconacetobacter xylinus) is a bacterium that produces extracellular cellulose under static culture conditions. The highly reticulated cellulose matrix along with the entrapped cellulose-forming bacteria is commonly referred to as a pellicle. The processed bacterial cellulose membrane/film was modified into a composite bacterial cellulose membrane (CBCM) for pervaporation separation of aqueous–organic mixtures. The CBCM was prepared by coating with alginate or alginate+polyvinylpyrrolidone and cross-linking with glutaraldehyde. The pervaporation performance was determined using aqueous–organic mixtures such as, 1:1 (v/v) water–ethanol, water–isopropanol and water–acetone. The pervaporation performance of the CBCM was more effective for zeotropic mixtures (water–acetone) in comparison to the investigated azeotropic mixtures (water–ethanol and water–isopropanol). The selectivity of CBCM was found to be 4.8, 8.8, 19.8 for water–ethanol, water–isopropanol and water–acetone mixtures, respectively. The permeation flux for the water–acetone mixture was found to be 235 ml/m²/h. The present investigation demonstrated that the CBCM could be employed to concentrate azeotropic as well as zeotrope forming binary mixtures by preferential pervaporation of water, with low energy requirements in contrast to the established method of distillation. In addition, the effects of feed composition, operating temperature, membrane thickness, and method of CBCM preparation on pervaporation performance have been evaluated. Investigations with the CBCM revealed that 94.5% ethanol, 98% acetone and 98.5% isopropanol concentrations could be attained from the initial 50% aqueous mixtures of these chemicals by way of pervaporation. In the case of the isopropanol–water mixture the resolving property of the membrane was more evident as the concentration arrived at was 98.5%, in contrast to other binary mixtures. The surface characteristics of the CBCM were revealed by scanning electron microscopy. In view of its properties the CBCM can be useful for pervaporation separation of these chemicals at moderate temperatures and pressure. The CBCM could be employed in the downstream processing of heat-labile and flavor-imparting volatile molecules in the field of food biotechnology and fabrication of membrane bioreactors for on-line product purification. Further studies are under progress to use the membrane for the immobilization of food processing enzymes.     

A comprehensive overview of elements in bioremediation

Sustainable development requires the development and promotion of environmental management and a constant search for green technologies to treat a wide range of aquatic and terrestrial habitats contaminated by increasing anthropogenic activities. Bioremediation is an increasingly popular alternative to conventional methods for treating waste compounds and media with the possibility to degrade contaminants using natural microbial activity mediated by different consortia of microbial strains. Many studies about bioremediation have been reported and the scientific literature has revealed the progressive emergence of various bioremediation techniques. In this review, we discuss the various in situ and ex situ bioremediation techniques and elaborate on the anaerobic digestion technology, phytoremediation, hyperaccumulation, composting and biosorption for their effectiveness in the biotreatment, stabilization and eventually overall remediation of contaminated strata and environments. The review ends with a note on the recent advances genetic engineering and nanotechnology have had in improving bioremediation. Case studies have also been extensively revisited to support the discussions on biosorption of heavy metals, gene probes used in molecular diagnostics, bioremediation studies of contaminants in vadose soils, bioremediation of oil contaminated soils, bioremediation of contaminants from mining sites, air sparging, slurry phase bioremediation, phytoremediation studies for pollutants and heavy metal hyperaccumulators, and vermicomposting.     

Divergent Metabolic Pathways for Propane and Propionate Utilization by a Soil Isolate

The metabolism of propane and propionate by a soil isolate (Brevibacterium sp. strain JOB5) was investigated. The presence of isocitrate lyase in cells grown on isopropanol, acetate, or propane and the absence of this inducible enzyme in n-propanol- and propionate-grown cells suggested that propane is not metabolized via C-terminal oxidation. Methylmalonyl coenzyme A mutase and malate synthase are constitutive in this organism. The incorporation of ¹⁴CO₂ into pyruvate accumulated during propionate utilization suggests that propionate is metabolized via the methyl-malonyl-succinate pathway. These results were further substantiated by radiorespirometric studies with propionate-1-¹⁴C, -2-¹⁴C, and -3-¹⁴C as substrate. Propane -2-¹⁴C was shown, by unlabeled competitor experiments, to be oxidized to acetone; acetone and isopropanol are oxidized in this organism to acetol. Cleavage of acetol to acetate and CO₂ would yield the inducer for the isocitrate lyase present in propane-grown cells.     

