Anaerobic Growth Rate Research Articles

The proposition of an inexpensive chemically defined culture medium (MycoDef) for yeast studies

Chemically defined culture media (CDCMs) have their compositions qualitatively and quantitatively known. They are constituted of components able to meet the nutritional requirements of microorganisms. This study evaluated the employability of a multivitamin as the basis for the elaboration of a CDCM for experiments with yeasts. Candida albicans reference strains ATCC®90028™ and SC5314 were used. YNB® without amino acid (Difco Co.) was used as the standard in evaluations. For the preparation of the experimental culture medium (MycoDef), commercial multivitamin tablets had their coatings removed and were crushed until obtaining a fine powder; missing constituents were added. Comparisons were carried out evaluating aerobic and anaerobic planktonic growth rate, biofilm formation rate, whole-cell protein patterns by SDS-PAGE, minimum inhibitory concentrations and minimal fungicidal concentrations for clotrimazole, fluconazole, nystatin, and griseofulvin. Growth in MycoDef and YNB® did not differ between them (p>0.05) in both normoxic and anoxic conditions. Regarding employability for sensitivity testing, MycoDef showed performance like YNB®. The protein profiles of cells grown on both media did not differ in the number and positioning of bands. The results obtained allowed us to infer that MycoDef is a reliable low-cost culture medium useful for trials involving C. albicans.

Distinguishing functional from structural roles of conserved pore residues during formate translocation by the FocA anion channel.

The formate‐specific anion channel FocA of Escherichia coli belongs to the superfamily of homopentameric formate‐nitrite transporters (FNT). Minimally nine amino acid residues are conserved in the formate translocation pore of each protomer of the pentamer, including a histidine (H209) and a threonine (T91), both of which are crucial for bidirectional formate translocation through the pore. Information regarding in vivo functional or structural roles for the other seven conserved residues is limited, or nonexistent. Here, we conducted an amino acid‐exchange analysis of these seven conserved residues. Using an established formate‐responsive lacZ‐based assay to monitor changes in intracellular formate levels and anaerobic growth rate due to the inhibitory formate analog hypophosphite, we identified five of the seven residues analyzed to be important for the structural integrity of the pentamer, in particular, two highly conserved asparagine residues, N213 and N262. The remaining two conserved residues, K156 and N172, were essential for formate/hypophosphite translocation. K156 is located on the periplasmic fringe of the pore and aids the attraction of formate to the channel. Here, we show that this residue is also important for formate efflux from the cytoplasm to the periplasm, suggesting a role in formate release from the pore. N172 could be replaced by alanine with retention of low‐level bidirectional anion translocation function; however, exchange for threonine abolished anion translocation. N172 is, therefore, crucial for bidirectional formate translocation, possibly through its interaction with the conserved pore residue, T91.

The Nonmevalonate Pathway of Isoprenoid Biosynthesis Supports Anaerobic Growth of Listeria monocytogenes.

Isoprenoids are an essential and diverse class of molecules, present in all forms of life, that are synthesized from an essential common precursor derived from either the mevalonate pathway or the nonmevalonate pathway. Most bacteria have one pathway or the other, but the Gram-positive, facultative intracellular pathogen Listeria monocytogenes is unusual because it carries all the genes for both pathways. While the mevalonate pathway has previously been reported to be essential, here we demonstrate that the nonmevalonate pathway can support growth of strains 10403S and EGD-e, but only anaerobically. L. monocytogenes lacking the gene hmgR, encoding the rate-limiting enzyme of the mevalonate pathway, had a doubling time of 4 h under anaerobic conditions, in contrast to the 45 min doubling time of the wild type. In contrast, deleting hmgR in two clinical isolates resulted in mutants that grew significantly faster, doubling in approximately 2 h anaerobically, although they still failed to grow under aerobic conditions without mevalonate. The difference in anaerobic growth rate was traced to three amino acid changes in the nonmevalonate pathway enzyme GcpE, and these changes were sufficient to increase the growth rate of 10403S to the rate observed in the clinical isolates. Despite an increased growth rate, virulence was still dependent on the mevalonate pathway in 10403S strains expressing the more active GcpE allele.

