Molar Growth Yield Research Articles

Insights into growth kinetics and roles of enzymes of Krebs' cycle and sulfur oxidation during exochemolithoheterotrophic growth of Achromobacter aegrifaciens NCCB 38021 on succinate with thiosulfate as the auxiliary electron donor.

Achromobacter aegrifaciens NCCB 38021 was grown heterotrophically on succinate versus exochemolithoheterotrophically on succinate with thiosulfate as auxiliary electron donor. In batch culture, no significant differences in specific molar growth yield or specific growth rate were found for the two growth conditions, but in continuous culture in the succinate-limited chemostat, the maximum specific growth yield coefficient increased by 23.3% with thiosulfate present, consistent with previous studies of endo- and exochemolithoheterotrophs and thermodynamic predictions. Thiosulfate oxidation was coupled to respiration at cytochrome c551, and thiosulfate-dependent ATP biosynthesis occurred. Specific activities of cytochrome c-linked thiosulfate dehydrogenase (E.C. 1.8.2.2) and two other enzymes of sulfur metabolism were significantly higher in exochemolithoheterotrophically grown cell extracts, while those of succinyl-transferring 2-oxoglutarate dehydrogenase (E.C. 1.2.4.2), fumarate hydratase (E.C. 4.2.1.2) and malate dehydrogenase (NAD+, E.C. 1.1.1.37) were significantly lower-presumably owing to less need to generate reducing equivalents during Krebs' cycle, since they could be produced from thiosulfate oxidation.

Integrated constraints based analysis of an engineered violacein pathway in Escherichia coli

Strategies towards optimal violacein biosynthesis, a potential drug molecule, need systems level coordination of enzymatic activities of individual genes in a multigene operon vioABCDE. Constraints-based flux balance analysis of an extended iAF1260 model (iAF1260vio) with a reconstructed violacein module predicted growth and violacein yields in Escherichia coli accurately. Shadow price (SP) analysis identified tryptophan metabolism and NADPH as limiting. Increased tryptophan levels in Δpgi & ΔpheA were validated using in silico gene deletion analysis. Phenotypic phase plane (PhPP) analysis highlighted sensitivity between tryptophan and NADPH for violacein synthesis at molar growth yields. A synthetic VioABCDE operon (SYNO) sequence was designed to maximize Codon Adaptive Index (CAI: 0.9) and tune translation initiation rates (TIR: 2–50 fold higher) in E. coli. All pSYN E. coli transformants produced higher violacein, with a maximum six-fold increase in yields. The rational design E. coli: ΔpheA SYN: gave the highest violacein titers (33.8 mg/l). Such integrated approaches targeting multiple molecular hierarchies in the cell can be extended further to increase violacein yields.

Sulfoquinovose degraded by pure cultures of bacteria with release of C3-organosulfonates: complete degradation in two-member communities

Sulfoquinovose (SQ, 6-deoxy-6-sulfoglucose) was synthesized chemically. An HPLC-ELSD method to separate SQ and other chromophore-free sulfonates, e.g. 2,3-dihydroxypropane-1-sulfonate (DHPS), was developed. A set of 10 genome-sequenced, sulfonate-utilizing bacteria did not utilize SQ, but an isolate, Pseudomonas putida SQ1, from an enrichment culture did so. The molar growth yield with SQ was half of that with glucose, and 1mol 3-sulfolactate (mol SQ)(-1) was formed during growth. The 3-sulfolactate was degraded by the addition of Paracoccus pantotrophus NKNCYSA, and the sulfonate sulfur was recovered quantitatively as sulfate. Another isolate, Klebsiella oxytoca TauN1, could utilize SQ, forming 1mol DHPS (mol SQ)(-1) ; the molar growth yield with SQ was half of that with glucose. This DHPS could be degraded by Cupriavidus pinatubonensis JMP134, with quantitative recovery of the sulfonate sulfur as sulfate. We presume that SQ can be degraded by communities in the environment.