Production of isopropanol by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

A genetically engineered strain of Escherichia coli JM109 harboring the isopropanol-producing pathway consisting of five genes encoding four enzymes, thiolase, coenzyme A (CoA) transferase, acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824, and primary–secondary alcohol dehydrogenase from C. beijerinckii NRRL B593, produced up to 227 mM of isopropanol from glucose under aerobic fed-batch culture conditions. Acetate production by the engineered strain was approximately one sixth that produced by a control E. coli strain bearing an expression vector without the clostridial genes. These results demonstrate a functional isopropanol-producing pathway in E. coli and consequently carbon flux from acetyl-CoA directed to isopropanol instead of acetate. This is the first report on isopropanol production by genetically engineered microorganism under aerobic culture conditions.     

Genomic tools in bioremediation

Bioremediation is a process that uses microorganisms or their enzymes to remove pollutants from the environment. Generally, bioremediation technologies can be classified as in situ or ex situ. In situ bioremediation involves treating the contaminated material at the site while ex situ involves the removal of the contaminated material to be treated elsewhere. Like so much else in biology, the ease and availability of genomic data has created a new level of understanding this system. Bioremediation capabilities of the microbial population can be analyzed; not only by physiological parameters, but also by the use of genomic tools, and efficient remediation strategies can be planned. PCR and DNA- or oligonucleotide-based microarray technology is a powerful functional genomics tool that allows researchers to view the physiology of a living cell from a comprehensive and dynamic molecular perspective. This paper explores the use of such tools in bioremediation process.     

Biodegradation of vinyl chloride, <Emphasis Type="Italic">cis</Emphasis>-dichloroethene and 1,2-dichloroethane in the alkene/alkane-oxidising <Emphasis Type="Italic">Mycobacterium</Emphasis> strain NBB4

Mycobacterium chubuense strain NBB4 can grow on both alkanes and alkenes as carbon sources, and was hypothesised to be an effective bioremediation agent for chlorinated aliphatic pollutants. In this study, the ability of NBB4 to biodegrade vinyl chloride (VC), cis-dichloroethene (cDCE) and 1,2-dichloroethane (DCA) was investigated under pure-culture conditions and in microcosms. Ethene-grown NBB4 cells were capable of biodegrading VC and cDCE, while ethane-grown cells could biodegrade cDCE and DCA. The stoichiometry of inorganic chloride release (1 mol/mol in each case) indicated that VC was completely dechlorinated, while cDCE and DCA were only partially dechlorinated, yielding chloroacetate in the case of DCA, and unknown metabolites in the case of cDCE. The apparent maximum specific activities (k) of whole cells against ethene, cDCE, ethane and DCA were 93 ± 4.6, 89 ± 18, 39 ± 5.5, and 4.8 ± 0.9 nmol/min/mg protein, respectively, while the substrate affinities (K_S) of whole cells with the same substrates were 2.0 ± 0.15, 46 ± 11, 11 ± 0.33 and 4.0 ± 3.2 μM, respectively. In microcosms containing contaminated aquifer sediments and groundwater, NBB4 cells removed 85-95% of the pollutants (cDCE or DCA at 2 mM) within 24 h, and the cells remained viable for >1 month. Due to its favourable kinetic parameters, and robust survival and biodegradation activities, strain NBB4 is a promising candidate for bioremediation of chlorinated aliphatic pollutants.     

Microbial reductive dechlorination of trichloroethene to ethene with electrodes serving as electron donors without the external addition of redox mediators

In situ bioremediation of industrial chlorinated solvents, such as trichloroethene (TCE), is typically accomplished by providing an organic electron donor to naturally occurring dechlorinating populations. In the present study, we show that TCE dechlorinating bacteria can access the electrons required for TCE dechlorination directly from a negatively polarized (?450 mV vs. SHE) carbon paper electrode. In replicated batch experiments, a mixed dechlorinating culture, also containing Dehalococcoides spp., dechlorinated TCE to cis‐dichloroethene (cis‐DCE) and lower amounts of vinyl chloride (VC) and ethene using the polarized electrode as the sole electron donor. Conversely, neither VC nor ethene formation occurred when a pure culture of the electro‐active microorganism Geobacter lovleyi was used, under identical experimental conditions. Cyclic voltammetry tests, carried out on the filter‐sterilized supernatant of the mixed culture revealed the presence of a self‐produced redox mediator, exhibiting a midpoint potential of around ?400 mV (vs. SHE). This yet unidentified redox‐active molecule appeared to be involved in the extracellular electron transfer from the electrode to the dechlorinating bacteria. The ability of dechlorinating bacteria to use electrodes as electron donors opens new perspectives for the development of clean, versatile, and efficient bioremediation systems based on a controlled subsurface delivery of electrons in support of biodegradative metabolisms and provides further evidence on the possibility of using conductive materials to manipulate and control a range of microbial bioprocesses. Biotechnol. Bioeng. 2009;103: 85–91. © 2008 Wiley Periodicals, Inc.     