An anaerobic membrane bioreactor using a hollow fiber membrane and biogas agitation

Many biomass disposal demonstration projects are based on anaerobic digestion. However, the excessively slow anaerobic microorganism growth rate is a drawback because a decreased anaerobic microorganism population limits methane fermentation's efficiency. To ensure operation at higher loads, this study used an anaerobic membrane bioreactor (AnMBR) for maintaining anaerobic microorganisms’ growth, and this article introduces a series of improvements to address the reactor's shortcomings. Finally, we chose to mix-the internal biogas and conducted the experiment using a hollow fiber AnMBR.•Introducing the design of a highly efficient and compact anaerobic membrane bioreactor (AnMBR).•Introducing the initial OLR and the changes of HRT, SRT, TS, and flux of the permeate in the AnMBR after gradually increasing the load.•Monitoring decomposition characteristics in the gas meter connection.

Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgaris Suf1 transporter.

Knowledge on the genetic factors important for the efficient expression of plant transporters in yeast is still very limited. Phaseolus vulgaris sucrose facilitator 1 (PvSuf1), a presumable uniporter, was an essential component in a previously published strategy aimed at increasing ATP yield in Saccharomyces cerevisiae. However, attempts to construct yeast strains in which sucrose metabolism was dependent on PvSUF1 led to slow sucrose uptake. Here, PvSUF1-dependent S.cerevisiae strains were evolved for faster growth. Of five independently evolved strains, two showed an approximately twofold higher anaerobic growth rate on sucrose than the parental strain (μ=0.19h-1 and μ=0.08h-1 , respectively). All five mutants displayed sucrose-induced proton uptake (13-50μmol H+ (g biomass)-1 min-1 ). Their ATP yield from sucrose dissimilation, as estimated from biomass yields in anaerobic chemostat cultures, was the same as that of a congenic strain expressing the native sucrose symporter Mal11p. Four out of six observed amino acid substitutions encoded by evolved PvSUF1 alleles removed or introduced a cysteine residue and may be involved in transporter folding and/or oligomerization. Expression of one of the evolved PvSUF1 alleles (PvSUF1I209F C265F G326C ) in an unevolved strain enabled it to grow on sucrose at the same rate (0.19h-1 ) as the corresponding evolved strain. This study shows how laboratory evolution may improve sucrose uptake in yeast via heterologous plant transporters, highlights the importance of cysteine residues for their efficient expression, and warrants reinvestigation of PvSuf1's transport mechanism.

Pseudomonas aeruginosa overexpression system of nitric oxide reductase for in vivo and in vitro mutational analyses

Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N2O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is produced from nitrite (NO2−) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study. Characterizing the variants of the five conserved Glu residues located around the heme/non-heme iron active center allowed us to establish how the anaerobic growth rate of cNOR-deficient strains expressing cNOR variants correlates with the in vitro enzymatic activity of the variants. Since bacterial strains require active cNOR to eliminate cytotoxic NO and to survive under denitrification conditions, the anaerobic growth rate of a strain with a cNOR variant is a good indicator of NO decomposition capability of the variants and a marker for the screening of functionally important residues without protein purification. Using this in vivo screening system, we examined the residues lining the putative proton transfer pathways for NO reduction in cNOR, and found that the catalytic protons are likely transferred through the Glu57 located at the periplasmic protein surface. The homologous cNOR expression system developed here is an invaluable tool for facile identification of crucial residues in vivo, and for further in vitro functional and structural studies.

Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield

BackgroundReduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast Saccharomyces cerevisiae. Anaerobic cultures of wild-type S. cerevisiae require formation of glycerol to maintain the intracellular NADH/NAD+ balance. Previously, functional expression of the Calvin-cycle enzymes ribulose-1,5-bisphosphate carboxylase (RuBisCO) and phosphoribulokinase (PRK) in S. cerevisiae was shown to enable reoxidation of NADH with CO2 as electron acceptor. In slow-growing cultures, this engineering strategy strongly decreased the glycerol yield, while increasing the ethanol yield on sugar. The present study explores engineering strategies to improve rates of growth and alcoholic fermentation in yeast strains that functionally express RuBisCO and PRK, while maximizing the positive impact on the ethanol yield.ResultsMulti-copy integration of a bacterial-RuBisCO expression cassette was combined with expression of the Escherichia coli GroEL/GroES chaperones and expression of PRK from the anaerobically inducible DAN1 promoter. In anaerobic, glucose-grown bioreactor batch cultures, the resulting S. cerevisiae strain showed a 31% lower glycerol yield and a 31% lower specific growth rate than a non-engineered reference strain. Growth of the engineered strain in anaerobic, glucose-limited chemostat cultures revealed a negative correlation between its specific growth rate and the contribution of the Calvin-cycle enzymes to redox homeostasis. Additional deletion of GPD2, which encodes an isoenzyme of NAD+-dependent glycerol-3-phosphate dehydrogenase, combined with overexpression of the structural genes for enzymes of the non-oxidative pentose-phosphate pathway, yielded a CO2-reducing strain that grew at the same rate as a non-engineered reference strain in anaerobic bioreactor batch cultures, while exhibiting a 86% lower glycerol yield and a 15% higher ethanol yield.ConclusionsThe metabolic engineering strategy presented here enables an almost complete elimination of glycerol production in anaerobic, glucose-grown batch cultures of S. cerevisiae, with an associated increase in ethanol yield, while retaining near wild-type growth rates and a capacity for glycerol formation under osmotic stress. Using current genome-editing techniques, the required genetic modifications can be introduced in one or a few transformations. Evaluation of this concept in industrial strains and conditions is therefore a realistic next step towards its implementation for improving the efficiency of first- and second-generation bioethanol production.

Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae

Anaerobic industrial fermentation processes do not require aeration and intensive mixing and the accompanying cost savings are beneficial for production of chemicals and fuels. However, the free-energy conservation of fermentative pathways is often insufficient for the production and export of the desired compounds and/or for cellular growth and maintenance. To increase free-energy conservation during fermentation of the industrially relevant disaccharide sucrose by Saccharomyces cerevisiae, we first replaced the native yeast α-glucosidases by an intracellular sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from Phaseolus vulgaris (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 ± 0.02h−1 (LmSPase) and 0.06 ± 0.01h−1 (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01h−1 and 0.08 ± 0.00h−1, respectively. Determination of the biomass yield in anaerobic sucrose-limited chemostat cultures was used to assess the free-energy conservation of the engineered strains. Replacement of intracellular hydrolase with a phosphorylase increased the biomass yield on sucrose by 31%. Additional replacement of the native proton-coupled sucrose uptake system by PvSUF1 increased the anaerobic biomass yield by a further 8%, resulting in an overall increase of 41%. By experimentally demonstrating an energetic benefit of the combined engineering of disaccharide uptake and cleavage, this study represents a first step towards anaerobic production of compounds whose metabolic pathways currently do not conserve sufficient free-energy.

The effects of elevated temperature and ocean acidification on the metabolic pathways of notothenioid fish.

The adaptations used by notothenioid fish to combat extreme cold may have left these fish poorly poised to deal with a changing environment. As such, the expected environmental perturbations brought on by global climate change have the potential to significantly affect the energetic demands and subsequent cellular processes necessary for survival. Despite recent lines of evidence demonstrating that notothenioid fish retain the ability to acclimate to elevated temperatures, the underlying mechanisms responsible for temperature acclimation in these fish remain largely unknown. Furthermore, little information exists on the capacity of Antarctic fish to respond to changes in multiple environmental variables. We have examined the effects of increased temperature and pCO2 on the rate of oxygen consumption in three notothenioid species, Trematomus bernacchii, Pagothenia borchgrevinki, and Trematomus newnesi. We combined these measurements with analysis of changes in aerobic and anaerobic capacity, lipid reserves, fish condition, and growth rates to gain insight into the metabolic cost associated with acclimation to this dual stress. Our findings indicated that temperature is the major driver of the metabolic responses observed in these fish and that increased pCO2 plays a small, contributing role to the energetic costs of the acclimation response. All three species displayed varying levels of energetic compensation in response to the combination of elevated temperature and pCO2. While P. borchgrevinki showed nearly complete compensation of whole animal oxygen consumption rates and aerobic capacity, T. newnesi and T. bernacchii displayed only partial compensation in these metrics, suggesting that at least some notothenioids may require physiological trade-offs to fully offset the energetic costs of long-term acclimation to climate change related stressors.

Magnesium capability to attenuate the toxicity of aluminum on the growth of Saccharomyces cerevisiae PE-2

Summary The magnesium (Mg) capability to attenuate the toxicity of aluminum (Al) for the trehalose content, anaerobic growth, viability and budding rate of Saccharomyces cerevisiae, was studied in this work. Fermentations were carried out in triplicate with sterilized and diluted sugar cane media (4% total reducing sugars/pH 4.0) containing different Al (0.0, 50, 100 and 150 mg L-1) and Mg (0.0, 50 and 100 mg L-1) concentrations. The media were inoculated with 1 mL of 1% (wet basis) yeast suspension and incubated at 30ºC, 70 rpm for 20 hours in orbital shaker. At specific times during fermentation portions of cell suspension were taken out and the biomass concentration, yeast viability, budding rate and trehalose content on cells determined. The increase of Al levels, from 0.0 up to 150 mg L-1, showed a reduction on the yeast growth of approximately 95%, 55% and 18% as Mg increased from 0.0 to 50 and 100 mg L-1, respectively. The trehalose content experienced its lowest reduction when greater amounts of Mg were added to the fermentation process. Cell viability showed greater reductions as the content of Al in the media increased. Magnesium effectively protected yeast cells against the deleterious effects of Al on cell growth, viability, budding and trehalose content.