The Regulatory C-Terminal Domain of Subunit ε of F o F 1 ATP Synthase Is Dispensable for Growth and Survival of Escherichia coli

The C-terminal domain of subunit ε of the bacterial F₀F₁ ATP synthase is reported to be an intrinsic inhibitor of ATP synthesis/hydrolysis activity in vitro, preventing wasteful hydrolysis of ATP under low-energy conditions. Mutants defective in this regulatory domain exhibited no significant difference in growth rate, molar growth yield, membrane potential, or intracellular ATP concentration under a wide range of growth conditions and stressors compared to wild-type cells, suggesting this inhibitory domain is dispensable for growth and survival of Escherichia coli.

Sulfoacetate Is Degraded via a Novel Pathway Involving Sulfoacetyl-CoA and Sulfoacetaldehyde in Cupriavidus necator H16

Bacterial degradation of sulfoacetate, a widespread natural product, proceeds via sulfoacetaldehyde and requires a considerable initial energy input. Whereas the fate of sulfoacetaldehyde in Cupriavidus necator (Ralstonia eutropha) H16 is known, the pathway from sulfoacetate to sulfoacetaldehyde is not. The genome sequence of the organism enabled us to hypothesize that the inducible pathway, which initiates sau (sulfoacetate utilization), involved a four-gene cluster (sauRSTU; H16_A2746 to H16_A2749). The sauR gene, divergently orientated to the other three genes, probably encodes the transcriptional regulator of the presumed sauSTU operon, which is subject to inducible transcription. SauU was tentatively identified as a transporter of the major facilitator superfamily, and SauT was deduced to be a sulfoacetate-CoA ligase. SauT was a labile protein, but it could be separated and shown to generate AMP and an unknown, labile CoA-derivative from sulfoacetate, CoA, and ATP. This unknown compound, analyzed by MALDI-TOF-MS, had a relative molecular mass of 889.7, which identified it as protonated sulfoacetyl-CoA (calculated 889.6). SauS was deduced to be sulfoacetaldehyde dehydrogenase (acylating). The enzyme was purified 175-fold to homogeneity and characterized. Peptide mass fingerprinting confirmed the sauS locus (H16_A2747). SauS converted sulfoacetyl-CoA and NADPH to sulfoacetaldehyde, CoA, and NADP(+), thus confirming the hypothesis.

Nonfermentative Thermoalkaliphilic Growth Is Restricted to Alkaline Environments

Caldalkalibacillus thermarum strain TA2.A1 grew in pH-controlled batch culture containing a fermentable growth substrate (i.e., sucrose) from pH 7.5 to 10.0 with no significant change in the specific growth rate, suggesting that this bacterium was a facultative alkaliphile. However, when strain TA2.A1 was grown on a nonfermentable carbon source, such as succinate or malate, no growth was observed until the external pH was >9.0, suggesting that this bacterium was an obligate alkaliphile. Succinate transport and sucrose transport by strain TA2.A1 showed pH profiles similar to that of growth on these carbon sources, and the molar growth yield on sucrose was higher at pH 9.5 than at pH 7.5, despite the increased energy demands on the cell for intracellular pH regulation. Succinate transport, succinate-dependent oxygen consumption, and succinate dehydrogenase and F(1)F(o)-ATPase specific activities were all significantly lower in cultures of strain TA2.A1 grown at pH 7.5 than in those cultured at pH 9.5. No significant ATP synthesis via the F(1)F(o)-ATP synthase was detected until the external pH was >8.5. On the basis of these results, we propose that nonfermentative thermoalkaliphilic growth is specialized to function at high pH values, but not at pH values near neutral pH.

Complete Fermentation of Xylose and Methylglucuronoxylose Derived from Methylglucuronoxylan by Enterobacter asburiae Strain JDR-1