Analysis of Novel Soluble Chromate and Uranyl Reductases and Generation of an Improved Enzyme by Directed Evolution

Most polluted sites contain mixed waste. This is especially true of the U.S. Department of Energy (DOE) waste sites which hold a complex mixture of heavy metals, radionuclides, and organic solvents. In such environments enzymes that can remediate multiple pollutants are advantageous. We report here evolution of an enzyme, ChrR6 (formerly referred to as Y6), which shows a markedly enhanced capacity for remediating two of the most serious and prevalent DOE contaminants, chromate and uranyl. ChrR6 is a soluble enzyme and reduces chromate and uranyl intracellularly. Thus, the reduced product is at least partially sequestered and nucleated, minimizing the chances of reoxidation. Only one amino acid change, ^Tyr128^Asn, was responsible for the observed improvement. We show here that ChrR6 makes Pseudomonas putida and Escherichia coli more efficient agents for bioremediation if the cellular permeability barrier to the metals is decreased.     

Bioremediation of radioactive waste: radionuclide-microbe interactions in laboratory and field-scale studies

Given the scale of the contamination associated with 60 years of global nuclear activity, and the inherent high financial and environmental costs associated with invasive physical and chemical clean-up strategies, there is an unparalleled interest in new passive in situ bioremediation processes for sites contaminated with nuclear waste. Many of these processes rely on successfully harnessing newly discovered natural biogeochemical cycles for key radionuclides and fission products. Recent advances have been made in understanding the microbial colonization of radioactive environments and the biological basis of microbial transformations of radioactive waste in these settings.     

Alicycliphilus: current knowledge and potential for bioremediation of xenobiotics

Alicycliphilus is a promising candidate for participating in the development of novel xenobiotics bioremediation processes. Members of the Alicycliphilus genus are environmental bacteria mostly found in polluted sites such as landfills and contaminated watercourses, and in sewage sludges from wastewater treatment plants. They exhibit a versatile metabolism and the ability to use oxygen, nitrate and chlorate as terminal electron acceptors, which allow them to biodegrade xenobiotics under oxic or anoxic conditions. Pure cultures of Alicycliphilus strains are able to biodegrade some pollutants such as industrial solvents (acetone, cyclohexanol and N-methylpyrrolidone), aromatic hydrocarbons (benzene, toluene and anthracene), as well as polyurethane varnishes and foams, and they can even transform Cr(VI) to Cr(III). In addition, Alicycliphilus has also been identified in bacterial communities involved in wastewater treatment plants for denitrification, and the degradation of emerging pollutants such as triclosan, nonylphenol, N-heterocyclic aromatic compounds (indole and quinoline), and antibiotics (tetracycline and oxytetracycline). This work summarizes the current knowledge on the Alicycliphilus genus, describing its different metabolic characteristics, focusing on its xenobiotic biodegradation abilities and examining the distinct pathways and molecular bases that sustain them. We also discuss the progress made in genetic manipulation and ‘omics’ analyses, as well as Alicycliphilus participation in novel bioremediation strategies.     

Anaerobic degradation of acetone by Desulfococcus biacutus spec. nov.

From anaerobic digestor sludge of a waste water treatment plant, a gram-negative, strictly anaerobic sulfate-reducing bacterium was isolated with acetone as sole organic substrate. The bacterium was characterized as a new species, Desulfococcus biacutus. The strain grew with acetone with doubling times of 72 h to 120 h; the growth yield was 12.0 (±2.1) g · [mol acetone]^-1. Acetone was oxidized completely, and no isopropanol was formed. In labelling studies with ¹⁴CO₂, cell lipids (including approx. 50% PHB) of acetone-grown cells became labelled 7 times as high as those of 3-hydroxy-buyrate-grown cells. Enzyme studies indicated that acetone was degraded via acetoacetyl-CoA, and that acetone was channeled into the intermediary metabolism after condensation with carbon dioxide to a C₄-compound, possibly free acetoacetate. Acetoacetyl-CoA is cleaved by a thiolase reaction to acetyl-CoA which is completely oxidized through the carbon monoxide dehydrogenase pathway. Strain KMRActS was deposited with the Deutsche Sammlung von Mikroorganismen, Braunschweig, under the number DSM 5651.