Removal and upgrading of lignocellulosic fermentation inhibitors by in situ biocatalysis and liquid-liquid extraction.

Hydroxycinnamic acids are known to inhibit microbial growth during fermentation of lignocellulosic biomass hydrolysates, and the ability to diminish hydroxycinnamic acid toxicity would allow for more effective biological conversion of biomass to fuels and other value-added products. In this work, we provide a proof-of-concept of an in situ approach to remove these fermentation inhibitors through constituent expression of a phenolic acid decarboxylase combined with liquid-liquid extraction of the vinyl phenol products. As a first step, we confirmed using simulated fermentation conditions in two model organisms, Escherichia coli and Saccharomyces cerevisiae, that the product 4-vinyl guaiacol is more inhibitory to growth than ferulic acid. Partition coefficients of ferulic acid, p-coumaric acid, 4-vinyl guaiacol, and 4-ethyl phenol were measured for long-chain primary alcohols and alkanes, and tetradecane was identified as a co-solvent that can preferentially extract vinyl phenols relative to the acid parent and additionally had no effect on microbial growth rates or ethanol yields. Finally, E. coli expressing an active phenolic acid decarboxylase retained near maximum anaerobic growth rates in the presence of ferulic acid if and only if tetradecane was added to the fermentation broth. This work confirms the feasibility of donating catabolic pathways into fermentative microorganisms in order to ameliorate the effects of hydroxycinnamic acids on growth rates, and suggests a general strategy of detoxification by simultaneous biological conversion and extraction.

Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways for cytosolic acetyl-CoA synthesis

Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1Δ acs2Δ S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27h−1 were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.20h−1) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While demonstrating that yeast ACS can be fully replaced, this study demonstrates that further modifications are needed to achieve optimal in vivo performance of the alternative reactions for supply of cytosolic acetyl-CoA as a product precursor.

Analysis of Gene Expression as Marker of Relevant Metabolisms, in Three <i>Acidithiobacillus ferrooxidans</i> Strains, in Different Growth Conditions

Acidithiobacillus ferrooxidansis a chemiolithoautotrophic Gram-negative bacterium widely spread in ambient temperature bioleaching processes. Several strains ofAt. ferrooxidanswere isolated and studied and, some time later, questions arise about whether it was a species with a wide metabolic variation or a group of closely related species. Advances in molecular biology, phylogeny and genomics have shed some light onAt. ferrooxidansstrains and allows their grouping according to their relations. However, significant challenges remains to be met, such as understanding how a particular strain faces environmental challenges and how a particular kind of adaptive response affects the growth and activity of the strain. The purpose of this study was to identify differential expression signals betweenAt. ferrooxidansstrains −with different abundances and dynamics− present in the bioleaching system at Escondida mine. Culture characterization and DNA macroarrays techniques provided some answers. Analysis of growth curves showed that IESL 32 had the highest anaerobic growth rate, while aerobic growth was similar for all strains. It was shown that though the phylogenetic analysis based on 16S rRNA sequences suggested a close relation between IESL 32 and the type strain ATCC 23270, the growth curves and the expression profile showed that the type strain and strain D2 had the closest similarity. Growth experiments under different conditions, together with the comparative analysis of gene expressions inAt. ferrooxidans, could be a springboard for future investigations of strain characterization to broaden our knowledge about adaptation, metabolic strategies, regulation and microbial diversity in industrial processes.