Acid pretreatment is commonly used to release pentoses from the hemicellulose fraction of cellulosic biomass for bioconversion. The predominant pentose in the hemicellulose fraction of hardwoods and crop residues is xylose in the polysaccharide methylglucuronoxylan, in which as many as one in six of the beta-1,4-linked xylopyranose residues is substituted with alpha-1,2-linked 4-O-methylglucuronopyranose. Resistance of the alpha-1,2-methylglucuronosyl linkages to acid hydrolysis results in release of the aldobiuronate 4-O-methylglucuronoxylose, which is not fermented by bacterial biocatalysts currently used for bioconversion of hemicellulose. Enterobacter asburiae strain JDR-1, isolated from colonized hardwood (sweetgum), efficiently ferments both methylglucuronoxylose and xylose, producing predominantly ethanol and acetate. (13)C-nuclear magnetic resonance studies defined the Embden-Meyerhof pathway for metabolism of glucose and the pentose phosphate pathway for xylose metabolism. Rates of substrate utilization, product formation, and molar growth yields indicated methylglucuronoxylose is transported into the cell and hydrolyzed to release methanol, xylose, and hexauronate. Enterobacter asburiae strain JDR-1 is the first microorganism described that ferments methylglucuronoxylose generated along with xylose during the acid-mediated saccharification of hemicellulose. Genetic definition of the methylglucuronoxylose utilization pathway may allow metabolic engineering of established gram-negative bacterial biocatalysts for complete bioconversion of acid hydrolysates of methylglucuronoxylan. Alternatively, Enterobacter asburiae strain JDR-1 may be engineered for the efficient conversion of acid hydrolysates of hemicellulose to biofuels and chemical feedstocks.

Use of benzoate as an electron acceptor by Syntrophus aciditrophicus grown in pure culture with crotonate

In methanogenic environments, the main fate of benzoate is its oxidization to acetate, H(2) and CO(2) by syntrophic associations of hydrogen-producing benzoate degraders and hydrogen-using methanogens. Here, we report the use of benzoate as an electron acceptor. Pure cultures of S. aciditrophicus simultaneously degraded crotonate and benzoate when both substrates were present. The growth rate was 0.007 h(-1) with crotonate and benzoate present compared with 0.025 h(-1) with crotonate alone. After 8 days of incubation, 4.12 +/- 0.50 mM of cyclohexane carboxylate and 8.40 +/- 0.61 mM of acetate were formed and 4.0 +/- 0.04 mM of benzoate and 4.8 +/- 0.5 mM of crotonate were consumed. The molar growth yield was 22.7 +/- 2.1 g (dry wt) of cells per mol of crotonate compared with about 14.0 +/- 0.1 g (dry wt) of cells per mol of crotonate when S. aciditrophicus was grown with crotonate alone. Cultures grown with [ring-(13)C]-benzoate and unlabelled crotonate initially formed [ring-(13)C]-labelled cyclohexane carboxylate. No (13)C-labelled acetate was detected. In addition to cyclohexane carboxylate, (13)C-labelled cyclohex-1-ene carboxylate was detected as an intermediate. Once almost all of the benzoate was gone, carbon isotopic analyses showed that cyclohexane carboxylate was formed from both labelled and non-labelled metabolites. Glutarate and pimelate were also detected at this time and carbon isotopic analyses showed that each was made from a mixture labelled and non-labelled metabolites. The increase in molar growth yield with crotonate and benzoate and the formation of [ring-(13)C]-cyclohexane carboxylate from [ring-(13)C]-benzoate in the presence of crotonate are consistent with benzoate serving as an electron acceptor.

Continuous production of biosurfactant with foam fractionation

AbstractA glucose‐limited chemostat was used to determine the growth parameters of BBK006 for continuous production of the biosurfactant surfactin. The continuous cultivation exhibited low maintenance metabolism (m = 0.39 mmolglucose gbacteria−1 h−1) and high molar growth yield ($Y_{x/S}^{\max} = 30.8$ gbacteria molglucose−1). It was found that the surfactin production rate in continuous culture was not only a function of dilution rate but also varied with the initial concentration of glucose in the feed. A high steady state concentration of surfactin (18 mg L−1) was maintained in the culture at a dilution rate of 0.2 h−1 when glucose concentration in the feed was 0.25 g L−1. This is the first demonstration of continuous surfactin production and recovery using glucose as a carbon source. The production of surfactin is known to be related to the age of the microorganisms and a simple mathematical model has been constructed to show how the age‐related production can be quantified. Copyright © 2006 Society of Chemical Industry

In vitro cell growth of marine archaeal‐bacterial consortia during anaerobic oxidation of methane with sulfate

Anoxic sediment from a methane hydrate area (Hydrate Ridge, north-east Pacific; water depth 780 m) was incubated in a long-term laboratory experiment with semi-continuous supply of pressurized [1.4 MPa (14 atm)] methane and sulfate to attempt in vitro propagation of the indigenous consortia of archaea (ANME-2) and bacteria (DSS, Desulfosarcina/Desulfococcus cluster) to which anaerobic oxidation of methane (AOM) with sulfate has been attributed. During 24 months of incubation, the rate of AOM (measured as methane-dependent sulfide formation) increased from 20 to 230 micromol day(-1) (g sediment dry weight)(-1) and the number of aggregates (determined by microscopic counts) from 0.5 x 10(8) to 5.7 x 10(8) (g sediment dry weight)(-1). Fluorescence in situ hybridization targeting 16S rRNA of both partners showed that the newly grown consortia contained central archaeal clusters and peripheral bacterial layers, both with the same morphology and phylogenetic affiliation as in the original sediment. The development of the AOM rate and the total consortia biovolume over time indicated that the consortia grew with a doubling time of approximately 7 months (growth rate 0.003 day(-1)) under the given conditions. The molar growth yield of AOM was approximately 0.6 g cell dry weight (mol CH(4) oxidized)(-1); according to this, only 1% of the consumed methane is channelled into synthesis of consortia biomass. Concentrations of biomarker lipids previously attributed to ANME-2 archaea (e.g. sn-2-hydroxyarchaeol, archaeol, crocetane, pentamethylicosatriene) and Desulfosarcina-like bacteria [e.g. hexadecenoic-11 acid (16:1omega5c), 11,12-methylene-hexadecanoic acid (cy17:0omega5,6)] strongly increased over time (some of them over-proportionally to consortia biovolume), suggesting that they are useful biomarkers to detect active anaerobic methanotrophic consortia in sediments.

Growth of Geobacter sulfurreducens under nutrient‐limiting conditions in continuous culture

A system for growing Geobacter sulfurreducens under anaerobic conditions in chemostats was developed in order to study the physiology of this organism under conditions that might more closely approximate those found in the subsurface than batch cultures. Geobacter sulfurreducens could be cultured under acetate-limiting conditions with fumarate or Fe(III)-citrate as the electron acceptor at growth rates between 0.04 and 0.09 h(-1). The molar growth yield was threefold higher with fumarate as the electron acceptor than with Fe(III), despite the lower mid-point potential of the fumarate/succinate redox couple. When growth was limited by availability of fumarate, high steady-state concentrations were detected, suggesting that fumarate is unlikely to be an important electron acceptor in sedimentary environments. The half-saturation constant, Ks, for acetate in Fe(III)-grown cultures (10 microM) suggested that the growth of Geobacter species is likely to be acetate limited in most subsurface sediments, but that when millimolar quantities of acetate are added to the subsurface in order to promote the growth of Geobacter for bioremediation applications, this should be enough to overcome any acetate limitations. When the availability of electron acceptors, rather than acetate, limited growth, G. sulfurreducens was less efficient in incorporating acetate into biomass but had higher respiration rates, a desirable physiological characteristic when adding acetate to stimulate the activity of Geobacter species during in situ uranium bioremediation. These results demonstrate that the ability to study the growth of G. sulfurreducens under steady-state conditions can provide insights into its physiological characteristics that have relevance for its activity in a diversity of sedimentary environments.

A probable pathway in the degradation of 2-methoxyethanol by <i>Pseudomonas</i> species strain vb

A novel bacterium, Pseudomonas species strain VB, able to degrade 2-methoxyethanol was isolated from anaerobic sewage sludge. The bacterium utilizes 2-methoxyethanol as sole source of carbon and energy. In a time course experiment Pseudomonas sp. strain VB was grown in 2-methoxyethanol, ethylene glycol, glycollate, glyoxylate, oxalate and methanol. Strain VB was able to utilize all proposed intermediates except methanol. Comparable growth characteristics of Pseudomonas sp. strain VB grown in 2-methoxyethanol and proposed intermediates showed significant molar growth yields. These results indicate that ethylene glycol, glycollate, glyoxylate and oxalate might be intermediates in the degradation pathway of 2-methoxyethanol. Thus a reaction sequence: 2-methoxyethanol - ethylene glycol - glycollate - glyoxylate - oxalate is been proposed in this study. KEY WORDS: Degradation, 2-methoxyethanol, Pseudomonas species strain VB, metabolic pathway. Global Jnl Pure & Applied Sciences Vol.10(2) 2004: 239-242

The ars detoxification system is advantageous but not required for As(V) respiration by the genetically tractable Shewanella species strain ANA-3.