Prediction of Weak Acid Toxicity inSaccharomyces cerevisiaeUsing Genome-Scale Metabolic Models

The use of lignocellulosic biomass is critical for the economic production of transportation fuels and chemicals in renewable bioprocesses. While biomass is an abundant resource, necessary pretreatment to yield fermentable monosaccharides produces toxic compounds that dramatically affect fermentation performance. Weak acids such as acetic acid play an important role in the toxicity of lignocellulosic hydrolysate to Saccharomyces cerevisiae, a commonly used industrial organism. In order to explore the ramifications of weak acid inhibition on cellular metabolism, we adapted a genome-scale metabolic model of S. cerevisiae to describe toxicity of acetic acid by a decoupling mechanism. We evaluated the performance of the model in predicting growth rates and ethanol production characteristics under aerobic and anaerobic cultivations. We found that the model was able to capture the decreased growth during aerobic cultivations in the presence of acetic acid, but was unable to capture the increase in ethanol yield observed. The model was able to predict anaerobic growth rates and ethanol yields; however, at conditions of higher toxicity levels, discrepancies arose. We expect that a model such as this may find application in the optimization of lignocellulose-based bioprocesses in which there exists a critical economic trade-off between neutralization costs and product yields.

Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae

Xylose is the main pentose and second most abundant sugar in lignocellulosic feedstocks. To improve xylose utilization, necessary for the cost-effective bioconversion of lignocellulose, several metabolic engineering approaches have been employed in the yeast Saccharomyces cerevisiae. In this study, we describe the rational metabolic engineering of a S. cerevisiae strain, including overexpression of the Piromyces xylose isomerase gene (XYLA), Pichia stipitis xylulose kinase (XYL3) and genes of the non-oxidative pentose phosphate pathway (PPP). This engineered strain (H131-A3) was used to initialize a three-stage process of evolutionary engineering, through first aerobic and anaerobic sequential batch cultivation followed by growth in a xylose-limited chemostat. The evolved strain H131-A3-ALCS displayed significantly increased anaerobic growth rate (0.203±0.006h−1) and xylose consumption rate (1.866gg−1h−1) along with high ethanol conversion yield (0.41g/g). These figures exceed by a significant margin any other performance metrics on xylose utilization and ethanol production by S. cerevisiae reported to-date. Further inverse metabolic engineering based on functional complementation suggested that efficient xylose assimilation is attributed, in part, to the elevated expression level of xylose isomerase, which was accomplished through the multiple-copy integration of XYLA in the chromosome of the evolved strain.

Increased Ethanol Productivity in Xylose-Utilizing Saccharomyces cerevisiae via a Randomly Mutagenized Xylose Reductase

Baker's yeast (Saccharomyces cerevisiae) has been genetically engineered to ferment the pentose sugar xylose present in lignocellulose biomass. One of the reactions controlling the rate of xylose utilization is catalyzed by xylose reductase (XR). In particular, the cofactor specificity of XR is not optimized with respect to the downstream pathway, and the reaction rate is insufficient for high xylose utilization in S. cerevisiae. The current study describes a novel approach to improve XR for ethanol production in S. cerevisiae. The cofactor binding region of XR was mutated by error-prone PCR, and the resulting library was expressed in S. cerevisiae. The S. cerevisiae library expressing the mutant XR was selected in sequential anaerobic batch cultivation. At the end of the selection process, a strain (TMB 3420) harboring the XR mutations N272D and P275Q was enriched from the library. The V(max) of the mutated enzyme was increased by an order of magnitude compared to that of the native enzyme, and the NADH/NADPH utilization ratio was increased significantly. The ethanol productivity from xylose in TMB 3420 was increased ∼40 times compared to that of the parent strain (0.32 g/g [dry weight {DW}] × h versus 0.007 g/g [DW] × h), and the anaerobic growth rate was increased from ∼0 h(-1) to 0.08 h(-1). The improved traits of TMB 3420 were readily transferred to the parent strain by reverse engineering of the mutated XR gene. Since integrative vectors were employed in the construction of the library, transfer of the improved phenotype does not require multicopy expression from episomal plasmids.

PGM2 overexpression improves anaerobic galactose fermentation in Saccharomyces cerevisiae

BackgroundIn Saccharomyces cerevisiae galactose is initially metabolized through the Leloir pathway after which glucose 6-phosphate enters glycolysis. Galactose is controlled both by glucose repression and by galactose induction. The gene PGM2 encodes the last enzyme of the Leloir pathway, phosphoglucomutase 2 (Pgm2p), which catalyses the reversible conversion of glucose 1-phosphate to glucose 6-phosphate. Overexpression of PGM2 has previously been shown to enhance aerobic growth of S. cerevisiae in galactose medium.ResultsIn the present study we show that overexpression of PGM2 under control of the HXT7'promoter from an integrative plasmid increased the PGM activity 5 to 6 times, which significantly reduced the lag phase of glucose-pregrown cells in an anaerobic galactose culture. PGM2 overexpression also increased the anaerobic specific growth rate whereas ethanol production was less influenced. When PGM2 was overexpressed from a multicopy plasmid instead, the PGM activity increased almost 32 times. However, this increase of PGM activity did not further improve aerobic galactose fermentation as compared to the strain carrying PGM2 on the integrative plasmid.ConclusionPGM2 overexpression in S. cerevisiae from an integrative plasmid is sufficient to reduce the lag phase and to enhance the growth rate in anaerobic galactose fermentation, which results in an overall decrease in fermentation duration. This observation is of particular importance for the future development of stable industrial strains with enhanced PGM activity.