Arsenate [As(V); HAsO(4)(2-)] respiration by bacteria is poorly understood at the molecular level largely due to a paucity of genetically tractable organisms with this metabolic capability. We report here the isolation of a new As(V)-respiring strain (ANA-3) that is phylogenetically related to members of the genus Shewanella and that also provides a useful model system with which to explore the molecular basis of As(V) respiration. This gram-negative strain stoichiometrically couples the oxidation of lactate to acetate with the reduction of As(V) to arsenite [As(III); HAsO(2)]. The generation time and lactate molar growth yield (Y(lactate)) are 2.8 h and 10.0 g of cells mol of lactate(-1), respectively, when it is grown anaerobically on lactate and As(V). ANA-3 uses a wide variety of terminal electron acceptors, including oxygen, soluble ferric iron, oxides of iron and manganese, nitrate, fumarate, the humic acid functional analog 2,6-anthraquinone disulfonate, and thiosulfate. ANA-3 also reduces As(V) to As(III) in the presence of oxygen and resists high concentrations of As(III) (up to 10 mM) when grown under either aerobic or anaerobic conditions. ANA-3 possesses an ars operon (arsDABC) that allows it to resist high levels of As(III); this operon also confers resistance to the As-sensitive strains Shewanella oneidensis MR-1 and Escherichia coli AW3110. When the gene encoding the As(III) efflux pump, arsB, is inactivated in ANA-3 by a polar mutation that also eliminates the expression of arsC, which encodes an As(V) reductase, the resulting As(III)-sensitive strain still respires As(V); however, the generation time and the Y(lactate) value are two- and threefold lower, respectively, than those of the wild type. These results suggest that ArsB and ArsC may be useful for As(V)-respiring bacteria in environments where As concentrations are high, but that neither is required for respiration.

The characterization of a (nutritionally important) proline iminopeptidase from Eikenella corrodens.

Eikenella corrodens generates energy primarily through the oxidative deamination of specific amino acids, a process that is coupled to dissimilatory nitrate reduction to nitrite. Cell yields resulting from chemostat-growth of the organism in simple, chemically defined media containing varying amounts of proline confirm that this amino acid is a likely source of energy for E. corrodens in the oral environment. The importance of proline in ATP generation by the organism is reflected in molar growth yields, which showed that biomass production per mole of this amino acid was significantly higher than that for other amino acids. The organism was found to express, constitutively, the enzyme proline iminopeptidase, which releases proline from the N-terminus of small peptides. The enzyme was partially purified and characterized and found to exist in the cytoplasm as a 35 kDa monomer. Inhibition studies showed that the enzyme, although classified as a serine protease, also appears to require thiol groups for activity, a finding which is consistent with previous reports. The enzyme obeyed Michaelis-Menten kinetics and was found to have a Km value of 0.223 mM for the substrate proline-p-nitroanilide.

Energy metabolism of Actinobacillus actinomycetemcomitans during anaerobic and microaerobic growth in low- and high-potassium continuous culture.

Actinobacillus actinomycetemcomitans, a member of the gamma subclass of the Proteobacteria, has been implicated as the agent responsible for human periodontitis. In this study, A. actinomycetemcomitans 301-b was grown in fructose-limited chemostat cultures under anaerobic [redox potential (E(h))<-400 mV] and microaerobic (E(h)= -200 mV) conditions to characterize its energy metabolism. Effects of K(+) and Na(+) on growth and metabolism were also examined. In a control medium containing 5.2 mM K(+) and 24 mM Na(+), the molar growth yield on fructose (Y(fructose)) of microaerobic cultures was 1.3 times higher than the yield of anaerobic cultures at D < or =0.10 h(-1), but the difference in the Y(fructose) between microaerobic and anaerobic cultures decreased at D< or =0.10 h(-1). When the ATP yield from fermentation was estimated from the amounts of fructose consumed and acetate formed, the value of the microaerobic culture (2.49 mol ATP produced per mol fructose consumed) was lower than the anaerobic value [3.13 mol ATP (mol fructose)(-1)]. Therefore, ATP production from fermentation could not account for the increase in the Y(fructose) at D > 0.10 h(-1) and thus additional ATP was expected to be generated via respiration. Assuming that the Y(ATP) (g cells formed per mol ATP synthesized) was similar between anaerobic and microaerobic cultures, the estimated ATP yield from respiration was between 1.2 and 2.0 mol ATP (mol fructose)(-1) below D=0.10 h(-1) and decreased to 0.3 mol ATP (mol fructose)(-1) when D was increased to 0.19 h(-1). Such growth-rate-dependent decreases in the Y(fructose) and the estimated ATP production from respiration were also observed in a high-Na(+) (5.2 mM K(+) and 106 mM Na(+)) culture but not in a high-K(+) (81 mM K(+) and 24 mM Na(+)) culture. In the high-K(+) culture, the microaerobic Y(fructose) was 1.4-2.0 times higher than the anaerobic value and the respiration-derived ATP yield was estimated to be between 1.2 and 1.9 mol ATP (mol fructose)(-1) over a wide range of dilution rate. These results suggest that higher concentrations of extracellular K(+) are required for the respiration to occur in rapidly growing cells of A. actinomycetemcomitans.

Metabolic flux in cellulose batch and cellulose-fed continuous cultures of Clostridium cellulolyticum in response to acidic environment.

Clostridium cellulolyticum, a nonruminal cellulolytic mesophilic bacterium, was grown in batch and continuous cultures on cellulose using a chemically defined medium. In batch culture with unregulated pH, less cellulose degradation and higher accumulation of soluble glucides were obtained compared to a culture with the pH controlled at 7.2. The gain in cellulose degradation achieved with pH control was offset by catabolite production rather than soluble sugar accumulation. The pH-controlled condition improved biomass, ethanol and acetate production, whereas maximum lactate and extracellular pyruvate concentrations were lower than in the non-pH-controlled condition. In a cellulose-fed chemostat at constant dilution rate and pH values ranging from 7.4 to 6.2, maximum cell density was obtained at pH 7.0. Environmental acidification chiefly influenced biomass formation, since at pH 6.4 the dry weight of cells was more than fourfold lower compared to that at pH 7.0, whereas the specific rate of cellulose assimilation decreased only from 11.74 to 10.13 milliequivalents of carbon (g cells)(-1) h(-1). The molar growth yield and the energetic growth yield did not decline as pH was lowered, and an abrupt transition to washout was observed. Decreasing the pH induced a shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation. The acetate/ethanol ratio decreased as the pH declined, reaching close to 1 at pH 6.4. Whatever the pH conditions, lactate dehydrogenase was always greatly in excess. As pH decreased, both the biosynthesis and the catabolic efficiency of the pyruvate-ferredoxin oxidoreductase declined, as indicated by the ratio of the specific enzyme activity to the specific metabolic rate, which fell from 9.8 to 1.8. Thus a change of only 1 pH unit induced considerable metabolic change and ended by washout at around pH 6.2. C. cellulolyticum appeared to be similar to rumen cellulolytic bacteria in its sensitivity to acidic conditions. Apparently, the cellulolytic anaerobes studied thus far do not thrive when the pH drops below 6.0, suggesting that they evolved in environments where acid tolerance was not required for successful competition with other microbes.

Interrelationships between Growth Yield, ATPase and Adenylate Kinase Activities inZymomonas mobilis

The presence of cytoplasmic and membrane-bound adenylate kinase (EC 2.7,4.3) as well as inorganic pyrophosphatase (EC 3.6.1.1) was detected in Zymomonas mobilis ATCC 29191. An increase in the molar growth yield (Y X/S ) of Z. mobilis under aerobic growth conditions appeared to be in proportion to a reduction of membrane-bound adenylate kinase (mAK) and ATPase activities and to an increase in cytoplasmic adenylate kinase (AK) activity. Significant (1 - P < 0.01) multiple regressions were observed between the values of Y x (dependent variable), ATPase and AK or AK and mAK as independent variables, suggesting that a combined operation of these phosphohydrolases and phosphotransferases would be responsible for the biomass yield in Z. mobilis.