An evolved Escherichia coli strain for producing hydrogen and ethanol from glycerol

Glycerol is an attractive feedstock for biofuels since it accumulates as a byproduct during biodiesel operations; hence, here we consider converting glycerol to hydrogen using the formate hydrogen lyase system of Escherichia coli which converts pyruvate to hydrogen. Starting with E. coli BW25113 frdC that lacks fumarate reductase (this mutation reduces repression of hydrogen synthesis during glycerol fermentation) and by using both adaptive evolution and chemical mutagenesis combined with a selection method based on increased growth on glycerol, we obtained an improved strain, HW2, that produces 20-fold more hydrogen in glycerol medium (0.68±0.16mmol/L/h). HW2 also grows 5-fold faster (0.25±0.01/h) than BW25113 frdC on glycerol, so it achieves a reasonable anaerobic growth rate. Corroborating the increase in hydrogen production, glycerol dehydrogenase activity in HW2 increased 4-fold compared to BW25113 frdC. In addition, a whole-transcriptome study revealed that several pathways that would decrease hydrogen yields were repressed in HW2 (fbp, focA, and gatYZ) while a beneficial pathway which encodes enolase was induced. Ethanol production was also increased 5-fold in the evolved HW2 strain.

Variations in the microbiology of peritonsillar abscess

Microbiologic studies are routinely performed in the assessment of peritonsillar abscess (PTA). Though the bacterial growth rates of PTA are expected to be uniform due to high accessibility and reasonable sterility, they demonstrate a vast range of results, which is partially explained by the differing culturing methods and incubation times. Our aim was to retrospectively examine the changing features identified in the occurrence of PTA bacterial growth rates over a period of seven years. A retrospective study was undertaken on all cases of PTA admitted from January 1996 to December 2002. Details regarding sex, age and country of birth were obtained. Population data and the maximum residue level (MRL) of antibiotics in food were collected. Bacteriologic studies were analysed for gram stain, aerobic and anaerobic culture results, and also antibiotic sensitivities, if obtained. Four hundred and fifty-seven consecutive hospitalisations due to PTA were identified; 281 patients who had 310 hospitalisations with known results of the microbiologic studies were included. The most common pathogens were Streptococcus pyogenes and Prevotella. A statistically significant escalation was seen in the anaerobic growth rate from 6.8% of cases in 1996 to 37% in 1999. A similar change, though not significant, was noticed with the polymicrobial growth rate. None of the parameters investigated showed any statistically significant influence on this tendency. These results may clarify the immense range of bacterial study results reported, suggest a change in the biologic behaviour of the studied bacteria and direct further research.

The role of the nitrate respiration element of Thermus thermophilus in the control and activity of the denitrification apparatus

The nitrate conjugative element (NCE) encodes the ability to respire nitrate in the facultative Thermus thermophilus NAR1 strain. This process is carried out by two heterotetrameric enzymes that catalyse the oxidation of NADH (Nrc) and the reduction of nitrate (Nar), whose expression is activated by the NCE-encoded transcription factors DnrS and DnrT. We report the presence of NCE in other facultative strains of T. thermophilus and analyse its role in subsequent steps of the denitrification pathway. We encountered that nrc mutants of denitrifying strains show a decrease in anaerobic growth rates not only with nitrate, but also with nitrite, NO and N(2)O, which is concomitant to their lower NADH oxidation activities in vitro. We show that nitrate, nitrite and NO are activating signals for transcription of nrc in these strains. Finally, we demonstrate that DnrS and DnrT are required for anaerobic growth not only with nitrate, but also with nitrite, NO and N(2)O. These data allow us to conclude that: (i) Nrc constitutes the main electron donor for the four reductases of the denitrification pathway, and (ii) the NCE controls the expression of the whole denitrification pathway and the repression of the aerobic respiration through the transcription factors DnrS and DnrT.