Interrelationships between Growth Yield, ATPase and Adenylate Kinase Activities in Zymomonas mobilis

The presence of cytoplasmic and membrane-bound adenylate kinase (EC 2.7.4.3) as well as inorganic pyrophosphatase (EC 3.6.1.1) was detected in Zymomonas mobilis ATCC 29191. An increase in the molar growth yield (YX/S) of Z. mobilis under aerobic growth conditions appeared to be in proportion to a reduction of membrane-bound adenylate kinase (mAK) and ATPase activities and to an increase in cytoplasmic adenylate kinase (AK) activity. Significant (1 — P < 0.01) multiple regressions were observed between the values of YX/S (dependent variable), ATPase and AK or AK and mAK as independent variables, suggesting that a combined operation of these phosphohydrolases and phosphotransferases would be responsible for the biomass yield in Z. mobilis.

Development of a Rhodococcus recombinant strain for degradation of products from anaerobic dechlorination of PCBs.

The Gram-positive bacterium Rhodococcus sp. strain RHA1, naturally containing the biphenyl pathway, was electroporated with a broad host range plasmid containing the 4-chlorobenzoate (4-CBA) degradation operon (fcb) isolated from Arthrobacter globiformis strain KZT1. The recombinant strain grew in medium containing 4-CBA and 4-chlorobiphenyl (4-CB) as the only source of carbon, with stoichiometric release of chloride and a molar growth yield on 4-CB that suggested utilization of both biphenyl rings. In resting cell assays, similar rates of degradation were observed for wild-type and recombinant strains for the most common eight congeners from the anaerobic dechlorination of Aroclor 1242, but the recombinant strain accumulated lower amounts of chlorinated meta-cleavage products and no 4-CBA. Recombinant cells inoculated at 10(4) cells/g into nonsterile soil amended with 4-CB grew to 6-10(5) cells/g, a density consistent with the 4-CB consumed. 4-CB was removed only in the inoculated soil, and the recombinant strain did not grow in the same soil when it was not amended with 4-CB. The fcb operon remained stable in the recombinant strain reisolated from soil after 60 days. This work provides proof of concept that a Rhodococcus strain constructed to grow on a PCB would grow in nonsterile soil if the appropriate chlorobiphenyl is available.

Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium.

A reinvestigation of cellulose degradation by Clostridium cellulolyticum in a bioreactor with pH control of the batch culture and using a defined medium was performed. Depending on cellulose concentration, the carbon flow distribution was affected, showing the high flexibility of the metabolism. With less than 6.7 g of cellulose liter(-1), acetate, ethanol, H(2), and CO(2) were the main end products of the fermentation and cellulose degradation reached more than 85% in 5 days. The electron flow from the glycolysis was balanced by the production of H(2) and ethanol, the latter increasing with increasing initial cellulose concentration. From 6.7 to 29.1 g of cellulose liter(-1), the percentage of cellulose degradation declined; most of the cellulase activity remained on the cellulose fibers, the maximum cell density leveled off, and the carbon flow was reoriented from ethanol to acetate. In addition to that of previously indicated end products, lactate production rose, and, surprisingly enough, pyruvate overflow occurred. Concomitantly the molar growth yield and the energetic yield of the biomass decreased. Growth arrest may be linked to sufficiently high carbon flow, leading to the accumulation of an intracellular inhibitory compound(s), as observed on cellobiose (E. Guedon, M. Desvaux, S. Payot, and H. Petitdemange, Microbiology 145:1831-1838, 1999). These results indicated that bacterial metabolism exhibited on cellobiose was distorted compared to that exhibited on a substrate more closely related to the natural ecosystem of C. cellulolyticum. To overcome growth arrest and to improve degradation at high cellulose concentrations (29.1 g liter(-1)), a reinoculation mode was evaluated. This procedure resulted in an increase in the maximum dry weight of cells (2,175 mg liter(-1)), cellulose solubilization (95%), and end product concentrations compared to a classical batch fermentation with a final dry weight of cells of 580 mg liter(-1) and 45% cellulose degradation within 18 days.